UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Spin labelling studies of carbohydrate polymers Aplin, John Dalzell 1979

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1979_A1 A64.pdf [ 12.35MB ]
Metadata
JSON: 831-1.0060837.json
JSON-LD: 831-1.0060837-ld.json
RDF/XML (Pretty): 831-1.0060837-rdf.xml
RDF/JSON: 831-1.0060837-rdf.json
Turtle: 831-1.0060837-turtle.txt
N-Triples: 831-1.0060837-rdf-ntriples.txt
Original Record: 831-1.0060837-source.json
Full Text
831-1.0060837-fulltext.txt
Citation
831-1.0060837.ris

Full Text

SPIN LABELLING STUDIES OF CARBOHYDRATE POLYMERS by JOHN DALZELL APLIN B.A., Oxford U n i v e r s i t y , 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1979 © J o h n D a l z e l l A p l i n , 1979 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t n f Chemistry  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 w e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 D a t e 15th Feb 1979 - i i -ABSTRACT N i t r o x i d e r e p o r t e r groups were used i n two d i f f e r e n t c a p a c i t i e s f o r esr s t u d i e s of n a t u r a l l y o c c u r r i n g carbohydrates. As 'probes'—-small molecules i n s o l u t i o n — i n aqueous polysaccharide s o l s and gels, t h e i r r o t a t i o n a l and t r a n s l a t i o n a l d i f f u s i o n was found to be independent of the presence of polymer d e s p i t e the pronounced e f f e c t of the l a t t e r on v i s c o s -i t y and other macroscopic p r o p e r t i e s . As ' l a b e l s ' — c o v a l e n t l y attached to the macromolecule—they were more u s e f u l , esr lineshape a n a l y s i s y i e l d i n g i n f o r m a t i o n about r o t a t i o n a l r e o r i e n t a t i o n , a c c e s s i b i l i t y from s o l u t i o n and p r o x i m i t y to neighbouring unpaired s p i n s . Cyanogen bromide a c t i v a t i o n , carbodiimide and chloroacetamide coupling and other methods were used to a f f i x s p i n l a b e l s to agarose, Sepharoses, a l g i n a t e , c e l l u l o s e , wood, and the t e r m i n a l non-reducing s i a l i c acids of f e t u i n , bovine submaxillary mucin, the outer surface of the human e r y t h r o c y t e , and t i s s u e s e c t i o n s from human colon. In the s o l u t i o n g l y c o p r o t e i n s and at the c e l l s u r f a c e , r o t a t i o n a l r e o r i e n t a t i o n of the l a b e l s was found to be l a r g e l y independent of the o v e r a l l r o t a t i o n a l d i f f u s i o n of the c e l l or macromolecule. The g e l - s o l - g e l t r a n s i t i o n of l a b e l l e d agarose was examined, and i t was found that the two forms, as defined m a c r o s c o p i c a l l y , are not c l e a r l y d i s t i n g u i s h -able at the microscopic l e v e l by the m o b i l i t y of the polymer backbone. The Sepharose 4B gel bead was s t u d i e d using n i t r o x i d e as a model l i g a n d . D i p o l a r i n t e r a c t i o n s between immobilised l a b e l s were observed, suggesting the p o s s i b i l i t y of m u l t i p l e s i t e b i n d i n g of a p r o t e i n i n s o l u t i o n - H i -to the s t a t i o n a r y phase i n chromatographic sepa r a t i o n s . The randomness of the d i s t r i b u t i o n of attachment s i t e s was t e s t e d using s p i n d i l u t i o n , s o l v e n t -mediated shrinkage of the beads and e l e c t r o n exchange w i t h paramagnetic ions i n the s o l u t i o n phase. Only the l a s t revealed any inhomogeneity. The dependence of r o t a t i o n a l c o r r e l a t i o n time (T) on the l e n g t h of the 'spacer arm' connecting the n i t r o x i d e to the matrix was s t u d i e d f o r alkylamine and o l i g o g l y c i n e l i n k i n g groups, each of which was found to behave i n s i m i l a r f a s h i o n , T decreasing towards an asymptote w i t h i n c r e a s i n g spacer l e n g t h . The data i n d i c a t e d that u n i t s c o n t a i n i n g ^ 12 bonds should be optimal f o r a f f i n i t y chromatography, and l i t e r a t u r e r e p o r t s provided ample c o r r o b o r a t i o n . The dependence on c o n c e n t r a t i o n of e l e c t r o n exchange between n i t r o x -ides a f f i x e d to the surface of c e l l u l o s e , and paramagnetic ions i n s o l u t i o n , provoked the d e f i n i t i o n and d e r i v a t i o n of a c c e s s i b i l i t y parameters (z/) which i n c r e a s e d , as expected, w i t h spacer l e n g t h . N e g a t i v e l y charged reducing"agents (ascorbate, d i t h i o n i t e ) gained access to n i t r o x i d e s even at ^ 0.35 nm from the s u r f a c e , w h i l e f e r r o u s ions achieved only p a r t i a l reduc-t i o n ; at ^ 1.8 nm the three agents were e q u a l l y e f f e c t i v e . These r e s u l t s were discussed i n terms of proposed models f o r the c e l l u l o s e - w a t e r i n t e r -face. Measurement of e l e c t r o n - e l e c t r o n d i p o l a r i n t e r a c t i o n s enabled a new c a l c u l a t i o n of surface area to be made and the e f f e c t of d r y i n g to be s t u d i e d . - i v -TABLE OF CONTENTS INTRODUCTION 1 CHAPTER I . BACKGROUND IA: S t r u c t u r e and Chemistry of N i t r o x i d e s . . . 6 IB: E l e c t r o n Spin Resonance 10 IC: N i t r o x i d e Esr 11 ID: Polysaccharides 39 IE: I n t e r a c t i o n s at Surfaces and Wi t h i n Gels 43 IF: Glycoproteins 47 IG: Previous Studies 52 IH: Or g a n i s a t i o n of the Thesis 60 References 62 CHAPTER I I . DIFFUSION OF NITROXIDES IN POLYSACCHARIDE SOLUTIONS AND GELS IIA: I n t r o d u c t i o n 69 IIB : Tumbling 72 IIC: Exchange 75 IID: D i s c u s s i o n . 76 References 80 CHAPTER I I I . GELLING PROCESSES I I I A : Agarose ( i ) S t r u c t u r e of Agarose Gels . . . . 82 ( i i ) Spin L a b e l l i n g of Agarose . . . . 89 ( i i i ) Chain M o b i l i t y i n Agarose . . . . 91 I I I B : A l g i n a t e ( i ) S t r u c t u r e of A l g i n i c A c i d and Agarose Gels ' 106 ( i i ) L a b e l l i n g Procedure 110 ( i i i ) Spectra 113 References 120 CHAPTER IV. SEPHAROSE 4B AS A MATRIX FOR CHROMATOGRAPHY IVA: I n t r o d u c t i o n 123 IVB: Cyanogen Bromide A c t i v a t i o n 128 -v-IVC: Esr Studies of Sepharose 4B ( i ) Spectra 131 ( i i ) Q u a n t i t a t i o n of the Label . . . . . . 139 ( i i i ) Spin D i l u t i o n 141 ( i v ) Solvent-Mediated Shrinkage . . . . 146 (v) S e l e c t i v e Broadening 148 ( v i ) Other Chemistry • 153 ( v i i ) Summary 157 IVD: N i t r o x i d e as a Model f o r Immobilised Ligands ( i ) Spacer E f f e c t s 159 ( i i ) Solvent Cycles . 168 ( i i i ) L i t e r a t u r e Survey 177 References 190 CHAPTER V. CELLULOSE VA: Occurrence, Importance and S t r u c t u r e . . . 193 VB: Cyanogen Bromide-Mediated L a b e l l i n g of C e l l u l o s e and Wood ( i ) Unperturbed Spectra 204 ( i i ) Broadening by Paramagnetic 'Probe' Ions i n S o l u t i o n . . . . 211 ( i i i ) Reduction 221 VC: Triazine-Mediated L a b e l l i n g 225 VD: Surface Area 229 VE: Summary and Prospects 232 References 239 CHAPTER VI. GLYCOPROTEINS VIA: I n t r o d u c t i o n 242 VIB: BSM and F e t u i n 243 VIC: Carbodiimide Coupling 246 VID: Carbodiimide-Mediated L a b e l l i n g ( i ) BSM; 249 ( i i ) F e t u i n 252 ( i i i ) Non-Glycoprotein C o n t r o l M a t e r i a l s . 255 ( i v ) Human Tissue Sections and Erythrocytes 256 VIE: Periodate Oxidation-Reductive Amination . 258 VIF: The M o b i l i t y of Sugars i n Glycoproteins . 262 References 268 CHAPTER V I I . SUMMARY 271 CHAPTER V I I I . EXPERIMENTAL VIIIA: E l e c t r o n Spin Resonance 278 VII I B : Chapter I I 280 - v i -V I I IC: Chapter I I I . . . 281 VIIID: Chapter IV 284 V I I I E : Chapter V 288 V I I I F : Chapter VI 289 References 293 APPENDIX 1 2 9 4 APPENDIX 2 • 3 0 1 - v i i -LIST OF TABLES Table CHAPTER I 1-1 Reactions of the n i t r o x i d e f u n c t i o n a l group 8 1-2 P r i n c i p a l tensor components f o r three n i t r o x i d e s . . 15 1-3 I s o t r o p i c n i t r o g e n h y p e r f i n e coupling constant of (2) as a f u n c t i o n of solvent 18 CHAPTER I I I I - l Saccharides used i n Chapter I I 70 II-2 Linewidths and s p l i t t i n g s f o r three concentrations of (2) i n water and agarose g e l 76 CHAPTER IV IV-1 Data obtained from m i c r o a n a l y s i s of l a b e l l e d Sepharose 4B and c o n t r o l s 140 IV-2 D e r i v a t i v e s of Sepharose 4B 165 IV-3 Data from Figure IV-18 - . 173 IV-4 L i t e r a t u r e r e p o r t s on spacer arms 184 CHAPTER V V - l Low- and hig h - c o n c e n t r a t i o n a c c e s s i b i l i t y parameters f o r probe ions at l a b e l l e d c e l l u l o s e surfaces . . . 220 CHAPTER VI VI-1 C o r r e l a t i o n times of n i t r o x i d e s attached to gl y c o p r o t e i n s • 263 CHAPTER VII VII-1 C o r r e l a t i o n times f o r l a b e l l e d g l y c o p r o t e i n s and polysaccharides i n s o l u t i o n . 272 APPENDIX 1 A-1 Rates of r e o r i e n t a t i o n f o r v a r i o u s chromatographic matri x polysaccharides l a b e l l e d using (2) a f t e r C N B r - a c t i v a t i o n . 298 - v i i i -LIST OF FIGURES Figure CHAPTER I 1-1 Bond lengths and bond angles from X-ray data f o r two n i t r o x i d e s 7 1-2 N i t r o x i d e s used i n the t h e s i s w i t h t h e i r code numbers 9 1-3 Schematic r e p r e s e n t a t i o n of the n i t r o x i d e group showing the unpaired e l e c t r o n i n the n i t r o g e n p z o r b i t a l 12 1-4 T- and g - a n i s o t r o p i e s i n d i - t - b u t y l n i t r o x i d e o r i e n t e d i n a host c r y s t a l 14 1-5 Esr s p e c t r a of m a g n e t i c a l l y d i l u t e d i - t - b u t y l n i t r o x i d e as a p o l y c r y s t a l l i n e s o l i d and i n viscous and non-viscous s o l u t i o n 17 1-6 The asymptotic approach of the s p l i t t i n g 2G to 2T zg w i t h decreasing temperature 19 I-;7 Esr s p e c t r a of (3) i n aqueous g l y c e r o l s o l u t i o n s . . .21 1-8 A n i s o t r o p i c r o t a t i o n of a n i t r o x i d e o c c u r r i n g as a r e s u l t of s t e r i c hindrance by a macromolecule i n s o l u t i o n to which i t i s attached 24 1-9 Simulated s p e c t r a f o r i s o t r o p i c a l l y tumbling n i t r o x i d e s 26 I-10 Powder spectrum showing the heights d i and d and the s p l i t t i n g 2T 29 1-11 The v a r i a t i o n of the parameter d i / d i n powder sp e c t r a w i t h c o n c e n t r a t i o n of (2) 30 1-12 Esr lineshape of (2) i n aqueous s o l u t i o n as a f u n c t i o n of c o n c e n t r a t i o n of n i c k e l sulphate . . . 36 1-13 Peak-to-peak l i n e w i d t h of the c e n t r e - f i e l d resonance of (2) as a f u n c t i o n of c o n c e n t r a t i o n of Ni(H 20)2+ 38 1-14 C l a s s i f i c a t i o n of d i f f e r e n t types of hydrated p o l y s a c c h a r i d e s t r u c t u r e s . . 41 1-15 S i m p l i f i e d schematic view of a f f i n i t y chromatography 44 CHAPTER I I I I - l Esr s p e c t r a of 0.5 mM s o l u t i o n s of (9) i n water, 285% aqueous sucrose, sodium and calcium a l g i n a t e s 73 - i x -I I - 2 Dependence of T on macroscopic v i s c o s i t y i n a s e r i e s of aqueous sucrose s o l u t i o n s . . . . . . . 74 I I - 3 Esr spec t r a showing exchange broadening of (2) at three d i f f e r e n t concentrations i n water and 3% agarose g e l 77 CHAPTER I I I I I I - l X-ray s t r u c t u r e s f o r agarose 85 I I I - 2 Proposed two-step mechanism f o r g e l a t i o n 88 I I I - 3 Esr s p e c t r a of s p i n l a b e l l e d agarose 93 I I I - 4 C o r r e l a t i o n time as a f u n c t i o n of temperature i n s p i n l a b e l l e d agarose 94 I I I - 5 Arrhenius p l o t of c o r r e l a t i o n times i n l a b e l l e d agarose 97 I I I - 6 Esr sp e c t r a of s p i n l a b e l l e d agarose i n the presence of NiSOij. 101 I I I - 7 Arrhenius p l o t of c o n t r i b u t i o n s to c o r r e l a t i o n time from polysaccharide backbone motions i n l a b e l l e d agarose 103 I I I - 8 F r a c t i o n a l c o n t r i b u t i o n of agarose backbone motions to o v e r a l l tumbling r a t e as a f u n c t i o n of temperature 104 I I I - 9 Proposed s t r u c t u r e s f o r a l g i n a t e 109 111-10 Esr spec t r a of l a b e l l e d a l g i n a t e s o l and g e l . . . 114 I I I - l l Esr spectrum of manganous a l g i n a t e g e l . . . . . . 117 CHAPTER IV IV- 1 Proposed mechanism f o r cyanogen bromide a c t i v a t i o n of h y d r o x y l i c polymers 130 IV-2 Esr spec t r a of (34) 132 IV-3 Esr spec t r a of (44) 133 IV-4 Esr spectrum of p r e c i p i t a t e d agarose, CNBr-a c t i v a t e d and l a b e l l e d w i t h (9) 135 IV-5 Esr sp e c t r a of (2) and (34) i n water and g l y c e r o l . 138 IV-6 Esr spec t r a r e s u l t i n g from s p i n d i l u t i o n i n l a b e l l i n g of Sepharose 4B 143 IV-7 Loading as a f u n c t i o n of the composition of the r e a c t i o n mixture i n s p i n d i l u t i o n 144 IV-8 Esr spec t r a of (34) at low temperature and a f t e r d r y i n g 147 IV-9 Esr sp e c t r a of (34) i n n i c k e l sulphate s o l u t i o n s . 149 IV-10 Spectra from previous f i g u r e showing r e l a t i v e a m p l i f i c a t i o n s 150 IV-11 Dependence of the c e n t r e - f i e l d l i n e w i d t h of (34) on c o n c e n t r a t i o n of n i c k e l ions 151 IV-12 Esr spec t r a of (34), (35) and (33) . . . . . . . . 156 IV-13 Esr sp e c t r a of (34), (35), (37) and (39) 161 -x-IV-14 Esr sp e c t r a of (44), (45), (46), (47) and (48) . . . 162 IV-15 Esr sp e c t r a of (34), (35), (38), (40), (41), ' (42) and (43) 163 IV-16 Dependence of mean c o r r e l a t i o n time on spacer le n g t h 164 IV-17 Esr spec t r a of (34) as a f u n c t i o n of solvent environment . . 170 IV- 18 Esr spec t r a of (39) as a f u n c t i o n of solvent environment 172 CHAPTER V V- l S t r u c t u r e of c e l l u l o s e . 195 V-2 Proposed s t r u c t u r e s f o r the c e l l u l o s e m i c r o f i b r i l . 198 V-3 Chemical and u l t r a s t r u c t u r a l makeup of wood . . . . 200 V-4 Esr spec t r a of c e l l u l o s e s l a b e l l e d using the CNBr method . . . 206 V-5 Esr spec t r a of (51) i n solve n t s of d i f f e r e n t s w e l l i n g a b i l i t y 209 V-6 N i c k e l ions as probes i n l a b e l l e d c e l l u l o s e s without and w i t h spacer arms 212 V-7 Spectra a and b from previous f i g u r e showing r e l a t i v e a m p l i f i c a t i o n s 213 V-8 F e r r i c y a n i d e as a probe i o n i n l a b e l l e d c e l l u l o s e s 216 V-9 Increase of n i t r o x i d e c e n t r e - f i e l d l i n e w i d t h as a f u n c t i o n of n i c k e l i o n co n c e n t r a t i o n i n l a b e l l e d c e l l u l o s e s 218 V-10 Increase of n i t r o x i d e c e n t r e - f i e l d l i n e w i d t h as a f u n c t i o n of f e r r i c y a n i d e i o n co n c e n t r a t i o n i n l a b e l l e d c e l l u l o s e s 219 V - l l The p a r t i a l reducing a c t i o n of ferrous ions on l a b e l l e d c e l l u l o s e 223 V-12 Esr sp e c t r a of t r i a z i n e - l a b e l l e d c e l l u l o s e s . . . . 227 V- 13 Schematic p i c t u r e of a simple g e n e r a l i z e d h y d r o x y l i c surface c o n t a i n i n g pores 234 CHAPTER VI VI- 1 Esr spec t r a of f e t u i n and BSM l a b e l l e d using the EDC coupling procedure 250 VI-2 O u t l i n e of experiments i n v o l v e d i n B S M s p i n l a b e l l i n g and assay . . 251 VI-3 O u t l i n e of experiments i n v o l v e d i n f e t u i n l a b e l l i n g and assay 254 VI-4 Esr sp e c t r a of l a b e l l e d t i s s u e s e c t i o n s and human ery t h r o c y t e s 257 VI-5 Esr spec t r a of f e t u i n , periodate o x i d i s e d and r e d u c t i v e l y aminated, i n s o l u t i o n and a f t e r i m m o b i l i s a t i o n 261 - x i -APPENDIX 1 A-1 Esr s p e c t r a of Sephadex G50 and Sepharoses 2B and 6B, a l l CNBr-actlvated and reacted w i t h (2) 295 A-2 Esr s p e c t r a of CL Sepharoses, CNBr-activated and reacted w i t h (2) ..' 296 A-3 Esr sp e c t r a of l a b e l l e d CL Sepharoses i n water and i n the presence of 2M NiSOit 297 A-4 Esr sp e c t r a of l a b e l l e d CL Sepharose 4B as a f u n c t i o n of solvent environment 299 A-5 Esr sp e c t r a of l a b e l l e d sodium carboxymethyl c e l l u l o s e and l o c u s t bean gum 300 - x i i -GLOSSARY OF TERMS, SYMBOLS AND ABBREVIATIONS Terms and u n i t s the usages of which are standard or infrequent i n the t h e s i s are omitted; i n the l a t t e r case a d e f i n i t i o n normally appears at the appropriate place i n the t e x t . Numbered compounds are given i n the t e x t i n parentheses ( 1 ) , equations as [1] and references s u p e r s c r i p t e d . ag i s o t r o p i c hyperfine coupling constant C c o n c e n t r a t i o n (also expressed using square brackets) D e l e c t r o n - e l e c t r o n d i p o l a r coupling tensor d,di peak-to-peak i n t e n s i t i e s i n powder s p e c t r a as defined i n Figure 1-10 E^ a c t i v a t i o n energy G gauss g g-tensor g Q i s o t r o p i c g-value 2 nuclear g-factor g„,,»-g„„»g„,, p r i n c i p a l values of the g-tensor p a r a l l e l and perpendicular components of an a x i a l l y sym-metr i c g-tensor H e x t e r n a l l y a p p l i e d magnetic f i e l d ; increases from l e f t to r i g h t i n a l l f i g u r e s Hi microwave f i e l d Hz Hertz fi Planck's constant d i v i d e d by 2TT h peak-to-peak height of a f i r s t d e r i v a t i v e esr s i g n a l h ( a f t e r a number) hours h(0,±l) values of h f o r the three n i t r o x i d e resonances corresponding to m = 0,±1 - x i i i -n u clear s p i n operator (vector) exchange i n t e g r a l degrees K e l v i n r a t e constant f o r e l e c t r o n exchange r a t e constant f o r molecular c o l l i s i o n magnetic quantum number a s s o c i a t e d w i t h the component of I along the d i r e c t i o n of H magnetic quantum number a s s o c i a t e d w i t h the component of S along the d i r e c t i o n of H p r o b a b i l i t y f a c t o r r e l a t i n g c o l l i s i o n s to e l e c t r o n exchange electron-nucleus vector mean nearest-neighbour d i s t a n c e between n i t r o x i d e s e l e c t r o n s p i n operator temperature hype r f i n e c o u p l i n g tensor s p l i t t i n g between outer s p e c t r a l features i n a powder spectrum = 2T„ = 2T when T -»- °° r zz p r i n c i p a l values of the h y p e r f i n e tensor a n i s o t r o p i c p a r t of the h y p e r f i n e tensor p a r a l l e l and perpendicular components of an a x i a l l y symmetric hype r f i n e tensor s p i n - l a t t i c e or l o n g i t u d i n a l r e l a x a t i o n time 'true' s p i n - s p i n or transverse r e l a x a t i o n time apparent s p i n - s p i n r e l a x a t i o n time volume to volume r a t i o weight to weight r a t i o i n i t i a l slope a c c e s s i b i l i t y parameter f o r a n i t r o x i d e at a surface - x i v -f i n a l s l o p e a c c e s s i b i l i t y p a r a m eter f o r a n i t r o x i d e a t a s u r f a c e a n o m e r i c c o n f i g u r a t i o n s o f g l y c o s i d i c l i n k a g e s Bohr magneton n u c l e a r Bohr magneton d i f f e r e n c e between t h e r e s o n a n t f r e q u e n c i e s o f two c o l l i d i n g s p e c i e s v i s c o s i t y e l e c t r o n exchange f r e q u e n c y m o l e c u l a r c o l l i s i o n f r e q u e n c y d e n s i t y o f n i t r o x i d e s i n an a r r a y i n one, two, o r t h r e e d i m e n s i o n s c o r r e l a t i o n t i m e f o r r o t a t i o n a l r e o r i e n t a t i o n mean c o r r e l a t i o n t i m e c o r r e l a t i o n t i m e f o r r o t a t i o n a l r e o r i e n t a t i o n o f a p o l y -s a c c h a r i d e c h a i n i n a g e l c o r r e l a t i o n t i m e f o r r e o r i e n t a t i o n o f a n i t r o x i d e about bonds j o i n i n g i t t o a ma c r o m o l e c u l e o r s u r f a c e c o r r e l a t i o n t i m e f o r r o t a t i o n o f a ma c r o m o l e c u l e c o r r e l a t i o n t i m e f o r r o t a t i o n a l r e o r i e n t a t i o n of a p o l y -s a c c h a r i d e c h a i n i n a s o l u t i o n o r s o l l i f e t i m e o f a c o l l i s i o n a l complex c o r r e l a t i o n t i m e s f o r r e o r i e n t a t i o n about p e r p e n d i c u l a r axes f r a c t i o n a l c o n t r i b u t i o n t o r e o r i e n t a t i o n r a t e o f a l a b e l made by t h e polymer ( s o l o r g e l ) t o w h i c h i t i s a t t a c h e d -XV-Ato(C) A w 0 d i r e c t l y a t t a c h e d l a b e l , d i r e c t l y l a b e l l e d d o u b l e h e l i x exchange e x t r a c y c l i c , e x o c y c l i c X - f o l d h e l i x f r e e ends g e l h y d r o g e l l a b e l , s p i n l a b e l l o a d i n g , e x t e n t of l a b e l l i n g , e f f i c i e n c y o f l a b e l l i n g p e a k - t o - p e a k w i d t h o f a f i r s t d e r i v a t i v e a d s o r p t i o n s i g n a l i n an e s r s p e c t r u m A w as a f u n c t i o n o f c o n c e n t r a t i o n o f a probe i o n A w f o r t h e c e n t r e - f i e l d n i t r o x i d e l i n e (m^ . = 0) sometimes used t o denote a s y s t e m where no s p a c e r group has been i n t r o d u c e d as a s e p a r a t e s t e p ; does n o t i n d i c a t e t h a t t h e n i t r o x i d e i s j o i n e d by o n l y one bond t o t h e c a r b o h y d r a t e a h e l i x c o m p r i s i n g two i n t e r w o v e n s i n g l e - s t r a n d e d h e l i c e s may i n v o l v e e l e c t r o n s and l e a d t o l i f e t i m e ( H e i s e n b e r g ) b r o a d e n i n g ; o r atoms o r m o l e c u l e s , as d u r i n g t h e exchange of w a t e r i n t h e f i r s t c o o r d i n a t i o n s p h e r e of a h y d r a t e d i o n , w h i c h i s c h e m i c a l exchange used i n t e r c h a n g e a b l y t o d e s c r i b e f u n c t i o n a l groups i n s u g a r s n o t a t t a c h e d t o r i n g c a r b o n atoms X r e p e a t u n i t s i n t h e polymer make up one c o m p l e t e t u r n t e r m i n a l o l i g o m e r i c u n i t s o f polymer m o l e c u l e s ( u s u a l l y i n g e l s ) t h o u g h t t o e x p e r i e n c e i n c r e a s e d m o b i l i t y owing t o t h e i r l a c k o f p a r t i c i p a t i o n i n i n t e r m o l e c u l a r a s s o c i a t i o n s p r e d o m i n a n t l y d i s o r d e r e d permanent c o n t i n u o u s n e t w o r k c o n -s i s t i n g o f more t h a n one component, w i t h t h e appearance of a s o l i d aqueous g e l n i t r o x i d e c o v a l e n t l y a t t a c h e d t o a m a c r o m o l e c u l e number of n i t r o x i d e s p e r m o n o s a c c h a r i d e r e s i d u e , o r number of m o n o s a c c h a r i d e s p e r n i t r o x i d e , i n a l a b e l l e d c a r b o h y d r a t e m a t r i x , s u p p o r t m e l t i n g , d e n a t u r a t i o n N i I : C , N i ( l T ) , n i c k e l ( I I ) , Nx , n i c k e l o u s Nuc n u c l e a t i o n -xvi-g e l o r s o l i d used i n a f f i n i t y and r e l a t e d chromato-g r a p h i c t e c h n i q u e s g e l ->- s o l t r a n s i t i o n f o r m a l i s m s f o r a d i v a l e n t i o n p i t c h p r o b e r h e o l o g y s e t t i n g , g e l a t i o n s o l s p a c e r arm, l i n k i n g u n i t , e t c . s p i n s y n e r e s x s n u c l e o p h i l e f o r m a t i o n o f m i c r o c r y s t a l l i t e s , s u f f i c i e n t l y l a r g e t o be s t a b l e , i n a homogeneous sy s t e m by s u c c e s s i v e b i m o l e c u l a r a s s o c i a t i o n s . I n pol y m e r s a s s o c i a t i o n i s u s u a l l y between r e g i o n s o f two m o l e c u l e s , n o t i n t h e f i r s t i n s t a n c e i n v o l v -i n g t h e e n t i r e c h a i n l e n g t h a x i a l d i s t a n c e between r a d i a l l y c o i n c i d e n t p o i n t s i n a h e l i x s m a l l n i t r o x i d e o r p a r a m a g n e t i c i o n p r e s e n t i n s o l u t i o n t h e s c i e n c e o f f l o w ; r e l a t i o n between s h e a r s t r e s s and s h e a r r a t e s o l -> g e l t r a n s i t i o n d i s p e r s i o n o f m i c r o s c o p i c p a r t i c l e s w i t h t h e appearance o f a l i q u i d b i f u n c t i o n a l group j o i n i n g t h e n i t r o x i d e - c o n t a i n i n g r i n g t o a s u r f a c e o r polymer used on o c c a s i o n t o denote t h e u n p a i r e d e l e c t r o n o f t h e n i t r o x i d e ( n o r m a l l y s l o w ) c o n t r a c t i o n o f a g e l w i t h i n c r e a s e i n i t s m e c h a n i c a l s t r e n g t h and e x p u l s i o n o f s o l v e n t t r a p p i n g , masking, o c c l u s i o n PRT TBA PRBA Ac pyr SL - x v i i -terms used to des c r i b e phenomena wherein l a b e l s attached to gels or s o l i d s become i n a c c e s s i b l e to reagents present i n s o l u t i o n , or experience l a r g e increases i n c o r r e l a t i o n time as a r e s u l t of increased s t e r i c hindrance by the pol y -saccharide p e r i o d a t e - r e s o r c i n o l c o l o u r i m e t r i c assay f o r s i a l i c a c i d t h i o b a r b i t u r i c a c i d assay f o r f r e e s s i a l i c a c i d p e r i o d a t e - r e s o r c i n o l assay adapted f o r k e t o s i d i c a l l y bouttd- " s i a l i c a c i d acetate CH3CO pyruvate k e t a l \ / J t polysaccharide " . DNA BSM BSA HSA EDC CMC D-Glcp deo x y r i b o n u c l e i c a c i d bovine submaxillary mucin bovine serum albumin human serum albumin N-ethyl-N'-dimethylaminopropyl carbodiimide h y d r o c h l o r i d e H CHjCH2N = C = N(CH 2)3 N(CH3)2 Cl" N-cyclohexyl-N'-(2-morpholinoethyl) carbodiimide metho-p-toluene sulphonate ^ ^ - N = C = N C H 2 C H 2 - N ^ J ) O j S r - ^ ^ - C H j D-glucopyranose H.OH D-Galp - x v i i i -D-galactopyranose CH,OH H HO D-GlcNAcp 1-deoxy-l-acetamido-D-glycopyranose CH 2OH H.OH D-GalNAcp 1-deoxy-l-acetamido-D-galactopyranose L-GulpA L-guluronic a c i d H •° T — r H.OH H NHAc D-Manp D-mannopyranose CH 2OH D-ManpA D-mannuronic a c i d C 0 2 H H.OH NANA sa N-Acetylneuraminic a c i d s i a l i c acids ( d e r i v a t i v e s of neuraminic acid) CO,H Asn Gin asparagme ^ C 0 2 H H 2 N C O C H 2 C H ^ ' • NH 2 glutamine HgNCOCHgCHgCH / C 0 2 H Ser s e r i n e HOCH 2CH: , C 0 2 H "NH, -xix-threonine CH,CHCI-k I ^NH 2 OH 2 C02H NH 2 a s p a r t i c a c i d H0 2CCH 2CH^ glutamic a c i d H0 2CCH 2CH 2CH^ l y s i n e H 2NCH 2CH 2CH 2CH 2CH^ ^C0 2H N H 2 .C02H NH 2 -XX-ACKNOWLEDGEMENT T h i s p r o j e c t c o u l d n o t have been c o m p l e t e d w i t h o u t t h e s y n t h e t i c e f f o r t s o f L i a n e E v e l y n n o r t h e f r i e n d s h i p o f Diane M i l l e r , f o r b o t h o f w h i c h I am most g r a t e f u l . M i c h a e l B e r n s t e i n gave generous a c c e s s t o u n p u b l i s h e d d a t a . S t i m u l a t i n g d i s c u s s i o n s a r e acknowledged w i t h D r s . John W a t e r t o n , who a l s o p r o v i d e d a c c e s s t o u n p u b l i s h e d d a t a and commented l i b e r a l l y on t h e m a n u s c r i p t , M i c h a e l Adam, G e o f f r e y H e r r i n g and o t h e r members o f t h e C h e m i s t r y Department. My th a n k s a r e a l s o due t o D r s . P h i l Read and Don B r o o k s , P r o f . C h a r l e s C u l l i n g and C h a r l e s Ramey f o r l a b o r a t o r y s p a c e , a d v i c e and a s s i s t a n c e d u r i n g t h e e x e c u t i o n o f e x p e r i m e n t s d e s c r i b e d i n C h a p t e r V I , and t o t h e I . W. K i l l a m Founda-t i o n f o r f i n a n c i a l s u p p o r t . My g r e a t e s t debt o f t h a n k s , however, i s t o Dr. L a u r i e H a l l , whose t i r e l e s s h e l p and encouragement made t h e r e s e a r c h d e s c r i b e d i n t h i s t h e s i s p l e a s u r a b l e and w o r t h w h i l e . - x x i -da bienes Fortuna que no estan e s c r i t o s L u i s de Gongora INTRODUCTION C a r b o h y d r a t e s a r e amongst t h e most abundant t y p e s o f m o l e c u l e on t h e p l a n e t . They a r e found as r l b o s e d e r i v a t i v e s w i t h i n t h e n u c l e i c acids''", 2 and as components o f a n t i b o d i e s , enzymes, and hormones ; t h e y p l a y an i m p o r t a n t and as y e t i l l - u n d e r s t o o d r o l e a t t h e o u t e r s u r f a c e o f t h e c e l l 3 membrane as g l y c o l i p i d s and g l y c o p r o t e i n s ; t h e y p r o v i d e p l a n t s , t h r o u g h 4 s t a b l e carbon-oxygen bonds i n s t a r c h , w i t h a means o f s t o r i n g t h e sun's e n e r g y , and o u r s e l v e s w i t h t h e a b i l i t y t o s t o r e c h e m i c a l energy as glycogen"', as w e l l as b e i n g t h e main s t r u c t u r a l m a t e r i a l s i n p l a n t s ^ and a r t h r o p o d s ^ , t h e l a r g e s t c l a s s o f o r g a n i s m . The c o n s t i t u t i o n o f c a r b o h y d r a t e p o l y m e r s can be b r o k e n down i n t o p r i m a r y s t r u c t u r e , w h i c h r e f e r s t o m o n o s a c c h a r i d e c o m p o s i t i o n , sequence, l i n k a g e p o s i t i o n and o r i e n t a t i o n , and s u b s t i t u t i o n o f f u n c t i o n a l groups by a c e t a t e s , s u l p h a t e s , p y r u v a t e s and o t h e r s ; and h i g h e r - o r d e r s t r u c t u r e , w h i c h r e f e r s t o t h e t h r e e - d i m e n s i o n a l arrangement o f t h e monomer i n t o t h e polymer and i n t e r m o l e c u l a r i n t e r a c t i o n s between polymer c h a i n s . As w i t h p r o t e i n s t h e s e h i g h e r - o r d e r s t r u c t u r e s a r e p a r t l y d e t e r m i n e d by t h e p r i m a r y s t r u c t u r e , b u t a r e a l s o dependent on e n v i r o n m e n t a l p a r a m e t e r s l i k e pH, t e m p e r a t u r e and i o n i c s t r e n g t h . A s o l u t i o n o f a t y p i c a l p o l y s a c c h a r i d e , however, i s l i k e l y t o c o n t a i n more d i f f e r e n t c o n f o r m a t i o n s t h a n a s o l u t i o n o f a g l o b u l a r p r o t e i n . The p o t e n t i a l v a r i e t y o f p o l y m e r i c c a r b o h y d r a t e s , w h i c h a r i s e s f r o m t h e number o f f u n c t i o n a l groups p r e s e n t i n t h e c o r r e s p o n d i n g monomers, can 1 2 be seen by comparing the number of d i f f e r e n t dimers that may be formed from a simple aminoacid l i k e g l y c i n e — o n e — w i t h the number from a hexose such as glucopyranose, which i s eleven, a l l of which have been prepared, and most of 8 which occur i n one form or another i n Nature . S i x d i f f e r e n t t r i m e r s may be prepared from three d i f f e r e n t aminoacids, w h i l e 1056 t r i m e r s are p o s s i b l e from three d i f f e r e n t hexoses! In a d d i t i o n , to t h i s , w h i l e polypeptides and n u c l e i c a c i d s (which are not henceforth included w i t h other carbohydrates) are normally l i n e a r polymers, i n s a c c h a r i d i c polymers branching may be considered to be the norm. Another f a c t o r c o n t r i b u t i n g to the complexity of carbohydrate-c o n t a i n i n g species i s r e l a t e d to the manner i n which b i o s y n t h e s i s occurs. U n l i k e n u c l e i c a c i d s and p r o t e i n s , which are synthesized u s i n g templates, monosaccharides are added s e q u e n t i a l l y or block-wise to polymers by enzymic means. S u b s t i t u t i o n s such as the a d d i t i o n or removal of acetate groups occur i n the same f a s h i o n . Such a process i s a good deal l e s s exact, and leads to a v a r i a b i l i t y of s t r u c t u r e which may be expressed as p o l y d i s p e r s i t y , i n which a mixture of molecules w i t h s l i g h t l y d i f f e r e n t sequence or w i t h d i f f e r e n t arrangements of s u b s t i t u e n t s or branch-points occurs, and p o l y m o l e c u l a r i t y , i n which a mixture of molecules e x i s t s , a l l w i t h the same 9 s t r u c t u r e but w i t h d i f f e r e n t molecular weights or degrees of p o l y m e r i s a t i o n . A l l these f a c t o r s c o n t r i b u t e to the d i f f i c u l t y of e s t a b l i s h i n g the primary s t r u c t u r e of many carbohydrate polymers and to the danger of assuming a p a r t i c u l a r s t r u c t u r e to be r e p r e s e n t a t i v e or general. Indeed, the erroneous g e n e r a l i s a t i o n that carbohydrates tend to be random polymers w i t h l i m i t e d a p t i t u d e f o r s p e c i f i c i n t e r a c t i o n l e d to t h e i r being neglected i n biochemistry f o r some years. Recently, however, i t has emerged that the v a r i a b i l i t y of carbohydrate s t r u c t u r e s gives r i s e to an immense and 3 complex coding p o t e n t i a l which may have reached i t s most s o p h i s t i c a t e d e v o l u t i o n a r y stage i n the development of s e l f - n o n s e l f r e c o g n i t i o n i n the mammalian immune s y s t e m ^ . I t may be c l o s e r to the t r u t h to say that the complexity of a c e r t a i n carbohydrate does not n e c e s s a r i l y r e f l e c t the q u a l i t y of i t s i n f o r m a t i o n content. A l l t h i s has meant that the examination of primary s t r u c t u r e has occupied carbohydrate chemists and biochemists to a r a t h e r l a t e r date than t h e i r contemporaries i n the p r o t e i n f i e l d , and that higher-order s t r u c t u r e s have been ra t h e r neglected. Nevertheless, i t i s c l e a r that s p e c i f i c i n t e r a c t i o n s depend u l t i m a t e l y upon three-dimensional shape and i f only by analogy w i t h the development of p r o t e i n and n u c l e i c a c i d chemistry, hi g h e r -order s t r u c t u r e s i n carbohydrate biopolymers w i l l assume an i n c r e a s i n g importance as s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s begin to be e l u c i d a t e d . I t i s e q u a l l y c e r t a i n that w h i l e chemical methods have been most important i n the determination of carbohydrate primary s t r u c t u r e , p h y s i c a l methods w i l l play the major part i n determining shapes. Ju s t as i n the former case,.formidable problems have to be overcome; molecular hetero-geneity i s one, but f o r the s p e c t r o s c o p i s t there are two e q u a l l y c h a l l e n g -in g d i f f i c u l t i e s ' : that of overlapping s i g n a l s i n complex mixtures, and that presented by the small amounts of m a t e r i a l a v a i l a b l e from many b i o -l o g i c a l sources. Both of these may be avoided by the i n c o r p o r a t i o n of a s e n s i t i v e e x t r i n s i c probe i n t o the system. In the present work t h i s 11 12 f u n c t i o n i s f u l f i l l e d by n i t r o x i d e - c o n t a i n i n g m o l e c u l e s — ' s p i n l a b e l s ' ' —6 whose unpaired e l e c t r o n s may be detected at concentrations > 10 M by e l e c t r o n s p i n resonance ' , , g i v i n g i n f o r m a t i o n p r i n c i p a l l y about the motion of the n i t r o x i d e and i t s microenvironment. This i n f o r m a t i o n , which may be r e l a t e d to the shape and i n t e r a c t i o n s of species i n the v i c i n i t y of 4 the unpaired e l e c t r o n , i s conveniently obtained, s i n c e few b i o l o g i c a l systems cont a i n paramagnetic species which resonate at the same frequency as the n i t r o x i d e . I t s p r i c e , however, i s the p e r t u r b a t i o n of the system from i t s n a t i v e s t a t e . In a d d i t i o n , i f the l a b e l i s to be c o v a l e n t l y attached to the macromolecule, i d e a l l y , s p e c i f i c chemistry i s r e q u i r e d . One suspects that i t i s t h i s l a t t e r problem, r a t h e r onerous i n carbohydrates where many hydroxyl s u b s t i t u e n t s of approximately equal r e a c t i v i t y are present, has i n h i b i t e d other s c i e n t i s t s i n t e r e s t e d i n the f i e l d , because very few previous s t u d i e s of carbohydrate polymers by s p i n l a b e l l i n g have been made. The work to be described i n the present t h e s i s deals w i t h s e v e r a l d i f f e r e n t n a t u r a l l y - o c c u r r i n g polymeric carbohydrates w i t h emphasis on d i s c o v e r i n g the u t i l i t y of the s p i n l a b e l l i n g method i n a v a r i e t y of three-dimensional contexts. I t i s hoped that the experiments which have been performed are u s e f u l from the poi n t of view of the fundamentals of carbo-hydrate s t r u c t u r e , but that they a l s o have r a m i f i c a t i o n s w e l l beyond i n the areas of the i n d u s t r i a l and biochemical uses of polymeric carbohydrates and s t i l l more g e n e r a l l y i n polymer sci e n c e . In order to provide the r e q u i s i t e background to a study which i s i n t e r d i s c i p l i n a r y i n nature and to d e l i n e a t e the connecting l i n k s between those d i s c i p l i n e s and the d i f f e r -ent p arts of the t h e s i s , i t has proved necessary to burden the more informed reader w i t h a r a t h e r long f i r s t chapter, w i t h p o r t i o n s of which he w i l l already be f a m i l i a r . I f t h i s be merely t o l e r a t e d , i t i s hoped that sub-sequent chapters provide more enjoyable substance. 5 References 1. E. Harbers, G. F. Domagk, and W. M u l l e r , ' I n t r o d u c t i o n to N u c l e i c A c i d s , ' Reinhold, New York, 1968. 2. M. I. Horowitz, and W. Pigman, Ed., 'The Glycoconjugates,' Academic, New York, 1977. 3. R. C. Hughes, 'Membrane Gl y c o p r o t e i n s , ' Butterworths, London, 1976. 4. R. L. W h i s t l e r , Ed., 'Methods i n Carbohydrate Chemistry,' V o l . IV, Academic, New York, 1963. 5. D. J . Manners, Adv. Carbohydr. Chem., 12, 262-298 (1957). 6. V o l . I l l of reference 4. 7. A. B. F o s t e r , and J . M. Webber, Adv. Carbohydr. Chem., 15, 371-393 (1960). 8. N. Sharon, 'Complex Carbohydrates,' Addison-Wesley, Reading, Massa-ch u s e t t s , 1975. 9. G. 0. Asp i n - a l l , 'Polysaccharides,' Pergamon, Oxford, 1970. 10. S. Hakomori, and A. Kobata i n 'The Antigens,' Ed. M. S e l a , V o l . 2, 79-140, Academic, 1974. 11. L. J . B e r l i n e r , Ed., 'Spin L a b e l l i n g , ' Academic, New York, 1976. 12. G. I. L i k h t e n s h t e i n , 'Spin L a b e l l i n g Methods i n Molecular B i o l o g y , ' W i l e y - I n t e r s c i e n c e , New York, 1976. CHAPTER I BACKGROUND IA: S t r u c t u r e and Chemistry of N i t r o x i d e s N i t r o x i d e s were f i r s t synthesized i n about 1960"^. They are amongst the most s t a b l e of f r e e r a d i c a l s and t h e i r convenience as s p i n l a b e l s d e r i v e s from an a b i l i t y to p a r t i c i p a t e i n r e a c t i o n s that do not i n v o l v e the unpaired e l e c t r o n . Two types of h e t e r o c y c l i c n i t r o x i d e have been used i n the present work: p y r r o l i d i n e - 1-oxyl d e r i v a t i v e s and p i p e r i d i n e - l - o x y l d e r i v a t i v e s X v r _ _ | 0 2 where X may be one of a v a r i e t y of f u n c t i o n a l groups. X-ray s t u d i e s have shown that such r i n g s are o f t e n deformed r e l a t i v e to corresponding c y c l o -9 pentane and cyclohexane d e r i v a t i v e s . The C - N - C group may be planar or 3 pyramidal. The shapes of 4- k e t o - 2 , 2 , 6 , 6 - t e t r a m e t h y l - p i p e r i d i n e - l - o x y l (1) 4 and 3 - c a r b o x y l - 2 , 2 , 5 , 5 - t e t r a m e t h y l - p y r r o l i d i n e - l - o x y l (10) are shown i n 2 Figure 1-1 . Table 1-1 shows the types of r e a c t i o n s that the n i t r o x i d e f u n c t i o n a l i t y can undergo"*. These may i n f l u e n c e the c o n d i t i o n s and sequences of r e a c t i o n s used i n s y n t h e s i z i n g l a b e l s and i n c o r p o r a t i n g them i n t o the system of i n t e r e s t . Reaction 1 i s avoided by the use of n i t r o x i d e s l a c k i n g a-hydrogens; s t e r i c hindrance accorded by the presence of four 6 7 Figure 1-1: Bond lengths and bond angles from X-ray data f o r two n i t r o x i d e s . ( a ) ( 1 0 ) 1 + ; (b)(1) (adapted from reference 2). 8 methyl s u b s t i t u e n t s at the two a-carbons may a l s o reduce the r e a c t i v i t y of the r a d i c a l centre i n other processes. O x i d a t i o n by the n i t r o x i d e of a number of reducing agents both organic and i n o r g a n i c has been used i n the present s t u d i e s to i n v e s t i g a t e the a c c e s s i b i l i t y of the group i n hetero-geneous systems. The s t a b i l i t y of the r a d i c a l centre means that the second f u n c t i o n a l group X may be v a r i e d c o n s i d e r a b l y ^ without the danger of i n t e r -molecular r e a c t i o n s i n any of c a t e g o r i e s 1-5. Thus X may be t a i l o r e d to create a s i t e - d i r e c t e d probe f o r the system of i n t e r e s t . The n i t r o x i d e s used i n the present work are shown i n Figure 1-2 w i t h t h e i r code numbers. The 2 , 2 , 6 , 6 - t e t r a m e t h y l p i p e r i d i n e - l - o x y l moiety i s abbreviated on occasion as SL; thus compound (2) becomes tL^N-SL. Table 1-1: Reactions of the n i t r o x i d e f u n c t i o n a l group"* (1) D i s p r o p o r t i o n a t i o n 2 0 OH + 0 (2) Free R a d i c a l r e a c t i o n OR 0 (3) Carbon-nitrogen bond cleavage 0 (4) Oxidation 2 + 2Br (5) Reduction S 6 OH (6) Reaction w i t h strong H OH CI CI 9 F i g u r e 1-2: N i t r o x i d e s used i n t h e t h e s i s w i t h t h e i r code numbers. 10 IB: E l e c t r o n Spin Resonance E l e c t r o n s p i n resonance (esr) occurs i n a paramagnetic molecule when t r a n s i t i o n s between the Zeeman l e v e l s , whose degeneracy may be l i f t e d by the a p p l i c a t i o n of a magnetic f i e l d H, are induced by an electromagnetic f i e l d Hi of frequency v, where the separation of the Zeeman l e v e l s = ggH [1] h being Planck's constant, g the Bohr magneton (eft/2m), m the mass of the e l e c t r o n , and g a dimensionless parameter r e l a t e d to the e f f e c t i v e magnetic moment of the e l e c t r o n (y^) by = -ges [2] where Sfi i s the s p i n angular momentum v e c t o r . D i f f e r e n c e s i n the Zeeman energy between d i f f e r e n t molecules are described as changes i n g from i t s f r e e e l e c t r o n s p i n - o n l y value of 2.00232, as a r e s u l t of s p i n - o r b i t c o u p l -i n g . Thus the g-value i s used to c h a r a c t e r i s e the p o s i t i o n of the reson-ance i n the frequency spectrum. As i s c l e a r from equation [ 1 ] , the frequency of resonance i s dependent upon the a p p l i e d f i e l d ; most esr experiments, i n c l u d i n g those described h e r e i n , are conducted at X-band, that i s , at about 9.5 GHz (or a wavelength of about 3 cm), which corresponds to an e x t e r n a l f i e l d of the order of 3.4 kG. The experiment takes place i n a resonant c a v i t y whose dimensions depend on the frequency of H j . In the simple l i n e a r response experiments described here, although upward and downward t r a n s i t i o n s are induced w i t h equal p r o b a b i l i t y , net absorption of microwave energy from H-^  occurs at resonance as a r e s u l t of the greater p r o p o r t i o n of spins present i n the lower energy s t a t e . Because H^ i s weak, r e l a x a t i o n occurs s u f f i c i e n t l y r a p i d l y to enable the s p i n system to remain c l o s e to i t s e q u i l i b r i u m energy d i s t r i b u t i o n . Absorption of energy i s monitored w i t h the a i d of phase-11 s e n s i t i v e d e t e c t i o n using f i e l d modulation at 1 0 5 Hz (provided by Helmholz c o i l s on each s i d e of the c a v i t y ) and recorded as the f i r s t d e r i v a t i v e of the absorption s i g n a l . IC: N i t r o x i d e Esr The s p i n Hamiltonian f o r a n i t r o x i d e e l e c t r o n i s given by i\ = ft (Zeeman) + ^ (hyperfine) + ( d i p o l a r ) + f| (exchange) = 3-H-g-S^ + S_*T*I ' ' + i>.D«_S + J'1'S [3] where ^ and 1 are the e l e c t r o n and nuclear s p i n operators r e s p e c t i v e l y , and the nuclear Zeeman term has been omitted. The e l e c t r o n - g - v a l u e , and T, the el e c t r o n - n u c l e a r h y p e r f i n e coupling, are r e q u i r e d to be expressed as second-rank tensors because they represent direction-dependent q u a n t i t i e s which i n t u r n r e f l e c t the symmetry of the molecule. The h y p e r f i n e c o u p l i n g c o n s i s t s of a contact term a g i - i [4] where the i s o t r o p i c coupling constant a A i s a s c a l a r defined by a Q =-fggg NB N U(0)| 2 [5] and f ( 0 ) i s the unpaired e l e c t r o n wave f u n c t i o n at the nucleus; and a d i p o l a r i n t e r a c t i o n given by ( I - S ) r 2 - 3 ( l y r ) ( S ' r ) I.T'.S = -gBg Ne N r 5 [6] where _r i s the electron-nucleus d i s t a n c e v e c t o r . While the contact term r e q u i r e s s - o r b i t a l character i n ¥ (r) ( |>(0) | 2 ^ 0),.the d i p o l a r term disappears i f the e l e c t r o n d i s t r i b u t i o n has s p h e r i c a l symmetry. In the present case (Figure 1-3) the p r i n c i p a l h y p e r f i n e i n t e r a c t i o n occurs between the e l e c t r o n , shown here i n the n i t r o g e n 2p^ o r b i t a l , and the n i t r o g e n nucleus (1=1) 12 Figure 1-3: Schematic r e p r e s e n t a t i o n of the n i t r o x i d e group showing the unpaired e l e c t r o n i n the ni t r o g e n p o r b i t a l (adapted from reference 6) 13 le a d i n g to the c h a r a c t e r i s t i c 3 - l i n e spectrum (Figure 1-4^) i n which t r a n s -i t i o n s obey the s e l e c t i o n r u l e s Am^ . = 0, Am^ = ±1, g i v i n g peaks correspond-i n g to m = 0, ±1, where m and m are values of the components of and I_ along the e x t e r n a l f i e l d d i r e c t i o n . Small h y p e r f i n e i n t e r a c t i o n s w i t h B-hydrogens, of which there are 15 or 16, a l s o occur, but these are not r e s o l v e d . Hyperfine i n t e r a c t i o n s w i t h a- 1 3C n u c l e i (1.1% n a t u r a l abundance) and even 1 5N (0.4% n a t u r a l abundance) are observable as s a t e l l i t e peaks at high s i g n a l - t o - n o i s e . The e l e c t r o n - e l e c t r o n d i p o l a r c oupling D i s a l s o t e n s o r i a l and given by a f u n c t i o n analogous to [ 6 ] . N e i t h e r t h i s nor the i s o t r o p i c exchange i n t e r a c t i o n , both of which are distance-dependent, c o n t r i b u t e to the time-independent Hamiltonian i n s o l u t i o n s c o n t a i n i n g n i t r o x i d e monoradicals. Both w i l l be considered s e p a r a t e l y i n due course. The direction-dependence of the Zeeman and h y p e r f i n e i n t e r a c t i o n s may most c l e a r l y be demonstrated experimentally by the esr spectrum of a dia. magnetic host c r y s t a l "doped" w i t h n i t r o x i d e (Figure 1-4^). The spec-trum at each o r i e n t a t i o n of the c r y s t a l c o n s i s t s of three sharp L o r e n t z i a n l i n e s ; however, both the p o s i t i o n and s p l i t t i n g of the l i n e s are d i r e c t i o n -dependent. The o r i e n t a t i o n of the trapped n i t r o x i d e w i t h respect to the host c r y s t a l and the p r i n c i p a l values (g ,g ,g ; T ,T ,T ) of the g-xx yy ^ ^  xx yy ^ ^  and T-tensors may be found by measuring g'.and T at s i x d i f f e r e n t c r y s t a l o r i e n t a t i o n s and d i a g o n a l i s i n g the r e s u l t a n t tensors. g and T have almost the same p r i n c i p a l a x i s systems , which correspond to a good approximation to the molecular axes shown i n Figure 1-3. The p r i n c i p a l values of the g and h y p e r f i n e tensors f o r three n i t r o x i d e s of s t r u c t u r e s i m i l a r to those used i n the present work are given i n Table 1-2. As might be expected from the molecular symmetry, both tensors are approximately a x i a l l y symmetric, 14 Figure 1.-4: T-and g - a n i s o t r o p i c s i n d i - t - b u t y l n i t r o x i d e (12) o r i e n t e d i n a host c r y s t a l (tetramethyl-1,3-cyclobutane dione) at^oom temperature. The c r y s t a l was r o t a t e d i n the molecular xz plane. At 0 and 90 the exte r n a l f i e l d l a y along the z and x axes r e s p e c t i v e l y (from reference 7). Table 1-2: P r i n c i p a l tensor components f o r f i v e n i t r o x i d e s S t r u c t u r e g g g T (G) T (G) & x x 6 y y & Z z x x y yy HO-( N - 0 ( 3 ) 2.0095 2.0064 2.0027 9 (1) 2.0104 2.0074 2.0026 5.2 5.2 6.5 6.7 4.7 4.7 0 that i s , and & x x - & y y B z z T % T ^ T xx yy zz These are sometimes given formal a x i a l symmetry by the n o t a t i o n 8 x x g y y g i T = T = T, XX yy •*• g z z = 8 „ [11] T z z = T H [12] I n a d d i t i o n t h e v a l u e s o f t h e p r i n c i p a l components o n l y depend t o a s m a l l e x t e n t on t h e n a t u r e o f t h e second f u n c t i o n a l group X (page 6) so t h a t f o r a p p r o x i m a t e work t h e y need n o t be d e t e r m i n e d f o r each n i t r o x i d e . The s p e c t r u m o b t a i n e d from a d i l u t e s o l u t i o n of n i t r o x i d e a l s o c o n -13 t a i n s t h r e e s h a r p l i n e s ( F i g u r e 1-5 ) b u t h e r e t h e g-and T - a n i s o t r o p i e s have been a v e r a g e d so t h a t o n l y t h e F e r m i c o n t a c t c o n t r i b u t i o n t o |, c h a r a c t e r i s e d by t h e i s o t r o p i c s p l i t t i n g c o n s t a n t a^, r e m a i n s . Thus a . = (1/3) (T + T + T ) = (1/3) (2T, + T„) [13] and t h e g - v a l u e i s s i m i l a r l y a v e r a g e d : g = (1/3) ( g x x + g y y + g z z ) = (1/3) (2g_, + g„) [14] The e x i s t e n c e of b o t h c o n t a c t and n o n - c o n t a c t terms i n T i n d i c a t e s t h a t b o t h s- and p - o r b i t a l c h a r a c t e r a r e c o n t a i n e d i n The p o l a r i t y o f t h e environment may a f f e c t t h e e l e c t r o n d i s t r i b u t i o n as a r e s u l t o f i t s i n f l u e n c e on t h e p o l a r i t y o f t h e N-0 bond, w h i c h may be d e s c r i b e d by t h e r e s o n a n c e forms N - 0 * + N * - 0~ T h i s change i n d i s t r i b u t i o n i s f e l t i n t h e magnitude of a^; p o l a r s o l -v e n t s s t a b i l i s e t h e more p o l a r f o r m , i n c r e a s i n g t h e s p i n d e n s i t y a t n i t r o g e n and hence a^ ( e q u a t i o n [ 5 ] ) ( g ^ i s s l i g h t l y d e c r e a s e d i n t h e same way). F i g u r e 1-3, i n w h i c h t h e e l e c t r o n i s shown i n t h e n i t r o g e n 2p^ o r b i t a l , i s 14 t h e r e f o r e i n e x a c t i n two r e s p e c t s . T a b l e 1-3 shows some t y p i c a l v a l u e s o f a n f o r n i t r o x i d e (2) i n s o l v e n t s o f d i f f e r e n t p o l a r i t y . 17 292 K Figure 1-5: Esr spectra of m a g n e t i c a l l y d i l u t e d i - t - b u t y l n i t r o x i d e (11). ( a ) i n a p o l y c r y s t a l l i n e s o l i d ; (b)a viscous s o l u t i o n and (c)a non-viscous s o l u t i o n showing g-and T- a n i s o t r o p i c s (adapted from reference 13). 18 Table 1-3: I s o t r o p i c n i t r o g e n h y p e r f i n e coupling constant of (2) as a f u n c t i o n of s o l v e n t 1 - 4 S t r u c t u r e Solvent D i e l e c t r i c constant of solvent a Q(G) n-hexane 1.88 15.13 d i e t h y l ether 4.34 15.33 A - 1,4-dioxane 2.21 15.45 H 2N-( N-0 acetone 20.70 15.53 methyl a l c o h o l 32.7 16.21 water 78.4 17.18 Figure 1-5 a l s o shows the spectrum o f a p o l y c r y s t a l l i n e array of n i t r o x i d e s , as might be obtained by r a p i d - f r e e z i n g a d i l u t e s o l u t i o n i n t o a g l a s s . A l l p o s s i b l e o r i e n t a t i o n s of the n i t r o x i d e c o n t r i b u t e to the spec-trum, which i s simply a sum of resonances due to the o r i e n t a t i o n s shown i n Figure 1-4 together w i t h a l l others. I t i s c l e a r , t h e r e f o r e , that w h i l e the c e n t r a l maximum contains c o n t r i b u t i o n s from a l l o r i e n t a t i o n s , the outer extrema of the spectrum are due to r a d i c a l s o r i e n t e d w i t h the molecular z-axis p a r a l l e l to the e x t e r n a l f i e l d , and that at the ' r i g i d l i m i t ' the s p l i t t i n g between these gives a measure of 2T„. Figure 1-6"^ shows the approach of the s p l i t t i n g (2T) between the outer features to 2T„ as temperature i s reduced i n a n i t r o x i d e - l a b e l l e d polymer. The r i g i d l i m i t occurs when T _ 1 < |T„ - T j <\, 7 x 10 7 s " 1 [15] and T _ 1 < |g„ - g j J aHh"1 * 3 x 1 0 7 s**1 [16] at 9.5 GHz whereT i s the c o r r e l a t i o n time f o r r o t a t i o n a l r e o r i e n t a t i o n . 19 Figure 1-6: The asymptotic approach of the s p l i t t i n g 2T to ^ z z w i t h decreasing temperature i n a n i t r o x i d e - l a b e l l e d p o l y s t y r e n e (from reference 15) . 20 Between the two extremes of non-viscous s o l u t i o n (where x <\> 10 s f o r a s mall molecule) and p o l y c r y s t a l l i n i t y (where x > ^10 ^ s ) , p a r t i a l averaging of a n i s o t r o p i c q u a n t i t i e s occurs (Figure I-5b). The u t i l i t y of the s p i n l a b e l l i n g method derives from the f o r t u i t o u s r e s u l t that the spec-trum i s s e n s i t i v e to motional e f f e c t s on a timescale which encompasses that of the motions experienced by many polymers. In the present work the f a c t that p r o v i d i n g motions i n the given frequency range occur, the p h y s i c a l form of the sample, whether s o l u t i o n or s o l , g e l or s o l i d , i s unimportant, i s of the greatest s i g n i f i c a n c e . A s e r i e s of s p e c t r a obtained i n a system where motion i s i s o t r o p i c and c o r r e l a t i o n time increases i s shown i n Figure 1-7 . The d i r e c t i o n of the g anisotropy i s such that the h i g h - f i e l d t r a n s i t i o n ( a l l s p e c t r a are p l o t t e d w i t h f i e l d i n c r e a s i n g to the r i g h t ) , which corresponds to m^  = - 1 , begins to broaden before the centre and l o w - f i e l d l i n e s as the tumbling r a t e of the r a d i c a l begins to decrease. Processes l e a d i n g to linebroaden-i n g are c h a r a c t e r i s e d by the transverse r e l a x a t i o n time T 2 which i s r e l a t e d to the peak-to-peak width ( A w ) of a L o r e n t z i a n d e r i v a t i v e curve by p - l _ i / 3 ) 2 T ? = - ^ A o ) [17] As the l i n e w i d t h i n c r e a s e s , the peak-to-peak height (h) decreases as the square of the width; thus Mmp" T 2 (mj). T 2 (mp [18] 16 Quoting the r e s u l t s of theory , i n the present case T 2 d i f f e r s f o r the d i f f e r e n t h y p e r f i n e components according to the r e l a t i o n [ T 2 ( m I ) ] " ; L = A + Bm]; + Cm^ [19] 21 F i g u r e 1-7: E s r s p e c t r a o f 2 , 2 , 6 , 6 - t e t r a m e t h y l - 4 - h y d r o x y p i p e r i d i n e -1 - o x y l (3) i n aqueous g l y c e r o l s o l u t i o n s . The c o r r e l a t i o n t i m e i n c r e a s e s as s o l u t i o n v i s c o s i t y i s r a i s e d and t e m p e r a t u r e lowered a - f (from r e f e r e n c e 6) . 22 where A includes c o n t r i b u t i o n s to t h e l l i n e w i d t h from unresolved h y p e r f i n e c o u p l i n g s , broadening by other paramagnetics (such as oxygen) present i n the s o l u t i o n , i n s t r u m e n t a l f a c t o r s and other mechanisms independent of the magnetic a n i s o t r o p i c s . This r e l a t i o n provides a way of c a l c u l a t i n g x from experimental s p e c t r a i n the 10 ^ - 10 ^ s range as f o l l o w s . Because of the square r e l a t i o n s h i p [18], peak-to-peak height i s a more s e n s i t i v e measure of l i n e w i d t h than the width i t s e l f , so i t i s con-v e n i e n t ^ to w r i t e [19] as T 2(0). where T 2(±1) T 2(0) T 2 (±1) - - 1 T 2(0) C + B h(0) h(±l) [20] [21] 16TT 45 16 T -1 [ T z z - i ( T _ + T _ ) ] H T B ^ [ g _ - i ( g _ + g _ ) ] [22] x x yy zz XX yy C = 77 2 [T - i ( T + T ) ] 2 72 zz xx yy T values are expressed i n MHz, and numbers i n parentheses are again m -values. By t a k i n g the sum and d i f f e r e n c e of T 2(0)/T 2(±1) and T 2 ( 0 ) / T 2 ( - 1 ) i t i s a l s o p o s s i b l e to o b t a i n independent values f o r x from the terms i n Bm^ . and Cm^ .. These tend to d i f f e r owing to the presence of both g and T tensor components i n B, w h i l e C contains elements only of T. Where c a l c u l a t i o n s based on equation [20] have been used here, the quad r a t i c term has been a r b i t r a r i l y chosen. S u b s t i t u t i n g values from Table 1-2, i t turns out that C ^  -B at H = 3.4 kG. The negative value of B lends greatest width to the h i g h - f i e l d l i n e (m^ = -1; equation [19]). This treatment assumes that molecular motion i s i s o t r o p i c ; that tumbling motion i s s u f f i c i e n t l y slow to i n f l u e n c e the l i n e w i d t h ; and that x 2 a Q 2 x> which e n s u r e s t h a t t h e t h r e e l i n e s do not o v e r l a p so t h a t l^Cm^) i s a m e a s u r a b l e p a r a m e t e r . Thus a t X-band t h e e q u a t i o n s a r e a p p l i c a b l e i n t h e range -11 -9 5 x 10 < T < 5 x 10 s. —7 —8 Ver y s l o w m o t i o n s (10 > x > 10 s) may be c h a r a c t e r i s e d by t h e s p l i t t i n g (2T) between t h e ou t e r m o s t extrema of t h e s p e c t r u m , as i m p l i e d by F i g u r e 1-6. V a r i o u s means have been used t o r e l a t e t h i s s p l i t t i n g t o c o r -r e l a t i o n t i m e , b u t o n l y t h e 2T parameter i t s e l f need be mentioned h e r e . F o r i n t e r m e d i a t e m o b i l i t i e s and i n g e n e r a l f o r a c c u r a t e d e t e r m i n a t i o n o f x, t h e r e i s no s u b s t i t u t e f o r t h e p r a c t i c e o f s p e c t r a l s i m u l a t i o n u s i n g 18 computer methods r e c e n t l y d e v e l o p e d f o r t h e e n t i r e r a n g e o f x v a l u e s . 19 S p e c t r a l s i m u l a t i o n i s used i n p a r t s of t h e p r e s e n t s t u d y . So f a r t h e d i s c u s s i o n has been r e s t r i c t e d t o i s o t r o p i c r e o r i e n t a t i o n o f n i t r o x i d e s , a s i t u a t i o n t h a t i s u n l i k e l y t o p r e v a i l when a m a c r o m o l e c u l e i s l a b e l l e d . To be p e r f e c t l y g e n e r a l , we may i m a g i n e an a r r a y o f n i t r o x -i d e s t o be o r i e n t e d i s o t r o p i c a l l y ( i n a l l o r i e n t a t i o n s a t any i n s t a n t ) o r a n i s o t r o p i c a l l y . The l a t t e r c a s e w i l l u s u a l l y r e f l e c t an a n i s o t r o p y i n t h e medium, as i n t h e c a s e o f o r i e n t e d b i l a y e r s ; a l l t h e systems d e s c r i b e d h e r e i n f a l l i n t o t h e f o r m e r c a t e g o r y . Each n i t r o x i d e w i t h i n an i s o t r o p i c d i s t r i b u t i o n may undergo i s o t r o p i c o r a n i s o t r o p i c r e o r i e n t a t i o n . A p e r t i n e n t example of t h e l a t t e r s i t u a t i o n o c c u r s when a l a b e l r e s i d e s w i t h i n t h e narrow b i n d i n g s i t e o f a g l o b u l a r p r o t e i n ( F i g u r e 1-8); r e o r i e n t a t i o n i s r a p i d about one a x i s b u t r e s t r i c t e d i n t h e two p e r p e n d i c -u l a r d i r e c t i o n s by s t e r i c h i n d r a n c e by p a r t s o f t h e p r o t e i n . Thus ( F i g u r e 1-8) x < x ^ x . I n a d d i t i o n , t u m b l i n g o f t h e whole macro-° y. x z m o l e c u l a r complex (T^) w i l l p r o v i d e a n o t h e r component t o T 2 r e l a x a t i o n . When a l i n e a r polymer i n s o l u t i o n i s l a b e l l e d one might e x p e c t t h e a n i s -o t r o p y t o be r e d u c e d , though c l e a r l y t h e number and n a t u r e of bonds i n t h e Figure 1-8: A n i s o t r o p i c r o t a t i o n of a n i t r o x i d e o c c u r r i n g as a r e s u l t of s t e r i c hindrance by a macromolecule i n s o l u t i o n to which i t i s attached. R e o r i e n t a t i o n i s expected to be r a p i d about the y a x i s , l e s s r a p i d about x and z, leading to anisotropy at large T ] n. i m i s the time constant f o r r o t a t i o n a l d i f f u s i o n of the macromolecule. 25 l i n k a g e group between l a b e l and polymer w i l l be o f i m p o r t a n c e . Many d i f f e r e n t and h i g h l y complex l i n e s h a p e s can a r i s e from a n i s o -t r o p i c r e o r i e n t a t i o n , and a g a i n t h e b e s t way t o q u a n t i t a t e t h e m o t i o n i s by s p e c t r a l s i m u l a t i o n . Programmes have been d e v e l o p e d f o r a x i a l l y s y m m e t r i c r e o r i e n t a t i o n i n w h i c h two c o r r e l a t i o n t i m e s , T„ and x_j_ a r e d e r i v e d ^ . I n p r a c t i c e , however, a t l e a s t where m o t i o n a l a n i s o t r o p y i s s m a l l and -9 m o t i o n s a r e r a p i d (x ^ x,, ^ T 1 < 10 s ) , x has o f t e n been c a l c u l a t e d u s i n g e q u a t i o n [20] o r u s i n g s i m u l a t i o n methods t h a t assume i s o t r o p i c r e o r i e n t a -t i o n . Some w o r k e r s have even d i s p e n s e d c o m p l e t e l y w i t h x, p r e f e r r i n g t o 20 use e i t h e r 2T o r , i n t h e f a s t e r m o t i o n a l r e g i m e , t h e q u a n t i t i e s h ( 0 ) / h ( - l ) 21 or h ( + l ) / h ( - l ) as e m p i r i c a l i n d i c e s o f m o b i l i t y . F o r p r e s e n t p u r p o s e s , i t i s u s e f u l t o b e a r i n mind a r e s u l t i l l u s -t r a t e d by t h e l i n e s h a p e s shown i n F i g u r e 1-9, w h i c h have been s i m u l a t e d on t h e a s s u m p t i o n of i s o t r o p i c t u m b l i n g and u s i n g i d e n t i c a l p a r a m e t e r s e x c e p t -9 -9 f o r c o r r e l a t i o n t i m e (x) w h i c h t a k e s t h e v a l u e s 2.0 x 10 ( a ) , 3.2 x 10 (b) -9 and 5.0 x 10 s ( c ) . The appearance o f two s p e c t r a l 'components' a t h i g h f i e l d i s n o t , as might a t f i r s t be t h o u g h t , n e c e s s a r i l y a s s o c i a t e d w i t h t h e e x i s t e n c e o f two d i s t i n c t c o r r e l a t i o n t i m e s o r w i t h t h e o c c u r r e n c e o f m o t i o n a l a n i s o t r o p y . I n g e n e r a l i n t h e p r e s e n t work, t h e e x a c t q u a n t i t a t -i v e n a t u r e o f t u m b l i n g m o t i o n s i s c o n s i d e r e d l e s s i m p o r t a n t t h a n changes i n t u m b l i n g r a t e s o c c a s i o n e d by e x t e r n a l p e r t u r b a t i o n s o r t h e p h y s i c a l s t a t e o f t h e system o f i n t e r e s t . To r e t u r n b r i e f l y t o an e a r l i e r p o i n t , i t i s c l e a r t h a t i n s i t u a t i o n s where l i n e s a r e broadened and o v e r l a p , a l t e r a t i o n s i n t h e h y p e r f i n e c o u p l i n g due t o t h e p o l a r i t y of t h e e n v ironment a r e d i f f i c u l t o r i m p o s s i b l e t o quan-t i t a t e . F o r t u n a t e l y , i n most o f t h e s t u d i e s d e s c r i b e d h e r e , l a b e l s a r e l o c a t e d i n p o l a r , h y d r o x y l i c e n v i r o n m e n t s w h i c h would n o t be e x p e c t e d 26 Figure 1-9: Simulated s p e c t r a f o r i s o t r o p i c a l l y tumbling n i t r o x i d e s . Programme parameters were i d e n t i c a l except f o r T . (a)2 ns; (b)3.2 ns; (c)5 ns. Note the appearance of a new f e a t u r e at high f i e l d i n (c) ( t h i s s t u d y ) . markedly to* a l t e r (Table 1-3). Just as the Zeeman and h y p e r f i n e i n t e r a c t i o n s may a f f e c t both the energy (equation [3]) and the r e l a x a t i o n behaviour (equations [20]-[23]) of the n i t r o x i d e e l e c t r o n , the presence of e l e c t r o n - e l e c t r o n i n t e r a c t i o n s , d i p o l a r or exchange, may a l s o i n f l u e n c e e i t h e r or both of these.'. We begin by c o n s i d e r i n g d i p o l a r i n t e r a c t i o n s . The e l e c t r o n - e l e c t r o n d i p o l a r Hamiltonian # D = S;D-S [24] may be r e w r i t t e n as flD = - M r " [ ( S ' S ) r 2 - 3 ( S . r ) ( S - r ) ] [25] where r i s the i n t e r e l e c t r o n i c d i s t a n c e . I t may now be seen that the i n t e r a c t i o n i s direction-dependent and i s averaged to zero by r a p i d tumbling, so that i t has an opposite T./T dependence to that of strong exchange (which, as we s h a l l see, depends on c o l l i s i o n frequency), i n c r e a s i n g as motional averaging becomes l e s s complete at high v i s c o s i t y to a l i m i t i n g value which depends on (1 - 3 c o s 2 8 ) / r 3 where 6 i s the angle between the v e c t o r _r and the e x t e r n a l f i e l d J . Thus f l u c t u a t i n g d i p o l a r i n t e r a c t i o n s may provide a r e l a x a t i o n c o n t r i b u t i o n whose magnitude may be used to d e r i v e d i s t a n c e i n f o r m a t i o n ; the present d i s c u s s i o n , however, w i l l focus i n s t e a d on s e c u l a r d i p o l a r i n t e r a c t i o n s between n i t r o x i d e s i n a r i g i d l a t t i c e , which may a l s o be analysed to give e l e c t r o n - e l e c t r o n d i s t a n c e s . In a p o l y c r y s t a l l i n e array of paramagnetic species the v a r i e t y of o r i e n t a t i o n s and d i s t a n c e s gives r i s e to a l a r g e number of d i f f e r e n t d i p o l a r couplings f o r d i f f e r e n t 'spin packets,' whose L o r e n t z i a n l i n e s thus overlap g i v i n g an inhomogen-eously broadened envelope. The i n t e r a c t i o n may be q u a n t i t a t e d i n a number of ways: using the second moment of the l i n e , by f u l l s i m u l a t i o n of the lineshape, or by e m p i r i c a l c a l i b r a t i o n methods. The l a s t of these i s used 28 here. 22 The parameter d\/d (Figure 1-10) was f i r s t used by Kokorin et a l on an e m p i r i c a l b a s i s to c h a r a c t e r i s e i n t e r a c t i o n s between n i t r o x i d e s ; i t 23 has s i n c e been confirmed i n our l a b o r a t o r y w i t h a i d of computer s i m u l a t i o n that d l --3 — = AiAw(dipolar) + A 2 =? A3 r' + A^ [26] 23 f o r l a b e l (2) i n 50% aqueous g l y c e r o l and l a b e l (1) i n methanol at 77K between 250 and 2mM, where A u(dipolar) i s the a d d i t i o n a l l i n e w i d t h of a s p i n packet due to the d i p o l a r c o u p l i n g , r i s the mean nearest-neighbour d i s t a n c e and the A^ are constants. At high r a d i c a l concentrations (sm a l l r ) d^/d i s no longer measurable; here Heisenberg exchange a l s o s t a r t s to a f f e c t the lineshape. At very low concentrations (<2mM) p a r t i a l s a t u r a t i o n could not be avoided even at the lowest a v a i l a b l e microwave -4 power (1.6 x 10 W). The c a l i b r a t i o n curve f o r s o l u t i o n s of (2) i n 50% aqueous g l y c e r o l i s shown i n Figure 1-11. I n t e r - e l e c t r o n i c distances are c a l c u l a t e d from concentrations i n a random u n c o r r e l a t e d three-dimensional array as f o l l o w s : ^ [ 2 7 ] r 3 0.28 where [C] i s the conc e n t r a t i o n of n i t r o x i d e . Equation [27] i s der i v e d i n Appendix 2. At high concentrations a c o r r e c t i o n can be.-made f o r p o s i t i o n a l c o r r e l a t i o n of the n i t r o x i d e s , but t h i s was not necessary i n the present circumstances where comparison of experimentalwi.th.standard data i s made. In any case the c o r r e c t i o n i s sm a l l i n the conc e n t r a t i o n range of i n t e r e s t . Otherwise the use of 50% aqueous g l y c e r o l as solvent wherever p o s s i b l e ( f a s t - f r o z e n i n t o a glass) ensured that p a r t i a l c r y s t a l l i z a t i o n phenomena, le a d i n g to d e v i a t i o n s from randomness i n the d i s t r i b u t i o n of r a d i c a l s , were 30 I . O i 0 . 8 d 0 . 6 0 . 4 r x l ' i i i i i i i * 1.0 1.4 ,1.8 2 . 2 2 . 6 r ( n m ) Figure I-11: The v a r i a t i o n of the parameter d../d i n powder spectra with concentration of 4-amino-2,2,6,6-tetrametnylpiperidine-l-oxyl (2) i n 50% aqueous g l y c e r o l at 77 K ( t h i s study). 31 avoided. Other advantages accrue from the use of t h i s e m p i r i c a l method: although the change of lineshape w i t h c o n c e n t r a t i o n has been discussed e x c l u s i v e l y i n terms of d i p o l a r i n t e r a c t i o n s (and t h i s i s p h y s i c a l l y reasonable at the distances measured, 1.0 - 2.4 nm, though not at s h o r t e r d i s t a n c e s , where exchange might be expected to c o n t r i b u t e ) and as r e s u l t i n g only from nearest-neighbour i n f l u e n c e s , the a n a l y s i s does not depend on these assumptions. Nor does i t s u f f e r from the occurrence of p a r t i a l s a t u r a t i o n at low r a d i c a l concentrations as long as standard and e x p e r i --4 mental s p e c t r a are recorded at the same power l e v e l s (1.6 x 10 W i n a l l cases). In a d d i t i o n i t i s convenient. The most important assumptions inherent i n the method are that the 'natural' l i n e w i d t h of the L o r e n t z i a n s p i n packets i n the absence of d i p o l a r e f f e c t s are the same i n sample and standard and that the p r i n c i p a l hyperfine-and g-tensor components, p a r t i c u l a r l y T^zz> are the same i n the two cases. In other words, i t i s necessary to assume that i n H 2NSL and ROCNHSL . „ , „. . , , ' . .. " (see Chapters IV and V) unresolved proton hyperfxne c o u p l i n g and NH other c o n t r i b u t i o n s to. 'Acd(natural) are the same, and that the p o l a r i t y of the environment of a l a b e l attached to a carbohydrate polymer (R) i s the same as that of a l a b e l i n 50% aqueous g l y c e r o l . The former i s adjudged a f a i r l y reasonable assumption, and the l a t t e r has been confirmed f o r 2T z z i n R0CH2C0NHSL, where R i s agarose, so i s l i k e l y to be a good approximation . ROCNHSL „ . T. . T . . . _ , . i n » , where R i s agarose or c e l l u l o s e . I t i s c l e a r from the c a l i -NH b r a t i o n curve shown i n Figure 1-11 that the lineshape becomes l e s s and l e s s s e n s i t i v e to change i n r as the l a t t e r i n c r e a s e s . Generally speaking only values of d^/d > 0.4 have been used. Values of r are compared w i t h t o t a l amounts of n i t r o x i d e obtained by double i n t e g r a t i o n of the esr s i g n a l as a means of g a i n i n g i n f o r m a t i o n on the d i s t r i b u t i o n of attachment s i t e s on 32 the carbohydrates of i n t e r e s t . F i n a l l y , i n t h i s part of the chapter, exchange i s considered. When the wave fu n c t i o n s of two d i f f e r e n t unpaired e l e c t r o n s o v e r l a p , e l e c t r o -s t a t i c i n t e r a c t i o n s tend to couple the s p i n s , producing s i n g l e t and t r i p l e t s t a t e s i n b i r a d i c a l s where e l e c t r o n s are present at nearby s i t e s . The exchange energy i s i s o t r o p i c and has the form J'S^i'Sz- In s o l u t i o n s c o n t a i n i n g monoradicals the exchange i n t e r a c t i o n i s turned on b r i e f l y as r a d i c a l s d i f f u s e together and c o l l i d e , then turned o f f as they d i f f u s e apart. This i s expressed as J ( r , t ) and enables the e l e c t r o n s to exchange s p i n s t a t e s r a p i d l y , shortening the l i f e t i m e of the s t a t e s without a f f e c t -ing the t o t a l energy of the system. This i s t h e r e f o r e a transverse r e l a x -a t i o n process. In d i l u t e s o l u t i o n s l i k e those considered i n the f i r s t p a rt of t h i s s e c t i o n such events occur s u f f i c i e n t l y r a r e l y that they do not c o n t r i b u t e s i g n i f i c a n t l y to the l i n e w i d t h s . The exchange frequency (v ) i s r e l a t e d to the frequency w i t h which two r a d i c a l s c o l l i d e (VQ) by a p r o b a b i l i t y f a c t o r (p) as f o l l o w s : v = f p v 0 [28] e u S i m i l a r l y , i n terms of r a t e constants,k, k e = f p k 0 [29] where k^ = v /[C], kg = VQ/[C], [C] i s the co n c e n t r a t i o n of r a d i c a l s , and f i s a s t a t i s t i c a l f a c t o r which a r i s e s because exchange between two species whose resonant frequency i s the same i s not de t e c t a b l e . I t i s convenient to d i s t i n g u i s h the cases of strong and weak exchange; i n the former, the p r o b a b i l i t y f a c t o r y p = 1, w h i l e i n the l a t t e r case, p<<l. For strong exchange, t h e r e f o r e , according to equation [28], v - fvg and exchange experiments give no in f o r m a t i o n about e l e c t r o n d i s t r i b u t i o n s w i t h i n the r a d i c a l s p e c i e s . This s i t u a t i o n occurs i n the case of n i t r o x i d e 33 monoradicals i n s o l u t i o n . v t h e r e f o r e has a l i n e a r dependence on T./T e where n i s the s o l u t i o n v i s c o s i t y and T the temperature. Figure I I - 3 , a-d, shows the e f f e c t of i n c r e a s i n g s o l u t e c o n c e n t r a t i o n on the esr spectrum of a n i t r o x i d e s o l u t i o n . Linebroadening begins when 2 i T V e ^ Aw 3 x 10 6 Hz f o r the n i t r o x i d e s used here, and l i n e s begin to overlap when 2 T r v e a0 DL 5 x 10 7 H z * When exchange i s very r a p i d ( v g >> a 0) s o - c a l l e d "exchange narrowing" occurs (Figure I I - 3 d ) ; w i t h i n i t s l i f e t i m e i n a s i n g l e m s t a t e the e l e c t r o n 'sees' a l l three m v a l u e s , causing averaging i n t o one l i n e of width Aw ^ a^/l'nv^. Above t h i s frequency (as w e l l as i n very slow exchange) the l i n e w i d t h i s determined by other processes. The f a c t o r f i n the case of a n i t r o x i d e i s 2/3, s i n c e 1/3 of the c o l l i s i o n s w i l l be w i t h species i n the same nuclear s p i n s t a t e . Given t h i s i n f o r m a t i o n , the l i f e -time (v ^) of any s p i n s t a t e can be c a l c u l a t e d at a given c o n c e n t r a t i o n from the a d d i t i o n a l l i n e w i d t h . I t i s worth n o t i n g that w h i l s t at lower concentrations of r a d i c a l s the magnetic a n i s o t r o p i e s which dominate T 2 are modulated by r o t a t i o n a l motion, exchange i s modulated by t r a n s l a t i o n a l d i f f u s i o n . Where in s t e a d exchange i s weak the parameters p and VQ i n equation [28] are a f f e c t e d by change i n n/T i n a manner opposite to each other s i n c e p i s now dependent on the d u r a t i o n of a c o l l i s i o n (T^_ ^ 10 ^ s) so the temperature dependence i s only s l i g h t . In other words there i s no s i t u a t i o n i n which exchange i s expected to increase markedly w i t h a l a r g e decrease i n temperature where mean nearest neighbour di s t a n c e remains constant. Hence the assumption of d i p o l a r i n t e r a c t i o n s i n linebroadening observed on reducing the temperature i n systems stud i e d i n Chapters IV and V. In the second c l a s s of exchange experiment u t i l i z e d i n the present s t u d i e s , paramagnetic metal ions are introduced i n s o l u t i o n i n t o systems 34 co n t a i n i n g m a g n e t i c a l l y d i l u t e n i t r o x i d e s , l e a d i n g , i n the cases s t u d i e d , to i n c r e a s e i n l i n e w i d t t i s , •. without a l t e r a t i o n i n the p o s i t i o n of the resonances. The idea behind these experiments i s to compare the broaden-i n g observed (or the r a t e constant k g) i n s o l u t i o n s p e c t r a of n i t r o x i d e s w i t h that obtained when the l a b e l i s attached to a macromolecule, drawing conclusions about the s t e r i c hindrance exerted upon the former by the l a t t e r (v i s expected to be reduced as a r e s u l t of decreases i n VQ and, perhaps, p) and adding e x t r a i n f o r m a t i o n to that already gained from x. Some 25 precedent e x i s t s i n the p r o t e i n l i t e r a t u r e f o r t h i s type of experiment , „• but i t has not been a p p l i e d to the study of s u r f a c e s , which i s i t s main deployment i n the work i n hand. The choice of paramagnetic metal ions to act as s o - c a l l e d 'spin probes' i s determined by the f o l l o w i n g c r i t e r i a : water s o l u b i l i t y , chemical i n a c t i v i t y towards the system s t u d i e d , charge, s i z e , e f f i c i e n c y as a re l a x a n t (p) and the l a c k of a competing esr s i g n a l (though i n w e l l -defined s t a b l e metal complexes w i t h n i t r o x i d e - c o n t a i n i n g l i g a n d s the metal 26 esr s i g n a l i s of i n t e r e s t i n determination of e l e c t r o n i c s t r u c t u r e ). The nature of d e c i s i o n s governing the f i r s t four c r i t e r i a w i l l be made c l e a r i n subsequent chapters; i t i s convenient to discus s the l a s t two immediately. The l i n e w i d t h of an esr s i g n a l may be decomposed as f o l l o w s : 7 ^ = ^ + "^- [30] 1 2 T 2 ZT± where T 2 i s the 'true' s p i n - s p i n or transverse r e l a x a t i o n time and the f a c t o r 2 appears because the mean s p i n l i f e t i m e i s 2T^. Thus s p i n -l a t t i c e r e l a x a t i o n (T^) can a f f e c t the l i f e t i m e of the s p i n s t a t e , causing Heisenberg broadening i n the same way as T 2• In many t r a n s i t i o n metal ions s p i n - o r b i t c oupling provides a h i g h l y e f f i c i e n t means of- s p i n - l a t t i c e r e l a x a t i o n , which becomes r a p i d and dominates the l i n e w i d t h : T 2 = T 1. 35 Thus most of the t r a n s i t i o n metal species used i n the present s t u d i e s have no observable esr s i g n a l , at l e a s t at room temperature. This i s the case I I -3 f o r N i , whose e f f e c t on the spectrum of a 10 M s o l u t i o n of n i t r o x i d e (2) i s shown i n Figure 1-12. T^  values f o r N i " ^ complexes have been 27 -12 -13 estimated as 10 - 10 s, which gives a l i n e w i d t h of about 10 5 - 10 6 G! In p r i n c i p l e , i n t e r a c t i o n s between adjacent paramagnetic metal ions and n i t r o x i d e s may be of two types, d i p o l a r or exchange, e i t h e r or both of which may c o n t r i b u t e i n a given s i t u a t i o n . The nature of the i n t e r a c t i o n i n a case where the metal i o n i s i n s o l u t i o n and the n i t r o x i d e a f f i x e d to a macromolecule (and indeed i n general) may depend on VQ, p, J, the t o t a l e l e c t r o n s p i n of the metal i o n (S), the c o l l i s i o n l i f e t i m e ( x t ) , the d i f -ference between the resonant frequencies of the c o l l i d i n g species (6) and the e l e c t r o n r e l a x a t i o n times T^  and T 2 f o r both the. n i t r o x i d e and the metal. No proper theory e x i s t s to cover the range of p o s s i b l e s i t u a t i o n s , though c e r t a i n l i m i t i n g cases have been considered, and the experimental 25 l i t e r a t u r e abounds w i t h c o n t r a d i c t o r y assumptions . The exchange i n t e g r a l J i s , of course, u s u a l l y unknown, but the r a t h e r s m a l l amount of accurate data a v a i l a b l e on e l e c t r o n r e l a x a t i o n times i s more s u r p r i s i n g . 26 28 The f i e l d has been reviewed twice ' . Only a few r e l e v a n t t h e o r e t i c a l and experimental.:results w i l l be c i t e d . The greatest experimental and t h e o r e t i c a l e f f o r t s i n the area have 29-32 been made by M o l i n and co-workers who found that f o r t r a n s i t i o n metal complexes and n i t r o x i d e s i n s o l u t i o n s of low v i s c o s i t y exchange i n t e r a c t i o n s always dominate the c o l l i s i o n a l broadening, even where bulky l i g a n d s might have been expected to reduce the exchange i n t e g r a l J , though d i p o l a r i n t e r -a c t i o n s are expected to increase w i t h the metal T 1. At very short T-i I 36 Figure 1-12: Esr lineshape of a s o l u t i o n of 4-amino-2,2,6,6-tetramethyl-p i p e r i d i n e - l - o x y l (2) (ImM i n water) as a f u n c t i o n of concentration of n i c k e l sulphate. Note that at higher concentrations (d and e) the l i n e w i d t h s of i n d i v i d u a l hyperfine components cannot be d i r e c t l y measured due to t h e i r overlapping ( t h i s study). 37 ("^T ) t h e exchange b r o a d e n i n g d e c r e a s e s , b u t t h e d i p o l a r c o n t r i b u t i o n i s r e l a t i v e l y s m a l l e r . Thus t h e p f a c t o r i s r e d u c e d f o r m e t a l i o n s w i t h short, r e l a x a t i o n t i m e s , and weak exchange can o c c u r . Hyde and S a r n a have r e c e n t l y c o n f i r m e d t h e s e b a s i c c o n c l u s i o n s i n copper ( I I ) complexes w h i c h , i n f a c t , have a r e l a t i v e l y l o n g T i , and p r e d i c t e d t h a t H e i s e n b e r g exchange s h o u l d s i m i l a r l y dominate i n t e r a c t i o n s between n i t r o x i d e s and o t h e r t r a n s -i t i o n m e t a l complexes, i n c o n t r a s t w i t h t h e c a s e o f g a d o l i n i u m ( I I I ) , where as a r e s u l t o f s h i e l d i n g o f f e l e c t r o n s t o g e t h e r w i t h a l a r g e m a g n e t i c moment and l o n g T i , exchange i s l e s s e f f i c i e n t t h a n d i p o l e - d i p o l e r e l a x a -t i o n . I n o r d e r t o s i m p l i f y t h e i n t e r p r e t a t i o n o f t h e e x p e r i m e n t s p e r f o r m e d w i t h l a b e l l e d c a r b o h y d r a t e s , i t was deemed i m p o r t a n t i n t h e p r e s e n t s t u d y t o choose probe i o n s w i t h s h o r t T i v a l u e s , m i n i m i s i n g t h e d i p o l a r c o n t r i b u t i o n t o b r o a d e n i n g even i f p was somewhat r e d u c e d . The two probe i o n s of c h o i c e were N i ( H 2 0 ) 6 + ( T i ^  1 0 ~ 1 2 - 1 0 ~ 1 3 s) and F e ( C N ) | ~ ( T i < 1 0 _ 1 ° s ) " . The o b s e r v e d b r o a d e n i n g o f t h e n i t r o x i d e s i g n a l ( F i g u r e 1-12; a s i m i l a r phenom-3-enon i s o b s e r v e d u s i n g Fe(CNg ) ) o c c u r s w i t h o u t a f r e q u e n c y s h i f t o r exchange n a r r o w i n g phenomenon; t h i s i s because t h e m e t a l i o n p r o v i d e s , t h r o u g h exchange, an e f f i c i e n t s p i n - l a t t i c e r e l a x a t i o n pathway f o r t h e n i t r o x i d e , f o r w h i c h now T 2 2L T\. R a p i d and i r r e v e r s i b l e l o s s o f phase c o h e r e n c e t h e r e f o r e o c c u r s between c o l l i s i o n s . k^ f o r N i ^ ^ (aq) c a l c u l a t e d f r o m t h e 8 -1 -1 g r a d i e n t o f F i g u r e 1-13 i s 1.6 x 10 £.mole s . A s i m i l a r s t r a i g h t 3- a -1 -1 l i n e p l o t i s o b t a i n e d f o r Fe(CN)6 , l e a d i n g t o a k^ of 1.7 x 10 £.mole s 24 F o r d i - t - b u t y l n i t r o x i d e (12) i n s o l u t i o n i n t e t r a h y d r o f u r a n , k. = 5.0 x 1 0 9 £.mole _ 1 s _ 1 . 38 8 r o o 3 0 0 . 0 4 0 . 0 8 [Ni"],M Figure 1-13: Peak-to-peak l i n e w i d t h of the centre l i n e ( A u) 0(C)) of. the esr spectrum of ImM aqueous n i t r o x i d e ( 2 ) as a f u n c t i o n of co n c e n t r a t i o n of n i c k e l o u s i o n . The s t r a i g h t l i n e dependence i s c o n s i s t e n t w i t h an exchange mechanism f o r broadening, w i t h a r a t e constant k g (equation [29]) of 1.6 x 10 8 l.mole 1 s " 1 ( t h i s study). 39 ID: Polysaccharides Carbohydrates occur i n o l i g o m e r i c and polymeric form both indepen-dently and as components of more complex molecules. In the l a t t e r c ate-gory we can place g l y c o l i p i d s and l i p o p o l y s a c c h a r i d e s i n which the carbo-hydrate i s l i n k e d to a l i p i d ; and g l y c o p r o t e i n s , peptidoglycans and proteo-glycans (or mucopolysaccharides) i n which i t i s j o i n e d to peptide or p r o t e i n . We s h a l l be concerned w i t h g l y c o p r o t e i n s , considered s e p a r a t e l y i n s e c t i o n I F , and polymers which c o n t a i n only carbohydrate, h e r e a f t e r r e f e r r e d to as polysaccharides. The bulk of the work described i s concerned w i t h two e x t r a - c e l l u l a r and i h t r a - . c e l l u l a r seaweed p o l y s a c c h a r i d e s — a g a r o s e and a l g i n i c a c i d — a n d c e l l u l o s e , the s t r u c t u r a l polysaccharide found i n p l a n t w a l l s . These three polysaccharides have d i f f e r e n t types of primary s t r u c t u r e , though a l l three are l i n e a r . C e l l u l o s e (49) i s A - G l c p ^ w h i l e agarose (18) (at l e a s t i n i t s i d e a l i s e d form; some sulphate and other s u b s t i t u e n t s are u s u a l l y present) has a d i s a c c h a r i d e repeat u n i t -f-^D-Galp y-4- 3,6-Anhydro-L-Galp^ n and a l g i n i c a c i d (19) has a block s t r u c t u r e H^L-GulpA 1] [4D-ManpAJh-. a n B m This v a r i a t i o n c o n t r a s t s w i t h some of the g l y c o p r o t e i n s to be discussed i n s e c t i o n IF which have a p e r i o d i c o l i g o s a c c h a r i d e u n i t s , and r e f l e c t s a f u n c t i o n a l d i s p a r i t y . Perhaps the most important i n f o r m a t i o n concerning the three-40 dimensional shapes of polysaccharides has come from X-ray d i f f r a c t i o n s t u d i e s of o r i e n t e d f i b r e s , though other p h y s i c a l methods have begun to be used. D e t a i l e d i n f o r m a t i o n about chain packing i n c e l l u l o s e has been obtained (Chapter V) and agarose has been shown to adopt a left-handed t h r e e - f o l d double h e l i c a l conformation i n the condensed phase (Chapter I I I ) . Further challenges remain, however, s i n c e even i n c e l l u l o s e f i b r e s regions of s u b s t a n t i a l d i s o r d e r occur which cannot be analysed by X-ray methods; i n the water-soluble polysaccharides on the other hand i t i s necessary to show how molecular shapes i n o r i e n t e d f i b r e s r e l a t e to those found i n more hydrated s t a t e s . A g e n e r a l i s a t i o n of the d i f f e r e n t p h y s i c a l forms found amongst polysaccharides i s shown i n Figure 1-14. Chain m o b i l i t y i s expected to decrease from a to d as s o l v a t i o n decreases. A f i f t h type of o r g a n i s a t i o n , that of a l i q u i d c r y s t a l l i n e phase, would f i t between gels and s o l i d s , but no polysaccharide e x h i b i t i n g t h i s type of behaviour has been found. For present purposes, a g e l , which i s e a s i e r to imagine than to d e f i n e , may be s a i d to be a predominantly d i s o r d e r e d , permanent, continuous network c o n s i s t i n g of more than one component, w i t h the appearance of a s o l i d . Thus agarose g e l s , which may be squeezed, bent or broken, can c o n t a i n over 99.9% water; t h e i r f u n c t i o n i n nature i s c l e a r l y to r e g u l a t e the surroundings of the parent organism v i a c o n t r o l of water. The systems chosen f o r study r e f l e c t a d e s i r e to compare polysacchar-ides from each of the four c l a s s i f i c a t i o n s . I t was f e l t that t h i s was more important, given the nature of the s p i n l a b e l l i n g method, than s e l e c t -i n g systems on the grounds of a c l a s s i f i c a t i o n based on primary s t r u c t u r e . I t i s c l e a r from t h i s type of c l a s s i f i c a t i o n that the extent of s o l v a t i o n , which may depend on d i f f e r e n t types of i n t e r m o l e c u l a r i n t e r a c t i o n i n d i f -f e r e n t p o l y s a c c h a r i d e s , i s of paramount importance. As solvent p o l a r i t y 41 s t r u c t u ^ , r °" ° f d f f e r e n t ^ P e s of hydrated polysaccharide ilntlZJ a t l 0 n . ^ c r e a s e s from a to d. Cooperative hydrogen bonding 0 f t 6 n t h e d o m i ^ n t i n t e r m o d u l a r i n t e r a c t i 0 n 3 3 F i n n e u t r a } p o l y s a c c h a r i d e s 1 42 decreases, most polysaccharides are expected to p r e f e r more condensed p h y s i c a l s t a t e s (though t h i s i s by no means u n i v e r s a l l y t r u e ) ; f r a c t i o n a l p r e c i p i t a t i o n using ethanol:water mixtures are commonly used as a means of p u r i f i c a t i o n . The e f f e c t - of solvent changes i n carbohydrate g e l s , which has not yet been widely s t u d i e d , i s one point of i n t e r e s t i n the # present t h e s i s . C e l l u l o s e , agarose and a l g i n i c a c i d , however, represent an a t y p i c a l s e l e c t i o n of polysaccharides i n that they are a l l l i n e a r . There are two reasons f o r t h i s . In the f i r s t p l a c e , because of the e f f i c i e n t packing of t h e i r chains, l i n e a r p olysaccharides tend to a s s o c i a t e 34 ( i n t o gels or s o l i d s ) more r e a d i l y than branched ones , which makes them more s u i t a b l e f o r study by s p i n l a b e l l i n g . Secondly, the behaviour of branched polymers i s l i k e l y to be more complex and l e s s r e a d i l y i n t e r p r e t -a ble. In s o l u t i o n , although i t may be expected that each monomer e x i s t s f o r most of the time i n a c h a i r conformation w i t h maximum e q u a t o r i a l s u b s t i t u -t i o n , r o t a t i o n s about the g l y c o s i d i c l i n k a g e s between neighbouring u n i t s are s t i l l g e n e r a l l y to be expected, g i v i n g r i s e to random c o i l conformations. These are p a r t i c u l a r l y l i k e l y i n 1-6-linked pyranosides l i k e dextran, and 35 i t i s p o s s i b l e to compute conformational energy maps which i n d i c a t e p r e f e r r e d conformations. Longer range order i s a l s o p o s s i b l e i n s o l u t i o n ; experimental data on s o l u t i o n behaviour are s t i l l s carce, however, because of the problems i n v o l v e d i n studying time-dependent aggregation and l o c a l c o n c e n t r a t i o n f l u c t u a t i o n s i n systems which are o f t e n p o l y d i s p e r s e and polymolecular. The m a j o r i t y of in f o r m a t i o n about the behaviour of polysaccharides i n more hydrated forms has come from s t u d i e s on v a r i o u s o r d e r - d i s o r d e r t r a n s i -36 t i o n s , such as that from g e l to s o l . I t has u s u a l l y not proved p o s s i b l e 43 to i d e n t i f y u n e quivocally conformations i n these ordered s t a t e s as the same as those seen i n X-ray f i b r e p a t t e r n s , but i n many cases the c i r c u m s t a n t i a l evidence from such techniques as o p t i c a l r o t a t i o n , c i r c u l a r dichrois'm, l i g h t s c a t t e r i n g , v i s c o s i t y , as w e l l as thermodynamic stu d i e s , i s q u i t e convincing. Thus, f o r example, i t i s probable that double h e l i c e s mediate g e l formation i n agarose (Chapter I I I ) . In n e u t r a l polysaccharides i t has g e n e r a l l y been found that i n t e r a c t i o n s i n gels and the s o l i d s t a t e , are 33 dependent on cooperative hydrogen-bonding i n t e r a c t i o n s (though t h i s may no longer be a f a i r g e n e r a l i s a t i o n ten years hence). I o n i c polysaccharides w i l l i n general need to bind counterions i n passing to a more ordered s t a t e , as i n the case of a l g i n a t e (Chapter I I I ) . ,37,39,52 IE: I n t e r a c t i o n s at Surfaces and Wi t h i n Gels In the l a s t ten years a f f i n i t y or b i o s p e c i f i c chromatography" has r e v o l u t i o n i s e d biochemical s e p a r a t i o n and p u r i f i c a t i o n . By immobilis-i n g on an i n s o l u b l e m a t r i x one of two (or more) species between which a s p e c i f i c b i n d i n g process occurs i t i s p o s s i b l e to s e l e c t the other from a complex mixture i n s o l u t i o n , d i s c a r d i n g components w i t h no s p e c i f i c b i n d i n g a c t i v i t y by e l u t i o n and l a t e r r e c o v e r i n g the bound species i n such a way as to render the immobilised conjugate reusable. An example from carbo-38 hydrate biochemistry i s that of the group of g l y c o p r o t e i n s c a l l e d l e c t i n s which have made an important c o n t r i b u t i o n to c e l l surface science by v i r t u e of t h e i r a b i l i t y to bind s e l e c t i v e l y to monosaccharides or s p e c i f i c sequences thereof. Very o f t e n these m a t e r i a l s have been i s o l a t e d using i n s o l u b l e 38 'supports 1' to which are a f f i x e d monosaccharides . A d d i t i o n of excess f r e e monosaccharide, a f t e r removal of non-binding m a t e r i a l , s h i f t s the e q u i l i b r i u m such that the l e c t i n may be recovered from the e l u a t e . The process i s represented s c h e m a t i c a l l y i n Figure 1-15. A host of d i f f e r e n t 44 / Figure 1-15: S i m p l i f i e d schematic view of a f f i n i t y chromatography, (a)ligand-support conjugate; (b)molecules with the a b i l i t y to bind the immobilised l i g a n d are s e l e c t e d from a complex mixture; (c)molecules remaining i n s o l u t i o n are e l u t e d ; ( d ) a d d i t i o n of excess f r e e l i g a n d d i s p l a c e s the bound sp e c i e s , which can be e l u t e d . 45 i n t e r a c t i o n s designed by Nature has been put to use i n t h i s way: antibody-39 , 37 ' . . . . , . 40 . antxgen , enzyme-substrate , and n u c l e i c a c i d base-pair i n t e r a c t i o n s are three other examples. The wide success of a f f i n i t y separations has given r i s e to s e v e r a l 41 other r e l a t e d methods. Hydrophobic chromatography r e l i e s on i n t e r a c t i o n s between immobilised a l k y l or aromatic species and hydrophobic domains w i t h i n 42 g l o b u l a r p r o t e i n s . Covalent chromatography uses t h i o l - d i s u l p h i d e i n t e r -change between an immobilised t h i o l and c y s t e i n e residues i n p r o t e i n s . Immobilised l i g a n d s l i k e EDTA (ethylenediaminetetraacetate) are f i n d i n g 43 44 a p p l i c a t i o n i n the bulk removal of heavy metal p o l l u t a n t s from water ' Attempts are being made to design c h i r a l macrocycles which, immobilised on 45 an i n s o l u b l e m a t r i x , w i l l e f f e c t o p t i c a l r e s o l u t i o n . Charge-transfer 46 i n t e r a c t i o n s have r e c e n t l y found s i m i l a r a p p l i c a t i o n . The l i t e r a t u r e on s c i e n t i f i c and t e c h n o l o g i c a l uses of immobilised enzymes i s undergoing an 47 exponential i n c r e a s e . Many s i m i l a r t r i c k s and v a r i a n t s on the method have developed and need not concern us here. Two other aspects are, however, r e l e v a n t : the f a c t that polysaccharides are commonly used as 48 support matrices , and the e m p i r i c a l r e s u l t (Chapter IV) that i t i s o f t e n necessary to use a 'spacer'' molecule between the support and the attached l i g a n d i n order to maximise the e f f i c i e n c y of s e p a r a t i o n , o r , i n other words, the stre n g t h of b i n d i n g . (This can a l s o be t r u e i n s o l u t i o n b i n d -49 i n g of macromolecules .) Support m a t e r i a l s from a l l four c l a s s e s of polysaccharide shown i n Figure 1-14 have been e x p l o i t e d i n t h i s f i e l d . Conjugation to s o l u b l e o l i g o - or poly-saccharides can permit molecular s e l e c t i o n by an antibody or l e c t i n at a l a t e r stage^^. S y n t h e t i c or n a t u r a l l y o c c u r r i n g hydrogels and g e l s , p a r t i c u l a r l y agarose under i t s commercial guise of Sepharose"'''", 46 are probably the most commonly used supports. However, s o l i d m a t e r i a l s 52 53 l i k e c e l l u l o s e and even glass can a l s o be used d e s p i t e the r e s e r v a t i o n that t h e i r e f f e c t i v e surface areas are l i k e l y to be s m a l l e r . Separate chapters i n the present t h e s i s are devoted both to Sepharose and to c e l l u l o s e . The need f o r a 'spacer uni t ' i n a f f i n i t y chromatography, even i n g e l s , i m p l i e s s t e r i c hindrance by the polysaccharide 'surface' of those s p e c i e s , p a r t i c u l a r l y l a r g e molecules, i n s o l u t i o n . I t was hoped that the combination of spacer arm v a r i a t i o n s w i t h esr would give some i n s i g h t i n t o t h i s phenomenon, from the standpoints of ease of b i n d i n g from s o l u t i o n , surface geometry, the d i s t r i b u t i o n of m o d i f i c a t i o n s i t e s (which may i n favourable cases lead to m u l t i p l e b i n d i n g of a p o l y f u n c t i o n a l 54 p r o t e i n ), and the r e l a t i o n s h i p of these v a r i a b l e s to the g e l s t r u c t u r e ; Chapter IV shows that these hopes were not without foundation. A development which i s undoubtedly r e l a t e d to advances i n chromato-55 graphy i s that of matrix-supported c a t a l y s t s ; the ease and e f f i c i e n c y w i t h which the (sometimes expensive) c a t a l y s t may be recovered i s a t t r a c t i v e to the chemical i n d u s t r y . The idea of a spacer arm has even been used i n the context of c a t a l y t i c a l l y a c t i v e metal complexes supported on amorphous 56 s i l i c a . This has a l s o meant that s t u d i e s of what may l o o s e l y be c a l l e d unmodified s u r f a c e s , that i s , of m a t e r i a l s where the support i s the c a t a l y s t , have assumed new importance. Organic s y n t h e s i s on s o l i d 57 58 supports , o r i g i n a l l y used by M e r r i f i e l d f o r polypeptides , and the use 59 of immobilised organic reagents , have been shown to hold c e r t a i n advan-tages f o r the chemist, as has the l o c a l l y high i o n i c s t r e n g t h at a charged 60 surface . Surface e l e c t r o c h e m i s t r y i s another e x c i t i n g , r a p i d l y expand-ing new f i e l d ^ . With these developments has come the need f o r fundamental 47 s t u d i e s on the nature of the i n t e r f a c e between s o l i d and s o l u t i o n phases. Techniques l i k e ESCA ( e l e c t r o n spectroscopy f o r chemical a n a l y s i s ) , LEED (low energy e l e c t r o n d i f f r a c t i o n ) , and EXAFS (extended X-ray a b s o r p t i o n f i n e s t r u c t u r e ) , which may be c o l l e c t e d under the heading of 'surface physics' may i n general only be performed at high vacuum. The presence of a s o l i d or g e l o f t e n i n h i b i t s the use of conventional s o l u t i o n s p e c t r o -scopic methods and most of these w i l l not s e l e c t surface species i n p r e f e r -ence to others. E l e c t r o n microscopy*^ i s u s e f u l i n the study of s t a t i c arrangements, and attenuated t o t a l r e f l e c t a n c e i n f r a - r e d s p e c t r o s c o p y ^ has been used to d i s t i n g u i s h f u n c t i o n a l groups at s u r f a c e s . For surface dynamics, however, although fluorescence spectroscopy and e l e c t r o c h e m i s t r y show promise, there i s a r e a l need f o r new methods. Thus'the present s t u d i e s were a l s o p a r t l y motivated by a d e s i r e to explore the p o t e n t i a l of esr i n i n v e s t i g a t i o n of the s o l u t i o n - s u r f a c e i n t e r f a c e . IF: G l y c oproteins Glycoproteins have been found w i t h i n most cate g o r i e s of p r o t e i n s , whether c l a s s i f i e d by s t r u c t u r e , f u n c t i o n , or place of o r i g i n ; they c o n t a i n carbohydrate l i n k e d to aminoacid residues i n the p r o t e i n by g l y c o s i d i c bonds. I t i s g r a d u a l l y becoming apparent that Nature has designed a range of protein-carbohydrate conjugates running the f u l l gamut from c l o s e 67 68 to 0 to almost 100% carbohydrate ' . Thus p r o t e i n s such as lysozyme and bovine serum albumin c o n t a i n no carbohydrate; at the other end of the spectrum, some membrane-bound blood group substances r e p o r t e d l y c o n t a i n 67 about 85% sugar by weight . Molecules c o n t a i n i n g more sugar than t h i s are g e n e r a l l y classed as polysaccharides. The carbohydrate may be present as short chains o c c u r r i n g r e l a t i v e l y o f t e n i n the aminoacid sequence, or as fewer, longer oligomers, commonly h a v i n g r a t h e r complex b r a n c h e d s t r u c t u r e s . To some extent carbohydrate content has been r e l a t e d to f u n c t i o n , but no g e n e r a l i s a t i o n s are i n order. Only a few d i f f e r e n t monosaccharides have been found i n g l y c o -p r o t e i n s ^ 7 , the main ones being g a l a c t o s e , mannose, fucose, N - a c e t y l -glucosamine, N-acetylgalactosamine, arabinose ( i n p l a n t s ) , and the acylneuraminic (or s i a l i c ) a c i d s . These may be l i n k e d to aminoacids i n two ways: O-and N - g l y c o s i d i c a l l y . The N-glycosides are u s u a l l y of the type where X may be one of s e v e r a l aminoacids. I t should be added that by no means a l l aminoacid t r i a d s of t h i s form are g l y c o s y l a t e d . O-Glycosides may be formed w i t h s e r i n e , threonine and the l e s s common aminoacids 5-hydroxylysine and 4-hydroxyproline; the sugars that have been found as aminoacid O-glycosides are g a l a c t o s e , arabinose, mannose, x y l o s e , N-acetylgalactosamine and N-acetylglucosamine. . -Ser-49 In r a r e cases t h i o g l y c o s i d i c l i n k a g e s to c y s t e i n e have been found. Both of the main types of g l y c o s i d e may occur together i n the same g l y c o p r o t e i n . G l y c o p r o t e i n b i o s y n t h e s i s i s only now being u n r a v e l l e d and w i l l not concern us here. I t i s i n t e r e s t i n g to note, however, that i t does not always occur by s e q u e n t i a l enzymic a d d i t i o n of monosaccharides, though the microheterogeneity seen i n polysaccharides i s s t i l l observed to a greater extent than i n the p r o t e i n p o r t i o n of the molecules. Many of the more complex sugar p r o s t h e t i c groups seem to conta i n a 'core region,'' next to the p r o t e i n , i n which the p r i n c i p a l residues are mannose and N - a c e t y l -glucosamine, and an 'outer region 1 i n which are found s i a l i c a c i d s and fucose as non-reducing t e r m i n a l r e s i d u e s , g a l a c t o s e , and others. Sequenc-in g these, e s p e c i a l l y i n cases where more than one d i f f e r e n t o l i g o -saccharide i s present, can be d i f f i c u l t , but recent work using nuclear 69 magnetic resonance (nmr) at high f i e l d has provided an important a d d i t i o n to e x i s t i n g chemical and enzymic methods. Chemical i d e n t i f i c a t i o n i s an important step i n assessing the s t r u c t u r e -f u n c t i o n r e l a t i o n s h i p . The carbohydrates of g l y c o p r o t e i n s have been i m p l i -cated i n a number of f u n c t i o n a l r o l e s , which may be d i v i d e d i n t o three main groups: ( i ) the c r e a t i o n of s p e c i f i c physico-chemical p r o p e r t i e s ; ( i i ) molecule-membrane i n t e r a c t i o n s ; and ( i i i ) membrane-membrane i n t e r a c -t i o n s (though a f o u r t h category, molecule-molecule i n t e r a c t i o n s , cannot be excluded as a p o s s i b i l i t y ) . Functions i n the f i r s t category g e n e r a l l y seem to be r e l a t e d to the h i g h l y hydrated nature of saccharides i n aqueous environments. Thus g l y c o p r o t e i n s are enabled to l u b r i c a t e the eye socket and to act as p r o t e c t i v e coatings i n the r e s p i r a t o r y and i n t e s t i n a l t r a c t s . Mucins, which t y p i c a l l y c o n t a i n many di s a c c h a r i d e s of the form 50 H R N H / l o R C0 2H OH S i a l i c a c i d s (13) (13a) R' = Ac, R = H : N-acetyl neuraminic a c i d (NANA) OR H are o f t e n r e s i s t a n t to p r o t e o l y t i c enzymes; i n the same way, human i n t e s -t i n a l enzymes, which c o n t a i n 30-40% carbohydrate, appear to be protected from p r o t e o l y s i s by a 'coating' of c a r b o h y d r a t e ^ . In many such cases, t e r m i n a l s i a l i c a c i d c o n t r i b u t e s to the p r o p e r t i e s of the system by v i r t u e of i t s n e g a t i v e l y charged C - l carboxylate group which, i t i s assumed, maximises h y d r a t i o n of the p r o s t h e t i c groups and causes them to take up a ' f u l l y extended' conformation. However, g a s t r i c mucus forms a k i n d of viscous g e l ^ , the major c o n s t i t u e n t of which, by weight, i s water, i n which i t i s obvious that i n t e r m o l e c u l a r i n t e r a c t i o n s are c o n s i d e r a b l e , so that counter^ions must play an important p a r t . Another example of sacc-harides h e l p i n g to confer unusual p h y s i c a l p r o p e r t i e s upon a system occurs i n the a n t i f r e e z e g l y c o p r o t e i n s found i n the sera of a n t a r c t i c fish^"*". The n e g a t i v e l y charged carboxylate groups of the s i a l i c acids are undoubt-72 edly important i n r e g u l a t i n g the adhesive p r o p e r t i e s of c e l l surfaces , and are known to bind calcium ions i n c o l l a b o r a t i o n w i t h the e x o c y c l i c The second, and, e s p e c i a l l y , the t h i r d groups of carbohydrate func-t i o n s are g e n e r a l l y much more d i f f i c u l t to i n v e s t i g a t e because s m a l l q u a n t i t i e s of m a t e r i a l o f t e n take part i n s u b t l e , s p e c i f i c and yet important i n t e r a c t i o n s . Glycoproteins are known to a s s o c i a t e w i t h the membrane i n a number of d i f f e r e n t ways, but i t i s important that the t r i o l 73 51 74 c a r b o h y d r a t e i s a l w a y s l o c a t e d on t h e e x t e r n a l s u r f a c e . The same i s 74 t r u e of t h e membrane g l y c o l i p i d s . The g l y c o p r o t e i n s have been u n e q u i v o c a l l y a s s o c i a t e d w i t h a number o f r e c e p t o r a c t i v i t i e s — f o r i n s u l i n , hormones, l e c t i n s , a n t i b o d i e s and o t h e r s , as w e l l as p l a y i n g r o l e s i n c e l l a d h e s i o n 7 " ' . There i s e v i d e n c e t o s u g g e s t t h a t c a r b o h y d r a t e e x p r e s s i o n a t 74 t h e c e l l s u r f a c e changes a f t e r m a l i g n a n t t r a n s f o r m a t i o n . P e r h a p s t h e most famous r e c e n t work i n t h i s f i e l d , r e l a t i n g b o t h t o s u g a r s a t t h e c e l l s u r f a c e and i n s o l u b l e g l y c o p r o t e i n s , has been on t h e e f f e c t of s i a l i c a c i d 7 6 on t h e c i r c u l a t o r y l i f e t i m e of serum g l y c o p r o t e i n s and e r y t h r o c y t e s Enzymic removal- of s i a l i c a c i d from a number of serum g l y c o p r o t e i n s was f i r s t shown t o l e a d t o a r a p i d i n c r e a s e i n t h e r a t e o f t h e i r r e m o v a l from c i r c u l a t i o n by t h e l i v e r . ( O t h e r s , s u c h as t r a n s f e r r i n , were u n a f f e c t e d . ) More r e c e n t l y , t h e h e p a t o c y t e membrane-bound s p e c i e s r e s p o n s i b l e f o r r e c o g -n i t i o n of t e r m i n a l g a l a c t o s e exposed as a r e s u l t of t h e r e m o v a l of s i a l i c a c i d has been i s o l a t e d and found t o be a g l y c o p r o t e i n 7 7 . Removal of i t s s i a l i c a c i d i n t h e same way l e a d s t o l o s s o f a c t i v i t y as a r e s u l t o f t h e a s s o c i a t i o n o f i t s newly-exposed g a l a c t o s e w i t h t h e b i n d i n g s i t e 7 7 ! The h e p a t i c b i n d i n g g l y c o p r o t e i n i n i t s u n m o d i f i e d form was found t o a g g l u t i n -a t e human and r a b b i t e r y t h r o c y t e s , and i t has been found t h a t t h e i r c i r c u l a t o r y l i f e t i m e , though not t h a t of c h i c k e n e r y t h r o c y t e s , i s s i m i l a r l y 7 8 a f f e c t e d by t r e a t m e n t w i t h n e u r a m i n i d a s e . As o l d e r c e l l s have been found to c o n t a i n fewer s i a l i c a c i d r e s i d u e s t h a n t h e i r younger c o u n t e r -p a r t s , t h i s may p r o v i d e a c l e a r a n c e mechanism. N o n - r e d u c i n g t e r m i n a l s i a l i c a c i d has a l s o been a s s o c i a t e d w i t h 'masking' of c a r b o h y d r a t e 74 a n t i g e n s The phenomenon o f l y mphocyte 'homing' i n w h i c h t h e c e l l s m i g r a t e t o the s p l e e n e x c e p t when t r e a t e d p r i o r t o i n j e c t i o n w i t h a f u c o s i d a s e , when they migrate i n s t e a d to the l i v e r ' J , belongs i n the same category. S i m i -l a r l y , periodate o x i d a t i o n of surface sugars, as w e l l as b i n d i n g by the l e c t i n concanavalin A, which i s d i r e c t e d p r i m a r i l y to a-mannosides, cause 6 7 mitogenic s t i m u l a t i o n of lymphocytes . I t i s not necessary to labour the p o i n t that carbohydrates, i n g e n e r a l , and s i a l i c a c i d i n p a r t i c u l a r , whether i n blood serum, mucous c o a t i n g s , or at the c e l l s u r f a c e , f u l f i l ' important and h i g h l y s p e c i f i c b i o l o g i c a l r o l e s . I t i s to be expected that the i n t r o d u c t i o n of a s p i n l a b e l , whose s i z e i s roughly that of a monosaccharide, w i l l perturb or even a b o l i s h some of these i n t e r a c t i o n s , g r e a t l y d i m i n i s h i n g the value of any environmental i n f o r m a t i o n that i t provides. However, there are important general ques-t i o n s which have not yet been r e s o l v e d and which the method i s w e l l adapted 80 to answer; although spectroscopic techniques such as nmr have given much in f o r m a t i o n about m o b i l i t y — s e g m e n t a l and o v e r a l l — i n p r o t e i n s , very l i t t l e i s known about the behaviour of carbohydrate p r o s t h e t i c groups. Are they c o n f o r m a t i o n a l l y r i g i d , or f l e x i b l e ? Does t h e i r m o b i l i t y depend to any extent on the p r o p e r t i e s of the macromolecule to which they are attached? Does i t depend on the length of the o l i g o s a c c h a r i d e ? An attempt to answer some of these questions i n Chapter VI represents a n a t u r a l extension both of the chemistry and of the spectroscopic approaches taken i n preceding chapters. IG: Previous Studies The l i t e r a t u r e on s p i n l a b e l l i n g i s enormous. Since McConnell's 81 o r i g i n a l paper w e l l over one thousand p u b l i c a t i o n s have concerned them-selves i n one way or another w i t h n i t r o x i d e s . The great m a j o r i t y of the s t u d i e s d e s c r i b e s p i n l a b e l l i n g of enzymes or other p r o t e i n s on the one 53 hand, or membranes on the other. D e s c r i p t i o n s of these two types of a p p l i -c a t i o n s , together w i t h chapters on the chemistry of s p i n l a b e l s and the d e t a i l e d theory r e q u i r e d f o r i n t e r p r e t a t i o n of t h e i r esr s p e c t r a are 82 brought together i n a b a s i c t e x t by B e r l i n e r . S i m i l a r m a t e r i a l i s • • i • u * • k . , 6,7,80,83 . , covered more c o n c i s e l y m a number of review a r t i c l e s . A comple-25 mentary monograph by L i k h t e n s h t e i n describes developments i n the Soviet Union which have been l a r g e l y ignored i n North America, i n c l u d i n g much work on i n t e r a c t i o n s between n i t r o x i d e s and other paramagnetic s p e c i e s . The purpose of the present s e c t i o n i s to review aspects of the l i t e r a t u r e which have not been included i n any of the above ref e r e n c e s , but which are p e r t i n e n t to the work described. Previous r e p o r t s of the use of s p i n l a b e l s i n carbohydrate systems are extremely sparse. O-Glycosides of galactose,. N-acetylglucosamine and 85 c h i t o b i o s e as substrates f o r 3-galaetosidase and g-galactoside permease , 86 and lysozyme have been reported by McConnell's group, though, the y i e l d s seemed to be low. In the l a t t e r case, nuclear s p i n r e l a x a t i o n of protons i n the p r o t e i n by the unpaired e l e c t r o n i n the l a b e l l e d , bound s u b s t r a t e was detected, and used to estimate d i s t a n c e s . In a d d i t i o n syntheses of two 1-thiogalactose d e r i v a t i v e s have been reported^. L a b e l l i n g at the e x t r a c y c l i c 5 - p o s i t i o n of aldehydo-furanose d e r i v a t i v e s using a 2,3-bis-hydroxylaminobutane d e r i v a t i v e to give 1-oxyl i m i d a z o l i n e s has been 87 88 89 achieved ' . Gagnaire and Odier have e s t e r i f i e d blocked glucose at the 1- and 6-positions w i t h a c i d c h l o r i d e d e r i v a t i v e s of n i t r o x i d e s , making two mono-nitroxide d e r i v a t i v e s , and have gone on to make doubly s u b s t i t u t e d glucose ( n i t r o x i d e s at p o s i t i o n s 2 and 3), as w e l l as doubly s u b s t i t u t e d methyl c e l l o b i o s i d e and maltoside w i t h a n i t r o x i d e e s t e r at each 6 - p o s i t i o n 90 91 and the other p o s i t i o n s a c e t y l a t e d . H a l l and Adam have made a number 54 of d i f f e r e n t 1, 2 and 6-spin l a b e l l e d monosaccharides using the 5 - t r i a z i n e moiety as a ' l i n k i n g ' group. Rassat and co-workers have reported the complexation of a number of 92 d i f f e r e n t n i t r o x i d e s , i n c l u d i n g a b i r a d i c a l , w i t h a- and g - c y c l o d e x t r i n Thermodynamic parameters f o r the i n c l u s i o n complex were able to be derived from esr s p e c t r a of the b i r a d i c a l . The h y p e r f i n e s p l i t t i n g s , were c o n s i s -tent w i t h a nonpolar environment f o r the complexed n i t r o x i d e . To the author's knowledge, only four r e p o r t s of s p i n l a b e l l e d carbo-hydrate polymers had appeared from o u t s i d e ' h i s own l a b o r a t o r y at the time 89 of w r i t i n g . Gagnaire and Odier attached n i t r o x i d e s to the 6-positions and to the reducing end of c e l l u l o s e acetate using chemistry s i m i l a r to that used i n t h e i r monosaccharide s t u d i e s , and observed motional anisotropy i n the rapid-tumbling region i n the l a t t e r case. Esr spectroscopic changes accompanying the enzymatic depolymerisation of s p i n l a b e l l e d amy-93 l o s e have been observed , tumbling becoming i n c r e a s i n g l y r a p i d as the enzymic a c t i o n progressed, as expected. L a b e l l i n g of the p o l y s a c c h a r i d e was accomplished by r e a c t i o n w i t h a 2-chloromethyl d i - N - o x y l - d i h y d r o i m i d -a z o l e , and s e p a r a t e l y w i t h an i s o t h i o c y a n a t o - p i p e r i d i n e - l - o x y l . In the t h i r d such r e p o r t , which appeared when the present s t u d i e s were i n progress, hydroxyl s u b s t i t u e n t s i n carboxymethyl c e l l u l o s e s o l u t i o n s were l a b e l l e d by means of r e a c t i o n w i t h cyanogen bromide followed by 4-amino-2,2,6,6-94 . t e t r a m e t h y l p i p e r i d i n e - l - o x y l (2) . L a s t l y , membrane preparations from Gaffkya homari were shown to c a t a l y s e the i n v i t r o b i o s y n t h e s i s of s p i n 95 l a b e l l e d peptidoglycan from a s p i n l a b e l l e d n u c l e o t i d e . Exchange i n t e r -a c t i o n s were observed between adjacent l a b e l s at room temperature, but disappeared on p a r t i a l r e d u c t i o n w i t h ascorbate. The spectrum a f t e r p a r t i a l r e d u c t i o n gave x , ' and t h i s was used, together w i t h an e m p i r i c a l 55 parameter c h a r a c t e r i s i n g exchange, to draw conclusions about the geometry of the polymer. Two groups have r e c e n t l y reported i n c o r p o r a t i o n of n i t r o x i d e s i n t o the carbohydrate component of the i s o l a t e d Fc r e g i o n of an antibody, as w e l l as i n t o the same region of the i n t a c t antibody, by periodate o x i d a t i o n 96 97 98 followed by r e d u c t i v e amination ' ' . Very d i f f e r e n t conclusions were reached about the extent of i m m o b i l i s a t i o n ; i n one case the s p i n l a b e l appeared to be r e o r i e n t i n g q u i t e r a p i d l y even i n the presence of bound antigen, w h i l e i n the other case r e o r i e n t a t i o n was a good deal slower. In n e i t h e r case were s i g n i f i c a n t d i f f e r e n c e s observed between m o b i l i t y i n i s o l a t e d Fc and i n t a c t IgG. F i n a l l y , Grant and Sharom have described a method whereby n i t r o x i d e s may be incorporated n o n - s p e c i f i c a l l y i n t o hydroxyl f u n c t i o n a l i t i e s i n -, • • „ , . 9 9 , 1 0 0 , ganglxoside head group regions . This i n v o l v e s the formation of phosphate d i e s t e r s w i t h primary hydroxyl groups i n the presence of t r i i s o p r o p y l b e n z e n e s u l p h o n y l c h l o r i d e and a phosphate d e r i v a t i v e of p i p e r i d i n e - l - o x y l . Headgroup m o b i l i t y was found to be great and not very dependent on the f l u i d i t y of the b i l a y e r . Three aspects of s p i n l a b e l l i n g s t u d i e s which have not been covered i n monographs are r e l e v a n t to the present work. These are s p i n l a b e l l i n g s t u d i e s of s y n t h e t i c polymers, of n u c l e i c a c i d s , and of s u r f a c e s , and they are b r i e f l y reviewed here; the i n t e n t i o n i s not to be comprehensive, but r a t h e r to convey the main l i n e s of approach to problems which are r e l a t e d to those encountered h e r e i n . In s o l u t i o n and i n the l i q u i d phase g e n e r a l l y , s y n t h e t i c polymers oft e n have more i n common w i t h polysaccharides than do g l o b u l a r p r o t e i n s 56 because t h e i r molecular conformations are u s u a l l y l e s s r i g o r o u s l y defined than those of the p r o t e i n s . Several groups are a c t i v e i n the f i e l d of polymer s p i n l a b e l s t u d i e s ; B u l l o c k and Cameron have summarized some of 101 102 t h e i r own c o n t r i b u t i o n s , as have Tormala and Lindberg . Studies may be d i v i d e d roughly i n t o two c l a s s e s : those using small molecular weight n i t r o x i d e s as 'probes' i n polymer s o l u t i o n s , melts, or s o l i d s , and those using l a b e l l e d polymers. In 'concentrated' s o l u t i o n s (§ 10% by weight of polymer) and melts c o n t a i n i n g p o l y v i n y l a c e t a t e i t has been shown that lineshapes r e f l e c t i n g s i m i l a r motional regimes, ( g e n e r a l l y r a t h e r s t r o n g l y 103 immobilized), r e s u l t i n both types of experiment . At low polymer co n c e n t r a t i o n , however, motions of probes are r a p i d and l e s s dependent 102 upon the presence of the polymer than are the motions of l a b e l s ; the l a t t e r , however, are r e l a t i v e l y independent of the polymer c o n c e n t r a t i o n at low concentrations. In gene r a l , i n v e s t i g a t o r s have been i n t e r e s t e d i n the dependence of T and, i n a few cases s p i n - s p i n i n t e r a c t i o n s , on s o l v e n t , polymer c o n c e n t r a t i o n , or molecular weight, and most o f t e n of a l l , tempera-tu r e (T). Arrhenius p l o t s of lnx vs T \ the gradients of which_give E /R where E i s the a c t i v a t i o n energy f o r r e o r i e n t a t i o n of the l a b e l and a a R i s the gas constant, deviate from l i n e a r i t y only when some form of phase t r a n s i t i o n , such as the formation of a g l a s s , o c c u r s ^ \ Such t r a n s i t i o n s have a l s o been c h a r a c t e r i s e d by the s p l i t t i n g between outer h y p e r f i n e 104 extrema . In l a b e l l e d polymers T tends to a maximum as molecular weight i n c r e a s e s ; f o r polystyrene at three d i f f e r e n t temperatures, the maximum, the magnitude of which was temperature dependent, occurred at about the same number-average molecular weight i n each case (5-10 x 10^)^^. This seemed to r e f l e c t the pro g r e s s i v e d i m i n u t i o n of c o n t r i b u t i o n s from 'whole-molecule' processes such as end-over-end r o t a t i o n w i t h i n c r e a s i n g 57 molecular weight, and the increase of segmental motions, and an attempt was made to f a c t o r out these c o n t r i b u t i o n s to T. The same workers a l s o went to some t r o u b l e to show that x r e f l e c t e d the dynamics of the polymer r a t h e r than a r i s i n g p a r t l y or wholly from independent r e o r i e n t a t i o n of the l a b e l by comparing r e s u l t s obtained ~ using meta- and para-l a b e l l e d polystyrene w i t h those from other techniques. However, t h i s connection has i n other cases been found to be l a c k i n g , as i n a study of l a b e l l e d polymethylmethacrylate by the same authors"'"^"'". Solvents at low c o n c e n t r a t i o n have been found to exert l i t t l e e f f e c t on n i t r o x i d e s p e c t r a of l a b e l l e d l i n e a r and c r o s s - l i n k e d polystyrene and polymethyl-methacrylate i n the s o l i d phase, r o t a t i o n of the l a b e l o c c u r r i n g about i t s l i n k a g e to the polymer"*"^. At intermediate solvent concentrations two types of motion were observed, one p o p u l a t i o n of n i t r o x i d e s experiencing the same slow motions, w h i l e another was a f f e c t e d by segmental ( l o c a l mode) motion. The populations were not i n t e r c o n v e r t i n g w i t h i n the esr ti m e s c a l e . At high d i l u t i o n a l l n i t r o x i d e s were rel a x e d by the f a s t e r l o c a l mode mechanism. The dependence of molecular motion i n l a b e l l e d c o l l a g e n on h y d r a t i o n shows s i m i l a r e f f e c t s A study of e n d - l a b e l l e d poly-y-benzyl-a,L-glutamate i n d i c a t e d a n i s o -t r o p i c r e o r i e n t a t i o n i n i s o t r o p i c , b i p h a s i c , and l i q u i d c r y s t a l l i n e s t a t e s i n the presence of dimethylformamide^' 7 and t h i s i n s p i r e d a thorough t h e o r e t i c a l treatment of a n i s o t r o p i c e f f e c t s i n n i t r o x i d e s p e c t r a by Freed A 1 1 0 8 and co-workers L i t t l e that i s conceptually d i f f e r e n t from the polymer s t u d i e s e x i s t s i n the l i t e r a t u r e on s p i n l a b e l l e d n u c l e i c a c i d s . Most s t u d i e s have used Arrhenius p l o t s to c h a r a c t e r i s e the 'melting' 1 or denaturation of h e l i c e s i n 25 109 s o l u t i o n ' . One other piece of work deserves mention as i t shows the 58 so f a r u n d e r u t i l i s e d p o t e n t i a l of s p i n - s p i n i n t e r a c t i o n s i n d e f i n i n g changes i n molecular geometry. Here the b i n d i n g of ethidium bromide and a c r i f l a v -in e to DNA s p i n l a b e l l e d using an ethyleneimine d e r i v a t i v e l e d to an increase i n the d i s t a n c e between labels''"''"^. This was i n t e r p r e t e d i n terms of a lengthening of the DNA molecule. The boundary between s t u d i e s of polymers i n the s o l i d s t a t e , and s u r f a c e s , i s b l u r r e d . What may be thought of as 'large holes' between chains i n c r o s s - l i n k e d polymers and r e s i n s become smaller as the extent of c r o s s - l i n k i n g i n c r e a s e s ; i n gels the c r o s s l i n k s may simply be hydrogen or other bonds between polymer chains; i n a t r u e s o l i d the c r o s s - l i n k s may be covalent (or other) bonds but small holes or pores o f t e n s t i l l e x i s t at the s u r f a c e , and the geometry of these may be solvent-dependent. I o n i c i n t e r a c t i o n s between i o n exchange r e s i n s and l a b e l s c a r r y i n g the opposite type of charge have been.studied and shown to be dependent on the extent of cross-linking"'"''""'". P r o g r e s s i v e i m m o b i l i z a t i o n was observed as the water 112, content was reduced e i t h e r by d r y i n g or replacement by nonaqueous s o l v e n t s -In n e u t r a l polystyrene r e s i n s solvent-induced s w e l l i n g s i m i l a r l y decreased 113 x , whereas on a s i l i c a surface the same author found a reduced solvent 114 dependency . Two s p i n probes w i t h d i f f e r e n t h y d r o p h i l i c i t y were used to d i s t i n g u i s h micro-environments w i t h i n a s o l i d phase copolymer of c h l o r o -methylated polystyrene w i t h d i v i n y l b e n z e n e , i n the presence of one and two-component solvent systems, on the b a s i s of t h e i r h y p e r f i n e s p l i t t i n g 115 constants In two e x c e l l e n t recent papers which appeared during the course of the work described i n t h i s t h e s i s , Whitesides' group reported a thorough study of the surface of-low-density polyethylene f i l m " ' ^ ' . In the f i r s t place the f i l m was o x i d i s e d w i t h chromic a c i d and the nature of the 59 f u n c t i o n a l groups thus created was e s t a b l i s h e d w i t h help of attenuated t o t a l r e f l e c t a n c e IR spectroscopy and chemical d e r i v a t i s a t i o n . They were found to be p r i m a r i l y c a r b o x y l i c a c i d s , aldehydes and ketones, and were assayed by f l u o r i m e t r i c methods. The nature of the surface was i n v e s t i -gated using c o v a l e n t l y attached n i t r o x i d e s and f l u o r e s c e n t l a b e l s . Although d i f f i c u l t i e s were experienced w i t h t e n a c i o u s l y adsorbed reagents and l a b e l s , s e v e r a l important conclusions were reached: that the m o b i l i t y of l a b e l s was dependent on s o l v a t i o n and that t h i s may be a slow process; that judging both by m o b i l i t y and a c c e s s i b i l i t y to r e d u c t i o n from s o l u t i o n , most, but not a l l l a b e l s were i n s u r f a c e - a c c e s s i b l e s i t e s ; that the surface was s t a b l e , that i s , groups d i d not appear to migrate from there to the i n t e r i o r ; and that the two kinds of r o t a t i o n a l freedom observed f o r l a b e l s may a r i s e from attachment to surface s i t e s i n c r y s t a l l i n e and amorphous regions. N i t r o x i d e s , both chemically bound and adsorbed, have a l s o been used to study the surfaces of s i l i c a , o x i d i s e d s i l i c o n , s i l i c a t e s , q u a rtz, a l u m i n o s i l i c a , alumina, g a l l i u m oxide, and other such m a t e r i a l s , u s u a l l y because of t h e i r importance i n chromatography, c a t a l y s i s or the e l e c t r o n i c s i n d u s t r y . S o l v a t i o n i s again a parameter of paramount importance. Two d i f f e r e n t groups have reported the observation of 2 7 A 1 h y p e r f i n e c o u p l i n g to the e l e c t r o n of an adsorbed nitroxide"'"''"^'"'""'"9; however t h i s only occurred a f t e r a c t i v a t i o n . a t high temperature had removed adsorbed water, l e a v i n g c o o r d i n a t i v e l y unsaturated aluminium. Otherwise H-bonding to surface hydroxyls appears to occur, w i t h considerable i m m o b i l i s a t i o n , both here and . ' .,. 120 , . . i n s i l i c a . In the l a t t e r case, the a c t i v a t i o n energy f o r r e o r i e n t a t i o n of a l a b e l at a dry surface decreased w i t h d i s t a n c e from the surface. A d e t a i l e d study has a l s o been made of s i l i c a using some s u b t l e chemical 60 arguments to d e l i n e a t e the d i s t r i b u t i o n and amounts of v i c i n a l and geminal d i o l s and i s o l a t e d hydroxyls present at the surface at d i f f e r e n t tempera-121 tures . Further experiments w i t h l a b e l l e d s i l i c a have made use of exchange and d i p o l a r i n t e r a c t i o n s to measure n i t r o x i d e - n i t r o x i d e d i s t a n c e s 23 and hence d e r i v e i n f o r m a t i o n about the d i s t r i b u t i o n of surface s i t e s B i l a y e r s , v e s i c l e s and c e l l s may a l s o be considered to have s u r f a c e s , and l a b e l s have been designed which l o c a t e at or near the po l a r head group 122 r e g i o n . Surface a c c e s s i b i l i t y i n such systems i s g e n e r a l l y considered to be r e l a t e d to the depth of immersion of the n i t r o x i d e i n the 123,124 b i l a y e r IH: Organisation of the Thesis N i t r o x i d e s have p r e v i o u s l y been used both as l a b e l s ( c o v a l e n t l y attached to the system of i n t e r e s t ) and probes (small r e p o r t e r molecules d i f f u s i n g w i t h i n , or associated w i t h , the system). Their use as probes i n c e r t a i n p olysaccharide s o l u t i o n s and gels i s explored i n Chapter I I as a way o f ^ r e l a t i n g macroscopic to microscopic p r o p e r t i e s . This r e l a t i o n s h i p i s f u r t h e r explored by means of l a b e l l i n g i n the remainder of the t h e s i s . The chemical methods by which the l a b e l i s a f f i x e d to the v a r i o u s carbohydrates are g e n e r a l l y discussed where they are most e x t e n s i v e l y used. S i m i l a r l y , the main spectroscopic emphasis changes from chapter to chapter depending on the system i n v o l v e d , although the same methods are a p p l i e d i n some cases to d i f f e r e n t systems, so that the demarcation of chapters f o l l o w i n g Chapter I I according to systems s t u d i e d r a t h e r than methods used to study them r e f l e c t s the b i a s of the i n v e s t i g a t o r . Chapter I I I i s devoted to g e l formation i n agarose; Chapter IV to Sepharose as a chromatographic support; Chapter V to c e l l u l o s e ; and Chapter VI to g l y c o p r o t e i n s i n s o l u t i o n and at 61 the c e l l s u r f a ce. An attempt to c o r r e c t any imbalance r e s u l t i n g from t h i s b i a s i s made i n Chapter V I I , which summarizes the r e s u l t s and draws connect-in g l i n e s between the d i f f e r e n t systems. F i n a l l y , a l l the experimental i n f o r m a t i o n i s c o l l e c t e d i n Chapter V I I I . 62 References 1. E.'G. Rozantsev, 'Free N i t r o x y l R a d i c a l s , ' Plenum, New York, 1970. 2. J . Lajzerowicz i n 'Spin L a b e l i n g , Theory and A p p l i c a t i o n s , ' Ed. L. J . B e r l i n e r , 239-249, Academic, New York, 1976. 3. S. Ament, J . Wetherington, J . Moncrief, K. F l o h r , M. Mochizuki, and E. K a i s e r , J . Am. Chem. S o c , 95_, 7896-7897 (1973). 4. D. Bordeaux, and J . La j z e r o w i c z , Acta C r y s t . , B30, 790-793 (1974). 5. B. J . Gaffney i n 'Spin L a b e l i n g , Theory and A p p l i c a t i o n s , ' Ed. L. J . B e r l i n e r , 183-238, Academic, New York, 1976. 6. I . C. P. Smith i n ' B i o l o g i c a l A p p l i c a t i o n s of E l e c t r o n Spin Resonance Spectroscopy,' Ed. J . R. Bo l t o n , D. Borg and H. Swartz, 483-539, W i l e y - I n t e r s c i e n c e , 1972. 7. P. J o s t , and 0. H. G r i f f i t h i n 'Methods i n Pharmacology,' V o l . I I , Ed. C. C h i g n e l l , 223-276, Appleton-Century-Crofts, New York, 1972. 8. L. J . L i b e r t i n i , and 0. H. G r i f f i t h , J . Chem. Phys., _53, 1359-1367 (1970). 9. D. Bordeaux, J . L a j z e r o w i c z , R. B r i e r e , H. Lemaire, and A. Rassat, Org. Mag:. Res, 5_, 47-52 (1973). 10. A. Capiomont, B. Chion, J . L a j z e r o w i c z , and H. Lemaire, J . Chem. Phys., 60, 2530-2535 (1974). 11. 0. H. G r i f f i t h , D. W. C o r n e l l , and H. M. McConnell, J . Chem. Phys., 43, 2909-2910 (1965). 12. W. Snipes, J . Cupp, G. Cohn, and A. K e i t h , Biophys. J . , JL4_, 20-32 (1974). 13. J . E. Wertz, and J . R. Bolto n , ' E l e c t r o n Spin Resonance, Elementary Theory and P r a c t i c a l A p p l i c a t i o n s , ' McGraw-Hill, New York, 1972. 14. B. R. Knauer, and J . J . Napier, J . Am. Chem. S o c , 98, 4395-4400 (1946). 15. A. T. B u l l o c k , G. G. Cameron, and P. M. Smith, J . Polymer S c i . , Polymer Physics Ed., 11, 1263-1269 (1973). 16. D. K i v e l s o n , J . Chem. Phys., 33, 1094-1106 (1960). 17. P. L. Nordio i n 'Spin L a b e l i n g , Theory and A p p l i c a t i o n s , ' Ed. L. J . B e r l i n e r , 5-52, Academic, New York, 1976. 18. J . H. Freed, i b i d . , 53-132. 63 19. P. Coffey, B. H. Robinson, and L. R. Dalton, Chem. Phys. L e t t s . , 35, 360-366 (1975). 20. S. A. Goldman, G. V. Bruno, and J . H. Freed, J . Chem. Phys., _59, 3071-3091 (1973). 21. F. J . Sharom, and C. W. M. Grant, Biochem. Biophys. Res. Commn., 74, 1039-1045 (1977). 22. A. I. Kokorin, K. I . Zamarayev, G. L. Grigoryan, V. P. Ivanov, and E. G. Rosantsev, B i o f i z i k a , 17, 34-41 (1972). 23. J . C. Waterton, unpublished,,results. 24. M. P. Eastman, R. G. Kooser, R. M. Das, and J . H. Freed, J . Chem. Phys., 51, 2690-2709 (1969). 25. G. I. L i k h t e n s h t e i n , 'Spin L a b e l i n g Methods i n Molecular B i o l o g y , ' W i l e y - I n t e r s c i e n c e , 1976. 26. S. S. Eaton, and G. R. Eaton, Coord. Chem. Rev., 2_6, 207-262 (1978). 27. W. B. Lewis, and L. 0. Morgan, Trans. Metal Chem., _4, 33-112 (1968). 28. A. V. K u l i k o v , and G. I. L i k h t e n s h t e i n , Adv. Mol. Relax. I n t . Proc. , 10, 47-79 (1977). 29. K. M. S a l i k h o v , A. B. Doctorov, Yu. N. M o l i n , and K. I . Zamarayev, J . Mag. Res., 5_, 189-205 (1971). 30. G. I. Skubnevskaya, and Yu. N. M o l i n , K i n e t . R a t a l . , 8^ , 1012-1017 (1967). 31. G. I. Skubnevskaya, K. M. Sa l i k h o v , S. M. Smirnova, and Yu. N. M o l i n , i b i d . , 11, 733-737 (1970). 32. 0. A. Anisimov, A. T. N i k i t a y e v , K. I . Zamarayev, and Yu. N. M o l i n , Teor. Eksp. Khim. , ]_, 556-559 (1971). 33. G. A. J e f f r e y , and L. Lewis, Carbohydr. Res., 60, 179-182 (1978). 34. R. J . Dobbins i n ' I n d u s t r i a l Gums,' Ed. R. L. W h i s t l e r , 19-25, Academic, New York, 1973. 35. T. L. Bluhm, and A. Sarko, Carbohydr. Res., _54, 125-138 (1977). 36. D. A. Rees, i n 'Biochemistry of Carbohydrates, ' Ed. , W. J-. Whelan, MTP I n t l . Rev. S c i . , S e r i e s One, V o l . 5, 1-42, B u t t e r w o r t h s , London, 1975. 37. P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. S c i . U.S.A., 61, 636-642 (1968). 64 38. I . J . G o l d s t e i n , and C. E. Hayes, Adv. Carbohydr. Chem. Biochem., 35, 127-340 (1978). 39. I . H. Silman, and E. K a t c h a l s k i , Ann. Rev. Biochem., 35, 873-908 (1966). 40. U. Lindberg, and T. Persson, Eur. J . Biochem., 31, 246-254 (1972). 41. Z. E r - e l , Y. Zaidenzaig, and S. S h a l t i e l , Biochem. Biophys. Res. Commn., 49, 383-390 (1972). 42. K. B r o c k l e h u r s t , J . Car l s s o n , and M. J . P. K i e r s t a n , Biochem. J . , 133, 573-584 (1973). 43. Z. Horvath, K. F a l b , and K. Fodor, Magy. Kern. F o l y . , 83, 254-257 (1977). 44. F. G a s p a r r i n i , G. P a l m i e r i , and G. C a n c e l l i e r e , Env. S c i . Technol., 10, 9310935 (1976). 45. G. D. Y. Sogah, and D. J . Cram, J . Am. Chem. S o c , 98, 3038-3041 (1976) . 46. J . Porath, J . Chromatog., 159, 13-24 (1978). 47. K. Mosbach, F.E.B.S. L e t t . , 62, E80-E95 (1976). 48. J . Porath, and R. Axen, Meths. Enzymol., 44, 19-45 (1976). 49. R. U. Lemieux, D. R. Bundle, and D. A. Baker, J . Am. Chem. S o c , 97, 4076-4083 (1975). 50. J . Lbnngren, I . J . G o l d s t e i n , and J . E. Niederhuber, Arch. Biochem. Biophys., 175} 661-669 (1976). 51. ' A f f i n i t y Chromatography, P r i n c i p l e s and Methods,' Pharmacia Fine Chemicals, 1974. 52. L. S. Lerman, Proc. Nat. Acad. S c i . U.S.A., 39, 232-236 (1953). 53. M. K. Weibel, E. R. Doyle, A. G. Humphrey, and H. J . B r i g h t , Biochem. Bioeng. Symp., 3, 167-171 (1972). 54. H. P. Jennissen, J . Chromatog., 159, 71-83 (1978). 55. F. R. H a r t l e y , and P. N. Vezey, Adv. Organometal. Chem., 15_, 189-234 (1977). 56. Z. B r z i n s k a , Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia, 1978:1. 57. J . I. Crowley, and H. Rapoport, Accts. Chem. Res., 9_, 135-144 (1976). 65 58. R. B. M e r r i f i e l d , J . Am. Chem. S o c , 85, 2149-2154 (1963). 59. S. L. Regen, and C. K o t e e l , i b i d . , 99, 3837-3838 (1977). 60. A. M. Klibanov, N. 0. Kaplan, and M. D. Kamen, Proc. Nat. Acad. S c i . U.S.A., 75, 3640-3643 (1978). 61. L. L. M i l l e r , and M. R. Van De Mark, J . Am. Chem. S o c , 100, 639-640 (1978). 62. L. L. Kesmodel, and G. A. Somorjai, A c c t s . Chem. Res., 9_, 392-398 (1976). 63. E. E. M u e t t e r t i e s , Agnew. Chem. I n t l . Ed., 17, 545-558 (1978). 64. A. L. Robinson, Science, 197, 34-36 (1977). 65. A. M. Grimstone, 'The E l e c t r o n Microscope i n Biology,' A r n o l d , London, 1977. 66. P. A. W i l k s , and T. H i r s c h f e l d , Appl. Spectrosc. Rev., 1, 99-130 (1967). 67. N. Sharon, 'Complex Carbohydrates,' Addison-Wesley, Reading, Massachusetts, 1975. 68. A. Gottschalk, Ed., 'Glycoproteins: Their Composition, S t r u c t u r e and Function,' E l s e v i e r , Amsterdam, 1972. 69. L. Dorland, J . Haverkamp, J . F. G. V l i e g e n t h a r t , B. Fournet, G. S t r e c k e r , G. Spik, J . M o n t r e u i l , K. Schmid, and J . P. B i n e t t e , F.E.B.S. L e t t . , _89, 149-152 (1978). 70. A. A l l e n , R. H. P a i n , and T. R. Robson, Nature, 264, 88-89 (1976). 71. Y. Yeh, and R. E. Feeney, Accts. Chem. Res., 11, 129-135 (1978). 72. M. G. V i c k e r , and J . G. Edwards, J . C e l l . S c i . , 10, 759-768 (1972). 73. L. W. Jaques, E. B. Brown, J . M. B a r r e t t , W. S. Brey, and W. Weltner, J . B i o l . Chem., 252, 4533-4538 (1977). 74. R. C. Hughes, 'Membrane Gl y c o p r o t e i n s , ' Butterworths, London, 1976. 75. K. M. Yamada, and K. Olden, Nature, 275, 179-184 (1978). 76. G. Ashwell, and A. G. M o r e l l , Adv. Enzymol., 41, 99-128 (1974). 77. R. J . S t o c k e r t , A. G. M o r e l l , and I . H. Scheinberg, Science, 197, 667-668 (1977). 78. G. Ashwell, and A. G. M o r e l l , Trends Biochem. S c i . , 76-78(1977). 66 79. G. M. Gesner, and V. Ginsburg, Proc. Nat. Acad. S c i . U.S.A., 52, 750-755 (1964). 80. R. A. Dwek, 'Nuclear Magnetic Resonance i n Biochemistry,' Clarendon, Oxford, 1973. 81. T. J . Stone, T. Buckman, P. L. Nordio, and H. M. McConnell, P r o c , Nat. Acad. S c i . U.S.A., 54, 1010-1017 (1965). 82. L. J . B e r l i n e r , Ed. 'Spin L a b e l i n g , Theory and A p p l i c a t i o n s , ' Academic, New York, 1976. 83. F. S. A x e l , Biophys. S t r u c t . Mech., 2, 181-218 (1976). 84. H. M. McConnell, and B. J . Gaffney, Quart. Rev. Biophys., _3, 91-136 (1970). 85. W. G. Struve, and H. M. McConnell, Biochem. Biophys. Res. Commn., 49, 1631-1637 (1972). 86. R. W. Wien, J . D. M o r r i s e t t , and H. M. McConnell, Biochemistry, 11, 3707-3716 (1972). 87. J . M. J . Tronchet, E. Mihaly, and M. Geoffroy, Helv. Chim. Acta.., 58, 1187-1191 (1975). 88. J . M. J . Tronchet, J . Ojha-Poncet, E. Winter-Mihaly, B. Kohler, and M. Geoffroy, i b i d . , 60, 888-891 (1977). 89. D. Gagnaire, and L. Odier, B u l l . Soc. Chim. F r . , 2325-2328 (1974). 90. L. Odier, These (Docteur Es-Scienees Physiques), Grenoble, 1975. 91. M. J . Adam, and L. D. H a l l , Carbohydr. Res. ( i n p r e s s ) . 92. J . M a r t i n i e , J . Michon, and A. Rassat, J . Am. Chem. S o c , 97_, 1818-1823 (1975). 93. R. Darcy, and K. F. McGeeney, E x p e r i e n t i a , 32, 1129-1131 (1976). 94. M. C. Cafe, N. G. Pryce, and I . D. Robb, Polymer, 17_, 91-92 (1976). 95. L. S. Johnston, and F. C. Neuhaus, Biochemistry, 16, 1251-1257 (1977). 96. K. J . W i l l a n , B. Golding, D. G i v o l , and R. A. Dwek, F.E.B.S. L e t t . , 80, 133-136 (1977). 97. V. P. Timofeev, I. V. Dudich, Yu. K. Sykulev, and R. S. N e z l i n , i b i d . , 90, 191-195 (1978). 98. R. S. N e z l i n , V. P. Timofeev, Yu. K. Sykulev, and S. E. Zurabyan, Immunochem., 15, 143-144 (1978). 67 99. F. J . Sharom, and C. W. M. Grant, J . Supramol. S t r u c t . , 6_, 249-258 (1977). 100. F. J . Sharom, and C. W. M. Grant, Biochem. Biophys. Res. Commn., 74, 1039-1045 (1977). 101. A. T. B u l l o c k , and G. G. Cameron i n ' S t r u c t u r a l Studies of Macro-molecules by Spectroscopic Methods,' Ed. K. J . I v i n , 273-315, Wiley, London, 1976. 102. P. Tormala, and J . J . Lindberg, i b i d . , 255-272. 103. A. M. Wasserman, T. A. Alexandrova, and A. L. Buchachenko, Eur. Polym. J . , 12, 691-695 (1976). 104. P. L. Kumler, and R. F. Boyer, Macromolecules, _9, 903-910 (1976). 105. Z. V e k s l i , and W. G. M i l l e r , i b i d . , 10, 686-692 (1977). 106. T. Nagamura, and A. E. Woodward, Biopolymers, 16, 907-919 (1977). 107. E. L. Wee, and W. G. M i l l e r , J . Phys. Chem. , 77., 182-189 (1973). 108. R. P. Mason, C. F. Polnaszek, and J . H. Freed, J . Phys. Chem.,78, 1324-1329 (1974). 109. H. Dugas, Accts. Chem. Res., 10, 47-54 (1977). 110. S. K. Zav r i e v , G. L. Grigoryan, and L. E. Minchenkova, Molekulyar-naya B i o l o g i y a , 10, 1387-1393 (1976). 111. D. B. Chesnut, and J . F. Hower, J . Phys. Chem., 75, 907-912 (1971). 112. R. Ramasseul, A. Rassat, P. Rey, and M. Rinaudo, Macromolecules, 9, 186-8 (1976). 113. S. L. Regen, J . Am. Chem. S o c , 96, 5275-5276 (1974). 114. S. L. Regen, i b i d . , 97, 3108-3112 (1975). 115. S. L. Regen, i b i d . , 99, 3838-3840 (1977). 116. J . R. Rasmussen, E. R. Stedronsky, and G. M. Whitesides, i b i d . , 99, 4736-4745 (1977). 117. J . R. Rasmussen, D. E. B e r g b r e i t e r , and G. M. Whitesides, i b i d . , 99, 4746-4756 (1977). 118. V. I. Evreinov, E. V. Lunina, and V. B. Golubev, Zh. F i z . Chim., 47, 1018-20 (1973). 119. G. P. Lozos, and B. M. Hoffman, J . Phys. Chem.,78, 2110-2116 (1974). 68 120. N. S i s t o v a r i s , W. 0. Riede, and H. S i l l e s c u , Ber. Bunsen-Gesellschaft, 79, 882-9 (1975). 121. B. E. Wagner, J . N. H e l b e r t , E. H. Poindexter, and R. D. Bates, Surface Science, 67, 251-268 (1977). 122. M. A. Schwartz, and H. M. McConnell, Biochemistry, 17_, 837-840 (1978). 123. C. A. Evans, and J . R. Bolt o n , J . Am. Chem. S o c , 99^ 4503-4504 (1977). 124. P. B r u l e t , and H. M. McConnell, Biochemistry, 16, 1209-1217 (1977). CHAPTER I I DIFFUSION OF NITROXIDES IN POLYSACCHARIDE SOLUTIONS AND GELS IIA: I n t r o d u c t i o n The f a s c i n a t i n g r h e o l o g i c a l p r o p e r t i e s that many polysaccharides impart to aqueous media have aroused con s i d e r a b l e a t t e n t i o n i n recent years as a r e s u l t of the abundance of p o s s i b l e i n d u s t r i a l applications"'": as non-t o x i c emulsion or foam s t a b i l i s e r s , t h i c k e n e r s , f i l m - f o r m i n g and g e l l i n g agents i n foods, p a i n t s and mining, as d r i l l i n g mud c o n s t i t u e n t s , c o a t i n g agents, chromatographic m a t e r i a l s and f o r a v a r i e t y of pharmaceutical purposes. Many of these uses, however, have developed r a t h e r f o r t u i t o u s l y or, at l e a s t , i n e m p i r i c a l f a s h i o n ; only very r e c e n t l y have attempts begun to be made to i n v e s t i g a t e the molecular i n t e r a c t i o n s which l e a d , f o r example, to non-Newtonian behaviour i n s o l u t i o n . The present chapter describes experiments that were designed to t e s t the r e l a t i o n s h i p between the motion of s m a l l , i d e a l l y n o n - i n t e r a c t i n g s o l u t e molecules ( n i t r o x i d e s ) and the bulk or macroscopic p r o p e r t i e s of s o l u t i o n s and gels of a number of polysaccharides of i n d u s t r i a l importance, two of which are s e l e c t e d f o r f u r t h e r study i n subsequent chapters. This behaviour i s contrasted w i t h that found i n s o l u t i o n s of sucrose. The primary s t r u c t u r e s of the saccharides u t i l i z e d are shown i n Table I I - l . 69 70 Table I I - l : Saccharides used i n Chapter I I sucrose starch.; l o c u s t bean gum xanthan gum agarose a l g i n i c a c i d " D-Glc-^D-Fru 3 a amylose -[^D-GIC^AD-GIC^1 J a an + amylopectin (branched chain polymers) (14) (15) lB Jn 6 1 D-Gal 3Jm 4 1 4 1 -FD-Glc^- J 1D-Glc- 5+ 3 Jn D-Man-6-0Ac 2 1 3 D-GlcA 4 1 D-Glc ^  pyr - A - G a l ^ S , 6-Anhydro-L-Gal-^r 3 a n -t-^D-MaiiAo] [ 4L-GulA^-p n a m (16) (17) (18) (19) *some 6-OCH3 su b s t i t u e n t s • Starch and agarose d i s s o l v e i n hot or b o i l i n g water, forming gels upon c o o l i n g . A l g i n i c a c i d i s s o l u b l e i n i t s s a l t form ( a l g i n a t e ) w i t h monovalent counterions. With d i - and t r i - v a l e n t c a t i o n s , of which calcium i s b i o l o g i c a l l y perhaps most important, i t forms g e l s . Locust bean (carob) gum i s f u l l y s o l u b l e only i n hot water but does not g e l . Xanthan gum i s s o l u b l e i n c o l d water and does not g e l ; s o l u t i o n s of xanthan gum co n t a i n i n g l o c u s t bean gum experience a s y n e r g i s t i c v i s c o s i t y i n c r e a s e 71 (maximised at 1:1 proportions) and g e l a f t e r heating to > 327 K and subse-quent c o o l i n g . Sucrose i s s o l u b l e i n water to about 300% weight/volume (above which s u p e r s a t u r a t i o n may occur) at which point the s o l u t i o n i s of a v i s c o s i t y roughly comparable to that of a 1% s o l u t i o n of a l g i n a t e ! A l l the polysaccharides form viscous s o l u t i o n s so that working at concentra-t i o n s above about 5% weight/volume i s o f t e n i m p r a c t i c a l as a r e s u l t of inhomogeneity. The molecular nature of agarose and calcium a l g i n a t e gels i s discussed i n some d e t a i l i n Chapter I I I . Amylose i s capable of c r y s t a l l i z i n g i n a 6 7 number of d i f f e r e n t conformations, at l e a s t some of which are h e l i c a l ' . However, the molecular nature of s t a r c h gels i s l i k e l y to be complex and 8 has not been pr o p e r l y c h a r a c t e r i s e d . I t has been suggested that the i n t e r a c t i o n between xanthan and l o c u s t bean gums which leads to g e l a t i o n i n v o l v e s s o - c a l l e d 'smooth' regions of the l a t t e r i n which no galactose s u b s t i t u e n t s are found on the backbone, i n t e r a c t i n g w i t h a p a r t i c u l a r ( p o s s i b l y h e l i c a l ) conformation. The exact d e t a i l s of the three-dimensional s t r u c t u r e of xanthan gum, a m i c r o b i a l product s p e c i f i c a l l y 9 developed f o r commercial purposes, are s t i l l a matter of controversy; three d i f f e r e n t models exist"*"^'"^>^>^ w I t seems c l e a r that the conform-a t i o n of the si d e - c h a i n — w h e t h e r extended or f o l d e d i n t o the backbone— depends upon the i o n i c s t r e n g t h of the s o l u t i o n . Not a c c i d e n t a l l y , the polysaccharides s e l e c t e d here represent some-t h i n g of a c r o s s - s e c t i o n i n terms of the complexity of t h e i r primary s t r u c t u r e s and t h e i r o r i g i n s . A l g i n a t e and agar, of which agarose i s the l e a s t sulphated, g e l l i n g f r a c t i o n , are w a t e r - r e t a i n i n g seaweed p o l y -saccharides whose b i o l o g i c a l r o l e probably i n v o l v e s both p h y s i c a l and chemical c o n t r o l of the weed's environment; xanthan i s the e x t r a c e l l u l a r polysaccharide of the bacterium Xanthomonas campestris; l o c u s t bean gum i s the seed gum of the carob t r e e , and s t a r c h i s used.for energy, storage i n p l a n t s . Xanthan and a l g i n a t e c o n t a i n a c i d i c f u n c t i o n a l i t i e s l i k e l y to give r i s e i n t h e i r i o n i s e d form to extended conformations and increased v i s c o s i t y , - w h i l e the other m a t e r i a l s are n e u t r a l . IIB: Tumbling The e f f e c t on the esr spectrum of a 0.5mM s o l u t i o n of l a b e l (9) of i n t r o d u c i n g a l a r g e (285% w/v) c o n c e n t r a t i o n of sucrose i s shown i n Figure I l - l a and b, and Figure I I - 2 shows the monotonic increase i n macroscopic v i s c o s i t y that accompanies the increase of sucrose c o n c e n t r a t i o n and con-comitant decrease of molecular tumbling r a t e . The l a t t e r i s c h a r a c t e r i s e d by the c o r r e l a t i o n time (x) c a l c u l a t e d using the r a t i o - o f - h e i g h t s method. This behavior p a r a l l e l s that of another n i t r o x i d e d e r i v a t i v e (3) i n g l y c e r o l shown as a f u n c t i o n of temperature i n Figure 1-7, and of l a b e l (2) as a f u n c t i o n of g l y c e r o l c o n c e n t r a t i o n i n Figure IV-5. No such (9) (2) (3) r e l a t i o n s h i p was found between micro- and macro-scopic v i s c o s i t y i n any of the polysaccharide systems chosen, whether i n the s o l u t i o n or g e l s t a t e . Figure I I - l c and d show l a b e l (9) tumbling, f o r example, i n 1% sodium a l g i n a t e (which has a greater macroscopic v i s c o s i t y than 285% sucrose) and a g e l formed by a d d i t i o n of calcium ions to t h i s s o l u t i o n . F i gure II-3e shows l a b e l (2) i n a 3% agarose g e l . I t i s obvious that i n the 73 Figure 11-1: Esr spectra of 0.5 M s o l u t i o n s of l a b e l ( 9 ) i n (a)water; (b)285% aqueous sucrose; ( c ) l % aqueous sodium a l g i n a t e s o l u t i o n ; (d)calcium a l g i n a t e g e l . 74 CD o Q_ C CD 10 O C J > CD O o C D 160 140 H 120 100 8 0 -6 0 40 2 0 1 0 0 5 10 ^5 — T " 20 10 ,9c(s) Figure 11-2: Dependence of T on macroscopic v i s c o s i t y i n a s e r i e s of aqueous sucrose s o l u t i o n s (100 - 200% w/w). 75 p o l y s a c c h a r i d e s , microscopic v i s c o s i t y i s q u i t e independent of the b u l k p r o p e r t i e s of the system; the gels can be cut w i t h a k n i f e ! The same was found to be t r u e at higher concentrations of a l g i n a t e and agarose ( i n s o l u t i o n s up to 5% w/v) and i n a l l the other polysaccharides i n s i m i l a r 14 c o n c e n t r a t i o n ranges . Here t h e r e f o r e the l a b e l r e p o r t s only l o c a l con-d i t i o n s . Rapid r e o r i e n t a t i o n probably a r i s e s as a r e s u l t of the l o c a l i s a -t i o n of the l a b e l i n solvent pockets the i n t e r i o r s of which are not s i g n i f -i c a n t l y i n f l u e n c e d by the i n t e r a c t i o n s (between macromolecules i n t h e i r s o l v a t i o n spheres) that are r e s p o n s i b l e f o r the macroscopic v i s c o s i t y of the system. IIC: Exchange I t might be expected that a more s e n s i t i v e index of t r a n s l a t i o n a l d i f f u s i o n of f r e e r a d i c a l s w i t h i n a g e l matrix or macromolecular s o l u t i o n may be obtained by measuring l i n e w i d t h s i n the absence of r e l a x a t i o n changes due to r o t a t i o n a l r e o r i e n t a t i o n (as i s shown i n IIB f o r v a r i o u s p o l y -saccharides, though not f o r sucrose) but i n the presence of s u f f i c i e n t l y high concentrations of r a d i c a l that e l e c t r o n exchange, a r i s i n g from c o l l i -sions between p a i r s of r a d i c a l s , c o n t r i b u t e s measurably to T 2. In Figure I I - l , c o l l i s i o n a l events occur s u f f i c i e n t l y r a r e l y that the exchange c o n t r i b u t i o n to the l i n e w i d t h i s n e g l i g i b l e . Three higher concentrations of l a b e l (2) were chosen f o r the l i n e w i d t h measurements: 0.025M, at the onset of exchange broadening; 0.26M, near, but not at the exchange narrow-i n g l i m i t ; and 0.09M, approximately i n the middle of the range i n which n i t r o x i d e l i n e w i d t h s are s e n s i t i v e to c o n c e n t r a t i o n i n aqueous s o l u t i o n at room temperature. The r e s u l t s of comparisons between the l i n e w i d t h s at these concentrations i n water and i n 3% agarose g e l at 300 K are shown i n Table I I - 2 , and the s p e c t r a are shown i n Figure I I - 3 . E s s e n t i a l l y the 76 Table I I - 2 : Linewldths and s p l i t t i n g s f o r three concentrations of n i t r o x i d e (2) i n water and 3% agarose g e l conce n t r a t i o n of (2) (M) i n water i n 3% agarose g e l width of centre l i n e (G) s p l i t t i n g of outer f e a t u r e s (G) width of centre l i n e (G) s p l i t t i n g of outer f e a t u r e s (G) 2.5 x 10" 2 3.4 ± 0.2 3.3 ± 0.2 9.0 x 10" 2 8.1 ± 0.5 32.0 ± 1.0 9.0 ± 0.5 32.2 ± 1.0 2.6 x 1 0 _ 1 8.8 ± 0.2 8.9 ± 0.2 same r e s u l t obtained f o r agarose i n s o l u t i o n , and f o r sodium a l g i n a t e s o l u -t i o n s and calcium a l g i n a t e gels up to 5% concentrations; namely that the l i n e w i d t h s were the same w i t h i n experimental e r r o r i n the presence as i n the absence of the pol y s a c c h a r i d e , i n d i c a t i n g that exchange a l s o occurs l a r g e l y independently of the macromolecules. IID: D i s c u s s i o n In the f u l l y hydrated polysaccharides used f o r these experiments, l a r g e pores e x i s t c o n t a i n i n g solvent and sm a l l s o l u t e molecules which behave i n much the same way as i n f r e e s o l u t i o n , d e s p i t e the r a d i c a l changes e f f e c t e d i n macroscopic p r o p e r t i e s . Thus t r a n s l a t i o n i s not u s u a l l y i n t e r r u p t e d between c o l l i s i o n s by polymer chains nor t h e i r h y d r a t i o n spheres, and f r i c t i o n a l i n t e r a c t i o n s (which i n f l u e n c e r o t a t i o n ) are unchanged. In the context of such systems, t h e r e f o r e , i t i s not adequate to continue to desc r i b e exchange r a t e s as i n v e r s e l y p r o p o r t i o n a l to v i s c o s i t y , but r a t h e r to the microscopic v i s c o s i t y experienced by the paramagnetic s p e c i e s . Previous work i n which n i t r o x i d e s were allowed to d i f f u s e w i t h i n the pores of polyacrylamide beads'^ suggests that only when pores become s u f f i c i e n t l y 77 Figure I I - 3 : Esr sp e c t r a showing exchange broadening of l a b e l ( 2 ) at 300 K. (a)5.0 * 10 _ 1 +M i n water; (b)2.5 x io" 2M i n water; (c)9.0 x io 2M i n water; (d)2.6 x 10 _ 1M i n water; (e)5.0 x'io _ l +M i n 3% agarose g e l ; ( f ) 2 . 5 x 10"2M i n 3% agarose g e l ; (g)9.0 x 10 _ 2M i n 3% agarose g e l ; (h)2.6 x 10 _ 1M i n 3% agarose g e l . s m a l l to exclude s p h e r i c a l molecules of molecular weight > ^6 x 10 3 does t r a n s l a t i o n a l d i f f u s i o n begin to be a f f e c t e d ; r o t a t i o n a l d i f f u s i o n i s r e l a t i v e l y i n s e n s i t i v e to the polymer w e l l below t h i s l i m i t . In commerc-i a l l y a v a i l a b l e bead form, 6% agarose (Sepharose 6B) excludes molecules > 4 x 10 5 molecular weight. Thus i t i s not unreasonable that pores w i t h i n gels c o n t a i n i n g the highest agarose concentrations used here (5%) are s u b s t a n t i a l l y l a r g e r than those r e q u i r e d f o r e f f e c t s to be measurable i n n i t r o x i d e s p e c t r a . The same c o n c l u s i o n can be drawn f o r a l g i n a t e . R o t a t i o n a l d i f f u s i o n of f l u o r e s c e n t - l a b e l l e d p r o t e i n molecules has a l s o been shown to be i n s e n s i t i v e to the presence of v a r i o u s polysaccharides 16 i n concentrations s i m i l a r to those used here , though at consid e r a b l y higher concentrations (20 - 30%) e f f e c t s begin to be seen. No doubt (and t h i s i s to be expected by analogy w i t h s p i n l a b e l l i n g s t u d i e s of s y n t h e t i c polymers''"7'"'"^ and collagen"^) at much lower degrees of h y d r a t i o n , the e f f e c t s of 'forcing' the l a b e l i n t o i n t e r a c t i o n w i t h the polysaccharide would be f e l t i n the esr spectrum, but the study of p a r t i a l l y hydrated systems was outside the scope of the present i n v e s t i g a t i o n . One p u b l i c a -t i o n , however, does re p o r t that f l u o r e s c e n c e d e p o l a r i s a t i o n of a s m a l l "probe" molecule (uranine) i s s e n s i t i v e to the presence of agarose at very 20 low co n c e n t r a t i o n (0 - 0.3% by weight) and i t s g e l a t i o n ; t h i s i s an anomaly i n the present l i t e r a t u r e , and the authors themselves suggest that b i n d i n g of the probe to the polymer may be r e s p o n s i b l e . 21 22 23 Studies of d i f f u s i o n ' and fluorescence d e p o l a r i s a t i o n of s o l u t e s w i t h i n polysaccharide gels other than those p r e s e n t l y of i n t e r e s t suggest that s o l u t e t r a n s l a t i o n a l , though not r o t a t i o n a l , d i f f u s i o n may be a f f e c t e d e i t h e r by l o c a l changes i n solvent v i s c o s i t y owing to o r d e r i n g near hydrated polymer chains or, at higher temperatures, by the motions of 79 t h e polymer c h a i n s t h e m s e l v e s even a t c o n c e n t r a t i o n s s i m i l a r t o t h o s e used h e r e . There i s a l s o e v i d e n c e f o r l o n g - r a n g e ( t e n t h s o f m i l l i m e t e r s ) and 24 s l o w ( t e n s o f m i n u t e s ) r e a r r a n g e m e n t s of g e l s t r u c t u r e i n a l g i n a t e and f o r v e r y s l o w (days) a g g r e g a t i o n - d i s p e r s i o n phenomena i n s o l u t i o n s o f 25 M e n i n g o c o c c a l p o l y s a c c h a r i d e s . . D a t a a r e as y e t so s c a r c e and f r a g m e n t a r y , however, t h a t no p i c t u r e can be b u i l t up of p o l y s a c c h a r i d e b e h a v i o u r i n s o l u t i o n and t h e i n f l u e n c e o f p r i m a r y s t r u c t u r e on i t . 21 22 23 P r e v i o u s d i f f u s i o n s t u d i e s ' ' used mono- o r o l i g b - s a c c h a r i d i c o r o t h e r w i s e h y d r o x y l i c s o l u t e s , and i t must be c o n c l u d e d t h a t t h e s p i n l a b e l i s n o t f o r p r e s e n t p u r p o s e s a good p r o b e , perhaps because i t does n o t , i n t h e p r e s e n c e o f e x c e s s s o l v e n t (> 95% by w e i g h t ) , compete e f f e c t i v e l y f o r s i t e s a d j a c e n t t o polymer m o l e c u l e s . I t was i n f e r r e d t h a t i n o r d e r t o g a i n i n s i g h t i n t o m o l e c u l a r p r o c e s s e s a s s o c i a t e d w i t h p o l y s a c c h a r i d e g e l a -t i o n , c o v a l e n t a t t a c h m e n t o f t h e l a b e l t o t h e polymer might p r o v i d e a more p r o f i t a b l e avenue. T h i s a p p r o a c h i s f o l l o w e d i n C h a p t e r I I I i n f u r t h e r i n v e s t i g a t i o n s o f two p o l y s a c c h a r i d e s , a g a r o s e and a l g i n a t e . 80 References 1. R. L. W h i s t l e r , Ed., ' I n d u s t r i a l Gums,' Academic, New York, 1973. 2. R. L. W h i s t l e r , Ed., 'Methods i n Carbohydrate Chemistry,' V o l . IV, Academic, New York, 1963. 3. P. E. Jansson, L. Kenne, and B. Lindberg, Carbohydr. Res., 45, 275-282 (1975). 4. M. Duckworth, and W. Yaphe, i b i d . , 16, 189-197 (1971). 5. J . Boyd, and J . R. Turvey, i b i d . , 6>6, 187-194 (1978). 6. H. H. Wu, and A. Sarko, i b i d . , 54, C3-C6 (1977). 7. K. Kainuma, and D. French, Biopolymers, 11_, 2241-2250 (1972). 8. I . C. M. Dea, E. R. M o r r i s , D. A. Rees, E. J . Welsh, H. A. Barnes, and J . P r i c e , Carbohydr. Res., 57_, 249-272 (1977). 9. A. Jeanes, J . E. P i t t s l e y , and F. R. S e n t i , J . Appl. Polym. S c i . , _5, 519-526 (1961). 10. G. Holzwarth, and F. G. P r e s t r i d g e , Science, 197, 757-759 (1977). 11. G. Holzwarth, Carbohydr. Res., 66, 173-186 (1978). 12. R. Moorhouse, M. D. Walkinshaw, and S. Arnott i n ' E x t r a c e l l u l a r M i c r o b i a l P o l y s a c c h a r i d e s ; ' ACS Symp. Ser. , 45^, 90-102 (1977). 13. C. J . Lawson, personal communication. 14. J . D. A p l i n , and L. D. H a l l , Carbohydr. Res., 59, C20-C24 (1977). 15. A. D. K e i t h , W. Snipes, R. J . Mehlhorn, and T. Gunter, Biophys. J . , 19, 205-217 (1977). 16. B. N. Preston, B. Obrink, and T. C. Laurent, Eur. J . Biochem., 33, 401-406 (1973). 17. A. M. Wasserman, T. A. Alexandrova, and A. L. Buchachenko, Eur. Polym. J . , 12, 691-5 (1976). 18. P. L. Kumler, and R. F. Boyer, Macromolecules, 9_, 903-910 (1976). 19. T. Nagamura, and A. E. Woodward, Biopolymers, 16, 907-919 (1977). 20. A. Hayashi, K. K i n o s h i t a , and M. Kuwano, Polymer J . , 9_, 219-225 (1977). 21. W. Brown, and K. Chitumbo, J . Chem. Soc. Faraday I , 7JL, 1-11 (1975). W. Brown, and K. Chitumbo, i b i d . , 7_1, 12-21 (1975). W. Brown, G. Klerow, K. Chitumbo, and T. Amu, i b i d . , 72_, 485-494 (1976). W. Mackie, D. B. S e l l e r s , and J . S u t c l i f f e , Polymer, 19, 9-16 (1978). S.Fujime, F.C.Chen, and B.Chu, Biopolymers, 16, 945-963 (1977). \ CHAPTER I I I GELLING PROCESSES I I I A : Agarose ( i ) S t r u c t u r e of Agarose Gels Agar, a mixture of polysaccharides e x t r a c t e d from Rhodophyta, or red seaweed, f i r s t found a p p l i c a t i o n as a c u l t u r e medium f o r microorganisms"'" but has sin c e become more f a m i l i a r to the biochemist as a source of mater-i a l s u s e f u l f o r the separation of macromolecules w i t h a wide range of molecular weights (Chapter I V ) . Agaropectin, the charged, n o n - g e l l i n g f r a c t i o n of polysaccharide has not been e x t e n s i v e l y c h a r a c t e r i s e d ; agarose (18) i s the name given to the least-charged, g e l l i n g f r a c t i o n . I t s i d e a l i z e d s t r u c t u r e has a l t e r n a t i n g residues of 1 — 4 - l i n k e d 3,6-anhydro-a-L-2 galactopyranose and 1 — 3 - l i n k e d g-D-galactopyranose ; i n p r a c t i c e , however, agarose (18) the g e l l i n g f r a c t i o n of agar i s a complex mixture e x h i b i t i n g a considerable p o l y d i s p e r s i t y , the exact nature of which i s dependent on the source of the agar, i t s stage of development when harvested, and the t r o u b l e taken i n i t s purification"'". Thus s m a l l q u a n t i t i e s of O-sulphate w i l l g e n e r a l l y be 82 83 present together w i t h methoxyl and pyruvate, the l a t t e r 4,6-linked to D-galactose. Sulphate and pyruvate s u b s t i t u e n t s lend an i o n exchange cap a c i t y to the polymer which i s not d e s i r a b l e f o r most biochemical separ-a t i o n s ; these as w e l l as the 0-methyl s u b s t i t u e n t s a f f e c t the g e l l i n g p r o p e r t i e s of the system i n ways which have been at l e a s t q u a l i t a t i v e l y elaborated"'". I n t e r e s t i n g comparisons based on primary and higher order s t r u c t u r e s can a l s o be made between the g e l l i n g p r o p e r t i e s of agaroses and 3 those of carrageenans, a c l o s e l y r e l a t e d f a m i l y of seaweed polysaccharides . This b a s i c primary s t r u c t u r e has been confirmed r e c e n t l y by means of 4 enzymic d i g e s t i o n s of agarose ; however, the r e l a t i v e s c a r c i t y of such enzymes i n Nature provides a d d i t i o n a l advantage to the biochemist, who wishes to avoid degradation during periods of storage. S i m i l a r l y advantageous i s the chemical s t a b i l i t y of agarose, which r e a d i l y t o l e r -ates pH values between 3 and 11"\ Under more a c i d i c c o n d i t i o n s reducing sugars are produced by h y d r o l y s i s of the g l y c o s i d i c l i n k a g e of anhydro-galactose r e s i d u e s . Under more b a s i c c o n d i t i o n s some 'peeling' ( l o s s of monosaccharide from the reducing terminus) and d i s c o l o u r a t i o n may occur. U n t i l about 10 years ago l i t t l e was known about the molecular i n t e r -a c t i o n s which cause g e l a t i o n i n aqueous po l y s a c c h a r i d e s , and s t u d i e s of the g e l phase were s t i l l l i m i t e d by t h e o r e t i c a l and p r a c t i c a l d i f f i c u l t i e s to a very few p h y s i c a l techniques. However, recent X-ray d i f f r a c t i o n s t u d i e s of the condensed s t a t e , when c o r r e l a t e d w i t h s t u d i e s of the g e l u s i n g other p h y s i c a l methods ( o p t i c a l r o t a t i o n , n.m.r., molecular weight, thermodynamic 6 measurements, microscopy ), have provided q u i t e good c i r c u m s t a n t i a l e v i -dence that i n t e r a c t i o n s are s i m i l a r i n the two cases, p a r t i c u l a r l y i n the 3 . 7 carrageenans , but a l s o xn agarose . According to the X-ray data^, which was i n t e r p r e t e d using computerised molecular model b u i l d i n g , f i l m s and f i b r e s of agarose are made up of thr e e -f o l d (that i s , three d i s a c c h a r i d e residues to one turn) double h e l i c e s i n which the i n d i v i d u a l chains are p a r a l l e l , . w i t h a p i t c h of 1.90 nm and t r a n s l a t e d a x i a l l y by 0.95 nm r e l a t i v e to each other. Two p r o j e c t i o n s of the double h e l i x are shown i n Figure I I I - l . Because the h e l i x i s r a t h e r more compressed than (say) those e x h i b i t e d by carrageenans, i t s i n t e r i o r i s not completely occupied by the two polymer chains, and i t i s thought that water molecules may r e s i d e there; convenient hydrogen-bonding schemes can be drawn up 7. Thus, the agarose h e l i x d i f f e r s fundamentally from those formed by amylose (15) i n s o f a r as the l a t t e r present non-hydrogen-bonding moieties (mainly C-H bonds) to the centre, enabling the accommoda-t i o n of non-polar species as i n c l u s i o n complexes. In agarose, the c a v i t y i s smaller (0.45 nm at the narrowest point between atomic centres, compared w i t h 0.8 nm i n V-amylose), and inclu d e s 0-2 of galactose and 0-5 of 3,6-anhydrogalactose; both l i k e l y to engage i n H-bonding. Much the same s t r u c t u r e was found to occur i n various s u b s t i t u t e d agaroses 7: those i n c o r p o r a t i n g 6-0-sulphate, 6-0-methyl and 4,6-0-pyruvate d e r i v a t i v e s of D-galactose, and the 2-0-sulphate and 2-0-methyl d e r i v a t i v e s of 3,6-anhydro-L-galactose, a l l present i n v a r y i n g degree 7. Of the four (nomin-a l l y ) u n s u b s t i t u t e d hydroxyl groups per d i s a c c h a r i d e , only the 2-0H of galactose faces the i n t e r i o r of the h e l i x ; s u b s t i t u t i o n of bulky or charged groups at t h i s p o s i t i o n might be expected to d e s t a b i l i s e i t . A r n o t t , Rees e t _ a l . 7 went on to p o s t u l a t e on the b a s i s of changes observed i n o p t i c a l r o t a t i o n at the g e l m e l t i n g and s e t t i n g p o i n t s that the left-handed double h e l i c e s present i n damp f i b r e s and f i l m s a l s o provide the means byywhich i n t e r c h a i n l i n k s form i n g e l s . For gels made from u n s u b s t i t u t e d as w e l l as the various s u b s t i t u t e d agaroses a h y s t e r e s i s 85 a b c Figure 111 -1: (a)Agarose s i n g l e h e l i c e s ; (b)agarose double h e l i c e s , sideways and (c)end-on p r o j e c t i o n s . Note the inward-pointing hydroxyls i n (c) (from reference 7 ) . i s observed i n o p t i c a l r o t a t i o n , the value remaining constant w i t h i n c r e a s -i n g temperature, u n t i l a sharp i n c r e a s e i s observed at a temperature at or near the g e l m e l t i n g point (the l a t t e r c h a r a c t e r i s e d by mechanical measure-ments). No f u r t h e r increase occurs above t h i s p o i n t . On c o o l i n g the s o l , the r o t a t i o n remains constant, f i n a l l y undergoing a sharp decrease at or about the g e l s e t t i n g temperature, which may be as much as 50 K below the melt i n g point f o r a given g e l . These two c h a r a c t e r i s t i c temperatures are i n general d i f f e r e n t f o r d i f f e r e n t agarose samples; thus, f o r example, g e l l i n g temperature increases w i t h the methoxyl content of the polymer''". Since at any given temperature one f o r m — t h e s o l or the g e l — i s l i k e l y to be more thermodynamically s t a b l e than the other, the occurrence of a h y s t e r e s i s , a r i s i n g from a l a c k of a b i l i t y to i n t e r c o n v e r t and form an e q u i l i b r i u m at the point where the f r e e energies of the two forms are the same, i m p l i e s the existence of k i n e t i c b a r r i e r s to m e l t i n g or s e t t i n g or 8 9 both. S e t t i n g , by analogy w i t h the behaviour of DNA double h e l i c e s ' and c o l l a g e n t r i p l e h e l i c e s " ^ , could be c o n t r o l l e d by ' n u c l e a t i o n ' , the r a t e of which increases w i t h decreasing temperature. However, l i g h t s c a t t e r i n g measurements"'"''" have shown a s i n u s o i d a l c o n c e n t r a t i o n v a r i a t i o n through the g e l , i n d i c a t i n g that separation i n t o domains of high concentra-t i o n by a s p i n o d a l mechanism may precede nucleated a s s o c i a t i o n . I t i s c l e a r that i s o l a t e d double h e l i c a l i n t e r a c t i o n s cannot alone account f o r the g e l l i n g phenomenon; one or both of two a d d i t i o n a l pro-cesses must be invoked. By analogy w i t h the carrageenans i t i s p o s s i b l e that simple p a i r - w i s e i n t e r a c t i o n s between molecules (such as that o c c u r r i n g i n DNA) are prevented by the in t r u s i o n ; , i n t o the sequence of a h e l i x -12 breaking sugar u n i t , perhaps a (non-anhydro-) L-galactose, which may have a 6-0-sulphate s u b s t i t u e n t (the l a t t e r i s a b i o s y n t h e t i c precursor of 87 3,6-anhydro-L-galactose). Such a residue would be c o n f o r m a t i o n a l l y unable to continue the h e l i c a l i n t e r a c t i o n , i n s t e a d branching away from i t s 12 neighbour . In t h i s way,a s i n g l e chain could p a r t i c i p a t e i n more than one " j u n c t i o n " r e g i o n , these being i n t e r s p e r s e d between regions of unassoci-ated chain, g i v i n g r i s e to a f u l l three-dimensional g e l network. However, although the magnitude of the o p t i c a l r o t a t i o n changes ( i n t e r p r e t e d i n terms of incremental p a i r w i s e i n t e r a c t i o n s ) imply that.most residues are present i n h e l i c a l a s s o c i a t i o n s 7 , i t i s l i k e l y that such a s s o c i a t i o n s do not dominate the g e l a t i o n process. Changes i n g e l t e x t u r e , o p t i c a l 3 13 d e n s i t y and l i g h t s c a t t e r i n g p r o p e r t i e s ' , the appearance of the g e l i n a 11 14 l i g h t microscope as w e l l as under the e l e c t r o n microscope , and i t s c h a r a c t e r i s t i c s as a g e l f i l t r a t i o n matrix"'""' a l l suggest l a r g e r aggregates, as does the c o n c e n t r a t i o n dependence of s p e c i f i c o p t i c a l rotation''""'", which suggests that conformational r e s t r a i n t s may a r i s e from i n t e r - h e l i x packing i n a d d i t i o n to h e l i x formation. A f u r t h e r and most important p o i n t i s that below 0.3% w/w, agarose s o l u t i o n s no longer g e l ; nor do they show a h y s t e r e s i s i n o p t i c a l r o t a t i o n , though a r e v e r s i b l e t r a n s i t i o n i s s t i l l observed near the s e t t i n g region''""'". Thus h y s t e r e s i s i s r e l a t e d to i n t e r -h e l i x a s s o c i a t i o n s of a k i n d that has not been c h a r a c t e r i s e d at the mole-3 7 c u l a r l e v e l . Rees ' has proposed a two-stage g e l a t i o n process as shown i n Figure I I I - 2 , i n which d i m e r i s a t i o n i s followed by formation of 'bundles' as a consequence of the s t i f f n e s s of the h e l i x , that i s , i t s l a c k of con-f o r m a t i o n a l entropy r e l a t i v e to random c o i l . A ' t h i r d t r a n s i t i o n ' between me l t i n g and g e l l i n g has been observed using fluorescence d e p o l a r i s a t i o n , o p t i c a l r o t a t i o n , reduced v i s c o s i t y and d i f f e r e n t i a l scanning c a l o r i m e t r y 16 by Hayashi et a l . and i d e n t i f i e d as a c o i l - h e l i x t r a n s i t i o n , but t h i s has not been detected by other workers. I-carrageenan, a s i m i l a r polysaccharide w i t h a high charge de n s i t y owing to the presence of O-sulphate s u b s t i t u e n t s , 88 89 can, i n c o n t r a s t , form s o l u b l e double helices"*"^. Furthermore, p a r t i a l degradation of carrageenans gives r i s e to n o n - g e l l i n g systems which show 3 the same o p t i c a l r o t a t i o n changes, but no h y s t e r e s i s . H y s t e r e s i s i n agarose can e q u a l l y be abolished by the presence of 2M potassium thiocyanate, which d i s t u r b s hydrogen bonding. These r e s u l t s may render the p o s t u l a t e of s p e c i a l h e l i x - b r e a k i n g residues as i n t e g r a l to the formation of a three-12 dimensional g e l network , as w e l l as the idea of s i n g l e chains p a r t i c i p a t -i n g i n many d i f f e r e n t h e l i c a l a s s o c i a t i o n s i n t e r s p e r s e d between lengths of 3 si n g l e - s t r a n d e d polymer (a d i f f i c u l t p h y s i c a l p r o c e s s ) , both unnecessary. I t i s p e r f e c t l y c l e a r i n the l i g h t of these published r e s u l t s that there i s a need f o r a d d i t i o n a l i n f o r m a t i o n on the molecular processes i n v o l v e d i n g e l formation and d i s s o l u t i o n . Published experiments w i t h 16 fluorescence spectroscopy use probes d i s s o l v e d i n the g e l or s o l u t i o n ; the use of the s p i n l a b e l l i n g technique, i n which n i t r o x i d e s are chemically attached to the polysaccharide molecules, was seen as an i d e a l method f o r probing changes i n m o b i l i t y i n i n t e r a c t i n g chains i n a r a t h e r more s p e c i f i c way than has p r e v i o u s l y been p o s s i b l e , s i n c e the problems i n v o l v e d i n i n t e r p r e t i n g macroscopic ( v i s c o s i t y , c a l o r i m e t r y ) measurements are l e g i o n , ( i i ) Spin L a b e l l i n g of Agarose An i d e a l procedure f o r a t t a c h i n g n i t r o x i d e to agarose would i n v o l v e m i l d c o n d i t i o n s w i t h the minimum of chemical p e r t u r b a t i o n and a low l e v e l of i n c o r p o r a t i o n or 'loading' i n order to preserve the n a t i v e s t r u c t u r e , i n s o f a r as t h i s i s commensurate w i t h workable s i g n a l - t o - n o i s e i n e s r . Cyanogen b r o m i d e - a c t i v a t i o n i s the most e f f i c i e n t , m i l d , aqueous method f o r m o d i f i c a t i o n of polysaccharides (discussed i n d e t a i l i n Chapter IV) but was found ;to r e s u l t i n c r o s s - l i n k i n g such that when p r e c i p i t a t e d agarose was l a b e l l e d i n c o l d water, i t was no longer s o l u b l e i n hot water. 90 Attempts made to l a b e l the gel form were not successful, probably because of the unfavourable competition between hydrolysis of the cyanogen bromide and i t s penetration i n t o , and subsequent reaction with, the g e l . Neither was reaction with the so l u t i o n form at 313 K successful, perhaps because of the requirement for two neighbouring hydroxyl f u n c t i o n a l groups being i n s u f f i c i e n t l y f u l f i l l e d i n i s o l a t e d chains, or because CNBr i s too r a p i d l y hydrolysed at such temperatures. Other unsuccessful attempts to l a b e l agarose i n aqueous bead form using non-crosslinking reagents are described i n Chapter IV. The method chosen for the present i n v e s t i g a t i o n consists of the reaction of hydroxyl groups on the polysaccharide i n the presence of strong base with 4-chloroacetamido-2,2,6,6-tetramethylpiperidine-l-oxyl (5), 18 synthesized according to McConnell . Although agarose i s rather stable to base, t h i s procedure i s not as mild as might have been desired; peeling was, however, minimised by using agarose i n the p r e c i p i t a t e d form wherein access i s more lim i t e d and degradation correspondingly r e s t r i c t e d . This was also convenient since the low s o l u b i l i t y of the l a b e l i n water necessitated the presence of a mixed solvent system (acetone/water). Furthermore, the l i m i t e d access also ensured low loading, and i t was reasonable to a n t i c i p a t e that h e l i c a l associations and aggregates present i n the l a b e l l e d s o l i d would be capable of reforming i n the gel phase. Th 91 acetamidoether l i n k a g e s thus created were s t a b l e under a l l c o n d i t i o n s used to study the behaviour of the l a b e l l e d agarose, no leakage being observed subsequent to washing the m a t e r i a l f r e e of unreacted l a b e l , even over periods of weeks. ( i i i ) Chain M o b i l i t y i n Agarose The esr spectrum of the l a b e l l e d agarose recorded at 293 K i s shown i n Figure I I I - 3 a . The l a b e l , presumed to be present at a c c e s s i b l e surfaces i n the s o l i d , tumbles at room temperature w i t h a c o r r e l a t i o n time (T) of -9 1.85 x 10 s, about 2 orders of magnitude more slo w l y than when i t i s unattached (Figure I I I - 3 a ) ( x ' s are c a l c u l a t e d using computer s i m u l a t i o n throughout the present s e c t i o n ) . I n t e g r a t i o n i n d i c a t e d that one n i t r o x i d e was present per approximately 1100 monosaccharide r e s i d u e s ; as would be expected on the b a s i s of t h i s f i g u r e n i t r o g e n was not detected by elemental a n a l y s i s . The spectrum of the l a b e l l e d m a t e r i a l at 77 K i s shown i n Figure I I I - 3 b and, again c o n s i s t e n t w i t h the above q u a n t i t a t i o n , no s p i n -s p i n d i p o l a r broadening i s d i s c e r n i b l e (d^/d < 0.4), which confirms that the average d i s t a n c e through space between nearest-neighbour r a d i c a l s i s > ^ 3.0 nm. This low l e v e l of m o d i f i c a t i o n ensures minimal p e r t u r b a t i o n . Figures I I I - 3 d and e show esr s p e c t r a of the l a b e l l e d agarose i n s o l (363 K, x = 3.0 x 1 0 ~ 1 0 s) and g e l (298 K, x = 9.2 x 10 _ 1° s) forms r e s p e c t i v e l y . ( E x a c t l y the same lineshape as Figure I I I - 3 b could be obtained by r a p i d - f r e e z i n g the g e l or s o l u t i o n forms to 77 K.) A small but s i g n i f i c a n t and r e p r o d u c i b l e increase i n the m o b i l i t y of the l a b e l occurs between the p r e c i p i t a t e d and the g e l forms, and another increase between g e l and s o l , at a given temperature (Figure I I I - 4 ) . The l a b e l l i n g procedure i s l i k e l y to have attached n i t r o x i d e s to sugar hydroxyl groups e i t h e r f a c i n g the outside of a double h e l i x { i . e . , those other than 2-OH F i g u r e I I I - 3 : E s r s p e c t r a o f s p i n l a b e l l e d a g a r o s e . ( a ) h y d r a t e d p r e c i p i t a t e d f o r m , 298 K; ( b ) p r e c i p i t a t e d f o r m , 77 K; ( c ) p r e c i p i t a t e d form, 298 K, a f t e r l y o p h i l i z a t i o n ; ( d ) s o l , 363 K; ( e ) g e l 298 K. 93 of D-galactose), or at the outside of a bundle of h e l i c e s , or i n a residue not i n v o l v e d i n a s s o c i a t i o n s at a l l , though such u n i t s are probably scarce i n the p r e c i p i t a t e d m a t e r i a l . However, i t i s q u i t e f e a s i b l e that a f t e r d i s s o l u t i o n and g e l l i n g , a l a b e l could f i n d i t s e l f i n a completely d i f f e r -ent environment w i t h respect to chain a s s o c i a t i o n s ; although a l a b e l at 2-OH of D-galactose could be expected to s t e r i c a l l y i n h i b i t d i m e r i s a t i o n 7 , n i t r o x i d e s s u b s t i t u t e d i n t o other p o s i t i o n s might be present i n h e l i c a l regions or i n s i n g l e chain regio n s , i n c l u d i n g 'free ends'. A t h i r d p o s s i b i l i t y must a l s o be considered, namely that a l a b e l could become 'trapped' as a r e s u l t of the aggregation of double h e l i c e s i n the second stage of the g e l l i n g process (Figure I I I - 2 ) . I t i s not c l e a r how the presence of a l a b e l might a f f e c t t h i s type of a s s o c i a t i o n ; however, although i t i s l i k e l y that the g e l - s t a t e spectrum represents the s u p e r p o s i t i o n of l i n e s from n i t r o x i d e s i n d i f f e r e n t s i t u a t i o n s , the line-shape changes are not commensurate w i t h the 'trapping' of anything but a small p r o p o r t i o n of the l a b e l s between chains. The spectrum of the g e l r e f l e c t s a l a b e l w i t h a degree of motional freedom which i s not s t r i k i n g l y d i f f e r e n t from that experienced i n the s o l i d or s o l s t a t e s . I t s m o b i l i t y i s a l s o roughly comparable to (and c e r t a i n l y not l e s s than) that of l a b e l s attached a f t e r  g e l a t i o n (see Chapter I V C ( i ) ) . Moreover, spe c t r a from 'trapped' l a b e l s i n agarose systems have been recorded and represent much l e s s mobile n i t r o x i d e s (see Chapter I V D ( i i ) ) . The apparent absence of t r a p p i n g may r e f l e c t s t e r i c hindrance by the n i t r o x i d e of a s s o c i a t i v e phenomena i n i t s v i c i n i t y ; but the apparent s i z e s of aggregates present i n the g e l , measured by l i g h t s c a t t e r i n g " ^ , e l e c t r o n microscopy"'"4 and g e l f i l t r a t i o n " ' " " ' suggest that these may c o n t a i n an average of only 7 to 11 h e l i c e s , so i t i s not i n c o n c e i v a b l e , given the present l e v e l of l o a d i n g , that n i t r o x i d e s may be confined to the outsides of the bundles without any great d i f f i c u l t y . The dependence of the r o t a t i o n a l c o r r e l a t i o n time x upon temperature f o r the gel->-sol->gel c y c l e ( d i r e c t i o n i n d i c a t e d by the arrows) i s shown i n Figure I I I - 4 and a h y s t e r e s i s i s c l e a r l y v i s i b l e . The dynamic m e l t i n g and g e l l i n g temperatures, determined macroscopically as the temperature at which surface deformation occurred upon removal of a thermometer from the l a b e l l e d agarose, were 361.0 and 307.5 K r e s p e c t i v e l y ( i n d i c a t e d on the curve), the same as f o r the n a t i v e , u n l a b e l l e d m a t e r i a l , though the l a t t e r formed a r a t h e r harder g e l . However, changes i n the gradient of the p l o t which might have been expected at or near these temperatures by analogy w i t h s t u d i e s that have been made of the m e l t i n g of s p i n l a b e l l e d n u c l e i c 19 acids are not p a r t i c u l a r l y w e l l defined e i t h e r i n Figure I I I - 4 or when the same data are presented as an Arrhenius p l o t (Figure I I I - 5 ) . I t i s suggested that two f a c t o r s c o n t r i b u t e to t h i s . (a) The p o l y d i s p e r s i t y of the polysaccharide. Thermodynamic parameters f o r the g e l l i n g process are most meaningfully t r e a t e d on a 'per r e s i d u e ' 20 b a s i s , and i t i s p o s s i b l e that a c t i v a t i o n energies f o r r e o r i e n t a t i o n of the n i t r o x i d e as c a l c u l a t e d from Figure I I I - 5 may be erroneous, at l e a s t i n s o f a r as they r e f l e c t chain motions (see l a t e r d i s c u s s i o n ) . The observed sp e c t r a at or near the g e l l i n g and m e l t i n g temperatures and, conceivably, elsewhere may be assumed to c o n s i s t of a sum of d i f f e r e n t • s p e c t r a l l i n e s corresponding to l a b e l s attached to molecules i n v o l v e d i n pre- or p o s t - g e l aggregates and those attached ;to . f u l l y s o l v a t e d chains. This point leads on to (b). 3 7 (b) The nature of the g e l l i n g process. Other s t u d i e s ' have i n d i c a t e d that polysaccharide g e l l i n g and melt i n g d i f f e r s i n a fundamental way from n u c l e i c a c i d denaturation. The f a c t s that many more p o s s i b i l i t i e s e x i s t c y c l e . f o r the l i n k i n g of two polysaccharide chains to form a dimer, and that a s i n g l e chain may be inv o l v e d i n j u n c t i o n zones w i t h s e v e r a l others mean that events may occur at the molecular l e v e l over a range of temperatures even i n a monomolecular, monodisperse system. H e l i c e s may unravel and reform i n t o more s t a b l e arrangements. Even at the macroscopic l e v e l d i f f e r e n t features may be i d e n t i f i e d at va r i o u s temperatures g i v i n g r i s e 21 to d i f f e r e n t d e f i n i t i o n s of g e l l i n g and melti n g p o i n t s . I f the esr. data can be i n t e r p r e t e d as r e f l e c t i n g aspects of the molecular motion of the polysaccharide r a t h e r than simply the tumbling of the l a b e l about i t s ' l i n k i n g u n i t ' (that i s , i t s r o t a t i o n a l r e o r i e n t a t i o n r e l a t i v e to the p o l y -saccharide) , then we may draw the co n c l u s i o n that the g e l s t a t e as defined m a c r o s c o p i c a l l y does not imply a c e r t a i n f i x e d microscopic molecular m o b i l i t y and that the aggregation phenomena in v o l v e d i n g e l a t i o n do not give r i s e to the l a r g e decrease i n polymer m o b i l i t y that might i n t u i t i v e l y be expected. The f o l l o w i n g d i s c u s s i o n addresses t h i s problem. Two processes c o n t r i b u t e to the tumbling r a t e (x "*") of a l a b e l attached to a polysaccharide molecule i n s o l u t i o n . The f i r s t of these i s r o t a t i o n about s i n g l e bonds i n i t s ' l i n k i n g u n i t ' (x^ ^ ) , of which there are e f f e c t i v e l y f o u r , s i n c e the amide C-N has double bond ch a r a c t e r . Hydrogen bonding to the s a c c h a r i d i c surface may f u r t h e r reduce T \ though 0 99 r e s u l t s discussed i n Chapter IVD(i) f o r l i n k i n g u n i t s (or 'spacer arms') of a s i m i l a r type to that shown above suggest that t h i s i s not so. The second type of motion i s that of the polymer i t s e l f (x , which comprises 'segmental' and 'whole molecule' or ' o v e r a l l ' r o t a t i o n s . Thus ' ' an approximate sum of r a t e s may be made: x" 1 = x^" 1 + x ^ 1 [31] S i m i l a r l y f o r the g e l . x 1 = x„ 1 + x 1 [32] where x ^ r e f e r s to r e o r i e n t a t i o n of polysaccharide molecules w i t h i n the g 25 g e l m a t r i x , segmental motions being expected to dominate I t i s reasonable to assume that motion of the polysaccharide chains i n p r e c i p i t a t e d agarose does not occur on the esr timescale ( i . e . , x > 10 7 s ) . Thus i n t h i s system, x = x A part of the temperature dependence of the c o r r e l a t i o n time f o r p r e c i p i t a t e d agarose i s shown i n Figure I I I - 4 ( f i l l e d p o i n t s ) . Above 313 K i t begins to s w e l l and d i s s o l v e as i n d i c a t e d by the d i s c o n t i n u i t y i n the p l o t , so that the measurement of x^ may only be made below t h i s temperature. However, the l i n e a r i t y of the Arrhenius p l o t f o r p r e c i p i t a t e d agarose below 313 K i s evidence of the v a l i d i t y of the assumption (Figure I I I - 5 ) ; i t leads to an a c t i v a t i o n energy f o r x^-processes of 19.5 kJ.mol \ which compares favourably w i t h a c t i v a t i o n energies of ^  8-40 kJ.mol ^ c a l c u l a t e d f o r r e o r i e n t a t i o n of 26 n i t r o x i d e s bound to s i l i c a surfaces , where again no motional component due to the surface i s to be expected. The f u r t h e r assumption must now be made that at a given temperature, x £ ( p r e c i p i t a t e ) = x £ ( g e l ) = x- ( s o l ) . [33] That a r e l a t i o n s h i p e x i s t s between s o l v a t i o n and m o b i l i t y ( e i t h e r of the x or x. ( i = g,s) type) i s reasonable, and, t r i v i a l l y , may be demonstrated A/ 1 100 (Figure I I I - 3 c ) by recording the spectrum of the l a b e l l e d agarose p r e c i p i -t a t e at room temperature a f t e r freeze d r y i n g . (Note, however, that the s p l i t t i n g (2T) between the outermost f e a t u r e s , 67G, i s l e s s than that i n Figure I I I - 3 b (73G), i n d i c a t i n g the presence of some r e s i d u a l motion.) The assumption i n [33], s t a t e d i n another way, i s that although s o l v a t i o n of the polysaccharide changes i n passing from s o l i d to g e l to s o l , the s o l v a t i o n of the l a b e l , together w i t h i t s l i n k i n g u n i t , remains the same i n the presence of excess water. This i s made p o s s i b l e i n the s o l i d by i t s presence only at the surface and, i n the g e l , by the absence of a 'trapping' phenomenon. That, n e v e r t h e l e s s , an approximation i s made may be seen from the f o l l o w i n g experiment. 2+ Ni(H20)g ions were introduced i n t o s o l u t i o n i n the presence of the l a b e l l e d s o l i d to a conc e n t r a t i o n of 2M. The spectrum i s compared w i t h that of the unperturbed system i n Figure I I I - 6 . As has been shown i n Chapter IC, at t h i s c o n c e n t r a t i o n of the metal i o n s , r a p i d e l e c t r o n exchange between them and n i t r o x i d e molecules f r e e i n s o l u t i o n causes a r e d u c t i o n i n the n i t r o x i d e T^ w i t h concomitant l i n e broadening beyond the d e t e c t i o n l i m i t . (The n i c k e l esr s i g n a l i s s i m i l a r l y i n v i s i b l e owing to i t s own r a p i d r e l a x -a t i o n . ) In t h i s case, where the n i t r o x i d e species i s tethered to the sur f a c e , the presence of a ' r e s i d u a l ' spectrum, a l b e i t at low s i g n a l - t o -n o i s e , i n d i c a t e s that s t e r i c hindrance prevents r a p i d exchange between at l e a s t a proportioncof the n i t r o x i d e s and the metal ions i n the s o l u t i o n phase. Unfor t u n a t e l y , the presence of 2M NiSO^ d e s t a b i l i s e s the g e l r e l a t i v e to the s o l form. Thus, when l a b e l l e d agarose was d i s s o l v e d i n hot 2M NiSO^ s o l u t i o n , no g e l a t i o n occurred on c o o l i n g ; when the metal s a l t was allowed to d i f f u s e i n t o the g e l , the l a t t e r slowly (days) r e d i s s o l v e d , w i t h 101 C Figure I I I - 6 : Esr spectra (300 K) of spi n l a b e l l e d agarose.' (a)hydrated, p r e c i p i t a t e d form(* 1); (b)as a i n the presence of ' 2M N i S 0 4 ( x 1 6 ) ; ( c ) s o l form i n the presence of 2M NiSO (x 24). R e l a t i v e a m p l i f i c a t i o n i n parentheses. 102 gradual r e d u c t i o n of the esr s i g n a l i n t e n s i t y (Figure I I I - 6 c ) . The s i g n a l completely disappeared only when complete d i s s o l u t i o n had occurred. Therefore i t i s c l e a r that a l l l a b e l s are a c c e s s i b l e to the paramagnetic ions i n the s o l , w h i l s t t h i s i s not so i n the s o l i d (nor, probably, the g e l ) , though the p r o p o r t i o n of i n a c c e s s i b l e spins appears to be s m a l l ; s p e c t r a shown i n Figure I I I - 6 a and b d i f f e r by a f a c t o r of s i x t e e n i n ampli-f i c a t i o n . The present approach can t h e r e f o r e be seen as an approximation, but one which i s not unreasonable. The phenomenon of s t e r i c hindrance or 'masking' at the surface of polysaccharides i n g e l and s o l i d s t a t e s i s explored f u r t h e r i n the next two chapters. Perhaps a more s e r i o u s objec-t i o n to [33] i s that water at the surface i s l i k e l y to be ordered i n a 27 f a s h i o n d i f f e r e n t to that i n bulk s o l u t i o n I t now becomes p o s s i b l e to c a l c u l a t e x g and x^ v a l u e s , using equations [31] and [32] and s u b s t i t u t i n g values of x^ measured f o r the p r e c i p i t a t e d , l a b e l l e d agarose at v a r i o u s temperatures. Above 313 K, where [33] cannot be assumed even•approximately to h o l d , values of x^ are c a l c u l a t e d from the e x t r a p o l a t e d Arrhenius p l o t (Figure I I I - 5 ) . Such values may be expected to be l e s s r e l i a b l e as the temperature i n c r e a s e s , e s p e c i a l l y above the m e l t i n g p o i n t . The e x t r a p o l a t e d l i n e i s a l s o shown i n Figure I I I - 4 . The Arrhenius p l o t of the dependence of polymer backbone motions x^ ( i = g,s) on temperature i s shown i n Figure I I I - 7 and has s i m i l a r form to Figure I I I - 5 . Figure I I I - 8 shows these motions p l o t t e d (as r a t e s ) as percentages of the t o t a l tumbling r a t e (x "*") . Although measurement e r r o r s f o r x_^  VT "" (±10%) together w i t h the approximations inherent i n [33] as w e l l as [31] and [32], and the assumption that motion i s i s o t r o p i c i n c a l c u l a t i n g x, are such that overmuch s i g n i f i c a n c e should not be placed upon the absolute v a l u e s , conclusions may j u s t i f i a b l y be drawn from trends 23 19 H 1 1 1 1 1 — — i 1 i — 27 2.9 3.1 3.3 3.5 10 3T"' (K _ L) Figure 111-7 : Arrhenius p l o t of c o n t r i b u t i o n s to c o r r e l a t i o n time from polysaccharide backbone motions i n l a b e l l e d agarose. T r e f e r s to the gel form while T r e f e r s to the s o l . o 105 i n the data. In the f i r s t p l a c e , i t i s amply c l e a r that marked d i f f e r e n c e s i n m o b i l i t y — p e r h a p s of almost an order of magnitude—can occur i n polymer molecules i n gels i n d i f f e r e n t s t a t e s of o r g a n i s a t i o n . I t i s even p o s s i b l e to o b t a i n e q u a l i t y between a T and a T , though at d i f f e r e n t temperatures s g (Figure I I I - 7 ) . Put another way, "gel" may not be an adequate, unambiguous means of d e s c r i b i n g the s t a t e of agarose at a microscopic l e v e l . Large d i f f e r e n c e s i n m o b i l i t y can a l s o occur between two gels at the same tem-perature, but w i t h d i f f e r e n t immediate h i s t o r i e s . Thus d i r e c t l y a f t e r s e t t i n g , at 303 K, x was found to be $ 7x smaller than at the same tempera-ture a f t e r remaining at room temperature overnight. Marked d i f f e r e n c e s i n x as a p r o p o r t i o n of x ^ (from ^ 25 to ^ 70%) at 303 K f o r c e the same co n c l u s i o n . W i t h i n the time p e r i o d of a s i n g l e h y s t e r e s i s c y c l e (1 - 2 hours) using the l a b e l l e d g e l i t i s not p o s s i b l e to r e t u r n , at low temperature, to x values which are as l a r g e as at the s t a r t of the e x p e r i -ment (Figure I I I - 4 ) or to x ^/x ^ values which are as l a r g e (Figure I I I - 8 ) . This i s i n keeping w i t h the idea that r e o r g a n i s a t i o n of the g e l to more s t a b l e forms, perhaps i n c l u d i n g changes i n h e l i x aggregation and even u n f o l d i n g of i n d i v i d u a l h e l i c e s occurs below the formal (macroscopic) g e l l i n g temperature. No anomaly i s observed during the c o o l i n g c y c l e i n the Arrhenius p l o t of x^ (Figure I I I - 7 ) ; a marked change i n gradient occurs at the g e l l i n g temperature. However, the change i n gradient which occurs i n the p l o t of x_^  "Vx versus temperature (Figure I I I - 8 ) , the p r o p o r t i o n of motion c o n t r i b u t e d by the backbone decreasing more r a p i d l y w i t h temperature below the s e t t i n g p o i n t , seems to i n d i c a t e a gradual molecular r e o r g a n i s a -t i o n . Although no s i g n i f i c a n c e can be a t t r i b u t e d to the decrease i n x ~ 1 / x ~ 1 below 320 K (Figure I I I - 8 ) , both Figures I I I - 7 and I I I - 8 show 106 that very considerable a l t e r a t i o n s occur i n microscopic s t r u c t u r e during the h e a t i n g c y c l e below the melti n g p o i n t . These processes appear to begin near 320 K, where each p l o t shows a n o t i c e a b l e change i n grad i e n t . This o b s e r v a t i o n , which i s q u i t e c o n s i s t e n t w i t h our p i c t u r e of the g e l as con t a i n i n g many i n t e r a c t i o n s of d i f f e r e n t strengths (corresponding, f o r example, to d i f f e r e n t lengths of a s s o c i a t i o n ) , underlines the advantage of the esr technique over that of o p t i c a l r o t a t i o n , which detects t r a n s i -t i o n s only at the macroscopic m e l t i n g and g e l l i n g p o i n t s . The l a c k of dependence of x ^/x ^ on temperature, and the l i n e a r i t y of x p l o t t e d according to Arrhenius are as expected f o r a homogeneous polymer system, and provide f u r t h e r evidence that changes i n x^ r e f l e c t a fundamental property of the g e l r a t h e r than an a r t i f a c t of the l a b e l l i n g experiment. I I I B : A l g i n a t e ( i ) S t r u c t u r e of A l g i n i c A c i d and A l g i n a t e Gels A l g i n i c a c i d i s another non-toxic n a t u r a l l y o c c u r r i n g polysaccharide 28 of considerable commercial importance . I t was o r i g i n a l l y , and i s s t i l l i n l a r g e part e x t r a c t e d from v a r i e t i e s of brown seaweed (Phaeophyta), though more r e c e n t l y m i c r o b i a l a l g i n a t e s have been developed whose s t r u c -29 t u r e and p r o p e r t i e s c l o s e l y resemble those of the seaweed m a t e r i a l The t h i c k e n i n g , suspending, e m u l s i f y i n g , s t a b i l i z i n g and gel-forming p r o p e r t i e s of a l g i n i c a c i d and i t s d e r i v a t i v e s have l e d to many a p p l i c a t i o n s i n the food, pharmaceutical, cosmetic, -coating, adhesive, p a i n t , p o l i s h , j t , * J . 3 0 p r i n t i n g , and other i n d u s t r i e s Although s t r u c t u r a l s t u d i e s have been i n progress s i n c e the e a r l y part of the present century, i t i s only very r e c e n t l y that some d e t a i l s have been c l a r i f i e d . Two types of monosaccharide r e s i d u e , g-D-manno-107 pyranosiduronic acid (M), and a-L-gulopyranosiduronic acid (G) form an ex c l u s i v e l y l i n e a r polymer (19) made up of three d i f f e r e n t regions or 'blocks' of primary s t r u c t u r e : poly-3-D-mannuronic acid (poly-M), poly-a-L-guluronic acid (poly-G) and a t h i r d region which contains roughly equal proportions of the two monomer u n i t s , apparently joined i n random 31-34 sequence (poly-MG). In a l l cases linkage p o s i t i o n s are 1—4 . I t i s thought that the average length of the poly-M blocks i s ^  24 u n i t s , and 34 that the standard deviation i s small . However, the M/G r a t i o d i f f e r s depending on the sources of the material and other v a r i a b l e s . - T l . a l g i n i c a c i d (19) Alginates are s a l t s of a l g i n i c a c i d . The l a t t e r i s an in s o l u b l e fibrous material, and when the pH of an alginate s o l u t i o n i s reduced to ^ 3.0 p r e c i p i t a t i o n occurs. In the presence of monovalent cations i n the pH range 3 - 11.5, viscous solutions are formed which show t y p i c a l poly-e l e c t r o l y t e properties. When one or more of many d i - or t r i - v a l e n t metal ions are added to alginate s o l u t i o n s , gels are formed; calcium alginate 2+ 2+ 2+ i s most commonly used i n industry, and Ca , Sr and Ba are probably the most common ions found i n association with alginate i n seaweed, where an ion-exchange equilibrium with seawater probably e x i s t s ; the s e l e c t i v i t y of the gel for counterions seems to depend on i t s exact composition, 35 though not on molecular weight Considerable e f f o r t to elucidate the structure of the gel followed on 108 the heels of advances i n the determination of i t s primary s t r u c t u r e i n the l a t e 1960's and e a r l y 1970's. Rees and co-workers proposed an ' egg-36 box' model , which, though based more on stereochemical arguments than on experimental evidence, i s g e n e r a l l y accepted. Studies of competetive b i n d -2+ + 37 2+ ing of Ca and Na , the b i n d i n g of Ca to o l i g o s a c c h a r i d e s of i n c r e a s -38 i n g degree of p o l y m e r i s a t i o n i n both the guluronate and mannuronate s e r i e s 39 and p r o p e r t i e s such as s t i f f n e s s of the g e l i n d i c a t e that a cooperative b i n d i n g phenomenon occurs in~poly-G u n i t s of chain length > ^ 20 u n i t s . Poly-M, on the other hand, shows no such c o o p e r a t i v i t y . A c o n s i d e r a t i o n (based on the r e s u l t s of X-ray f i b r e d i f f r a c t i o n ) of the conformations of 40 41 the homopolymeric b l o c k s , Figure I I I - 9 ' , shows that a c o o r d i n a t i o n s i t e f o r d i v a l e n t c a t i o n s i n v o l v i n g two monosaccharide residues can be r e a d i l y 42 envisaged i n the guluronate, though not i n the mannuronate s e r i e s . This alone, however, cannot e x p l a i n the c o o p e r a t i v i t y , which, i t was suggested, could best be accounted f o r by c h a i i r ' a s s o c i a t i o n between polyguluronate blocks (Figure I I I - 9 ) , c r e a t i n g three-dimensional b i n d i n g s i t e s f o r the d i v a l e n t i o n s , each i n v o l v i n g four guluronate r e s i d u e s , two from each 36 37 chain ' . In t h i s way, i n theory, any number of such chains may be a s s o c i a t e d i n two dimensions, forming an i n f i n i t e c r o s s - l i n k e d array or g e l by v i r t u e of the non-associating poly-M and poly-MG bloc k s which 43 interconnect associated poly-G . Recently published r e s u l t s , however, suggest on the b a s i s of the dependence of i n t e n s i t y i n c i r c u l a r d i chroism 2+ upon the s t o i c h i o m e t r y of Ca b i n d i n g that the dominant a s s o c i a t i o n i s a d i m e r i s a t i o n process between poly-G b l o c k s , though a f t e r maximising dimeric 2+ a s s o c i a t i o n continued a d d i t i o n of Ca may provoke f u r t h e r o l i g o m e r i c 44 i n t e r a c t i o n s . That the i n i t i a l d i m e r i s a t i o n i s more e n e r g e t i c a l l y favourable than any f u r t h e r a s s o c i a t i o n may be r a t i o n a l i s e d on simple 109 Figure I I I - 9 : Proposed a l g i n a t e s t r u c t u r e s . (a)poly-M; (b)poly-G; ( c ) s o l ; ( d ) g e l ; (e)calcium i on b i n d i n g s i t e i n g e l . Oxygens supposed to c o n t r i b u t e to bind i n g are f i l l e d i n . The s t r u c t u r e i s expanded i n the v e r t i c a l d i r e c t i o n (adapted from reference 36). 110 e l e c t r o s t a t i c grounds; the presence of bound calcium i n the dimer reduces the e f f e c t i v e charge de n s i t y 'seen' by an unbound c a t i o n at the polyanion. Hydrodynamic evidence suggests that p o l y g u l u r o n i c a c i d has a 45 s t i f f r i b b o n - l i k e conformation ; the r i g i d i t y of a l g i n a t e gels i s mainly 46 a s s o c i a t e d w i t h poly-G dimers , which may rupture when the g e l i s deformed 47 w i t h consequent increase i n entropy and i n t e r n a l energy . E l e c t r o n microscopic examination of the g e l i s a l s o c o n s i s t e n t w i t h there being an absence of j u n c t i o n zones of dimension greater than that expected f o r small T 46 oligomers ( i i ) L a b e l l i n g Procedure Chemical m o d i f i c a t i o n of a l g i n i c a c i d was one of the most d i f f i c u l t challenges faced during the course of the work, p r i n c i p a l l y because of the r e l a t i v e f r a g i l i t y of the polymer to a c i d i c and b a s i c c o n d i t i o n s . Two options were open: to a t t a c h a n i t r o x i d e to hydroxyl or to c a r b o x y l f u n c t i o n a l i t i e s . The m o d i f i c a t i o n of the l a t t e r would be expected to exert a greater p e r t u r b a t i o n on the a s s o c i a t i v e processes i n v o l v e d i n g e l formation as a r e s u l t of the removal of negative charges; however, on the b a s i s of the s t r u c t u r a l model described i n the preceding s e c t i o n , a t t a c h -ment of a n i t r o x i d e at four of the a v a i l a b l e hydroxyls on a p a i r of neighbouring g u l u r o n i c a c i d residues would a l s o be expected to i n h i b i t d i m e r i s a t i o n ; change of conformation as a r e s u l t of the presence of the n i t r o x i d e could provide an e x t r a , unknown p e r t u r b a t i o n . N e i t h e r of the f u n c t i o n a l groups, t h e r e f o r e , was s i g n i f i c a n t l y p r e f e r a b l e as a s i t e f o r chemical m o d i f i c a t i o n , and attempts were made to a t t a c h n i t r o x i d e s to both, w h i l s t m aintaining low degrees of s u b s t i t u t i o n and m i l d r e a c t i o n c o n d i t i o n s . Chapter VI describes the use of a m i l d aqueous method f o r formation of c a r b o x y l i c amides using a water-soluble c o u p l i n g agent, N-ethyl-N'-I l l dimethylaminopropyl carbodiimide h y d r o c h l o r i d e (EDC) (20) at pH values i n the range A.5 - 6.0. This reagent was used i n attempts to promote the r e a c t i o n of 4-amino-2,2,6,6-tetramethylpiperidine-l-oxyl (2) w i t h u r o n i c acids i n a l g i n a t e under a v a r i e t y of r e a c t i o n c o n d i t i o n s without success, the l a t t e r being measured as before by the presence or absence of a workable esr s i g n a l . The same r e a c t i o n was attempted i n the presence of a s i m i l a r reagent, N-cyclohexyl-N'-(2-morpholinoethyl) carbodiimide p-toluenesulphonate (CMC) (21) w i t h s i m i l a r r e s u l t s . E v i d e n t l y t h i s f a i l u r e i s r e l a t e d to a s p e c i a l property of a l g i n a t e , s i n c e carboxymethyl c e l l u l o s e (see Appendix 1) and g l y c o p r o t e i n s (see Chapter VI) were s u c c e s s f u l l y l a b e l l e d i n t h i s way. Unsuccessful attempts were a l s o made to modify the hydroxyl groups of a l g i n a t e under heterogeneous c o n d i t i o n s using 2,4-dichloro-6-(2',2',6,'6'-t e t r a m e t h y l - l ' - o x y l - p i p e r i d i n e - 4 ' - o x y ) - s - t r i a z i n e (8) and 4-chloroacetamido-2 , 2 , 6 , 6 - t e t r a m e t h y l p i p e r i d i n e - l - o x y l . (5) i n aqueous acetone mixtures i n the presence of s t r o n g * (NaOH) and weak (Na^CO^) bases and i n r e f l u x i n g hexane i n (20) (2) Cl (5) (8) *M. J . Adam, personal communication. 112 the absence of base. The r e a c t i o n s f a i l e d i n a l l cases, and i t was decided to t r y the r e a c t i o n of neighbouring hydroxyls w i t h cyanogen bro-mide, which had proved s u c c e s s f u l w i t h agarose (see Chapter IV f o r a f u l l e r d e s c r i p t i o n of the r e a c t i o n ) . This r e a c t i o n used sodium carbonate, pH 9.5, so was not expected to cause s u b s t a n t i a l base-catalysed degradation. Under homogeneous c o n d i t i o n s attempts to react sodium a l g i n a t e w i t h aqueous cyanogen bromide followed by l a b e l ( 9 ) : f a i l e d to give adequate s i g n a l to noise and the same was true when calcium a l g i n a t e g e l , d i v i d e d i n t o s m a l l p i e c e s , was used. Reaction of other gums (e.g., xanthan) i n s o l u t i o n w i t h CNBr a l s o f a i l e d (see Appendix 1). In s o l u t i o n , l a b e l l i n g may be l i m i t e d by the l a c k of opportunity f o r i n t e r -molecular r e a c t i o n i n v o l v i n g hydroxyl groups on d i f f e r e n t chains. In the g e l , p e n e t r a t i o n of CNBr may be slow r e l a t i v e to i t s r a t e of h y d r o l y s i s ; i t i s a l s o l i k e l y that hydroxyls brought together as a r e s u l t of chain a s s o c i a t i o n s are u n a v a i l a b l e f o r r e a c t i o n , being occupied i n metal b i n d i n g . Some success was encountered, however, i n attempts to l a b e l a l g i n i c a c i d i n heterogeneous mixtures. The y i e l d of the r e a c t i o n was extremely s e n s i t i v e to i t s c o n d i t i o n s . The f i r s t step, a c t i v a t i o n w i t h CNBr, was performed using a c e t o n i t r i l e - a q u e o u s carbonate mixtures. The water swelled the polysaccharide without d i s s o l v i n g i t during the two minutes required f o r the a c t i v a t i o n . A f t e r removal of the supernatant by f i l t r a t i o n , aqueous NH (19) 0 113 solutions of l a b e l (9) were added, allowing f u l l d i s s o l u t i o n of the a l g i n -ate to occur during the second stage of the r e a c t i o n . If CNBr concentra-t i o n was too high ( > 2 g/ml) c r o s s - l i n k i n g occurred, r e s u l t i n g i n an insoluble polymer. (This was also observed i n other systems: agarose ( t h i s chapter), locust.bean gum (see Appendix 1).) If the concentration was too low, the y i e l d was reduced. The rate of d i s s o l u t i o n of the a l g i n a t e , which was probably governed p r i m a r i l y by the composition of solvent used i n the f i r s t step, appeared to be c r u c i a l ; i f t h i s occurred to too great an extent during t h i s step, a c t i v a t i o n , which c l e a r l y depends on there being more a v a i l a b l e hydroxyl pairs than e x i s t on a si n g l e i s o l a t e d chain, was i n e f f i c i e n t ; i f , on the other hand, swelling during the f i r s t step was l i m i t e d , penetration of the CNBr suffered and the y i e l d was reduced. Optimal reaction conditions were never achieved; the r e s u l t s reported were obtained on a si n g l e preparation of l a b e l l e d alginate, ( i i i ) Spectra Figure III-10a shows the spectrum of an aqueous s o l u t i o n of sodium algi n a t e , l a b e l l e d using the cyanogen bromide method, at 298 K with T = 6.0 x 10 ^ s, a value not d i s s i m i l a r to those obtained i n agarose; at i t s s e t t i n g temperature of 307.5 K, the agarose s o l has T 3.5 x 10 ^ s. while at 298 K the newly set gel has T 6.0 x 10 ^ s. Unfortunately, exact comparison of these data i s d i f f i c u l t because of the d i f f e r e n t l i n k i n g units which are present i n the two cases: agarose: l-O a l g i n a t e : O 114 Figure I I I - 1 0 : Esr spectra of l a b e l l e d a l g i n a t e , (a)sodium s a l t i n s o l u t i o n ; (b)calcium a l g i n a t e g e l . 115 I t i s to be expected, t h e r e f o r e , that s l i g h t l y d i f f e r e n t c o n t r i b u t i o n s to T are present i n the two systems. Increase of the a l g i n a t e s o l u t i o n c o n c e n t r a t i o n by a d d i t i o n of u n l a b e l l e d sodium a l g i n a t e d i d not a f f e c t the spectrum measurably up to 5% w/v, which suggests that t-^-type motions and ' l o c a l mode' motions may both be important c o n t r i b u t o r s to x; 'end-over-end' motions should become l e s s frequent under c o n d i t i o n s of i n c r e a s i n g polymer con c e n t r a t i o n . A d d i t i o n of calcium ions to the l a b e l l e d a l g i n a t e gave a g e l w i t h spectrum as shown i n Figure III-10b. The g e l was somewhat s o f t e r than that obtained using the n a t i v e a l g i n a t e as a r e s u l t of i t s modi-f i c a t i o n ( c o n t r o l samples t r e a t e d only w i t h base gave the same g e l s t r e n g t h as i n the n a t i v e a l g i n a t e ) and the spectrum i s complex, apparently a r i s i n g from the s u p e r p o s i t i o n of spectra from two populations of spins having -9 r e s p e c t i v e l y greater and l e s s e r m o b i l i t i e s , both w i t h x > 10 s. The s p e c t r a l broadening a s s o c i a t e d w i t h the g e l a t i o n process i s a good deal more marked than i n the case of agarose, which i s rat h e r s u r p r i s i n g c o n s i d e r i n g that d i m e r i s a t i o n i s thought to be the primary process i n a l g i n a t e , w h i l e higher aggregates c e r t a i n l y form i n agarose. This provides f u r t h e r c i r -c u m s t a n t i a l evidence that l a b e l s are not trapped i n aggregates during agarose g e l a t i o n . In a l g i n a t e , according to Figure I I I - 9 , i t i s p o s s i b l e f o r a l a b e l to be present on a guluronate residue which p a r t i c i p a t e s i n a c r o s s - l i n k , on a hydroxyl on the si d e away from the metal b i n d i n g s i t e (Figure I I I - 9 ) . To f u r t h e r i n v e s t i g a t e t h i s p o s s i b i l i t y , a g e l was formed between the l a b e l l e d a l g i n a t e and a d i v a l e n t paramagnetic i o n , manganese(II). This i o n was s e l e c t e d on the b a s i s of ( i ) i t s i o n i c r adius (80 nm), which i s not d i s s i m i l a r to that of calcium (99 nm), ( i i ) i t s having the same charge as calcium, ( i i i ) the p r o b a b i l i t y that 2+ . , 48 bound and unbound Mn would provide r e a d i l y d i s t i n g u i s h a b l e esr s i g n a l s 116 Manganous a l g i n a t e gels are s o f t e r than t h e i r calcium counterparts; i t was hoped that f a c t o r s ( i ) and ( i i ) would lead to s i m i l a r b i n d i n g s i t e geometries, but even i f t h i s were not so, i t was reasonable to assume that the b i n d i n g s i t e s s t i l l formed between guluronate b l o c k s . The spectrum of l a b e l l e d Mn^* a l g i n a t e i s shown i n Figure I I I - l l a and expanded i n Figure I l l - l i b i n the n i t r o x i d e r e g i o n . Despite lengthy d i a l y s i s against 2+ deionised water, the c h a r a c t e r i s t i c s i x l i n e spectrum of Mn(H20)g i s s t i l l present. The g e l r e d i s s o l v e d a f t e r very long periods (weeks) of d i a l y s i s , so that the r e s u l t s are c o n s i s t e n t w i t h a slow e q u i l i b r i u m 49 between f r e e and bound manganese, as was found i n a previous study D i a l y s i s against the disodium s a l t of ethylenediamine t e t r a a c e t i c a c i d caused more r a p i d (hours) d i s s o l u t i o n of the g e l w i t h the reappearance of spectrum a i n Figure 111-10. The most s i g n i f i c a n t f e a t u r e of Figure I I I - l l a i s the presence of n i t r o x i d e s i g n a l s ; the l i n e w i d t h of the centre n i t r o x i d e l i n e can be roughly estimated as 1.7 G compared w i t h 3.0 G i n the calcium analogue (Figure I I I - 1 0 b ) , and 1.5 G i n the sodium analogue (Figure I I I - 1 0 a ) . We can t h e r e f o r e say that n i t r o x i d e broadening does not appear to have occurred as a r e s u l t of the presence of the paramagnetic i o n ; the manganous a l g i n a t e g e l appears to be more f l e x i b l e at the molecular as w e l l as the macroscopic l e v e l . Under r i g i d - l a t t i c e c o n d i t i o n s i n which d i p o l a r i n t e r a c t i o n s between Mh ^ and n i t r o x i d e are not averaged by motion or r e l a x a t i o n i t i s p o s s i b l e to observe decreases i n s i g n a l i n t e n s i t y without broadening"^'"'"'". Such i n t e r a c t i o n s are a l s o , of course, dependent on d i s t a n c e . In the present case i n which n i t r o x i d e motion i s known not to be completely frozen on the esr t i m e s c a l e , and where n i t r o x i d e s are probably randomly o r i e n t e d and d i s t r i b u t e d w i t h respect to paramagnetic i o n b i n d i n g s i t e s , a number of 117 Figure I I I - l l : (a)Esr spectrum of manganous a l g i n a t e ; (b)expanded c e n t r a l p o r t i o n showing superimposed n i t r o x i d e resonances. 118 d i f f e r e n t types of i n t e r a c t i o n may be p o s s i b l e between the paramagnetic centres. The most th a t can be s a i d i n c o n c l u s i o n i s that not a l l of the n i t r o x i d e s are s u f f i c i e n t l y c l o s e to manganous ions to cause the complete disappearance of the n i t r o x i d e s i g n a l , which i s e x a c t l y what i s expected i f the a l g i n a t e i s l a b e l l e d at random, si n c e some l a b e l s w i l l be found i n poly-M and poly-MG b l o c k s . I t i s p o s s i b l e that Figure III-10b represents a super— p o s i t i o n of s p e c t r a from n i t r o x i d e s i n d i f f e r e n t environments. This type of experimental approach was not, t h e r e f o r e , thought to be worthy of f u r t h e r a t t e n t i o n ; i t i s worth n o t i n g , however, that other magnetic resonance techniques may be capable of determining d e t a i l s of metal b i n d i n g 49 s i t e geometries i n a l g i n a t e s a l t s of paramagnetic c a t i o n s and those w i t h non-zero nuclear s p i n One i s l e f t , t h e r e f o r e , w i t h the observation that a l g i n a t e g e l a t i o n appearsrto cause a s u b s t a n t i a l l y greater r e d u c t i o n i n m o b i l i t y than agarose g e l a t i o n , d e s p i t e the smaller s c a l e of the chain aggregates and the p r o b a b i l i t y that more non-junction, f u l l y hydrated s e c t i o n s are present i n the former system. Even t h i s c o n c l u s i o n must be q u a l i f i e d i n the l i g h t of the d i f f e r e n t l i n k i n g u n i t s used, though f r e e energy c a l c u l a t i o n s suggest 53 that the poly-G blocks are s t i f f ( considerably more so than poly-M). I t i s a l s o probable that segmental motions are q u i t e r e s t r i c t e d i n a l g i n a t e compared t o , say, dextran, which i s 1-6 l i n k e d (and where segmental motions 25 have been stud i e d using nmr ). The i n i t i a l impetus f o r the present study o r i g i n a t e d i n the idea that i f the s p e c t r a l changes as s o c i a t e d w i t h g e l a t i o n of l a b e l l e d a l g i n a t e were s u f f i c i e n t l y marked, i t might be p o s s i b l e to l a b e l p a r t i a l l y hydrolysed, p u r i f i e d o l i g o s a c c h a r i d e fragments (poly-M and poly-G*) and study t h e i r *Obtained as a: g i f t from Tate and L y l e , U. K. 119 behaviour i n the presence of calcium ions i n s o l u t i o n , thus being able to provide d i r e c t evidence that guluronate a s s o c i a t i o n , and not that of mannuronate, i s r e s p o n s i b l e f o r g e l a t i o n . Unfortunately the l a c k of a s u f f i c i e n t l y m i l d and r e p r o d u c i b l e method f o r a t t a c h i n g the l a b e l rendered such a study i m p r a c t i c a b l e . 120 References 1. K. G. Gu i s e l e y , and D. W. Renn, 'Agarose: P u r i f i c a t i o n , P r o p e r t i e s and Biomedical A p p l i c a t i o n s , ' Marine C o l l o i d s Division,,FMC Corpora-t i o n , 1977. 2. C. A r a k i , and K. A r a l , B u l l . Chem. Soc. Jpn., 30, 287-293 (1957). 3. D. A. Rees, I . W. S t e e l e , and F. B. Williamson, J . Polymer S c i . , Part C, 28, 261-276 (1969). 4. M. Malmqvist, Carbohydr. Res., 62, 337-348 (1978). 5. J . Porath, and R. Axen, Meths. Enzymol., 44, 19-45 (1976). 6. D. A. Rees, Biochem. J . , 126, 257-273 (1972). 7. S. A r n o t t , A. Fulmer, W. E. S c o t t , I . C. M. Dea, R. Moorhouse, and D. A. Rees, J . Mol. B i o l . , 90, 269-284 (1974). 8. J . Marmur, and P. Doty, i b i d . , 3, 585-594 (1961). 9. J . G. Wetmur, and N. Davidson, i b i d . , 31, 349-370 (1968). 10. P. J . F l o r y , and C. S. Weaver, J . Am. Chem. S o c , 82, 4518-4525 (1962). 11. E. P i n e s , and W. P r i n s , Macromolecules, _6, 888-895 (1973). 12. I . C. M. Dea, A. A. McKinnon, and D. A. Rees, J . Mol. B i o l . , 68, 153-172 (1972). 13. B. Obrink, J . Chromatog., 37_, 329-330 (1968). 14. A. Amsterdam, Z. E r - e l , and S. S h a l t i e l , Arch. Biochem. Biophys., 171, 673-677 (1975). 15. T. C. Laurent, Biochim. Biophys. Acta, 136, 199-205 (1967). 16. A. Hayashi, K. K i n o s h i t a , and M. Kuwano, Polymer J . , 9_, 219-225 (1977). 17. R. A. Jones, E. J . S t a p l e s , and A. Penman, J . Chem. S o c , P e r k i n I I , 1608-1612 (1973). 18. H. M. McConnell, W. Deal, and R. T. Ogata, Biochemistry, 8, 2580-2585 (1969). 19. H. Dugas, Accts. Chem. Res., 10, 47-54 (1977). 20. T. A. Bryce, A. A. McKinnon, E. R. M o r r i s , D. A. Rees, and D. Thorn, Faraday Discus. Chem. S o c , 57_, 221-229 (1974).. 121 21. W. Yaphe, and M. Duckworth, Proc. 7th I n t . Seaweed Symp., Ed. K. Nisizawa, 15-22, Wiley, New York, 1972. 22. E. T. F o s s e l , K. R. K. Easwaran, and E. R. B l o u t , Biopolymers, 14, 927-935 (1975). 23. I. V. Dudich, V. P. Timofeev, M. V. Vo l ' k e n s h t e i n , and A. Yu.Misharin, Mol. B i o l . , 11, 685-693 (1977). 24. A. T. B u l l o c k , and G. G. Cameron i n ' S t r u c t u r a l Studies of Macro-molecules by Spectroscopic Methods,' Ed. K. J . I v i n , 273-315, Wiley, New York, 1976. 25. A. J . Benesi, and J . T. G e r i g , Carbohydr. Res., 53_, 278-283 (1977). 26. N. S i s t o v a r i s , W. 0. Riede, and H. S i l l e s c u , Ber. Bunsen-Gesellschaft, 79, 882-889 (1975). 27. W. Drost-Hansen, Ind. Eng. Chem., 61, 10-47 (1969). 28. K. B. Gu i s e l e y , 'Seaweed C o l l o i d s , ' Kirk-Othmer E n c y c l . Them. Technol., 2nd E d i t i o n , 17_, 763-784, -Wiley, New York, 1968. 29. L. Deavin, T. R. Jarman, C. J . Lawson, R. C. R i g h e l a t o , and S. Slocombe, ACS Symp. Ser., 45, 14-26 (1977). 30. W. H. McNeely, and D. J . P e t t i t i n ' I n d u s t r i a l Gums,' Ed. R. L. W h i s t l e r , 49-82, Academic, New York, 1973. 31. A. Haug, B. Larsen, and 0. Smidsr^d, Acta Chem. Scand., 21, 691-704 (1967). 32. A. Haug, B. Larsen, and 0. Smidsrtfd, i b i d . , 20, 183-190 (1966). 33. A. Penman, and G. R. Sanderson, Carbohydr. Res., 25, 273-282 (1972). 34. J . Boyd, and J . R. Turvey, Carbohydr. Res., 6<5, 187-194 (1978). 35. A. Haug, Acta Chem. Scand., 15, 1794-1795 (1961). 36. G. T. Grant, E. R. M o r r i s , D. A. Rees, P. J . C. Smith, and D. Thorn, F.E.B.S. L e t t . , 32, 195-198 (1973). 37. D. A. Rees i n 'Biochemistry of Carbohydrates,' Ed. W. J . Whelan, MTP I n t l . Rev. S c i . , Series I , V o l . 5, 1-42, Butterworths, London, 1975. 38. R. Kohn, and B. Larsen, Acta Chem. Scand., 26, 2455-2468 (1972). 39. 0. Smidsr«5d, and A. Haug, i b i d . , 26, 79-88 (1972). 40. E. D. T. A t k i n s , I . A. Nieduszynski, W. Mackie, K. D. Parker, and E. G. Smolko, Biopolymers, 12, 1879-1887 (1973). 122 41. E. D. T. A t k i n s , I. A.. Nieduszynski, W. Mackie, K. D. Parker, and E. E. Smolko, i b i d . , 12, 1865-1878 (1973). 42. T. Anthonsen, B. Larsen, and 0. Smidsr^d, Acta Chem. Scand., 27, 2671-2673 (1973). 43. E. R. M o r r i s , D. A. Rees, and D. Thorn, Chem. Commn., 245-246 (1973). 44. E. R. M o r r i s , D. A. Rees, D. Thorn, and J . Boyd, Carbohydr. Res., 66, 145-154 (1978). 45. S. G. W h i t t i n g t o n , Biopolymers, 10, 1481-1489 (1971). 46. 0. Smidsr^d, Faraday D i s c . Chem. S o c , 57, 263-274 (1974). 47. I. L. Andresen, and 0. Smidsrtfd, Carbohydr. Res., 58, 271-279 (1977). 48. R. A. Dwek, 'Nuclear Magnetic Resonance i n Biochemistry,' Clarendon, Oxford, 1973. 49. J . Oakes, and T. F. C h i l d , Native P h y s i c a l Science, 244, 107-108 (1973). 50. J . S. L e i g h , J . Chem. Phys.,_52, 2608-2612 (1970). 51. J . S. T a y l o r , J . S. Lei g h , and M. Cohn, P r o c Nat. Acad. S c i . , 6 4 , 219-226 (1969). 52. S. Forsen, Chemistry i n B r i t a i n , 14, 29-35 (1978). 53. E. B a i l e y , J . R. M i t c h e l l , and J . M. V. Blanshard, C o l l o i d Polym. S c i . , 255, 856-860 (1977). CHAPTER IV SEPHAROSE 4B AS A MATRIX FOR CHROMATOGRAPHY IVA: I n t r o d u c t i o n A number of d i f f e r e n t m a t e r i a l s have been used as media f o r g e l f i l -t r a t i o n , chromatography"'" and e l e c t r o p h o r e t i c s e p a r a t i o n s ^ and, more r e c e n t l y , i o n exchange"^, a f f i n i t y 4 , c o v a l e n t " ' , m e t a l c h e l a t e ^ , h y d r o p h o b i c 7 8 9 and c h a r g e t r a n s f e r c h r o m a t o g r a p h i e s and f o r t h e i m m o b i l i s a t i o n o f enzymes . The r e q u i r e m e n t s " ^ o f a 's u p p o r t m a t r i x ' — p e r m e a b i l i t y and h i g h s u r f a c e a r e a , m e c h a n i c a l s t r e n g t h , good f l o w c h a r a c t e r i s t i c s , c h e m i c a l s t a b i l i t y , ease o f d e r i v a t i s a t i o n , c o n v e n i e n c e o f h a n d l i n g , l ow p r i c e and re a d y a v a i l a b i l i t y — a r e n o t , i n g e n e r a l , a l l f u l f i l l e d a t once. Amongst n o n - c a r b o h y d r a t e s u b s t a n c e s polyacrylamide"'"''" i s perhaps most w i d e l y u s e d, b u t s u f f e r s f r o m poor f l o w c h a r a c t e r i s t i c s and n o n - s p e c i f i c a d s o r p t i o n phenomena; g l a s s 12 beads have e x c e l l e n t m e c h a n i c a l and f l o w p r o p e r t i e s b u t low s u r f a c e a r e a and r a t h e r u n p r e d i c t a b l e s u r f a c e c h e m i s t r y ; new m a t e r i a l s , s u c h as p o l y m e r -13 14 c o a t e d g l a s s and t i t a n i u m d i o x i d e g e l a r e under i n v e s t i g a t i o n b u t have so f a r n o t s u p e r s e d e d t h e p o l y s a c c h a r i d e s . Of t h e l a t t e r , c e l l u l o s e , d e x t r a n , s t a r c h and agar a r e most i m p o r t a n t , though a v a r i e t y o f o t h e r s have been u s e d , p a r t i c u l a r l y i n ca s e s where a s u g a r - b i n d i n g p r o t e i n i s s e p a r a t e d on a polymer whose n a t i v e s t r u c t u r e i n c l u d e s t h e l i g a n d ^ ' "^. C e l l u l o s e and s t a r c h a r e cheap and abundant, b u t have p o o r p o r o s i t y and f l o w p r o p e r t i e s , e s p e c i a l l y when packed i n columns. I n a d d i t i o n t h e y a r e s u s c e p t i b l e t o m i c r o b i a l d e g r a d a t i o n . The m a t e r i a l s w h i c h most e f f e c t i v e l y 123 124 combine the abovementioned features are dextran (-r^Glcp^j- , c r o s s - l i n k e d using e p i c h l o r o h y d r i n and u s u a l l y s o l d as Sephadex) and agarose, e i t h e r i n c r o s s - l i n k e d form or as the n a t i v e g e l (Sepharose). In t e c h n i c a l agarose (18) a p p l i c a t i o n s crude u n f r a c t i o n a t e d agar i s o f t e n s u i t a b l e , f o r b i o s p e c i f i c adsorbents, agarose (that f r a c t i o n of agar having fewest charged residues) i s u s u a l l y chosen and i s the main subject of the present chapter. I t i s allowed to g e l i n suspension i n such a way as to form s p h e r i c a l beads at one of s e v e r a l concentrations up to a maximum of 6%, thus c r e a t i n g pores which allow access to solvent molecules but not to species of diameter greater than a c e r t a i n defined v a l u e , which f o r molecules of a given shape, may be c o r r e l a t e d w i t h a molecular w e i g h t — t h e e x c l u s i o n l i m i t . Thus Sepharose 2B, 4B and 6 B ^ correspond to 2, 4 and 6% agarose gel s and have e x c l u s i o n l i m i t s of 40 x 10 6, 20 x 10 6 and 4 x 10 6 r e s p e c t i v e l y f o r g l o b u l a r p r o t e i n s ; f o r polysaccharides the f i g u r e s are 20 x 10 e, 5 x 10 e and 1 x 10 6. The g e l s t r u c t u r e i s w e l l adapted to the demands of molecular s i e v e chromatography and, because of the l a r g e s urface area ( i f indeed 'surface' can be d e f i n e d ) , b e t t e r s u i t e d to a f f i n i t y methods than m a t e r i a l s l i k e c e l l u l o s e . I t has a l s o been found that enzymes a f f i x e d to Sephadex e x h i b i t 18 higher r e l a t i v e a c t i v i t y than the corresponding cellulose-bound enzymes A p r i n c i p a l disadvantage of the polysaccharide matrices l i e s i n t h e i r 125 i n c o m p a t i b i l i t y w i t h organic s o l v e n t s , which cause shrinkage ( i n t e r - c h a i n bonding being p r e f e r r e d , i n instances where i t i s not overcome by s t e r i c s t r a i n , over the greater s o l v a t i o n enjoyed i n water) and consequent r e d u c t i o n i n the number of groups ^ a v a i l a b l e f o r r e a c t i o n , narrowing of pores and red u c t i o n of flow r a t e s . This phenomenon i s of i n t e r e s t i n i t s e l f and i s explored i n the present chapter. For the purposes of chromatography, how-ever, i t has been l a r g e l y overcome by the simple expedient of chemical c r o s s -l i n k i n g , which a l s o leads to increased g e l r i g i d i t y , chemical and thermal 19 s t a b i l i t y . Thus the Sepharose CL s e r i e s , c r o s s - l i n k e d w i t h 2,3-dibromo-propanol under s t r o n g l y a l k a l i n e c o n d i t i o n s : j-OH HO-| BrCH2CH(Br)CH2OH , ^ C H ^ C H ^ OH i s amenable to a greater v a r i e t y of chemical procedures f o r l i g a n d immobilis-a t i o n , i n c l u d i n g r e a c t i o n s o c c u r r i n g i n organic media and i n a r a t h e r wider pH range than i s compatible w i t h Sepharose. The extent of the c r o s s -l i n k i n g i s p r o p r i e t a r y i n f o r m a t i o n , but i t i s estimated that coupling y i e l d s are reduced i n CL Sepharoses by up to 50% owing to the re d u c t i o n i n numbers 19 of a v a i l a b l e hydroxyls . I t i s thought that the c r o s s - l i n k s occur between chains already a s s o c i a t e d i n t o ' j u n c t i o n zones' (Figure I I I - 2 ) , and indeed no 20 a l t e r a t i o n i n the e x c l u s i o n l i m i t r e s u l t s . I t has been found that p l a c i n g f i v e atoms between the two carbohydrate oxygens confers the maximum mechan-21 i c a l s t a b i l i s a t i o n , and that b i f u n c t i o n a l aromatic molecules are s i m i l a r l y e f f e c t i v e ^ , but i t i s a l s o necessary to ensure p r e s e r v a t i o n of a uniform, h y d r o p h i l i c m i c r o s t r u c t u r e w i t h i n the bead, so that short c r o s s - l i n k s are to be p r e f e r r e d . Sepharose CL and Sephadex are b r i e f l y discussed i n Appendix 1. A wealth of chemistry has been developed f o r the d e r i v a t i s a t i o n of 126 polysaccharides f o r chromatography, some adapted from s o l i d phase peptide synthesis (though matrix requirements being d i f f e r e n t , s y n t h e t i c polymers have u s u a l l y been p r e f e r r e d i n the l a t t e r a r ea). Although each agarobiose u n i t has 4 h y d r o x y l groups, degree of s u b s t i t u t i o n i n aqueous s o l u t i o n never approaches t h e o r e t i c a l l i m i t s except perhaps i n the presence of very strong base, presumably because of the involvement of many hydroxyls i n i n t e r - c h a i n hydrogen-bonding, or t h e i r s u b s t i t u t i o n by small amounts of sulphate, pyruvate and methyl. An important e x t r a f a c t o r shaping the chemistry of a f f i n i t y chromatography has been the discovery of the need f o r 22 23 a 'spacer arm' to separate the l i g a n d from the matrix ' . For the most par t t h i s phenomenon has not been s y s t e m a t i c a l l y s t u d i e d ; r e p o r t s of spacer e f f e c t s from the l i t e r a t u r e are discussed i n the l i g h t of present r e s u l t s i n s e c t i o n I V D ( i i i ) . What can one hope to l e a r n from esr s t u d i e s of Sepharose, and, more imp o r t a n t l y , what conclusions can be drawn f o r the b e n e f i t of p r a c t i t i o n e r s of a f f i n i t y chromatography? I t i s c l e a r that the character of the m a t r i x m a t e r i a l , the extent of c r o s s - l i n k i n g and the e f f i c i e n c y and nature of the d e r i v a t i s a t i o n chemistry w i l l a l l i n f l u e n c e the number of immobilised l i g a n d s ( l a b e l s ) and t h e i r d i s t r i b u t i o n . Thus we may hope to f i n d simple means to q u a n t i t a t e b i n d i n g s i t e s and i n cases where these are s u f f i c i e n t l y densely d i s t r i b u t e d to enable l a b e l s to i n t e r a c t w i t h each other, measure t h e i r s e p a r a t i o n . The question i s a l s o r a i s e d of whether the l i g a n d s are randomly or non-randomly d i s t r i b u t e d . I f i t i s p o s s i b l e to measure d i s -t r i b u t i o n using e s r , then one may go f u r t h e r , and r e l a t e that d i s t r i b u t i o n to a one-, two- or three-dimensional array of l a b e l s . I f d i m e n s i o n a l i t y can be i d e n t i f i e d , i t may be p o s s i b l e to deduce that the bead s t r u c t u r e c o n s i s t s mainly of i s o l a t e d s i n g l e - s t r a n d e d polymer c h a i n s ( I D ) , can be 127 approximated as a surface (2D), or i s best approached as a three-dimensional array of s a c c h a r i d i c f u n c t i o n a l groups. The question may a l s o be answered of whether any heterogeneity e x i s t s i n the m o b i l i t i e s of the p o p u l a t i o n of l a b e l s , how these m o b i l i t i e s are r e l a t e d to the distance the l a b e l s are extended from the polysaccharide chains, and how solvent environment a f f e c t s t h i s dependency. A l l may then be t e s t e d against the current model, which p o s t u l a t e s the existence both of ' j u n c t i o n ' and 'non-junction' zones i n which polysaccharide molecules are r e s p e c t i v e l y a s s o c i a t e d by cooperative hydrogen-bonding or present as s i n g l e strands (Chapter I I I ) . Where c o n s i d e r a t i o n s of fundamental s t r u c t u r e are paramount, as i n the preceding chapter, the s p i n l a b e l l i n g method b e n e f i t s from a h i g h sen-s i t i v i t y of e s r , which enables low l e v e l s of m o d i f i c a t i o n to be sustained. In the present chapter, cyanogen bromide-mediated chemistry i s used to a t t a c h l a b e l s to Sepharose 4B, because i t i s t h i s r e a c t i o n that i s most commonly used to immobilise l i g a n d s f o r a f f i n i t y chromatography. I t i s the most e f f i c i e n t method f o r coupling n u c l e o p h i l i c species to polysaccharides i n aqueous s o l u t i o n , and gives more h i g h l y modified products than would have been d e s i r a b l e i n Chapter I I I . In a d d i t i o n i t i s c l e a r from the e f f o r t , not yet wholly s u c c e s s f u l , that has been expended i n attempting to c h a r a c t e r i s e the r e a c t i o n products (see next s e c t i o n ) that t h i s i s not an i d e a l r e a c t i o n from the standpoint of the chemist. I t i s u n s a t i s f a c t o r y to pursue s t r u c t u r a l s t u d i e s on a system which i s not f u l l y c h e m i c a l l y c h a r a c t e r i s e d . However, such o b j e c t i o n s are outweighed by the s t a t e d object of s c r u t i n y of the chromatographic process, and d i s c u s s i o n of the r e s u l t s obtained using the cyanogen bromide a c t i v a t i o n procedure to a t t a c h l a b e l s to Sepharose 4B forms the major part of t h i s chapter. Again i n the l i g h t of t h i s aim, the substance of the chapter i s d i v i d e d i n :the f o l l o w i n g 128 way. A f t e r a review of published work on the mechanism of cyanogen bromide a c t i v a t i o n i n IVB, s e c t i o n I V C ( i ) - ( v ) considers'the r e s u l t s obtained using the method to s p i n l a b e l Sepharose 4B and describes a number of p h y s i c a l manipulations which together c o n s t i t u t e a d e t a i l e d a p p r a i s a l of the esr data. In s e c t i o n IVC(vi) i n f o r m a t i o n obtained using other l a b e l l i n g methods i s added, and i n I V C ( v i i ) elements from the whole which have a d i r e c t bearing on our p i c t u r e of Sepharose 4B are summarized f o r the b e n e f i t of the reader who i s not a s p e c t r o s c o p i s t . S e c t i o n IVD contains r e s u l t s from systems i n which l a b e l s are conjugated to the support v i a spacer arms of v a r i o u s lengths and goes on to consider the i m p l i c a t i o n s of these r e s u l t s i n a f f i n i t y chromatography. F i n a l l y , Appendix 1 deals i n p a r t w i t h pre-l i m i n a r y r e s u l t s using other matrices: Sepharoses 2B and 6B, c r o s s - l i n k e d (CL) Sepharose 2B, 4B and 6B, and Sephadex G25. Two batches of Sepharose 4B from Pharmacia were used throughout: one purchased p r e - a c t i v a t e d and f r e e z e - d r i e d , and the other as the unmodified hydrated g e l . IVB: Cyanogen Bromide A c t i v a t i o n Despite the complexity of the products, the cyanogen bromide a c t i v a -t i o n procedure has remained the method of choice f o r d e r i v a t i s i n g p o l y -saccharides f o r chromatographic and other purposes s i n c e i t s i n t r o d u c t i o n by Porath, Axen and Ernback i n 1 9 6 7 " ^ W h i l e e a r l i e r methods of p o l y -saccharide d e r i v a t i s a t i o n used s t r i n g e n t c o n d i t i o n s to enable hydroxyls (weak n u c l e o p h i l e s ) to react w i t h e l e c t r o p h i l e s i n a s i n g l e step process, t h i s method r e s u l t s i n the i n t r o d u c t i o n of an e l e c t r o p h i l i c group which may subsequently be attached by a n u c l e o p h i l e such as amine which i s r p r e s e n t i n the l i g a n d . The r e a c t i o n i s thought to proceed mainly as shown i n 129 Figure IV-1. N u c l e o p h i l i c a t t a c k of hydroxyl on CNBr proceeds w i t h displacement of Br forming a r e a c t i v e cyanate (22) to which hydroxyl may add, e i t h e r g i v i n g an unreactive carbamate (23) or an imidocarbonate. The l a t t e r may be c y c l i c (24), or a c y c l i c (29) i n which case a c r o s s - l i n k i s created between polysaccharide strands. That such c r o s s - l i n k s are formed i s evident because- a number of polysaccharides so t r e a t e d i n t h i s l a b o r a t o r y became i n s o l u b l e even i n b o i l i n g water: agarose (Sepharose), guar gum, and a l g i n i c a c i d are three examples. This phenomenon ex p l a i n s why CNBr chemistry was not used i n Chapter I I I . The a c y c l i c imidocarbonate may be l a r g e l y present i n the case of agarose as a 4,6-0-substituted galactose d e r i v a t i v e (24a), which has a s i m i l a r s t r u c t u r e to the n a t u r a l l y o c c u r r i n g 25 4,6-0-pyruvate . In Sephadex, glucose residues are probably 2,3-0-s u b s t i t u t e d g i v i n g a 5-membered c y c l i c intermediate. The imidocarbonate may react i n a v a r i e t y of ways: w i t h l i g a n d (RNB^), where R may be anything from a p r o t e i n to a spacer molecule, to give immobilised products (26), (27), and (28), or w i t h base and water to carbonate (25), carbamate and even back to the o r i g i n a l polysaccharide w i t h e v o l u t i o n of carbonate and ammonium. Cyanogen bromide s t a r t i n g m a t e r i a l may a l s o be hydrolysed by base. The presence of c y c l i c and a c y c l i c imidocarbonates, carbamates and carbonates was suggested by i n f r a - r e d spectroscopy, mass spectrometry, and 26 nmr on small model compounds . In p r a c t i c e the r e a c t i o n proceeds i n two stages: the f i r s t c o n s i s t s of a c t i v a t i o n i n strong carbonate b u f f e r using a s o l u t i o n of CNBr i n water at 278 K ( i n the present s t u d i e s a m o d i f i c a t i o n of 27 the method of Cuatrecasas was used). F o l l o w i n g t h i s step, which i s performed i n ^ 4 minutes, the r e a c t i o n s o l u t i o n c o n t a i n i n g unreacted CNBr i s f i l t e r e d o f f , the Sepharose washed, and the amine-containing l i g a n d ( i n t h i s case a n i t r o x i d e ) added i n pH 8 b u f f e r , a f t e r which c o u p l i n g proceeds 130 HO" + NH% + I ^C-0 (253 corbonote C233 OH", Hji -OH H (-0H (B) + oo|"* N H ; (ii) inter - c h o i n |-OH HO-] B r C " » (-O-CsN HO-| (183 (22) i NH f - o - c ^ -4 (2SD acyclic imidocorbonale I RNH, | - o - c - o - J NR I II (313 (-0-C303 ^NHR 'NH -4 HO-| (263 Figure IV-1: Proposed mechanism f o r cyanogen bromide a c t i v a t i o n of agarose(18) and other h y d r o x y l i c polymers. Reaction i n v o l v i n g p a i r s of neighbouring hydroxyls may occur i n e i t h e r i n t r a - ( i ) or i n t e r - m o l e c u l a r ( i i ) f a s h i o n , the l a t t e r g i v i n g r i s e to cro s s -linkages . 131 at room temperature for 20 hours. I t i s probable that the proportions of the d i f f e r e n t products depend on the exact conditions of concentration, pH, time and temperature i n each of the two steps; thus ligand incorporation increases with pH for some ligands but decreases for others; p o s i t i v e charge i n the der i v a t i s e d matrices (measured by the binding of radio-l a b e l l e d phosphate) varies by a factor of about f i v e depending on the 28 ' ligand and the reaction conditions IVC: Esr Studies of Sepharose 4B ( i ) Spectra Figures IV-2 and IV-3 show serie s of spectra due to samples of Sepharose 4B activated with cyanogen bromide and reacted with l a b e l s (2) and (9) re s p e c t i v e l y under e s s e n t i a l l y the same conditions. With one exception, namely spectrum a i n Figure IV-3, which came from Sepharose activated i n the laboratory, a l l the spectra are due to samples from the batch of Sepharose which had been CNBr-activated by Pharmacia. They r e f l e c t moderately immobile labels and two p a r t i a l l y - r e s o l v e d s p e c t r a l components are v i s i b l e at low and, sometimes, high f i e l d . Signal-to-noise v a r i a t i o n s are not s i g n i f i c a n t , as the amount of sample i n the cavity was not always the same. The quite s i g n i f i c a n t differences i n lineshape were unable to be correlated with any one of a number of parameters thought to a f f e c t the deta i l e d course of the r e a c t i o n — s l i g h t differences i n tempera-ture (the f i r s t step was performed while s t i r r i n g on a bed of ice-water, the second at ambient temperature), concentration of CNBr (2g.ml -1 ± 20%), duration of the a c t i v a t i o n step (4 ± 1 min.) and, perhaps the completeness (9) 0 132 a b c d Figure IV-2: Esr spectra of (34), Sepharose 4B, CNBr-activated and reacted w i t h n i t r o x i d e ( 2 ) , i n water. 134 of removal of unreacted CNBr, may a l l c o n t r i b u t e . These d i f f e r e n c e s , however, bore no apparent r e l a t i o n to whether the a c t i v a t i o n step was performed by Pharmacia or i n our l a b o r a t o r y . Neither d i d any c o n s i s t e n t d i f f e r e n c e between p y r r o l i d i n y l and p i p e r i d i n y l d e r i v a t i v e s emerge. Figure IV-4 shows the spectrum obtained by r e a c t i o n of a sample of the p r e c i p i t a t e d agarose used i n Chapter I I I w i t h CNBr and l a b e l (9). The lineshape appears r a t h e r s i m i l a r to those i n Figures IV-2 and IV-3, though an a d d i t i o n a l 'sharp' component i s superimposed which i n d i c a t e s the presence of some adsorbed l a b e l . This was more d i f f i c u l t to remove from the p r e c i p i t a t e d m a t e r i a l than from the g e l beads. Otherwise one tends to the same co n c l u s i o n as was drawn i n Chapter I I I , namely that m o b i l i t y of species at the surface of the s o l i d does not d i f f e r r a d i c a l l y from that of those immobilised w i t h i n the g e l . This spectrum serves to i l l u s t r a t e another p o i n t , that i t was easy to d i s t i n g u i s h the presence of unreacted l a b e l s i n the products, so that washing procedures were able to be devised to thoroughly r i d gels of t h i s r e s i d u e . The r e l a t i v e narrowness of esr l i n e s due to f r e e (or adsorbed) l a b e l makes a s e n s i t i v e i n d i c a t o r . In cases where d e r i v a t i v e s were made whose spec t r a were i n s u f f i c i e n t l y broad to enable a d i s t i n c t i o n to be made between f r e e and bound (see IVD) the same washing procedures were adopted. In t h i s way i t was a simple matter to detect 'leakage' of bound l a b e l from the ma t r i c e s ; h y d r o l y s i s occurs, presumably at the imidocarbonate l i n k a g e , g i v i n g r i s e to f r e e s p i n l a b e l . This was n o t i c e a b l e over a pe r i o d of weeks, e s p e c i a l l y i n gels stored at room temperature, normal procedure being to s t o r e them at 278 K. However, i t was not s i g n i f i c a n t during the l i f e t i m e of a s i n g l e experiment. Exper-iments were performed i n a l l cases on samples l e s s than 2 weeks o l d l e s t leakage should, i n the long run, a f f e c t the lineshape. 10G Figure IV-4: Esr spectrum of aqueous p r e c i p i t a t e d agarose (as used i n Chapter I I I ) , CNBr-activated and reacted w i t h n i t r o x i d e ( 9 ) . Note the presence of sharper resonances due. to adsorbed l a b e l . Although to the untutored eye the two p a r t i a l l y r e s o l v e d components to high and, sometimes, low f i e l d i n Figures IV-2 and IV-3 might suggest the e x i s t e n c e of two d i s t i n c t populations of spins of d i f f e r e n t l i n e w i d t h , such features can a r i s e from a homogeneous po p u l a t i o n of i s o t r o p i c a l l y r e o r i e n t i n g s p i n s , as shown by the simulated s p e c t r a i n Figure 1-9. However, none of the lineshapes i n Figures IV-2 and IV-3 can i n f a c t be simulated under t h i s assumption, so i t may be taken that e i t h e r motional a n i s t r o p y or s i t e inhomogeneity, or both of these, are present. I t i s u n l i k e l y that v a r i a t i o n s i n motional a n i s t r o p y are r e s p o n s i b l e f o r the d i f f e r e n c e s i n lineshape between samples given the s i m i l a r i t y of t h e i r r e s p e c t i v e h i s t o r i e s . On the other hand, p o s s i b i l i t i e s f o r s i t e v a r i a t i o n s a r i s e both from the nature of the g e l s t r u c t u r e (Chapter I I I ) and the probable product mixture from the l a b e l l i n g chemistry (Figure IV-1); per-haps the s p e c t r a l changes i n Figures IV-2 and IV-3 could a r i s e from v a r i a -t i o n s i n the r e l a t i v e p r o p o r t i o n of two populations of n i t r o x i d e s , under-going s i t e exchange l e s s than about 10^ times per second. Two simple experiments were i n i t i a l l y attempted. F i r s t l y , i n the hope of d i s t i n g u i s h i n g l a b e l s i n ' j u n c t i o n ' and 'non-junction' zones, denaturants commonly used i n p r o t e i n chemistry were added to the g e l : 8M urea, 100% dimethyl sulphoxide and 1M sodium dodecyl sulphate. No change i n lineshape was observed a f t e r t r e a t i n g the l a b e l l e d Sepharose 4B w i t h any of these s o l u t i o n s , then washing i t f r e e again w i t h water. Observed lineshape changes even i n t h e i r presence were l i m i t e d , and i n the case of 8M urea, unable to be detected at a l l . No p r o g r e s s i v e changes occurred during s e v e r a l hours' exposure. These experiments provided c o n f i r m a t i o n that no adsorbed l a b e l was present i n the g e l , s i n c e a l l three d i s r u p t non-covalent bonds. 137 A f u r t h e r method was t r i e d , that of thermal denaturation. As a r e s u l t of c r o s s - l i n k i n g by CNBr, the g e l s t r u c t u r e was only n o t i c e a b l y d i s -rupted a f t e r b o i l i n g i n water f o r s e v e r a l minutes, a f t e r which p a r t i a l d i s s o l u t i o n appeared to have occurred. The esr spectrum i n d i c a t e d that very extensive h y d r o l y s i s of bound l a b e l had r e s u l t e d , so again no con-c l u s i o n was drawn. However, these experiments serve to i l l u s t r a t e the marked d i f f e r e n c e s between higher-order s t r u c t u r e s i n g l o b u l a r p r o t e i n s , most of which would have been r a p i d l y denatured i n any of the above con-d i t i o n s , and p o l y s a c c h a r i d e s . In the second type of experiment, water was replaced i n the i n t e r i o r of the g e l beads by 50% aqueous g l y c e r o l and by 100% g l y c e r o l . The d i e l e c -t r i c constant of g l y c e r o l (42.5 as opposed to 78.4 f o r water) i s such that the i s o t r o p i c h y p e r f i n e c o u p l i n g constant ( a g ) - i s reduced by 0.7 G (Chapter I C ) , but the r e s u l t a n t change i n g e l volume was not d e t e c t a b l e using s o l -vent displacement on about 0.5 ml. of g e l . Thus the g e l s t r u c t u r e i s assumed to remain e s s e n t i a l l y the same, so that the s p e c t r a l changes shown i n Figure IV-5 (and compared w i t h those f o r n i t r o x i d e i n s o l u t i o n ) can be assumed to be the consequence of motional changes a r i s i n g from l a b e l - s o l v e n t or label-polymer i n t e r a c t i o n s . Spectrum IV-5d serves to reemphasize the p o i n t that the two s p e c t r a l components cannot be i d e n t i f i e d w i t h two phys-i c a l l y d i s t i n c t populations of l a b e l s , s i n c e the apparent r a t i o of these has changed as a r e s u l t of changes associated only w i t h motion. At 100% g l y c e r o l (Figure IV-5e), two overlapping features are no longer apparent; e q u a l l y , t h i s does not e l i m i n a t e the p o s s i b i l i t y that s i t e heterogeneity e x i s t s , s i n c e s p e c t r a l s i m u l a t i o n s can r e a d i l y be employed to show that d e t a i l e d lineshapes are l e s s s e n s i t i v e to changes i n c o r r e l a t i o n time near the r i g i d l i m i t than i n mid-range (see, f o r example, Figure 1-6). I t i s 138 Figure IV-5: Esr spectra of ( a ) l a b e l ( 2 ) , 0.5mM i n 50% aqueous g l y c e r o l ; (b)(2) i n 100% g l y c e r o l ; ( c ) ( 3 4 ) , CNBr-activated Sepharose 4B, l a b e l l e d w i t h ( 2 ) , i n water; (d)(34) i n 50% aqueous g l y c e r o l : (e)(34) i n 100% g l y c e r o l . 139 u s e f u l to poi n t out at t h i s stage that line-broadening such as that observed i n Figure IV-5c, d and e could a r i s e not only from an increase of c o r r e l a -t i o n time (T) (shown i n Figure IV-5a and b f o r a model s o l u t i o n system of d i l u t e spins) but a l s o from l e s s complete averaging of s p i n - s p i n d i p o l a r i n t e r a c t i o n s as the c o r r e l a t i o n time i n c r e a s e s , ( i i ) Q u a n t i t a t i o n of the Label Although not c e n t r a l to the r e s o l u t i o n of the nature of the complex lineshape obtained by cyanogen bromide-mediated attachment of n i t r o x i d e s to Sepharose 4B, i t i s h e l p f u l f o r some of the f u r t h e r experiments to be d i s -cussed to have some idea of the extent to which the n a t i v e s t r u c t u r e of the ge l has been modified. I t was p o s s i b l e to q u a n t i t a t e l a b e l s present i n the p olysaccharide i n two d i f f e r e n t ways—by elemental a n a l y s i s , and by double i n t e g r a t i o n of the f i r s t d e r i v a t i v e spectrum and comparison w i t h a standard.* Both methods s u f f e r e d from important drawbacks, but f u l f i l l e d t h e i r primary purpose, which was to o b t a i n an order-of-magnitude f i g u r e to compare w i t h the more r e l i a b l e d i s t a n c e measurements discussed l a t e r . The elemental a n a l y s i s data f o r a t y p i c a l sample of Sepharose 4B, p r e - a c t i v a t e d by Pharmacia and su p p l i e d i n f r e e z e - d r i e d form, and reacted w i t h 4-amino-2,2,6,6-tetramethylpiperidine-l-oxyl (2), together w i t h data f o r c o n t r o l s performed on the same sample and a non-activated 4B provided by Pharmacia as a swollen aqueous g e l , are shown i n Table IV-1. Each sample was f r e e z e - d r i e d , then heated i n vacuo i n a dr y i n g p i s t o l at 384 K fo r 18 hours and immediately, r a p i d l y and t i g h t l y capped to minimise water adsorption (though i t i s very l i k e l y that some water remains t i g h t l y bound at 384 K, 0.01 t o r r ) . The q u a n t i t a t i o n i s based on n i t r o g e n a n a l y s i s . *A t h i r d and more s e n s i t i v e , though very expensive method would have been to use t r i t i u m - l a b e l l e d n i t r o x i d e . 140 Although a complex mixture of products may r e s u l t from the r e a c t i o n , i t i s f a i r to assume that a l l the n i t r o g e n present i n excess of that found when water, rather than s p i n l a b e l , was present as the n u c l e o p h i l e , r e s u l t e d from the two n i t r o g e n atoms present i n the l a b e l . Thus by comparing n i t r o g e n data i n (a) and (b) of Table IV-1 w i t h the f i g u r e s f o r carbon, i t Table IV-1: Data obtained from m i c r o a n a l y s i s of l a b e l l e d Sepharose 4B and c o n t r o l s mono- mono- mono-saccharide saccharide saccharide residues residues residues per l a b e l per reac- per l a b e l t i o n from i n t e -Sample C H N g r a t i o n (a) Sepharose/CNBr/SL 45.86 2.26 6.86 36 19 (b) Sepharose/CNBr/H 20 41.83 1.64 6.46 5 (c) Sepharose g e l 45.25 6.78 0 (d) Sepharose/CNBr 43.38 6.53 0 (e) t h e o r e t i c a l , dry agarose 47.06 5.88 0 i s p o s s i b l e to compute the number of galactose residues present per l a b e l — 36 to the nearest i n t e g e r . A l s o , from the same data, assuming one n i t r o g e n atom per a c t i v a t e d s i t e , i t may be seen that one i n every f i v e galactose residues i s ' a c t i v a t e d ' (though more than one sugar may be in v o l v e d i n one ' s i t e ' ) . As expected, no n i t r o g e n i s found i n n a t i v e Sepharose 4B, but i n view of the f i g u r e s f o r sample (b), i t i s s u r p r i s i n g to f i n d no n i t r o g e n i n (d) , CNBr-activated 4B sup p l i e d by Pharmacia i n f r e e z e - d r i e d form. This must have been l o s t i n processes l i k e |> N H_JHa?_^J-O oH • C 0 2 + N H 3 141 but the reason why t h i s does not happen to the same extent a f t e r r e a c t i o n w i t h water (18 h, Table I V - l ( b ) ) i s not c l e a r . D i f f e r e n c e s i n carbon and hydrogen contents from the c a l c u l a t e d values even i n n a t i v e 4B can be a s c r i b e d to bound water present a f t e r heating i n vacuo or absorbed from the a i r during the a n a l y t i c a l procedures. The main e r r o r here, however, comes from the m i c r o a n a l y s i s : ± 0.3 i n each f i g u r e , which i n the case of n i t r o g e n could come to ± 20%. The i n t e g r a t i o n procedure, s i n c e i t depended on weighing of samples, s u f f e r e d more from the phenomenon of atmospheric water adso r p t i o n . In a d d i t i o n , the s m a l l samples that could be accommodated w i t h i n the resonant c a v i t y , a f t e r d r y i n g , n e c e s s i t a t e d weighing on a very s m a l l s c a l e (± 4 mg). (Elemental a n a l y s i s could not be used i n the s p i n d i l u t i o n experiments described l a t e r , so that there i n t e g r a t i o n had to be performed on samples w i t h low s i g n a l - t o - n o i s e . ) Worst of a l l , the second i n t e g r a t i o n was c a r r i e d out by means of peak-cutting i n the presence of poor b a s e l i n e s . E r r o r s were hard to determine, but ± 50% represents an upper l i m i t . As shown i n Table IV-1, a f i g u r e of 19 r e s i d u e s / s p i n was obtained f o r the same sample as was used i n the elemental a n a l y s i s . Thus the i n t e g r a t i o n and m i c r o a n a l y s i s are w i t h i n experimental e r r o r of each other. ( i i i ) Spin D i l u t i o n In order to increase the average distance between spins the n i t r o x i d e molecule (2) was d i l u t e d w i t h i t s diagmagnetic analogue (32) i n r e a c t i o n s w i t h CNBr-activated Sepharose 4B under otherwise i d e n t i c a l c o n d i t i o n s , and (2) (32) 142 the e f f e c t on the esr spectrum monitored. In the experiment the r e s u l t s of which are shown i n Figure IV-6 t h i s was e f f e c t e d at a constant t o t a l c o n centration ( [ ( 2 ) ] + [(32)] = 0.06M), and constant amount of Sepharose 4B. In a d d i t i o n to the p r o p o r t i o n of n i t r o x i d e i n the r e a c t i o n mixture, two pieces of i n f o r m a t i o n could be obtained: the number of residues per n i t r o x i d e by s p e c t r a l i n t e g r a t i o n (elemental a n a l y s i s not i n t h i s case being s u f f i c i e n t l y s e n s i t i v e ) and the average nearest-neighbour d i s t a n c e between spins ( r ) , which may be obtained from the d i / d parameter defined i n Chapter IC, and measured from powder spec t r a recorded at 77 K. That 'loading' turned out to be such that d i p o l a r i n t e r a c t i o n s were measurable i n n i t r o x i d e - l a b e l l e d Sepharose 4B was f o r t u i t o u s . D i s c u s s i o n presupposes that the para- and dia-magnetic analogues have equal r e a c t i v i t i e s , but the v a l i d i t y of the assumption can i n any case be confirmed w i t h i n experimental e r r o r by p l o t t i n g percentage of n i t r o x i d e i n the r e a c t i o n mixture against the number of unpaired spins per monosaccharide residue i n the product, the l a t t e r obtained from i n t e g r a t i o n data; t h i s i s shown i n Figure IV-7. I t must a l s o be assumed that the a l t e r a t i o n i n net charge of the modified m a t r i x caused by s u b s t i t u t i o n of amine f o r n i t r o x i d e does not a f f e c t the f i n a l d i s t r i b u t i o n s i g n i f i c a n t l y . A l l experiments were conducted using a l a r g e excess of l a b e l s r e l a t i v e to the number of b i n d i n g s i t e s on the polymer. F i n a l l y , i t i s assumed that the motional p r o p e r t i e s of the n i t r o x i d e s are not changed by the s p i n d i l u t i o n . Thus the observed s p e c t r a l narrowing (Figure IV-6) occurs because s p i n - s p i n i n t e r a c t i o n s decrease as the n i t r o x i d e s i t e s are i n c r e a s i n g l y s u b s t i t u t e d . Again none of the s p e c t r a can be simulated assuming a s i n g l e , i s o t r o p i c a l l y tumbling p o p u l a t i o n of n i t r o x i d e s , and again i t i s tempting to conclude from the appearance of the s p e c t r a that two populations are present, the broader Figure IV-6: Esr spectra r e s u l t i n g from sp i n d i l u t i o n i n CNBr-mediated l a b e l l i n g of Sepharose 4B with (2). D i l u t i o n was e f f e c t e d using the diamagnetic analogue(32). Percentage of n i t r o x i d e i n the l i g a n d mixture: (a)17; (b)33; (c)67; (d)83; (e)100. -P-l O C f 75r-tl o 5Cf-25h l _ 0.01 0.02 -I 0.03 L _ 0.04 s p i n s / r e s i d u e l _ 0.05 Figure IV-7. Number of n i t r o x i d e s ( 2 ) per monosaccharide (from i n t e g r a t i o n ) i n the product as a f u n c t i o n of proportion of n i t r o x i d e i n the r e a c t i o n mixture i n sp i n d i l u t i o n experiments with CNBr-activated Sepharose 4B, 145 l i n e decreasing i n i n t e n s i t y as the p r o p o r t i o n of n i t r o x i d e i n the r e a c t i o n mixture decreases. However, i f such a d i s t r i b u t i o n a l heterogeneity was present, i t was not able to be detected i n q u a n t i t a t i v e experiments, which were as f o l l o w s . We may d e f i n e a parameter p as the d e n s i t y of spins i n the system, i n -3 -1 u n i t s of (say) s p i n nm . This i s then p r o p o r t i o n a l to s p i n g from i n t e g r a t i o n and (according to Figure IV-7) to the p r o p o r t i o n of n i t r o x i d e i n the r e a c t i o n mixture. Depending on the nature of the system, p may be r e l a t e d to r i n three ways: p = (r) ^ (one-dimensional) [34] 1/2 p <* (r) (two-dimensional) [35] 1/3 p <* (r) (three-dimensional) [36] U n f o r t u n a t e l y , the data obtained could, i n p l o t s of (% n i t r o x i d e ) against r , be f i t t e d almost e q u a l l y w e l l to a l l three r e l a t i o n s ! This u n d e r l i n e s the considerable l i m i t a t i o n s of the method. As the n i t r o x i d e i s progres-s i v e l y d i l u t e d , s i g n a l - t o - n o i s e i n esr decreases p r o p o r t i o n a l l y (Figure IV-6). Thus dj/ d i s measured w i t h p r o g r e s s i v e l y lower accuracy. Secondly, as r increases and the d i p o l a r i n t e r a c t i o n becomes weaker, the l a t t e r becomes l e s s and l e s s s e n s i t i v e to changes i n the former, which tends to an asymptote (< ^ 0.4) at h i g h s p i n d i l u t i o n (Figure 1-11). The l i m i t a t i o n s of i n t e g r a -t i o n as a means to q u a n t i t a t i o n h a v e been discussed. The value of r increases from ^ 1.2 to ^ 2.0 nm on passing from 100 to 17% n i t r o x i d e i n the r e a c t i o n mixture. Given approximate f i g u r e s of 20-40 r e s i d u e s / s p i n at 100% n i t r o x i d e , i t i s evident that the system does not con-s i s t mainly of d i l u t e , extended, s i n g l e - s t r a n d e d p o l y s a c c h a r i d e chains, s i n c e 20 residues would, i n that case, encompass a d i s t a n c e of about 10 nm. I t i s a l s o evident from the distances involved that at l i q u i d n i t r o g e n temperature 146 the s p i n - s p i n i n t e r a c t i o n i s d i p o l a r i n o r i g i n . As to the d i s t r i b u t i o n of n i t r o x i d e s and i t s r e g u l a r i t y , the d\/d parameter i s able only to i n d i c a t e an average d i s t a n c e , and i t i s c l e a r that p as measured here i s not very s e n s i t i v e to what may be q u i t e s m a l l d e v i a t i o n s from homogeneity, ( i v ) Solvent-Mediated Shrinkage In t h i s experiment the shrinkage caused i n the g e l beads by exposure to solvents l e s s p o l a r than water was used as a means to explore f o r pos-s i b l e inhomogeneities i n the d i s t r i b u t i o n of l a b e l s . The change i n bead volume was measured by solvent displacement. A f t e r measuring volume (at room temperature) and r (at 77 K) the l a b e l l e d beads were subjected to change of solvent through a m i s c i b l e s e r i e s of decreasing p o l a r i t y : 50% aqueous g l y c e r o l , e thanol, 1,4-dioxan, n-heptane, dioxan, e t h a n o l , 50% aqueous g l y c e r o l . In each case the change was e f f e c t e d v i a 30:70 and 70:30 i n t e r -mediate solvent mixtures, though i t was shown that r values were not depend-ent on the d e t a i l e d course of the solvent t r a n s i t i o n . The measurements of volume and r were repeated at the heptane stage and again a f t e r the r e t u r n to aqueous g l y c e r o l . I t was a l s o p o s s i b l e to dry the beads a f t e r shrinkage by a l l o w i n g the heptane to f u l l y evaporate overnight, a f t e r which r (though not the volume) was again determined. Spectra are shown i n Figure IV-8. Bead volume was found to decrease by one t h i r d (a ' s w e l l i n g f a c t o r ' of 1.50) on passing from water to heptane, r e t u r n i n g again to the same value on passing back to water. Unless a s i g n i f i c a n t d e v i a t i o n from randomness 1/3 occurs i n the d i s t r i b u t i o n , r i s expected to decrease as (volume) , and t h i s was found to occur w i t h i n experimental e r r o r . Thus i n one t y p i c a l sample r decreased from 1.40 to 1.19 nm (+ 0.03). A f u r t h e r decrease to 1/3 • 1.11 nm accompanied evaporation of the s o l v e n t . (1.50) = 1.15, and 1.19 x 1.15 = 1.34 nm, which i s w i t h i n experimental e r r o r of the observed a b c d Figure IV-8: Esr spe c t r a of (34), CNBr-activated Sepharose 4B, l a b e l l e d w i t h (2). ( a ) i n 50% aqueous g l y c e r o l , 77 K; ( b ) i n n-hexane, 77 K; (c)dry powder, 298 K; (d)dry powder, 77 K. 4>-148 value of 1.40. Some i n s i g h t i n t o the r e l a t i v e p o l y s a c c h a r i d e - s o l v a t i n g power of n-heptane and water can be gained by comparing the value of r f o r dry beads w i t h that f o r beads i n the two s o l v e n t s . I t can a l s o be seen by comparing the spectrum of dry beads at room temperature w i t h that at 77 K that some motion of the l a b e l occurs on the esr timescale even i n the dry beads, l e a d i n g to a 6% re d u c t i o n i n s p l i t t i n g (2T) between the outer f e a t u r e s . (v) S e l e c t i v e Broadening By i n t r o d u c i n g i n c r e a s i n g c o n c e n t r a t i o n of n i c k e l sulphate i n t o the g e l beads, causing exchange-broadening of a c c e s s i b l e n i t r o x i d e s , i t was p o s s i b l e to show that d e s p i t e the l a c k of evidence f o r inhomogeneity i n the popula t i o n from the experiments described i n previous s e c t i o n s , such inhomo-geneity does e x i s t , at l e a s t to a l i m i t e d extent. The r e s u l t s of the experiment are shown i n Figures IV-9 and IV-10. Whi l s t i n s o l u t i o n s of 2+ n i t r o x i d e s exchange occurs to the extent t h a t , above 0.5M Ni(H 20)g , spec-t r a l l i n e s are no longer d e t e c t a b l e even at high microwave power and modula-t i o n amplitude (Figure 1-12), the spectrum of l a b e l l e d Sepharose 4B shows a 2+ r e s i d u a l s i g n a l even at 2M Nl(H20)g whose i n t e n s i t y i s roughly twelve times l e s s than that i n the absence of the paramagnetic i o n . The depend-ence of the width of the c e n t r e - f i e l d peak (AOJQ) on conc e n t r a t i o n of added n i c k e l ( I I ) i s shown i n Figure IV-11 and i s complex and q u i t e d i f f e r e n t from the p l o t shown i n Figure 1-13 f o r n i t r o x i d e homogeneously broadened by n i c k e l i o n s . The l a c k of dependence of ACJQ o n n i c k e l c o n c e n t r a t i o n above 0.5M confirms the existence of a subpopulation of n i t r o x i d e s whose transverse r e l a x a t i o n appears not to be a f f e c t e d by the presence of paramagnetic ions i n the s o l u t i o n phase. That the subpopulation represented by t h i s r e s i d u a l s i g n a l belongs under the envelope represented by Figure IV-9a can be seen i n 149 Figure IV-9: Esr spectra of (34), CNBr-activated Sepharose 4 l a b e l l e d with (2), i n suspension i n aqueous solutions contain various concentrations of n i c k e l sulphate. (a)0; (b)0.01M; (c)0.10M; (d)0.50M; (e)l.OOM; (f)2.00M. Figure IV - 1 0 : (a)Spectrum a from previous f i g u r e ; (b)spectrum f from previous f i g u r e , showing r e l a t i v e a m p l i f i c a t i o n s . O 151 Figure IV-11: Dependence of the c e n t r e - f i e l d l i n e w i d t h AOJ q (C ) on the concentration of n i c k e l ions present i n (34). 152 Figure IV-10, where Figures IV-9a and f are reproduced together w i t h t r a c e s of the outer wings of the aqueous spectrum at the same a m p l i f i c a t i o n as the nickel-broadened spectrum. Although the r e s i d u a l spectrum cannot be e x a c t l y simulated assuming a s i n g l e i s o t r o p i c a l l y r e o r i e n t i n g p o p u l a t i o n of sp i n s , c e r t a i n features are unmistakeable: the wider s e p a r a t i o n of the wings i n Figure IV-9f than i n IV-9a i n d i c a t e s an increase i n c o r r e l a t i o n time (T); and the r e l a t i v e i n t e n s i t i e s of the l i n e s and t h e i r r e s o l u t i o n suggest the presence of s p i n - s p i n i n t e r a c t i o n s . I t can be estimated from approximate s i m u l a t i o n s that the c o r r e l a t i o n time, which c l e a r l y represents an average i n Figure IV-9a, may increase by approximately a two f a c t o r on passing to IV-9f, w h i l e the l i n e w i d t h of the s p i n packets (a parameter used i n simula-t i o n , and a l s o an average value i n Figure IV-9a) may s i m i l a r l y i n c r e a s e by four times. Thus, although the r e s i d u a l spectrum represents only about 20% of the t o t a l n i t r o x i d e p o p u l a t i o n , i t i s c l e a r that heterogeneity e x i s t s both i n tumbling rates and i n s p i n - s p i n i n t e r a c t i o n s . I t i s reasonable that l a b e l s l e s s a c c e s s i b l e to species i n s o l u t i o n are a l s o l e s s mobile than the average; they may be supposed to be occluded w i t h i n small 'pores' i n the g e l s t r u c t u r e . The r e l a t i o n s h i p between a c c e s s i b i l i t y and m o b i l i t y i s developed i n more d e t a i l i n s e c t i o n I V D ( i ) . The question remains open as to whether they experience a sho r t e r apparent s p i n - s p i n r e l a x a t i o n time because they are a c t u a l l y i n c l o s e r proximity than the average to other l a b e l s , or whether they experience s e c u l a r d i p o l a r i n t e r a c t i o n s which are l e s s completely averaged because of the increase i n c o r r e l a t i o n time. A very rough c a l c u l a t i o n , i n v o l v i n g comparison of the magnitude of the d i p o l a r i n t e r a c t i o n measured at 77 K (x assumed to be i n f i n i t e ) w i t h the tumbling r a t e r e quired to average i t , i n d i c a t e s that averaging should be e f f e c t e d at -9 -9 T ^ 5 x 10 s; the best s i m u l a t i o n f o r Figure IV-9f gives T W x 10 s. 153 Thus i t i s not unreasonable that s p i n - s p i n i n t e r a c t i o n s observed i n l a b e l l e d Sepharose 4B at room temperature are d i p o l a r i n o r i g i n . An attempt was made to confirm t h i s by i n c r e a s i n g the temperature i n the esr c a v i t y i n the presence of the aqueous l a b e l l e d m a t e r i a l (as i n Figure IV-9a). Exchange and d i p o l a r mechanisms have the opposite temperature dependence (Chapter I ) . No increase i n l i n e w i d t h was observed but even below 320 K, sharp l i n e s due to hydrolysed n i t r o x i d e i n s o l u t i o n begin to appear, presumably a f f e c t i n g any s p i n - s p i n i n t e r a c t i o n s at the matrix. In a d d i t i o n matrix s w e l l i n g may under these c o n d i t i o n s a c t u a l l y e f f e c t a change i n the d i s t a n c e between s i t e s . However, the r e s u l t i s not i n c o n s i s t e n t w i t h the e x i s t e n c e of unresolved d i p o l a r broadening i n the room-temperature spectrum of l a b e l l e d Sepharose 4B. ( v i ) Other Chemistry I t has already been shown that chemical methods other than the reac-t i o n w i t h cyanogen bromide are a v a i l a b l e f o r modifying p o l y s a c c h a r i d e s . In the present study, i t was thought d e s i r a b l e to seek other means of a t t a c h i n g the s p i n l a b e l i n order to t e s t whether the nature of the chemical l i n k a g e between i t and the saccharide was of importance i n determining i t s motional behaviour. C e r t a i n l i m i t a t i o n s , however, had to be placed upon the types of r e a c t i o n used i n a d d i t i o n to those i n e v i t a b l y imposed by the nature of f u n c t i o n a l groups a v a i l a b l e i n agarose and the demands of n i t r o x i d e chem-i s t r y . I t was d e s i r a b l e to reproduce the c o n d i t i o n s of the CNBr a c t i v a t i o n as c l o s e l y as p o s s i b l e . In p a r t i c u l a r the c o n d i t i o n s had to be aqueous to avoid any s i t e s e l e c t i v i t y based on solvent-mediated shrinkage of the Sepharose. In the second p l a c e , although n e u t r a l , b a s i c or perhaps m i l d l y a c i d i c c o n d i t i o n s were n e c e s s i t a t e d by the a c i d - i n s t a b i l i t y of the n i t r o x i d e f u n c t i o n a l i t y , strong base i s known to cause s w e l l i n g of polysaccharides as 154 a r e s u l t of hydrogen bond-breakage, again suggesting that the i n t e g r i t y of the g e l bead might be threatened, or, at l e a s t , new hydroxyl f u n c t i o n a l i t i e s exposed. The chloracetamide l a b e l l i n g procedure described i n Chapter I I I was i n i t i a l l y attempted using water as solvent ( i n s t e a d of water/acetone) both w i t h sodium carbonate and w i t h r e l a t i v e l y d i l u t e (0.5M) sodium hydroxide as base. Neither procedure gave an esr s i g n a l a f t e r washing the beads. The low s o l u b i l i t y of l a b e l (5) i n water may have c o n t r i b u t e d to t h i s . The same bases were used to promote n u c l e o p h i l i c displacement of c h l o r i d e , t h i s time from l a b e l (8) and again no s i g n a l r e s u l t e d . A s i m i l a r f a t e was met by an attempt to promote the l o s s of imidazole from d e r i v a t i v e (7) i n the presence of sodium carbonate, or d i l u t e NaOH at pH 9.0 as w e l l as (18) (7) pH 10.5 ( s i m i l a r l y b a s i c c o n d i t i o n s a l s o promoted r a p i d l o s s of the CNBr-attached l a b e l from the g e l beads). Reverting to the cyanogen bromide method, l a b e l (6) was reacted w i t h 155 a c t i v a t e d Sepharose 4B to give (33) whose spectrum i s compared i n Figure IV-12 w i t h those of (34) and (35) (see s e c t i o n I V D ( i ) ) . T f o r (33), —9 1.7 x 10 s, i s of about the same order of magnitude as that f o r (35), (34) which has g l y c i n e interposed between the matrix and the l a b e l , and there i s nothing to suggest the presence of s p i n - s p i n i n t e r a c t i o n s . This l a t t e r f e a t u r e may be r e l a t e d both to the increase i n m o b i l i t y and an increase i n average i n t e r - l a b e l d i s t a n c e . The greater m o b i l i t y on the other hand i s not simply i n t e r p r e t a b l e , s i n c e the f l e x i b i l i t y of the l i n k i n g group i n (33) can be assumed equal to that i n (34). However, r e s u l t s presented i n s e c t i o n IVD(i) show that motional freedom i s r a p i d l y c u r t a i l e d as l a b e l s approach more c l o s e l y to the matrix. I t i s u s e f u l to compare here the spectrum obtained a t t a c h i n g l a b e l (5) to p r e c i p i t a t e d agarose i n the presence of strong base and acetone (Figure I I I - 3 ) w i t h the s p e c t r a obtained i n the g e l using the CNBr method and l a b e l ( 2 ) , even bearing i n mind the r e s e r v a t i o n s expressed at the beginning of the present s e c t i o n . On the b a s i s of a c o n s i d e r a t i o n of the two l i n k i n g groups: 156 157 (34) (36) greater m o b i l i t y i s expected and observed i n (36); x, c a l c u l a t e d u s i n g the -9 r a t i o - o f - h e i g h t s method, decreases from 2.6 x 10 s i n (34), using spectrum - 9 a i n Figure IV-6, where s p i n - s p i n e f f e c t s are minimised, to 1.4 x 10 s i n (36), using spectrum e of Figure I I 1 - 3 . In the g e l phase of agarose at _9 room temperature x f o r (36) v a r i e s from 1.2 to 0.7 x 10 (Figure I I I - 4 ) . Secondly, no d i p o l a r i n t e r a c t i o n s are observed i n the l a b e l l e d agarose (36) even at 77 K; t h i s can be r e l a t e d to the reduced e f f i c i e n c y of the l a b e l l i n g r e a c t i o n r e l a t i v e to CNBr. CNBr-mediated l a b e l l i n g of the p r e c i p i t a t e d agarose used i n Chapter I I I gives r i s e to a lineshape (Figure IV-4) q u i t e s i m i l a r to those shown i n Figures IV-2 and IV-3. I t i s p o s s i b l e that had the 'loading' been increased i n Chapter I I I to the l e v e l s observed i n the present chapter, (an increase of about a 5 0 - f a c t o r ) , no g e l a t i o n phenomenon would have been observed! However, the same s o r t of phenomenon of a r e s i d u a l 'masked' po p u l a t i o n of l a b e l s , l e s s mobile than the average, i s observed i n both cases, des p i t e the one atom d i f f e r e n c e i n l i n k i n g groups, ( v i i ) Summary When n i t r o x i d e s are attached to Sepharose 4B using the cyanogen bro-mide a c t i v a t i o n procedure a complex lineshape r e s u l t s , c o n t a i n i n g inhomo-geneous broadening from unaveraged e l e c t r o n - e l e c t r o n d i p o l a r e f f e c t s and at l e a s t a l i m i t e d amount of motional heterogeneity (that i s , l a b e l s i n 158 d i f f e r e n t s i t e s having d i f f e r e n t r a t e s of r e o r i e n t a t i o n ) . However, i t i s not yet c l e a r how the d i f f e r e n t s i t e s are r e l a t e d e i t h e r to the product d i s t r i b u t i o n or the s t r u c t u r e of the ma t r i x . D i f f e r e n c e s i n lineshape observed i n d i f f e r e n t samples of the l a b e l l e d m a t r i x (Figures IV-2 and IV-3) are perhaps best a s c r i b e d to d i f f e r e n t extents of l a b e l l i n g , g i v i n g r i s e to changes i n the magnitude of the d i p o l a r i n t e r a c t i o n . This i s c o n s i s t e n t w i t h changes observed (Figure IV-6) upon d i l u t i n g the n i t r o x i d e w i t h a diamagnetic analogue, which a l s o reduces s p i n - s p i n i n t e r a c t i o n s . S h r i n k i n g of the matrix under the i n f l u e n c e of nonaqueous solvent causes the s i t e s of l a b e l attachment to approach each other more c l o s e l y . Looking forward to the i m p l i c a t i o n s of these r e s u l t s to the a f f i n i t y chromatography method and the next s e c t i o n , the l o c a t i o n of l a b e l s at average nearest-neighbour distances of only about 1.4 nm from each other c l e a r l y suggests the p o s s i b i l i t y of m u l t i p l e - s i t e i n t e r a c t i o n s of immobilised species w i t h macromolecules i n the bead pores. However, although the l a b e l s are immobilised under aqueous c o n d i t i o n s , aqueous metal ions are not able to gain access to a l l of them from s o l u t i o n , which sug-gests that the same might w e l l be true f o r a macromolecule. Separation e f f i c i e n c y might then be d r a s t i c a l l y reduced at low l i g a n d l e v e l s . The observation that about s i x times as many s i t e s are a c t i v a t e d as are eventu-a l l y l a b e l l e d may even r e f l e c t l i m i t e d a c c e s s i b i l i t y of some s i t e s to the l a b e l i t s e l f . However, the s e n s i t i v i t y of lineshape to the nature of the l i n k i n g group, explored more f u l l y i n the next s e c t i o n , suggests a way i n which a c c e s s i b i l i t y might be improved. The s t a b i l i t y of the ma t r i x to d e r i v a t i s a t i o n confirms that chromatographic separations may be performed on Sepharose under q u i t e s t r i n g e n t c o n d i t i o n s . 159 IVD: N i t r o x i d e as a Model f o r Immobilised Ligands ( i ) Spacer E f f e c t s In order to examine the i n f l u e n c e of the carbohydrate polymer on the environment of the l a b e l , two types of spacer groups, each of whose length could be v a r i e d , were introduced between the l a b e l and the m a t r i x , r e s u l t i n g i n the p r o g r e s s i v e extension of the l a b e l i n t o the solvent pockets present i n the g e l . This was achieved by r e a c t i n g Sepharose 4B w i t h CNBr as before followed by b i f u n c t i o n a l compounds of general form ^N^A/vCC^H. The termin-a l c a r b o x y l moiety could then be reacted w i t h amine-containing l a b e l s i n the presence of a w a t e r - s o l u b l e carbodiimide, i n t h i s case N-ethyl-N'-dimethyl-aminopropyl carbodiimide h y d r o c h l o r i d e (EDC) (20) to from a s t a b l e amide l i n k a g e : The l a t t e r r e a c t i o n i s discussed i n more d e t a i l i n Chapter VI. The two s e r i e s of b i f u n c t i o n a l molecules u t i l i s e d i n the present study were the to-aminocarboxylic acids (H 2N(CH 2)pC0 2H, 1 <_ p <_ 10) and the o l i g o - g l y c i n e s (H 2N(CH 2C0NH)^CH 2C0 2H, 0 £ q < 5). These two groups of compounds represent more and l e s s hydrophobic spacers commonly used i n a f f i n i t y chromatography experiments. Series of spectra from Sepharose 4B conjugated through w-amino c a r b o x y l i c a c i d s (p = 1,3,5 and 10) to l a b e l s (2) and (9) NH - C - N H NH 0 - C - N H OH C O N H S L 160 and through o l i g o g l y c i n e s (q = 0,1,2,3,4, and 5) to l a b e l (2) are shown i n Figures IV-13, IV-14 and IV-15 r e s p e c t i v e l y together w i t h examples of t h e i r counterparts i n the absence of any spacer arm. In Figure IV-16 mean c o r r e l a t i o n times (T, c a l c u l a t e d by the r a t i o - o f - h e i g h t s method) f o r each of the spacer conjugates are p l o t t e d against the number of bonds i n the spacer moiety (n). A l l of these d e r i v a t i v e s together w i t h t h e i r n-values are represented i n Table IV-2. Each d e r i v a t i v e was made on at l e a s t three separate occasions, so that no poi n t on Figure IV-16 i s averaged over l e s s than three values of T. E r r o r bars represent ± (standard d e v i a t i o n ) . As the tendency i s f o r s c a t t e r to increase w i t h decreasing n, more data were obtained at low n: f o r n £ 11 no point represents l e s s than f i v e separate x— values. The sp e c t r a used i n the Figures IV-13, 14 and 15 were r e p r e s e n t a t i v e (centre-range) i n each i n s t a n c e and x was always -9 < % 2 x 10 s. No evidence f o r d i p o l a r i n t e r a c t i o n s between n i t r o x i d e s was found i n any spacer-conjugated product even at 77 K, probably because of the reduced l o a d i n g r e s u l t i n g from a two-step i n s t e a d of a one-step r e a c t i o n . Although s i g n a l - t o - n o i s e of i n d i v i d u a l t r a c e s i n the f i g u r e s i s not s i g n i f i c a n t as the sample qu a n t i t y was not c o n t r o l l e d , i t was found that i n two-step r e a c t i o n s i n v o l v i n g cyanogen bromide a c t i v a t i o n followed by carbodiimide-mediated c o u p l i n g , y i e l d tended to decrease w i t h decreasing n, though t h i s was not thought s u f f i c i e n t l y important to q u a n t i t a t e . I t was c l e a r , however, that the increase i n s c a t t e r of x as n decreased was not a r e s u l t of the lower i n t e n s i t y of the spectrum l e a d i n g to greater e r r o r i n measuring s p e c t r a l parameters; i n the f i r s t p l a c e , at the s i g n a l - t o - n o i s e r a t i o s used (> 50:1) t h i s e r r o r i s dominated by the e r r o r i n measuring l i n e -width (rather than h e i g h t ) , which increases as n increases and the l i n e s become narrower, but never exceeds . 10%. Secondly, no decrease i n 161 Figure IV-13: Esr spectra of (a)(34); (b)(35); ( c ) ( 3 7 ) ; (d)(39) i n water (see Table IV-2 f o r s t r u c t u r e s ) . 162 Figure IV-14: Esr spectra of (a)(44); (b)(45); ( c ) ( 4 6 ) ; (d)(47); (e)(48) i n water (see Table IV-2 f o r s t r u c t u r e s ) . 163 Figure IV-15: Esr spectra of (a)(34); (b)(35); ( c ) ( 3 8 ) ; (d)(40); (e)(41); (f) (42) ; (g) (43) i n water (see Table IV-2 f o r s t r u c t u r e s ) . 164 20r 15 ID IV s o K)h 10 15 20 n Figure IV-16: Dependence of mean c o r r e l a t i o n time (T) f o r r o t a t i o n a l r e o r i e n t a t i o n on number of bonds (n) connecting the n i t r o x i d e - c o n t a i n i n g r i n g to the matrix monosaccharide, D : p y r r o l i d i n e - 1 - o x y l d e r i v a t i v e s , alkylamine spacers; p i p e r i d i n e - 1 - o x y l d e r i v a t i v e s , alkylamine spacers; p i p e r i d i n e - 1 - o x y l d e r i v a t i v e s , o l i g o g l y c i n e spacers. Note that p o i n t s at n=7 belong to both spacer s e r i e s . Table IV-2: D e r i v a t i v e s of Sepharose 4B (|r=OH) 2,2,6,6-tetramethylpiperidine-l-oxy (SL-6) d e r i v a t i v e 2,2,5,5-tetramethylpyrrolidine-l-oxy (SL-5) d e r i v a t i v e n NH |—0—C—NH—SL-6 NH (34) |—0—C—NH—SL-5 (44) 4 ffi—CH2—CO—NH—SL-6 (35) Mi—CH 2—CO—NH—SL-5 (45) 7 NH NH i i [—0—C-^H(CH2)3CO—NH— SL-6 (37) ' |—0—C—NHCCHg^CO—NH—SL-5 (46) 9 NH — 0 — C — ( N H CH 2C0) 2—NH—SL-6 (38) 10 NH t i NH i i — 0 — C — N H ( C H 2 ) 5 C 0 — N H — S L - 6 (39) |—0—G—NH(CH 2) 5C0—NH—SL-5 (47) 11 | — 0 — C — ( N H CH 2CO) 3—NH—SL-6 (40) 13 NH | — 0 — C — (NH CH^O)^—NH—SL-6 (41) |—0—G—NH(CHg) 1 0CO—NH—SL-5 (48) 16 NH I I — 0 — C — ( N H C H 2 C 0 ) 5 — N H — SL-6 (42) 19 NH II — 0 — C — ( N H CH 2C0) 6—NH—SL-6 (43) 22 166 standard d e v i a t i o n r e s u l t e d , f o r example, from d i s c a r d i n g three out of seven x-values (those obtained from lower s i g n a l - t o - n o i s e spectra) i n the case of (35) and i n the case of (46) two out of s i x data p o i n t s were d i s -carded r e s u l t i n g i n a decrease of 30% i n the standard d e v i a t i o n , the remaining value s t i l l being l a r g e r than that f o r any sample w i t h a greater n-value. Decreasing y i e l d and i n c r e a s i n g s c a t t e r as n decreases must the r e f o r e be considered s e p a r a t e l y . The most p l a u s i b l e e x p l a n a t i o n f o r the former observation i s that the y i e l d of the second s t e p — t h e carbo-d i i m i d e c o u p l i n g — d e c r e a s e s , suggesting the p o s s i b i l i t y that s t e r i c h i n -drance a f f e c t s t e r m i n a l c a r b o x y l groups i n short spacers ( g l y c i n e and y-aminobutyric acid) which are sm a l l e r than, or roughly the same s i z e as, the s p i n l a b e l s , not an unreasonable p o s s i b i l i t y c o n s i d e r i n g the observed hindrance of d i r e c t l y attached l a b e l s (IVC). On the other hand, i t does seem unreasonable to propose that the decrease of y i e l d r e s u l t s from d i f f e r -ences i n the y i e l d of the f i r s t c o upling step s i n c e smaller spacer molecules might be expected, i f anything, to gain access to more, not fewer, a c t i v a t e d s i t e s . However, i n view of the p r o b a b i l i t y of sm a l l d i f f e r e n c e s i n pK amongst the spacer u n i t s employed, and v a r i a t i o n s i n coupling y i e l d s w i t h 28 d i f f e r e n t l i g a n d s reported by other workers , t h i s remains a p o s s i b i l i t y . The increased s c a t t e r d i d not r e s u l t from the presence of f r e e n i t r o x i d e i n any instance—inr_.cases of doubt (e.g. , immediately a f t e r s y n t h e s i s ) , samples were washed w i t h b u f f e r s o l u t i o n s u n t i l a constant lineshape was obtained, that i s , u n t i l x's were the same w i t h i n measurement e r r o r s . Instead i t may be suggested that s u b t l e changes in.-reaction c o n d i t i o n s a f f e c t s p e c t r a l lineshapes at small n as was seen f o r d i r e c t l y attached l a b e l s ( I V C ( i ) ) , perhaps as a r e s u l t of d i f f e r e n t product;:ratios. This would not be expected to be observed at high n-values s i n c e here the motion of the l a b e l 167 i s l e s s s e n s i t i v e to the nature of i t s attachment s i t e , having c o n s i d e r a b l y more degrees of freedom as a r e s u l t of motion about the bonds i n the spacer u n i t . Values of x f o r s p e c t r a of d i r e c t l y attached l a b e l s have been omitted from Figure IV-16, s i n c e a s i n g l e i s o t r o p i c c o r r e l a t i o n time i s not adequate to describe motion i n these cases. However, 'best f i t ' approximate simu-l a t i o n s give values of 'x' at n=4 which are i n a l l cases greater than those -9 f o r systems w i t h n > 4 (thus, f o r example x = 2.6 x 10 s i s quoted i n IVC(vi) f o r (34), which i s - q u i t e c o n s i s t e n t w i t h the f i g u r e s presented i n Figure IV-16). Two general conclusions are j u s t i f i e d by the r e s u l t s presented i n F i g u r e IV-16: that the m o b i l i t y of l a b e l s increases w i t h spacer l e n g t h , approaching a l i m i t i n g v a l u e ; and that t h i s behaviour i s not i n i t s general features very d i f f e r e n t f o r e i t h e r of the two n i t r o x i d e species or e i t h e r of the two types of spacer. Thus, hindrance by the m a t r i x i s not, at l e a s t at n _> 7, very d i f f e r e n t i n the p i p e r i d i n e and the p y r r o l i d i n e s e r i e s . There i s no evidence that m o b i l i t y i s a f f e c t e d by an i n c r e a s e i n r i g i d i t y due to p a r t i a l -rr-character i n the C-N bonds of peptide l i n k a g e s i n o l i g o - g l y c i n e s , nor i s there evidence that hydrogen bonding occurs between spacers and the m a t r i x , nor f o r any f o l d i n g back of the spacers upon themselves. The increase of m o b i l i t y w i t h spacer l e n g t h , approaching a x value of about 4 x 10 s, has i n t e r e s t i n g i m p l i c a t i o n s f o r a f f i n i t y chromatography. We may d i v i d e the curve i n t o two p o r t i o n s , one at n < 10 where the depen-dence of x on n i s steep, and one at n >^  10 where t h i s dependence i s not marked.. (These p o i n t s , t r e a t e d to a l i n e a r r e g r e s s i o n , give a gradient of -0.15 and a c o r r e l a t i o n c o e f f i c i e n t of 0.6. A l l the e r r o r bars save one (n=13) l i e on the l i n e . ) In a f f i n i t y chromatography experiments i t i s 168 necessary to maximise the a v a i l a b i l i t y of the immobilised species w h i l e minimising the non-r.specif i c b i n d i n g c a p a c i t y of the spacer u n i t . The present experiments show that an optimal balance between these two f a c t o r s may be obtained at n=10 or 11, that i s by using e-aminocaproic a c i d or d i g l y c i n e as spacers. (In a d d i t i o n , n i s s u f f i c i e n t l y l a r g e that y i e l d s would not s u f f e r , assuming the same chemistry as used here.) As w i l l be shown, t h i s c o n c l u s i o n i s completely c o n s i s t e n t w i t h e m p i r i c a l observations of spacer e f f i c a c y i n a f f i n i t y separations and r e l a t e d processes, ( i i ) Solvent Cycles A d d i t i o n of l a r g e proportions of m i s c i b l e organic s o l v e n t s to aqueous s o l u t i o n s of polysaccharides g e n e r a l l y causes p r e c i p i t a t i o n to occur, even i f the solvent i s as p o l a r as ethanol. In the case of Sepharose g e l beads, what may be considered a true p r e c i p i t a t e or s o l i d i s prevented from forming by e x i s t i n g i n t e r m o l e c u l a r f o r c e s . Although loose aggregates sometimes form, and beads become more l i a b l e to adhere to g l a s s , i n d i v i d u a l beads r e t a i n t h e i r i n t e g r i t y and the main e f f e c t i s one of shrinkage, reducing pore s i z e and the molecular e x c l u s i o n l i m i t . The i n i t i a l m o t i v a t i o n i n the present work f o r i n t r o d u c i n g non-aqueous solv e n t s i n t o s p i n l a b e l l e d beads was to attempt to show at the molecular l e v e l how immobilised s p e c i e s , p a r t i c u l a r l y those prone i n s o l u t i o n to solvent denaturation, might behave when immobilised at a h y d r o p h i l i c surface and exposed to the same s o l v e n t s . Unfortunately i n t e r p r e t a t i o n of the r e s u l t s i s l i m i t e d by the l a c k of independent data from other techniques on how, at the molecular l e v e l , p o lysaccharide gels respond to non-aqueous s o l v e n t . This i s the f i r s t such study, and cannot pretend to more than s c r a t c h the surface. The present s e c t i o n deals w i t h r e s u l t s ; some of the i m p l i c a t i o n s f o r immobil-i s e d enzyme technology are discussed i n I V D ( i i i ) . Two d e r i v a t i v e s of Sepharose 4B :—with n i t r o x i d e ' d i r e c t l y attached' ( i . e . , n=4) v i a cyanogen bromide a c t i v a t i o n (34) and w i t h an e-amino cap r o i c a c i d spacer group between the l a b e l and the m a t r i x (39)—were passed through a solvent c y c l e s i m i l a r to the one described i n s e c t i o n I V C ( i v ) : water(78.5), ethanol(24.3), l,4-dioxan(2.2), n-heptane(^l.9), dioxan, ethanol, water, the esr spectrum being recorded at each stage. D i e l e c t r i c constants, measured at 298 K, have been given i n parentheses. Each solvent i s m i s c i b l e w i t h i t s successor and predecessor. A l a r g e excess of each solvent was washed through the beads p r i o r to making any s p e c t r a l measurement, and each solvent t r a n s i t i o n proceeded through two intermediate mixtures of 70:30 and 30:70 composition, though i t was found that the r e s u l t s were not s e n s i t i v e to the exact nature of the t r a n s i t i o n . These are d i s p l a y e d i n Figures IV-17 and IV-18. In d i s c u s s i n g them, i t should be r e a l i s e d that at l e a s t three d i f f e r e n t parameters may i n f l u e n c e the lineshape: m o b i l i t y , s p i n - s p i n i n t e r a c t i o n s , and the p o l a r i t y of the environment. We are f a m i l i a r w i t h the f i r s t two, and w i t h the increase i n s e c u l a r d i p o l a r i n t e r a c t i o n which occurs i n the d i r e c t l y - l a b e l l e d beads (34) as a r e s u l t of solvent-mediated shrinkage ( I V C ( i v ) ) . The assumption has been i m p l i c i t i n preceding s e c t i o n s that a p o l y s a c c h a r i d e environment w i l l not e x h i b i t a p o l a r i t y markedly d i f f e r e n t from that i n aqueous s o l u t i o n , and c l e a r l y when l a b e l s are present i n d i f f e r e n t s i t e s which are l i k e l y to experience d i f f e r e n t extents of s o l v a t i o n (judging at l e a s t by t h e i r d i f f e r i n g a c c e s s i b i l i t i e s to paramagnetic ions i n s o l u t i o n ) , t h i s f a c t o r i s d i f f i c u l t to q u a n t i t a t e , as are the r e l a t i v e c o n t r i b u t i o n s of the other two i n the absence of exact s i m u l a t i o n s . However, i n the l i g h t of previous d i s c u s s i o n , the main features of the s p e c t r a shown i n Figure IV-17 are r e a d i l y i d e n t i f i a b l e : as the solvent environment becomes Figure IV-17: Esr spectra of (34) as a f u n c t i o n of solvent environment. (a)water; (b)70:30 water:ethanol; (c)30:70 water:ethanol; (d)ethanol; ( e ) l , 4 - d ioxan; (f)n-heptane; (g)dioxan; (h)ethanol; ( i ) w a t e r . 171 l e s s and l e s s p o l a r and the beads s h r i n k , m o b i l i t y decreases and l i n e -broadening from s p i n - s p i n i n t e r a c t i o n s increases ( i t i s i n t e r e s t i n g to note the s i m i l a r i t y between the spectrum i n heptane, Figures IV-17f, and 2+ the r e s i d u a l spectrum a f t e r a d d i t i o n of 2M Ni(H20)g , Figure I V - 9 f ) . I t would seem that bead c o n t r a c t i o n must be accompanied by a 'trapping' or 'folding-back' of the l a b e l i n t o the matrix. Further and more convincing evidence to t h i s e f f e c t i s shown i n Figure IV-18. . In the presence of a 10-atom spacer arm, two superimposed sp e c t r a are unmistakably present i n dioxan and ethanol, one whose features resemble those of the aqueous spec-trum and another, broader spectrum which c l e a r l y represents a l e s s mobile p o p u l a t i o n of s p i n s . In n-heptane only one, h i g h l y immobile component i s r e s o l v e d ; no r a p i d l y r e o r i e n t i n g l a b e l s are present. Probably owing to the lower l o a d i n g , no d i p o l a r i n t e r a c t i o n s are present i n t h i s system, even at 77 K a f t e r s olvent shrinkage; thus only environmental p o l a r i t y and m o b i l i t y need be considered. I t i s a l s o evident that a h y s t e r e s i s occurs. This i s a l s o j u s t d i s c e r n i b l e i n Figure IV-17; here i t i s much more pro-nounced. On the 'return l e g ' from heptane to water, d i f f e r e n c e s i n r e l a t i v e heights of the two components compared to those on the 'outward l e g ' are e s p e c i a l l y easy to d i s t i n g u i s h at low f i e l d i n ethanol and dioxan. The same lineshape as was observed at the beginning, c o n s i s t i n g only of a rapid-tumbling component, obtains i n water. The change i n r e l a t i v e amounts of these two components, together w i t h t h e i r widely divergent c o r r e l a t i o n times, mean that the o v e r a l l l i n e -shape cannot be confused w i t h those due to s i n g l e homogeneous populations of a n i s t r o p i c a l l y tumbling l a b e l s . However, i t a l s o means that x c a l c u -l a t i o n s using the r e l a t i v e heights method (Chapter I C ) , and measurements of i s o t r o p i c h y p e r f i n e s p l i t t i n g constants (arj), both f o r the more r a p i d l y Figure IV-18: Esr spectra of (39) as a f u n c t i o n of solvent environment. (a)water; (b)ethanol; (c)1,4-dioxan; (d)n-heptane; (e)dioxan; ( f ) e t h a n o l ; (g)water. Note the two overlapping components i n (b), ( c ) , ( e ) , and (f) . 173 tumbling component, are not accurate, even w i t h i n the experimental e r r o r given i n Table IV-3, where some data c u l l e d from Figure IV-18 are reproduced. Table IV-3: Data from Figure IV-18 spectrum solvent h ( + l ) / h ( - l ) a 0(G) (measured) a o ( G ) 29 published f o r (2) 2T(G) I a water 2.2 ± 0.1 16.8 ± 0.3 16.99 ± 0.01 -b ethanol 2.9 15.3 16.08 -c dioxan 3.2 15.3 15.54 59.5 ± 0.5 d heptane - - 15.2 64.7 e dioxan 7.0 15.4 15.54 63.6 f ethanol 5.1 15.9 16.08 62.5 8 water 2.1 16.8 16.99 -However, the s p l i t t i n g s between outer (para l l e l ) components (2T) are as quoted, s i n c e c o n t r i b u t i o n s from the sharper components of the s p e c t r a are s m a l l at these frequencies. Values of arj f o r IV-18 b,c,e and f are w i t h i n experimental e r r o r of each other, and although lower than those f o r s p e c t r a a and g, as expected, a f i r m c o n c l u s i o n cannot be drawn. V a r i a t i o n s i n 2T could be a s c r i b e d e i t h e r to the p o l a r i t y dependence of the h y p e r f i n e tensor component T , or to d i f f e r e n c e s i n m o b i l i t y , but the nature of the Li Li changes c l e a r l y suggests that the l a t t e r may be dominant, as a h y s t e r e s i s i s again observed, as i t i s i n the r a t i o h ( + l ) / h ( - l ) , which i s used- to charac-t e r i s e changes i n the c o r r e l a t i o n time of the sharper component (h's are peak-to-peak h e i g h t s ; again the h ( + l ) / h ( - l ) parameter i s only u s e f u l as a very approximate i n d i c a t i o n of tumbling r a t e . hg cannot be used because of the build-up of i n t e n s i t y from both components i n the c e n t r e ) . Thus three i n d i c a t i o n s of h y s t e r e s i s are claimed. 174 In order to suggest an i n t e r p r e t a t i o n of these r e s u l t s i t i s necessary to consider on the molecular s c a l e the l i k e l y r e s u l t of exposure of the g e l beads to non-aqueous s o l v e n t . The nature of the f u n c t i o n a l groups i n the polysaccharide i s such that even i n water under c e r t a i n c o n d i t i o n s i n t e r -chain hydrogen bonding i s p r e f e r r e d to f u l l s o l v a t i o n . I t i s l i k e l y that as the solvent environment becomes l e s s p o l a r , s o l v a t i o n w i l l become a l e s s and l e s s favourable a l t e r n a t i v e to s e l f - a g g r e g a t i o n . Increases i n i n t e r -chain a s s o c i a t i o n lead to reductions i n volume of the beads. I t i s not l i k e l y that s u b s t a n t i a l r e o r g a n i s a t i o n of hydrogen bonds w i t h i n an already extant j u n c t i o n zone w i l l be able to occur during shrinkage, which i s r a p i d compared with,.say, the syneresis described i n Chapter I I I . Chemical c r o s s -l i n k s introduced during the cyanogen bromide a c t i v a t i o n w i l l i n any case i n h i b i t such r e o r g a n i s a t i o n . Thus we may imagine, on the b a s i s of the pre-v a i l i n g model f o r the g e l , two ways i n which shrinkage may occur: by the extension of j u n c t i o n zones, and by the a s s o c i a t i o n of j u n c t i o n zones i n t o l a r g e r bundles. Extension of j u n c t i o n zones i s envisaged as a c o n t i n u a t i o n of hydrogen bonding between already a s s o c i a t e d chains to encompass other, p r e v i o u s l y s i n g l e - s t r a n d e d regions. This would be d i f f i c u l t i f , as has been proposed, the a s s o c i a t i o n i s h e l i c a l . Furthermore, i t i s l e s s l i k e l y to occur i n places which are l a b e l l e d and hence perhaps s t e r i c a l l y i n h i b i t e d from a s s o c i a t i n g . On the other hand i t has already been suggested that a s s o c i a t i o n of h e l i c e s i n t o m u l t i p l e j u n c t i o n zones occurs normally during g e l a t i o n and s y n e r e s i s , though no model f o r t h i s a s s o c i a t i o n e x i s t s . In i n t e r p r e t i n g the s p i n l a b e l l i n g r e s u l t s one encounters the t r a d i -t i o n a l o b s t a c l e of whether the r e p o r t e r species a c t u a l l y r e f l e c t s the n a t i v e system or only i t s own ' a r t i f i c i a l ' environment. Here an a d d i t i o n a l d i f f i c u l t y occurs, that of d e c i d i n g whether the idea of i n t e r r e l a t i n g the 175 behaviour of two modified systems i s indeed v a l i d ; that i s to say, the question i s r a i s e d as to whether the spacer-conjugated system (39) responds d i f f e r e n t l y to solvent c y c l e s as a r e s u l t simply of the s p e c i a l p r o p e r t i e s of the spacer, which one expects to be more r e a d i l y s o l v a t e d i n non-aqueous s o l u t i o n s , and which presents a r a t h e r more s e r i o u s s t e r i c impedi-ment to chain a s s o c i a t i o n , d e s p i t e the low l e v e l of l o a d i n g . The f o l l o w -i n g d i s c u s s i o n assumes that the two systems behave i n broadly s i m i l a r f a s h i o n . Considering f i r s t the d i r e c t l y - l a b e l l e d m a t e r i a l (34), which has a spacer arm that i s q u i t e h y d r o p h i l i c , i t i s l i k e l y that replacement of water by l e s s p o l a r s o l v e n t s encourages the formation of spacer-matrix hydrogen bonds, s e v e r a l of X v r t i i c h can be shown to be f e a s i b l e - by the use of molecular models. Thus s o l v a t i o n and m o b i l i t y decreases; at the same time, entrapment of l a b e l s i n the polymer during a s s o c i a t i v e processes i s l i k e l y to occur. No obvious d i v i s i o n of n i t r o x i d e s i n t o components i n d i f f e r e n t motional regimes i s detected, u n l i k e the system (39) i n which the l a b e l i s separated from the matrix by a spacer arm, where the evidence suggests that l a b e l s are entrapped, c r e a t i n g a second, l e s s mobile popula-t i o n as aggregation occurs. Then i t f o l l o w s that the p r o p o r t i o n of the more mobile, s o l v a t e d component decreases w i t h solvent p o l a r i t y u n t i l , at the point of maximal shrinkage and aggregation, no l a b e l s are s o l v a t e d . Having formed a t i g h t aggregation i n h e p t a n e — a s t a t e of metastable e q u i l i b r i u m — r e s o l v a t i o n cannot occur to the same extent as on the 'outward l e g ' u n t i l the environment i s again aqueous.* Thus each intermediate stage *Indeed, i f beads c o n t a i n i n g heptane are resuspended i n water, hydra-t i o n does not occur w i t h i n the pores: the beads simply f l o a t , i n t h e i r shrunken s t a t e , upon the surface! 176 on the 'outward l e g ' a l s o represents a metastable e q u i l i b r i u m . * Such a h y s t e r e s i s no doubt a l s o occurs i n the d i r e c t l y - l a b e l l e d system, but i t would seem that the short and more h y d r o p h i l i c spacer arm, i n t h i s case, d i c t a t e s a l e s s e r s e n s i t i v i t y to environment. P a r a l l e l s f o r t h i s type of behaviour can be found i n the l i t e r a t u r e on s p i n - l a b e l l e d c o l l a g e n and s y n t h e t i c polymers. In the case of c o l l a -gen, s o l v a t e d (sharp) and non-solvated (broad) s p e c t r a are superimposed, 30 t h e i r r e l a t i v e i n t e n s i t y determined by the amount of water i n the f i l m , . The same has been found f o r polymers both i n l i n e a r and c r o s s - l i n k e d form w i t h a range of s o l v e n t s : at high polymer d i l u t i o n a l l l a b e l s are s o l v a t e d and tumble r a p i d l y ; at low solvent c o n c e n t r a t i o n n i t r o x i d e motion i s slow; 31 and i n the intermediate range both types of motion are superimposed M o b i l i t y has been shown to be r e l a t e d to the degree of solvent-induced s w e l l i n g of l a b e l l e d polystyrene beads, and the l i k e l i h o o d of hydrogen bonding of the n i t r o x i d e oxygen to hydroxyl s u b s t i t u e n t s at the surface i n 32 the absence of hydrogen bonding solvent shown . In the spacer-conjugated, l a b e l l e d Sepharose 4B system (39), the m o b i l i t y of the 'solvated' component does, apparently, depend on s o l v e n t . In l a b e l l e d ion-exchange r e s i n s 33 m o b i l i t y has been shown to be dependent on extent of c r o s s - l i n k i n g The main question r a i s e d by t h i s s e r i e s of experiments i s as f o l l o w s : i f s p i n l a b e l s are trapped w i t h i n a matrix of a s s o c i a t e d polysaccharide chains when solvent-induced shrinkage occurs, what would be the e f f e c t on an immobilised macromolecule? I t might be expected that entrapment might reduce the a c c e s s i b i l i t y of the macromolecule to small molecules i n s o l u t i o n *Although another view of t h i s phenomenon might have i t that r e s i d u a l t r a c e s of t i g h t l y - b o u n d water account f o r the d i f f e r e n c e between the two halves of the c y c l e . 177 i n a non-aqueous phase, w h i l e at the same time s t a b i l i s i n g i t r e l a t i v e to i t s s o l u t i o n form, i f only by v i r t u e of the r e l a t i v e s i m i l a r i t y of a p o l y -saccharide environment to an aqueous one, but probably a l s o by mechanical means. This p o i n t i s discussed i n the next s e c t i o n , ( i i i ) L i t e r a t u r e Survey In Table IV-4 a summary has been attempted of some of the most r e l e -vant published reports of spacer arm v a r i a t i o n i n a f f i n i t y s e p a r a t i o n s . Only papers i n which the e f f e c t s of systematic changes i n the spacer u n i t have been s t u d i e d are i n c l u d e d . No c l a i m to comprehensiveness i s made, the aim being simply to show to what extent i t i s v a l i d to equate ' m o b i l i t y ' w i t h ' a c c e s s i b i l i t y ' . In p a r t i c u l a r , a l a r g e l i t e r a t u r e on immobilised enzymes (which, i n t e r e s t i n g l y i n the l i g h t of the f o l l o w i n g d i s c u s s i o n , are u s u a l l y s t a b i l i s e d by i m m o b i l i s a t i o n ) i s omitted as i s an i n c r e a s i n g body of research i n t o hydrophobic chromatography. However, the impression created by the t a b l e that few s t u d i e s have concentrated on the i n f l u e n c e of the spacer i s c o r r e c t . Several p o i n t s should be made before proceeding to an e v a l u a t i o n of i t s content. The s p i n l a b e l l i n g experiments described here represent an attempt to f a c t o r out one p arameter—the a c c e s s i b i l i t y of the spacer t e r m i n u s — f r o m the l a r g e number which may c o n t r i b u t e to the success of a p a r t i c u l a r s e p a r a t i o n . The i n t e n t i o n i s to make a u s e f u l g e n e r a l i s a -t i o n as f a r as that i s p o s s i b l e i n view of the v a r i e t y of ligand-acceptor systems. No such g e n e r a l i s a t i o n can be made about the e f f e c t on l i g a n d b i n d i n g to an enzyme of the chemical and environmental p e r t u r b a t i o n i m p l i e d by spacer attachment and subsequent i m m o b i l i s a t i o n (though i n some cases b i n d i n g of ligand-spacer conjugates to enzyme i n s o l u t i o n has been com-34 35 pared ' ); nor can the present r e s u l t s throw l i g h t upon the p o s s i b i l i t y of n o n - s p e c i f i c i n t e r a c t i o n s , p o l a r or hydrophobic, between spacer u n i t s 178 and macromolecules i n s o l u t i o n , nor between l i g a n d s or s p e c i f i c spacers (other than those used here) and the matrix m a t e r i a l , although i t i s worth p o i n t i n g out that s e v e r a l separations i n i t i a l l y described as b i o s p e c i f i c have si n c e turned out "to have been based on the i n t e r a c t i o n of a macro-i i - ^ .36,37,38 molecule w i t h a spacer u n i t In Table iV- 4 , the q u a l i t a t i v e conclusions from a number of st u d i e s are l i s t e d together w i t h the matrix m a t e r i a l , immobilised and s o l u t i o n m o i e t i e s , and the length (n) and nature of the spacer. A q u a l i t a t i v e approach was necessary s i n c e assay methods and c r i t e r i a of success d i f f e r . A l l the examples c i t e d use (by chance) the CNBr a c t i v a t i o n procedure together w i t h spacers of the form RNH2, so that n, as before, i s c a l c u l a t e d by counting bonds from the d e r i v a t i s e d saccharide r i n g i n the support as f a r as the l i g a n d , assuming products to be of the form NH [-0-C-NHR Thus, f o r example, where the l i g a n d i s 1-amino glucose, the l a s t bond to be counted i s that to the g l y c o s i d i c n i t r o g e n : y spacer^ N H - ( ^ ) s i n c e the l a t t e r i s thought of as a par t of the s u b s t r a t e . In 8-linked adenosine 5'-monophosphate bonds are counted up to the r i n g carbon atom: NH 2 ' \ ^ — N H / \ T N N H 2 N ribose 5 - phosphate 179 In cases where aromatic groups and/or unsaturated u n i t s are incorporated i n t o the spacer n i s required to be more r i g o r o u s l y defined e i t h e r i n terms of a c c e s s i b i l i t y or extension from the matri x (n = no. of bonds) or i n terms of m o b i l i t y (n = no. of s i n g l e bonds). For present purposes the former d e f i n i t i o n i s adopted and i t i s assumed that a benzene r i n g i s equivalent to one a d d i t i o n a l bond. I t would be of great i n t e r e s t i n f u t u r e experiments to examine more c l o s e l y the r e l a t i o n s h i p between a c c e s s i b i l i t y and m o b i l i t y by comparing r e s u l t s using s p i n l a b e l s w i t h those on an a f f i n i t y system w i t h the same unsaturated spacer u n i t . Up to the present however few a f f i n i t y experiments have been performed using t h i s type of extension arm. Examination of Table IV-4 i n d i c a t e s that r e s u l t s from a number of d i f -f e r e n t l a b o r a t o r i e s v e r i f y the present p r o p o s i t i o n — t h a t p r o v i d i n g m o b i l i t y can be equated w i t h a c c e s s i b i l i t y , e f f i c i e n c y of se p a r a t i o n on Sepharose a f f i n i t y columns should approach a maximum at n = 10-11 (see Figure I V - 1 6 ) — i n very s a t i s f a c t o r y f a s h i o n . In a l l the cases considered, where i n t e r -a c t i o n decreased markedly ( r e l a t i v e to s o l u t i o n ) on i m m o b i l i s a t i o n of one p a r t i c i p a n t , t h i s was r e s t o r e d , at l e a s t i n p a r t , by i n t e r p o s i t i o n of a spacer between i t and the matrix. In some cases no more d e t a i l e d a n a l y s i s was attempted; where there was such a n a l y s i s , three d i s t i n c t types of behaviour emerged. In the m a j o r i t y of cases, b i n d i n g s t r e n g t h or separa-t i o n e f f i c i e n c y increased to a maximum 'plateau' l e v e l w i t h i n c r e a s i n g n. Generally spacer lengths have been increased not by one or two atoms, but by l a r g e r increments, so that accurate values of n corresponding to the onset of the p l a t e a u r e g i o n have not been obtained; nevertheless values of 10, 11 and 12 are by f a r the most common. In one or two s t u d i e s i t has • • • t. n .. ,36,-37,39 been found that b i n d i n g continues to increase w i t h n at values > 13 ; these have a l l been shown to be due to n o n - s p e c i f i c b i n d i n g i n v o l v i n g the 180 spacer u n i t . The t h i r d type of behaviour has been seen i n only one case; here b i n d i n g increased to a maximum at n = 13, decreasing at higher 40 values . This was not due to i n t e r a c t i o n w i t h the spacer and has not been s a t i s f a c t o r i l y e x p l a i n e d , though c o n t r o l experiments i n which assay of the b i n d i n g of ligand-spacer conjugates i n s o l u t i o n to the enzyme of i n t e r e s t i s compared w i t h the case where the conjugates are immobilised were not performed. Even t h i s type of c o n t r o l can be misleading s i n c e e n t r o p i c c o n t r i b u t i o n s may vary, a f a c t not g e n e r a l l y recognised. The evident u t i l i t y of the proposed rule-of-thumb prompts a f u r t h e r g e n e r a l i s a t i o n : i f the a f f i n i t y of a s o l u t i o n species f o r an immobilised l i g a n d continues to increase f o r n > 13, then i t i s l i k e l y that non-spec-i f i c e f f e c t s are coming i n t o p l a y . I t should be noted however that the present model does not take i n t o c o n s i d e r a t i o n the p o s s i b i l i t y of a deeply b u r i e d b i n d i n g s i t e i n the g l o b u l a r p r o t e i n , which might be expected to inc r e a s e the value of n at the onset of the plateau r e g i o n . The s p i n l a b e l l i n g data suggest simply that l i g a n d s escape the most marked i n f l u e n c e of the Sepharose 4B matrix at n - 12. Two apparent anomalies remain i n the m a t e r i a l presented i n Table 39 IV-4. In the f i r s t p l a c e , O'Carra et a l . achieved s i m i l a r r e s u l t s using immobilised oxamate b i n d i n g to l a c t a t e dehydrogenase w i t h n = 7 and 14 i n h y d r o p h i l i c spacers. I t should a l s o be pointed out that many separations have been s u c c e s s f u l l y performed on agarose matrices without the use of any spacer at a l l ( i . e . , at n = 4). This must r e f l e c t a r e a d i l y a c c e s s i b l e macromolecular b i n d i n g s i t e as w e l l as an a b i l i t y on the part of the species i n s o l u t i o n to c l o s e l y approach the 'surface' of the matrix as a r e s u l t of i t s p a r t i c u l a r s i z e , shape and h y d r o p h i l i c i t y . Such features are outside the scope of the present d i s c u s s i o n . The data obtained f o r d i r e c t l y attached l a b e l s (IVC), however, suggests;the l i k e l i h o o d that even i n the presence of a s o l u t i o n species i n which the above parameters are optimised not a l l of the s i t e s where a small l i g a n d may r e s i d e may be e q u a l l y acces-s i b l e . This may e x p l a i n r e s u l t s which suggest two d i s t i n c t r a t e constants 41 f o r enzyme bi n d i n g to immobilised l i g a n d , though i t has a l s o been suggested that b i n d i n g to the surface and the i n t e r i o r of the bead may occur at d i f f e r e n t r a t e s The second anomaly which r e q u i r e s d i s c u s s i o n a r i s e s from the r e s u l t s 43-46 of Lowe , who conducted experiments w i t h an enzyme, lipoamide dehydro-genase, immoblised on Sepharose v i a a s e r i e s of spacer u n i t s w i t h 12 <_ n 20, i n which the s t a b i l i t y of the enzyme to thermal and solvent (<. 80% aqueous dioxan)-mediated denaturation was i n v e s t i g a t e d . In the former case attachment to Sepharose s t a b i l i s e d the enzyme r e l a t i v e to i t s s o l u t i o n s t a t e f o r n <_ 19. In the l a t t e r , s t a b i l i s a t i o n r e l a t i v e to s o l u -t i o n was observed f o r a l l n, though as w i t h the thermally-mediated process, the enzyme became more s t a b l e w i t h i t s approach to the pol y s a c c h a r i d e . C l e a r l y i n these experiments i t i s no longer a c c e s s i b i l i t y that i s at stake (unless t h i s i s taken to mean solvent a c c e s s i b i l i t y to the Sepharose s u r f a c e , which i s not the present sense of the term) and perhaps the i n s i g h t gained from the esr experiment i s l e s s d i r e c t l y r e l e v a n t . Spin l a b e l l i n g cannot r e v e a l the s u b t l e balance of thermodynamic q u a n t i t i e s which determine thermal s t a b i l i t y ; no doubt, however, the entropy change accompanying denaturation w i l l d i f f e r f o r the immobilised and s o l u t i o n s i t u a t i o n s , 46 47 e s p e c i a l l y i n cases where m u l t i s i t e attachment occurs ' A c c e s s i b i l i t y , however, was i n v e s t i g a t e d by studying the dependencies on spacer length of r a t e s of r e a c t i o n of 2-mercaptoethanol w i t h the d i s u l -phide bond between the enzyme and the spacer terminus ( r e l e a s i n g the former 182 i n t o s o l u t i o n ) , and of a p r o t e o l y t i c enzyme, thermolysin, w i t h the immobil-i s e d species. The r a t e of the f i r s t process increased f o r a l l n _^  19, w h i l e i n the second case the r a t e i n c r e a s e d , reaching a constant value at n ^ 15. The s t a b i l i s a t i o n to denaturation by 30% aqueous dioxan seen even at l a r g e values of n, may r e a d i l y be explained i n terms of the entrapment phenomenon described i n the previous s e c t i o n . One may imagine two conse-quences of i n t r o d u c t i o n of the organic s o l v e n t : the development of m a t r i x -enzyme contacts s i n c e the polysaccharide i s made more congenial to the p o l a r groups on the enzyme e x t e r i o r r e l a t i v e to s o l v e n t ; and the shrinkage of the p o l y s a c c h a r i d e , tending, as w i t h the l a b e l , to occlude the enzyme, reducing solvent access. Both processes should, according to r e s u l t s presented i n I V D ( i i ) , be more e f f e c t i v e at low values of n. The model i s a l s o c o n s i s -tent w i t h the observation that s t a b i l i s a t i o n r e l a t i v e to s o l u t i o n does not 48 occur when a polystyrene matrix i s used . A l a y e r of t i g h t l y - b o u n d water at the surface of a h y d r o p h i l i c m a t r i x may be another important c o n t r i b u t i n g f a c t o r . This d i s c u s s i o n , by means of a p l a u s i b l e working hypothesis, helps to l a y the groundwork f o r the improved design of experiments i n which the a b i l i t y of support matrices to s t a b i l i s e enzymes i n harsh c o n d i t i o n s i s u t i l i z e d . F i n a l l y , on the b a s i s of the s p i n l a b e l l i n g r e s u l t s one or two p o i n t s 35 a r i s i n g from previous s t u d i e s can be c l a r i f i e d . Holroyde et a l . , comparing i n t e r a c t i o n s of glucokinase w i t h Sepharose-immobilised glucose found that a p o l y a l k y l spacer arm (n = 10) enhanced b i n d i n g r e l a t i v e to d i -and t r i - g l y c i n e spacers and suggested that t h i s could be accounted f o r by hydrophobic i n t e r a c t i o n between.the p r o t e i n and the spacer or by l i g a n d -m a t r i x or spacer-matrix i n t e r a c t i o n i n the g l y c i n e s e r i e s l e a d i n g to reduced 183 a c c e s s i b i l i t y . The f i r s t i n t e r p r e t a t i o n now seems l i k e l y . G u i l f o r d i n a 49 review a r t i c l e r a i s e d the p o s s i b i l i t y that some data obtained using systems i n which l i g a n d s have been immobilised v i a p o l y a l k y l spacer u n i t s can be i n t e r p r e t e d i n terms of folded or c o i l e d conformations at high n, forming a hydrophobic 'pocket' i n a c c e s s i b l e to aqueous s o l u t i o n . This i s not c o n s i s -34 tent w i t h present observations. In a r a t h e r thorough study, Lowe tethered AMP (adenosine monophosphate) to Sepharose by means of four spacers of i n c r e a s i n g hydrophobicity but constant n, and found that dehydrogenase bindi n g increased w i t h spacer hydrophobicity d e s p i t e the s i m i l a r a f f i n i t i e s of d i f f e r e n t spacer-AMP conjugates i n s o l u t i o n f o r the p r o t e i n s . T h i s , i t was suggested, i m p l i e d that.Migand a c c e s s i b i l i t y was lower i n the case where a more h y d r o p h i l i c spacer was used. As the spacers as w e l l as the l i g a n d were d i f f e r e n t from those used i n the present study, no f i n a l e v a l u a t i o n of Lowe's c o n c l u s i o n can be made. However, the present s t u d i e s c l e a r l y sug-gest that other f a c t o r s (such as entropy of binding) may be i n o p e r a t i o n . Table IV-4: L i t e r a t u r e r e p orts on spacer arms* immobilised .  s o l u t i o n r e f e r r l i g a n d species n spacer matrix comments ence b i o t i n a v i d i n HN(CH 2) 3CH(NH 2) CO-c e l l u l o s e weak bindin g ; K<j i n s o l u - 50 t i o n = 1 0 " 1 5 Sepharose strong binding 51 4B pyridoxamine 5 '-phosphate t y r o s i n e amino-t r a n s f e r a s e Sepharose 4B 12 HN(CH 2) 2NHC0(CH 2) 2C0NH 17 HN(CH 2) 3NH(CH 2) 3NHC0(CH 2) 2 HNCO poor separation good separation equally good s e p a r a t i o n 52,53 pyridoxamine 5 '-phosphate apo-glutamic 4 o x a l a c e t i c transaminase 11 17 Sepharose 4B HN(CH 2) 6NH HN(CH 2) 1 2NH no separation good separation equal l y good s e p a r a t i o n 54 AMP dehydro-genases 7-15 HN(CHo) NH m m = 2 - 10 11(a) HN(CH 2) 6NH 11(b) HN(CH 2)C0NH(CH 2) 3NH 11(c) HN(CH 2) 2C0NH(CH 2) 2NH 11(d) HNCH2C0NHCH2CHCH2NH OH Sepharose 4B Sepharose 4B binding increases to a maximum value which remains constant f o r n _> 12 binding increased w i t h hydrophobicity of spacer; b and d have s i m i l a r enzyme-binding p r o p e r t i e s i n s o l u t i o n , but b > d a f t e r i m m o b i l i s a t i o n 55 34 Table IV-4 continued immobilised l i g a n d s o l u t i o n species spacer matrix comments r e f e r -ence 5-13 HN(CH 2) NH m m = 0 - 8 no c o r r e l a t i o n observed between enzyme b i n d i n g i n s o l u t i o n and a f t e r i m m o b i l i s a t i o n , but binding increases w i t h n to an approximately con-stant value at n = 10 deoxythymidine staphylo-3'-p-amino- c o c c a l phenyl nuclease phosphate 5 '-phosphate 10 '16' 17 HN(CH 2) 2NHCOCH 2 HNCH2C0NHCH2C0 rN-^-CH; 2 CHNH HO C0 2H HN(CH 2) 3NH(CH 2) 3NH C0(CH 2) 2C0 Sepharose e f f e c t i v e but low b i n d -4B i n g capacity higher capacity no higher than at n = 10 no higher than at n = 10 56 ATP(adenosine triphosphate) N A D + ( n i c o t i n -amide adenine d i n u c l e o t i d e ) dehydro-genases and kinases 8-16 NH(CH 2) CO ^ m m = 2-10 Sepharose maximum binding achieved 4B at n = 12-13; decreased bindi n g n > 13 40 Table IV-4 continued immobilised l i g a n d s o l u t i o n species spacer matrix comments r e f e r -ence NAD dehydro-genases '16' NH(CH2)6NHC0 N=N ' 20' OH NHCH2CHCH2NHC0CH2 C0HNCH2CHCH2NH N=N 13' NHCH2CHCH2NHCO N rN Sepharose n o n - s p e c i f i c adsorption 4B at low i o n i c strength no n o n - s p e c i f i c e f f e c t s i n more h y d r o p h i l i c d e r i v a t i v e s 39 deamino- a l c o h o l 14 NH(CH2)6NHC0S Sepharose b i n d i n g , no n o n - s p e c i f i c 39 NAD+ dehydro-genase 11 OH HNCH2CHCH2NHCOCH2S 4B e f f e c t s Table IV-4 continued immobilised l i g a n d s o l u t i o n species n spacer m a t r i x comments r e f e r -ence oxamate l a c t a t e dehydro-genase 10 HN(CH 2) 6 14 OH HNCH2CHCH2NHCO I CHoCHCH2NHCH2 6H 7 OH HNCH2CHCH2 Sepharose n o n - s p e c i f i c b i n d i n g 4B e f f e c t s no n o n - s p e c i f i c e f f e c t s i n more h y d r o p h i l i c d e r i v a t i v e s 39 lipoamide dehydro-genase 12-21 NHAc HN(CH 2) NHCOCH m , SSCH 2CH 2 m = 2-10 Sepharose s t a b i l i t y of enzyme to 43,45 4B denaturation by heat or 30% aqueous dioxan as a fu n c t i o n of n; i n former case, more s t a b l e than i n s o l u t i o n f o r n < 18, i n l a t t e r case plateau at n > 15, but never l e s s s t a b l e than i n s o l u t i o n ; s t a b i l i t y decreases w i t h i n c r e a s i n g n f o r s m a l l n. A c c e s s i b i l i t y to t h i o l reagent i n s o l u t i o n i n -creases w i t h n f o r a l l n; a c c e s s i b i l i t y to a proteo-l y t i c enzyme increases w i t h n f o r n < 15, then remains constant Table IV-4 continued immobilised s o l u t i o n r e f e r -l i g a n d species n spacer matrix comments ence 9-phenyl guanine 4 Sepharose no binding 57 guanidine deaminase 4B 7 NHCH2CH20 binding D—tryptophan a-chymo- 4 Sepharose incomplete separation 58 methyl ester t r y p s i n 4B 11 HN(CH 2) 6C0 complete separation glucose glucokinase 4 Sepharose no i n t e r a c t i o n 35 4B 10 HN(CH 2) 5NH binds a l l spacer-10 (HNCH 2C0) 2 weak a f f i n i t y l i g a n d con-13 (HNCH 2C0) 3 weak a f f i n i t y jugates s t r o n g l y i n h i b i t enzyme i n s o l u t i o n p-amino g-galacto- 4 Sepharose no bindi n g 36 thiophenol s i d a s e 4B weak binding g-galactoside 10 HN(CH2)2NHCOCH2NH C0 2H very strong b i n d i n g '15' (HNCH2CO)2NHCH2 r e s u l t s reevaluated 37 N-N and found to be the same wi t h no l i g a n d Table IV-4 continued immobilised s o l u t i o n l i g a n d species n spacer matrix . comments ence xanthine 7 HN(CH 2) 3CH 3 Sepharose adsorption, hydrophobic 59 (hydrophobic oxidase 4B reversed i n and p o l a r chromato- l a c t a t e 1M NaCl i n t e r a c t i o n graphy) dehydro- 11 HN(CH 2)7CH 3 i r r e v e r s i b l e w i t h genase adsorption spacers deoxyribo-nuclease I a l l c a l i n e phosphatase urease *Spacer arms are not given where d i r e c t c oupling of an amine-containing l i g a n d to the cyanogen bromide-activated m a t r i x has occurred; the product i s assumed to be NH | — 0 — C — N H — l i g a n d , f o r which n = 4. 190 References 1. 'Sephadex Gel F i l t r a t i o n i n Theory and P r a c t i c e , ' Pharmacia Fine Chemicals P u b l i c a t i o n , 1975. 2. 'Pharmacia Agarose f o r E l e c t r o p h o r e s i s and Immunological Techniques,' i b i d . , 1977. 3. 'Sephadex Ion Exchangers. A Guide to Ion Exchange Chromatography,' i b i d . , 1974. 4. ' A f f i n i t y Chromatography P r i n c i p l e s and and Methods,' i b i d . , 1974. 5. K. B r o c k l e h u r s t , J . Ca r l s s o n , and M. P. J . K i e r s t a n , Biochem. J . , 133, 537-540 (1975). 6. B. Lonnerdal, J . Car l s s o n , and J . Porath, F.E.B.S. L e t t . , 7_5, 89-92 (1977). 7. Z. E r - e l , Y. Zaidenzaig, and S. S h a l t i e l , Biochem. Biophys. Res. Commn., 49, 383-390 (1972). 8. J . Porath, J . Chromatog., 159, 13-24 (1978). 9. 0. Zabo r s k i , 'Immobilized Enzymes,' CRC Press, Cleveland, Ohio, 1973. 10. J . Porath, and R. Axen, Meths. Enzymol., 44, 19-45 (1976). 11. A. P o l l a c k , R. L. Baughn, 0. A d a l s t e i n s s o n , and G. M. Whitesides, J . Am. Chem. S o c , 100, 302-304 (1978). 12. 'Bibliography on C o n t r o l l e d Pore Glass Chromatography and Related Subjects,' E l e c t r o - N u c l e o n i c s Inc., F a i r f i e l d , New Je r s e y , 1977. 13. 'Glycophase G,' P i e r c e Handbook and General Catalogue, P i e r c e Chemical Co., Rockford, I l l i n o i s , 1978. 14. J . F. Kennedy, S. A. Barker, and C. A. White, Carbohydr. Res., 54, 1-12 (1977). 15. J . Lonngren, I. J . G o l d s t e i n , and R. Bywater, F.E.B.S. L e t t . , 68, 31-34 (1976). 16. L. Rexova-Benkova, 0. Markovic, and M. J . F o g l i e t t i , C o l l e c t . Czech Chem. Commn., 42, 1736-1741 (1977). 17. 'Beaded Sepharose 2B-4B-6B,' Pharmacia Fine Chemicals P u b l i c a t i o n , 1973. 18. R. Axen, J . Porath, and S. Ernback, Nature, 214, 1302-1304 (1967). 191 19. 'Sepharose CL f o r Gel F i l t r a t i o n and A f f i n i t y Chromatography,' Pharm-a c i a Fine Chemicals P u b l i c a t i o n , 1976. 20. J . Porath, T. Laas, and J . C. Janson, J . Chromatog., 103, 49-62 (1975) . 21. T. Laas, P r o t i d e s B i o l . F l u i d s , 2_3, 495-503 (1975). 22. P. Cuatrecasas i n 'Biochemical Aspects of Reactions on S o l i d Supports,' Ed. G. R. Stark, 79-109, Academic, New York, 1971. 23. J . D. A p l i n , and L. D. H a l l , J . Am. Chem. S o c , 99, 4162-4163 (1977). 24. J . Porath, R. Axen, and S. Ernback, Nature, 215, 1491-1492 (1967). 25. M. Duckworth, and W. Yaphe, Carbohydr. Res., 16, 189-197 (1971). o 26. L. Ahgren, L. K&gedal, and S. Akerstrdm, Acta Chem. Scand., 26, 285-288 (1972). 27. S. C. March, I. P a r i k h , and P. Cuatrecasas, Anal. Biochem., 60, 149-152 (1974). 28. R. L. Schnaar, T. F. Sparks, and S. Roseman, i b i d . , 7_9, 513-525 (1977). 29. B. R. Knauer, and J . J . Napier, J . Am. Chem. S o c , 98^ , 4395-4400 (1976) . 30. T. Nagamura, and A. E. Woodward, Biopolymers, 16, 907-919 (1977). 31. Z. V e k s l i , and W. G. M i l l e r , Macromolecules, 10, 686-692 ' (1977). 32. S. L. Regen, J . Am. Chem. S o c , 97, 3108-3112 (1975). 33. D. B. Chesnut, and J . F. Hower, J . Phys. Chem., 7_5, 907-912 (1971). 34. C. R. Lowe, Eur. J . Biochem., 7_3, 265-274 (1977). 35. M. J . Holroyde, J . M. E. Chesher, I . P. Trayer, and D. G. Walker, Biochem. J . , 153, 351-361 (1976). 36. E. Steers, P. Cuatrecasas, and H. B. P o l l a r d , J . B i o l . Chem., 246, 196-200 (1971). 37. P. O'Carra, S. Barry, and T. G r i f f i n , Biochem. Soc. Trans., 1, 289-290 (1973). 38. J . E. Rood, and R. G. W i l k i n s o n , Biochim. Biophys. Acta, 334, 168-178 (1974). 39. P. O'Carra, S. Barry, and T. G r i f f i n , F.E.B.S. L e t t . , 43_, 169-175 (1974). 192 40. C. R. Lowe, M. J. Harvey, D. B. Craven, and P. 0. G. Dean, Biochem. J . , 133, 499-506 (1973). 41. C. R. Lowe, Biochem. Soc. Trans., j>» 251-253 (1977). 42. G. S. David, T. H. Chino, and R. A. R e i s f e l d , F.E.B.S. L e t t . , 43^ , 264-266 (1974). 43. C. R. Lowe, Biochem. Soc. Trans, _5, 253-255 (1977). 44. C. R. Lowe, Eur. J . Biochem., 76, 391-399 (1977). 45. C. R. Lowe, i b i d . , 76^ , 401-409 (1977). 46. C. R. Lowe, i b i d . , 76, 411-417 (1977). 47. J . Lasch, and R. Koelsch, i b i d . , 82, 181-186 (1978). 48. G. Manecke, J . Pure Appl. Chem., 4_, 507-514 (1962). 49. H. G u i l f o r d , Chem. Soc. Rev., 2, 249-270 (1973). 50. D. B. McCormick, Anal. Biochem., 13, 194-198 (1965). 51. P. Cuatrecasas, and M. Wilchek, Biochem. Biophys. Res. Commn., 3^3 235-239 (1968). 52. J. V. M i l l e r , P. Cuatrecasas, and E. B. Thompson, Proc. Nat. Acad. S c i . U.S. A., 68, 1014-1018 (1971). 53. J . V. M i l l e r , P. Cuatrecasas, and E. B. Thompson, Biochim. Biophys. Acta, 276, 407-415 (1972). 54. R. C o l l i e r , and G. Kohlaw, Anal. Biochem., 42_, 48-53 (1971). 55. M. C. Hipwell, M. J . Harvey, and P. D. G. Dean, F.E.B.S. L e t t . , 42, 355-359 (1974). 56. P. Cuatrecasas, J. B i o l . Chem., 2_45, 3059-3065 (1970). 57. B. R. Baker, and H. U. Siebeneick, J . Med. Chem., 14, 799-801 (1974) 58. P. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. S c i . U.S.A., 61, 636-643 (1968). 59. B. H. J . Hofstee, and N. F. O t i l l i o , Biochem. Biophys. Res. Commn., 53, 1137-1144 (1973). CHAPTER V CELLULOSE VA: Occurrence, Importance and Stru c t u r e C e l l u l o s e i s the most abundant n a t u r a l product on earth and i t would 1 2 not be wholly s u r p r i s i n g to f i n d i t elsewhere . I t has been estimated that 10 1 1 tonnes are produced per annum i n p l a n t s , and that humans create 10 9 tonnes of c e l l u l o s i c waste i n the same pe r i o d . I t i s common to f o l l o w such f i g u r e s by estimates of the length of time r e q u i r e d to coat the surface of the eart h w i t h the m a t e r i a l i n question; i n the case of c e l l u l o s e t h i s i s h ardly necessary, s i n c e much of the land surface of the eart h i s already thus coated. P l a n t c e l l u l o s e s are g e n e r a l l y found i n complex a s s o c i a t i o n w i t h other substances, forming the backbone m a t e r i a l of the c e l l w a l l ; the f i b r e c e l l s of wood, f o r example, have t h i c k w a l l s c o n s i s t i n g of c e l l u l o s e 2 3 and h e m i c e l l u l o s e s embedded i n a l i g n i n 'glue' ' (Figure V-3). The importance of the cotton p l a n t stems, i n c o n t r a s t , from i t s producing n e a r l y pure c e l l u l o s e i n . f i b r o u s form; wood c e l l u l o s e s must undergo a 4 vigorous and expensive e x t r a c t i o n p r i o r to use . However, the multi t u d e of a p p l i c a t i o n s devised by human ingenuity f o r c e l l u l o s e j u s t i f y t h i s con-s i d e r a b l e e f f o r t . The l i s t i s almost endless. F i b r e s , t e x t i l e s , p r o p e l -l a n t s , flame r e t a r d a n t s , f i l m s , c o a t i n g s , cosmetics, e x p l o s i v e s , foods, pharmaceuticals, adsorbents, a r t i f i c i a l tobacco, and s o i l s t a b i l i s e r s are a few of the areas i n which c e l l u l o s e and i t s d e r i v a t i v e s ( n i t r a t e s , xanthates, a c e t a t e s , ethers and others) are i n common use. I t i s i n e v i t a b l e that 193 194 c e l l u l o s e w i l l assume i n c r e a s i n g importance i n the f u t u r e both as a cheap raw m a t e r i a l and as a renewable energy resource. C e l l u l o s e (49) i s a l i n e a r poly-D-glucose w i t h 31-4 g l y c o s i d i c l i n k -ages (Figure V-la) u s u a l l y found at l e a s t i n higher p l a n t s w i t h a degree of p o l y m e r i s a t i o n i n excess of 1500 5. The conformation of the chain i s that of a f l a t r ibbon (Figure V-lb) which i t i s q u i t e n a t u r a l to a s s o c i a t e w i t h the e f f i c i e n t packing of chains and many i n t e r - c h a i n hydrogen bonds that c h a r a c t e r i s e c e l l u l o s e and account f o r i t s mechanical s t r e n g t h , s t a b i l i t y and i n s o l u b i l i t y i n conventional solvent systems^. In f a c t the ribbons are packed i n t o sheets which are then placed on top of each other i n such a way 9 as to stagger residues (Figure V - l c and d ) . I t i s not yet c l e a r whether 8 9 the chains are packed i n p a r a l l e l or a n t i p a r a l l e l ' . S u r p r i s i n g l y , the sheets appear to be a s s o c i a t e d only through Van der Waal's i n t e r a c t i o n s . 8 9 This s t r u c t u r e , the r e s u l t of X-ray f i b r e d i f f r a c t i o n s t u d i e s ' , i s thought to c h a r a c t e r i s e c e l l u l o s e I , the n a t u r a l l y - o c c u r r i n g form. Two other c r y s t a l l i n e forms have been c h a r a c t e r i s e d i n the same f a s h i o n : c e l l u l o s e 11"^, which r e s u l t s from m e r c e r i s a t i o n (treatment w i t h strong base), and which has a n t i p a r a l l e l chains packed on top of each other; and c e l l u l o s e III"'""'", prepared by treatment w i t h l i q u i d ammonia at 223 K, which has a s t r u c t u r e not u n l i k e that of c e l l u l o s e I. I t i s the n a t i v e m a t e r i a l which w i l l concern us here. From the p o i n t of view of the c r y s t a l l o g r a p h e r i t i s unfortunate that the s t r u c t u r e of c e l l u l o s e i s not wholly ordered, but i s thought to c o n s i s t of domains, some of which ( m i c r o c r y s t a l l i t e s ) c o n t a i n chains packed i n the manner described and give r i s e to the X-ray f i b r e p a t t e r n , w h i l s t others (amorphous regions) have a more di s o r g a n i s e d makeup which i s not amenable to d e t a i l e d s c a t t e r i n g a n a l y s i s . One way i n which t h i s can be demonstrated Figure V - l : S t r u c t u r e of c e l l u l o s e : (a)sequence; (b)chains resemble a f l a t ribbon; (c),(d)proposed chain packing (from reference 9 ). 196 i s by deuterium-hydrogen exchange, q u a n t i t a t e d using i n f r a - r e d s p e c t r o -4 12 scopy ' ; hydroxyl f u n c t i o n a l i t i e s i n the i n t e r i o r of c r y s t a l l i n e regions do not exchange even on prolonged exposure to deuterium oxide, w h i l s t those i n amorphous domains, wherein solvent i s capable of p e n e t r a t i n g , do so more o r — l e s s r a p i d l y . Approximate estimates of the c r y s t a l l i n i t y of a c e l l u l o s e sample may a l s o be obtained by p a r t i a l a c i d h y d r o l y s i s , the c r y s t a l l i t e s 3 being again i n a c c e s s i b l e , and by X-ray methods . Several methods have been proposed f o r the three-dimensional s t r u c t u r e of c e l l u l o s e which t r y to i n c o r p o r a t e both m i c r o c r y s t a l l i t e s and amorphous 13 regions. Two types of s t r u c t u r e hold currency ; both of these are about twenty years o l d , and i t t e s t i f i e s to the complexity of the (nominally simple) c e l l u l o s e molecule that f u r t h e r e l u c i d a t i o n has not occurred s i n c e , de s p i t e the i n c r e a s i n g i n s i g h t i n t o the makeup of the c r y s t a l l i n e regions provided by X-ray s c a t t e r i n g . The two are i l l u s t r a t e d i n Figure V-2. In 14 V-2a, s t r u c t u r e s resembling which have been proposed by Preston and Cronshaw , 15 16 Frey-Wyssling , and others , the m i c r o f i b r i l i s shown i n c r o s s - s e c t i o n . Here an ordered c r y s t a l l i n e 'core' i s surrounded by a domain whose order decreases w i t h distance from the core and i n which, i n wood, c e l l u l o s e might be found i n a s s o c i a t i o n w i t h other c o n s t i t u e n t molecules. In b, the c r y s -t a l l i n e and amorphous regions i n s t e a d a l t e r n a t e along the m i c r o f i b r i l l a r a x i s , c r y s t a l l i n e regions being perhaps 60 nm long by 6 nm across. This 17 18 type of model was o r i g i n a l l y proposed by Hess et a l . and Ranby 19 Figure V-2c shows a model which amalgamates features of a and b . Here three types of surface are proposed. Those of type a are h i g h l y ordered and not a c c e s s i b l e except to s t r o n g l y denaturing reagents. Type b surfaces are l e s s ordered, though s t i l l p h y s i c a l l y c l o s e to c r y s t a l l i n e r e g i o n s , and l i k e l y to c o n t a i n a considerable number of i n t e r - c h a i n hydrogen bonds. Figure V-2: Proposed s t r u c t u r e s f o r the c e l l u l o s e m i c r o f i b r i l , i n c o r p o r a t i n g regions of order and d i s o r d e r , ( a ) c r o s s - s e c t i o n of f i b r i l ; order increases towards a c e n t r a l core (from reference 5) ( b ) a l t e r n a t i o n of amorphous regions and m i c r o c r y s t a l l i t e s w i t h i n a s i n g l e m i c r o f i b r i l ; (c)model i n c o r p o r a t i n g elements of a and b. Three d i f f e r e n t types of surface are envisaged, l a b e l l e d A, B, and C (from reference 43) . 198 a i 50X ~ I ' 1 IO0A — / / /-> *~7 <-, * '—, <-7 .<-> '—> '-7 f '—1 f — 3 f i— j i Y 1/ * I—, '—i L—> £ — 3 i t i I t—f I— <—, <—}' <—> '—1 f r — 3 f 1 • . t—, l—i '—> •—I '—> '-> E—1 '—> 7 <~7 •-> 4 i— t i / b , £/*'// ******** f\ ' " ******** I'''''' *,,* ****\\'" " * * * * * * * * i ^ * ' ' ' * -'*'**** A—*•*•*—<rr /-l-s-i-r I-*******^>*/*** *.* ****** l i t * * * * * * * * * * * I w* * * * * ****** * * *\t * * * * / * * * * * * * * }} y * * ******** X* f * * ' • " ' " ' 4 * * * * * * - | - / - * - V - V - l - | - / - / ^ / — / — I-*-/-199 Type c surfaces are much l e s s ordered owing to t h e i r l o c a t i o n on segments of the f i b r i l which are d i s t o r t e d by t i l t and t w i s t . I f u n c e r t a i n t i e s s t i l l pervade i n c e l l u l o s e s t r u c t u r e determination, very much more remains to be learned about wood. E l e c t r o n microscopy has 3 enabled some of the l a r g e r - s c a l e features to be observed ; Figure V-3 2 3 shows the p o s t u l a t e d makeup of the c e l l w a l l of a wood f i b r e c e l l ' . Layers of m i c r o f i b r i l s are deposited at v a r i o u s angles to the d i r e c t i o n of growth, a l l o w i n g f o r subsequent c e l l expansion w i t h s t r e t c h i n g , of the w a l l s and alignment of the m i c r o f i b r i l s . The b i o s y n t h e t i c mechanisms in v o l v e d 5 20 21 i n the b u i l d i n g process are complex and p o o r l y understood ' ' From previous chapters i t w i l l be c l e a r to the reader )that s p i n l a b e l l i n g can make no pretence to compete w i t h the X-ray method f o r d e t e r -mination of three-dimensional s t r u c t u r e i n ordered systems. One h i g h l y important aspect of c e l l u l o s e which i s not capable of examination by s c a t -t e r i n g methods, and to which s p i n l a b e l l i n g i s w e l l s u i t e d , however, i s the nature of i t s surface a r c h i t e c t u r e , and i t s i n t e r a c t i o n s w i t h species i n a s o l u t i o n phase w i t h which i t makes contact. This has wide r a m i f i c a -t i o n s i n the t e x t i l e and p r i n t i n g i n d u s t r i e s where dyeing processes occur at the surface and f i b r e f i n i s h i s u s u a l l y a f u n c t i o n of surface treatment; i n d e r i v a t i s a t i o n i n c l u d i n g g r a f t i n g , and chemical breakdown, where the a c c e s s i b i l i t y of the polymer molecules to reagent i s a l s o c r u c i a l ; i n the study of the s t r u c t u r e of wood, where c e l l u l o s e surfaces i n t e r a c t w i t h other polymeric s p e c i e s ; i n bonding, coating and otherwise f i n i s h i n g x y l o i d products; i n the use of c e l l u l o s e d e r i v a t i v e s as chromatographic matrices i n i o n exchange, removal of heavy i o n p o l l u t a n t s from water, a f f i n i t y chromatography and r e l a t e d processes; and, perhaps most important i n the f u t u r e , i n understanding the mechanism of enzymatic breakdown of c e l l u l o s e . 200 Figure V-3: Chemical and u l t r a s t r u c t u r a l makeup of wood Note that only some of the co n s t i t u e n t s are marked. In p a r t i c u l a r , l i g n i n (black i n C) binds and s t a b i l i s e s the whole (from reference 2) 201 Obviously,'in the f i n a l a n a l y s i s , d e s c r i p t i o n s of the nature of t h i s ( i n t e r f a c e must be dependent on the o v e r a l l three-dimensional s t r u c t u r e . So f a r , however, although a considerable body of published research e x i s t s , i t has not been p o s s i b l e to i n t e g r a t e the f i n d i n g s of the surface e x p e r i -ments w i t h those of experiments aimed more at the o v e r a l l s t r u c t u r e , i n t o a s i n g l e u n i f i e d p i c t u r e . Because many of the s u r f a c e - o r i e n t e d experiments have, l i k e the aforementioned deuterium-hydrogen exchange i n v o l v e d the idea of 'penetration' i n t o the c e l l u l o s e m a t r i x , i t has been convenient to d i s -cuss the r e s u l t s i n terms of 'pores' or ' i n t e r n a l s u r f a c e s ' . This a p p l i e s i n part to the present work. I t i s reasonable to assume that such surfaces e x i s t i n the amorphous and not i n the c r y s t a l l i n e p o r t i o n s of the micro-f i b r i l , that i s , as a r e s u l t of d i s c o n t i n u i t i e s of molecular packing; beyond t h i s l i t t l e can at present be s a i d . I t i s necessary s i n c e the experiments to be d e s c r i b e d , though r e f l e c t i n g a very d i f f e r e n t approach to the problem from that p r e v i o u s l y used, nonetheless i n v o l v e 'penetration' of s o l u t i o n components more or l e s s deeply i n t o the c e l l u l o s e m a t r i x , to b r i e f l y review the l i t e r a t u r e on the c e l l u l o s e surface. A f i r s t - r a t e 22 review a r t i c l e has r e c e n t l y appeared I t i s c l e a r from measurements of adsorption of n i t r o g e n , vapours of water and v a r i o u s organic solvents that surface area of c e l l u l o s e depends on the ' s w e l l i n g ' or hydrogen-bonding c a p a c i t y of the s o l v e n t . Surface 23 2A 25 areas measured by n i t r o g e n ' and water adsorption may d i f f e r by as 26 much as two powers of ten, though t h i s change i s l a r g e l y r e v e r s i b l e W h i l s t as many as 50% of hydroxyl groups may be a c c e s s i b l e to water, only about 2.5% are a c c e s s i b l e to Diphenyl Fast Red 5BL-, a dye of molecular 27 weight 676, i n d i l u t e aqueous s o l u t i o n . Dry f i b r e s probably do not c o n t a i n a s u b s t a n t i a l pore s t r u c t u r e . Taken to a l i m i t , i n s t r o n g l y 202 hydrogen bond-disrupting s o l v e n t s , changes i n c r y s t a l l i n i t y and, e v e n t u a l l y , -, • 7 d i s s o l u t i o n may occur . Thus i t would appear that pores of v a r i o u s s i z e are present. Several workers have followed t h i s discovery up by measuring the amount of water a v a i l a b l e to a m u l t i f o r m i t y of s o l u t e molecules i n the presence of , , , - . i . - , , , 28,29 , 28 29 c e l l u l o s e : polyethylene g l y c o l s , dextrans , polyammes , uronic 29 30 31 32 a c i d s , i m i d a z o l i d i n o n e s , as w e l l as v a r i o u s small ions ' ' , i n f e r r i n g that i n a c c e s s i b l e water i s present i n pores of dimension smaller than the s o l u t e . Experiments w i t h charged species are g e n e r a l l y more d i f f i c u l t 'to i n t e r p r e t than those w i t h n e u t r a l s o l u t e s due to concentration-dependent e f f e c t s and a d s o r p t i o n ; i n p a r t i c u l a r c a t i o n s appear to be more s t r o n g l y 29 adsorbed than anions , perhaps because of the net negative charge of the 33 surface . When s a c c h a r i d i c or polyhydroxylated s o l u t e s are used, i t would appear that ever greater f r a c t i o n s of the water present at the surface become a v a i l a b l e as' solvent as the s i z e of the s o l u t e decreases. Another phenomenon must here be d i s t i n g u i s h e d , however: that of 'bound water'. 34 Evidence f o r t h i s has come from a number of measurements: d e n s i t y , 35 36 d i f f e r e n t i a l heats of s o r p t i o n , the phenomenon of non-freezing water , . 37 . . 38,39,40 thermal expansion , and, most r e c e n t l y , nmr r e l a x a t i o n . There i s l i t t l e q u a n t i t a t i v e c o r r e l a t i o n between the d i f f e r e n t measurements, but taken together they are s u f f i c i e n t to j u s t i f y the idea that d i f f e r e n c e s i n 41 behaviour e x i s t between surface and bulk solvent . In p a r t i c u l a r , i t i s l i k e l y that the e f f e c t i v e pore s i z e a v a i l a b l e to a s o l u t e w i l l depend upon, i n a d d i t i o n to molecular s i z e , i t s a b i l i t y to compete w i t h c e l l u l o s e f o r water molecules, that i s , upon i t s hydrogen-bonding a b i l i t y . Some s t u d i e s , p a r t i c u l a r l y those us i n g n m r ^ ' ^ , have i n d i c a t e d the presence of two c l a s s e s of bound water at the surface. This i s c o n s i s t e n t w i t h recent work using a chemical l a b e l l i n g technique which i s perhaps 42 43 c l o s e s t to the approach used h e r e i n . Roberts and Rowland ' reacted c e l l u l o s e w i t h N , N - d i e t h y l a z i r i d i n i u m c h l o r i d e i n the presence of strong base, i n t r o d u c i n g d i e t h y l a m i n o e t h y l groups at low degrees of s u b s t i t u t i o n (< 0.1). No change i n s t r u c t u r e accompanied t h i s m o d i f i c a t i o n , The l a b e l l i n g was coupled w i t h p a r t i a l a c i d h y d r o l y s i s f o r d i f f e r e n t periods of time followed by X-ray a n a l y s i s of the m a t e r i a l which remained undis-s o l v e d , and elemental a n a l y s i s f o r n i t r o g e n . Two types of a c c e s s i b l e surface were observed, e x i s t i n g i n p u r i f i e d n a t i v e cotton i n the r a t i o 0.36:0.64. The l e s s abundant of these had more hydroxyl groups a v a i l a b l e f o r s u b s t i t u t i o n and contained residues more r e a d i l y h y d r o l y s a b l e by a c i d . This was i d e n t i f i e d w i t h amorphous regions i n which few i n t e r - c h a i n hydrogen bonds e x i s t . The more abundant surface had fewer a v a i l a b l e h y d r o x y l s , presumably as a r e s u l t of i n t e r - c h a i n hydrogen bonds, and was l e s r e a d i l y hydrolysed and more i n t i m a t e l y connected w i t h regions of order. The r e s u l t s were i n t e r p r e t e d i n terms of the model shown i n Figure V-2c, the former surfaces being those of type c and the l a t t e r those of type b. I f d i f f e r e n t strengths of water b i n d i n g to c e l l u l o s e occur, i t i s l i k e l y that type c surfaces may provide the strongest b i n d i n g s i t e s owing to the greater number of a v a i l a b l e hydroxyl groups and the p o s s i b l e r e l e a s e of s t r a i n occasioned by h y d r a t i o n of the dry f i b r e w i t h i n such re g i o n s . One previous p u b l i c a t i o n notes the non-covalent i n t e r a c t i o n of n i t r o x i d e r a d i c a l s w i t h the surface of a cotton c e l l u l o s e , w i t h subsequent 44 observation of two types of r e o r i e n t a t i o n . No i n t e r p r e t a t i o n was attempted. VB: Cyanogen Bromide-Mediated L a b e l l i n g of C e l l u l o s e and Wood ( i ) Unperturbed Spectra Three d i f f e r e n t c e l l u l o s e - c o n t a i n i n g samples were l a b e l l e d using the r e a c t i o n described i n Chapter IV: NH OH (DCNBr Uo-C-NHSL OH (ii)H 2NSL(2) UOH (49),(49a) (51),(51a) or (50) or (52) The m a t e r i a l s so t r e a t e d are henceforward r e f e r r e d to as being ' d i r e c t l y l a b e l l e d ' . One was a p u r i f i e d chromatographic c e l l u l o s e powder (49), the second was the same m a t e r i a l p r e t r e a t e d w i t h 10% sodium hydroxide f o r 3 hours at room temperature to s w e l l (49a), and the t h i r d was a 50y v e r t i c a l microtome s e c t i o n taken from the annual growth r i n g of a Douglas f i r t r e e (50). The untreated c e l l u l o s e powder was a l s o l a b e l l e d v i a a spacer arm as before: l-OH (i) CNBr U-O-C L-OH (ii) H 2 N ( C H 2 ) 5 C 0 2 H r |—OH NH C-NH(CH2)5C02H (49) EDC HjNSLte) N H 0-C-NH(CH 2) 5 CONHSL OH (53) F i n a l l y , two samples of a water-soluble c e l l u l o s i c gum, O-carboxymethyl c e l l u l o s e , were l a b e l l e d . These are not p e r t i n e n t to the present d i s c u s -s i o n and are included i n the appendix to the t h e s i s . Figure V-4 shows the room temperature esr spectra i n the presence of Figure V--4: Esr spectra of c e l l u l o s e s s p i n l a b e l l e d using the CNBr method. (a)(51) i n the presence of some unattached l a b e l ( 2 ) (sharp resonances); (b) (51) ; ( c ) ( 5 3 ) ; (d)(51a) i n the presence of some unattached l a b e l ; (e)(52); ( f ) a b s o r p t i o n mode spectrum of (51). Note the poorer r e s o l u t i o n i n the wings. The s c a l e marker i s again 30 G. 207 water of the four w a t e r - i n s o l u b l e d e r i v a t i v e s , together w i t h an example of a r e a c t i o n mixture (Figure V-4a, that f o r the d i r e c t l y - l a b e l l e d n a t i v e c e l -l u l o s e powder) c o n t a i n i n g a p r o p o r t i o n of ' f r e e ' , unattached n i t r o x i d e . This can r e a d i l y be d i s t i n g u i s h e d on the ba s i s of l i n e w i d t h from the bound l a b e l , and washing procedures were r e a d i l y designed to remove unbound l a b e l . When the c e l l u l o s e was swollen w i t h NaOH p r i o r to r e a c t i o n , however, i t was found that a p r o p o r t i o n of unbound n i t r o x i d e remained adsorbed to the surface even a f t e r extensive washing w i t h 2.0M sodium c h l o r i d e , 8.0M urea and other denaturing s o l u t i o n s (Figure V-4d). In view of t h i s o b s e r v a t i o n , the untreated powder was chosen as the most convenient m a t e r i a l w i t h which to pursue f u r t h e r experiments. I t i s nevertheless evident beneath the 'adsorbed' s i g n a l i n Figure V-4d that the bound n i t r o x i d e i s s e n s i t i v e to changes at the surface e f f e c t e d by the strong base, the und e r l y i n g s i g n a l being n o t i c e a b l y broader than that of the unswollen m a t e r i a l (Figure V-4b). I t i s reasonable to be o p t i m i s t i c , t h e r e f o r e , about f u t u r e s t u d i e s of surface changes caused by s w e l l i n g reagents using bound n i t r o x i d e s ; i t i s a l s o c l e a r that a d s o r p t i o n of s p i n l a b e l s may provide another i n t e r e s t i n g way of probing the surface. The broad 'bound' s i g n a l i n d i r e c t l y - l a b e l l e d untreated c e l l u l o s e powder (51) was found upon i n t e g r a t i o n to co n t a i n an average of one n i t r o x i d e f o r each ^ 160 glucose r e s i d u e s . Adjustment f o r i n a c c e s s i b l e r e s i d u e s , of course, causes t h i s r a t i o to increase (see s e c t i o n VD). The absor p t i o n mode spectrum i s a l s o shown i n Figure V-4f; i t i s i n t e r e s t i n g to note the l o s s of r e s o l u t i o n i n the outer wings compared w i t h the d e r i v a t i v e mode (b). The 'bound' spectrum i s not u n l i k e that of l a b e l l e d Sepharose 4B, though the increased i n t e n s i t y of the outer ( p a r a l l e l ) components suggests a somewhat reduced m o b i l i t y , or greater hindrance at the sur f a c e ; x(average) i s probably 208 -8 -9 between 10 and 10 s. The complexity of the spectrum again r a i s e s the p o s s i b i l i t y t hat a n i s o t r o p y of r e o r i e n t a t i o n , e l e c t r o n - e l e c t r o n i n t e r a c -t i o n s , or heterogeneities'amongst the p o p u l a t i o n of l a b e l s are present. Small d i f f e r e n c e s i n lineshape (though not as n o t i c e a b l e as i n Sepharose) can a l s o be detected i n d i f f e r e n t l a b e l l e d powders from the same o r i g i n a l batch, which i s again most e a s i l y explained on the b a s i s of v a r i a t i o n s i n l o a d i n g g i v i n g r i s e to changes i n extents of unaveraged d i p o l a r broadening. The e f f e c t of i n t e r p o s i n g a spacer arm between the l a b e l and the m a t r i x i s , as expected, to decrease T' ( t y p i c a l l y to 7.0 x 10 s) such that i t f a l l s n i c e l y i n t o the range observed i n Chapter IV f o r the analogous d e r i v a t i v e of Sepharose 4B (39). Thus although the nature of hindrance to r e o r i e n t a t i o n may be d i f f e r e n t i n the two cases, i t would seem that i t s d i s t a n c e dependence i s roughly the same, which tends to argue, i n agreement w i t h r e s u l t s c i t e d i n l a t e r s e c t i o n s , against long and narrowly s t r u c t u r e d pores at the surface. Y i e l d i s again reduced i n t h i s two-step r e a c t i o n r e l a t i v e to the d i r e c t l a b e l l i n g process. Figure V-5 shows the e f f e c t of a solvent c y c l e (of the type described i n the preceding chapter) on the d i r e c t l y l a b e l l e d c e l l u l o s e (51). Here the bulk volume change i s very small i n comparison w i t h Sepharose (as a r e s u l t of the denser packing of the c e l l u l o s e chains) but e q u a l l y substan-t i a l changes i n n i t r o x i d e m o b i l i t y occur, l e a d i n g to s p e c u l a t i o n that a 'trapping' phenomenon may a l s o occur here, i n which surface pores c o n t r a c t , maximising glucose-glucose hydrogen bonds as hydrogen bonding to solvent i s decreased. Under these c o n d i t i o n s the n i t r o x i d e f u n c t i o n a l i t y may f o l d 42 back i n such a way as to hydrogen bond to a surface hydroxyl ; t h i s l a t t e r p o i n t i s of i n t e r e s t f a r t h e r on and w i l l be r e v i v e d as a p p r o p r i a t e . The spectrum of l a b e l l e d wood, as w e l l as showing a very small f r a c t i o n a b c d e g h F i g u r e V-5: E s r s p e c t r a o f d i r e c t l y l a b e l l e d c e l l u l o s e (51) i n s o l v e n t s o f d i f f e r e n t s w e l l i n g a b i l i t y . ( a ) w a t e r ; (b)70:30 water e t h a n o l ; (c)30:70 w a t e r : e t h a n o l ; ( d ) e t h a n o l ; ( e ) 1 , 4 - d i o x a n ; ( f ) n - h e p t a n e ; ( g ) 1 , 4 - d i o x a n ; ( h ) e t h a n o l ; ( i ) w a t e r . 210 of t i g h t l y adsorbed n i t r o x i d e , r e f l e c t s a stronger i m m o b i l i s a t i o n than i n the case of the p u r i f i e d c e l l u l o s e s . I t seems u n l i k e l y that t h i s i s the r e s u l t of reduced m o b i l i t y i n the polymer chains themselves, s i n c e i t i s thought that n e i t h e r i n c e l l u l o s e nor wood i s an important c o n t r i b u t i o n o 45 made at 10°Hz by backbone motions (though whatever motions do e x i s t are indeed l i k e l y to be of lower frequency i n wood than i n c e l l u l o s e ) . More probably, a fundamental change i n surface a r c h i t e c t u r e accounts f o r the reduced freedom of the l a b e l . I t should be made c l e a r that any a c c e s s i b l e n u c l e o p h i l e — n o t only hydroxyls of c e l l u l o s e — m a y be l a b e l l e d i n the wood sample. The experiment described here i l l u s t r a t e s that s p i n l a b e l l i n g , i n c l u d i n g experiments of the type to be described l a t e r i n the current chapter, may be r e a d i l y a p p l i e d to n a t i v e wood surf a c e s . I t w i l l be the purpose of the chapter to i l l u s t r a t e the u t i l i t y of such an approach i n p u r i f i e d c e l l u l o s e , thus preparing the ground f o r f u t u r e work w i t h wood i t s e l f . The p o s s i b i l i t y that the spectrum of the d i r e c t l y l a b e l l e d c e l l u l o s e powder (51) (Figure V-4b), contains a heterogeneity of c o n t r i b u t i n g n i t r o x -ide species i s , i n view of models proposed f o r the a r c h i t e c t u r e of the su r f a c e , of great i n t e r e s t . Given that the idea of a s u r f a c e - s o l u t i o n i n t e r f a c e i s more w e l l - d e f i n e d i n c e l l u l o s e than i n the g e l systems d e a l t w i t h i n previous chapters, the n o t i o n of i n t e r a c t i o n between a s p i n tethered to the surface and a species i n s o l u t i o n i s employed as a b a s i s f o r the i n v e s t i g a t i o n of the c e l l u l o s e - w a t e r i n t e r f a c e . The use of n i c k e l ions i n s o l u t i o n provides a s t a r t i n g point or i n t e l l i g e n c e r . The r e l a t i o n s h i p of the adherent s p i n to the i n t e r f a c e i s explored using the spacer arm idea of Chapter IV. 211 ( i i ) Broadening by Paramagnetic 'Probe' Ions i n S o l u t i o n The d i r e c t l y l a b e l l e d c e l l u l o s e powder (51) and the spacer-conjugated m a t e r i a l (53) were suspended, f i l t e r e d , and resuspended s e v e r a l times i n s o l u t i o n s c o n t a i n i n g v a r i o u s concentrations of n i c k e l sulphate, and t h e i r esr s p e c t r a then obtained. These are shown, i n 2.0M NiSO^, and compared w i t h those of the two purely aqueous suspensions i n Figure V-6. I n t e r e s t -i n g s p e c t r a l changes can be seen to have occurred, and no change i n the lineshape could be detected when 2.0M calcium c h l o r i d e was introd'uced i n s t e a d of n i c k e l sulphate. In the case of the d i r e c t l y l a b e l l e d sample, line-broadening i s observed w i t h a considerable (^  12x) r e d u c t i o n i n s i g n a l - t o - n o i s e r a t i o . I t would seem that a p r o p o r t i o n of the n i t r o x i d e s i s i n a c c e s s i b l e to the 'probe' i o n , w h i l e o t h e r s — t h e m a j o r i t y — a r e able to undergo e l e c t r o n exchange w i t h the s o l u t i o n s p e c i e s , producing a con-comitant line-broadening. For a n i t r o x i d e i n s o l u t i o n , no esr s i g n a l i s d e t e c t a b l e i n the presence of 2.0M Ni^" 1. That the' r e s i d u a l s i g n a l f i t s w i t h i n the envelope represented by the p u r e l y aqueous ' n a t i v e ' spectrum can be seen i n Figure V-7; i t appears to represent the broadest c o n s t i t u e n t of the n a t i v e spectrum. I t s lineshape i s r a t h e r s i m i l a r to that observed i n the analogous experiment i n Sepharose 4B and the d i s c u s s i o n i n Chapter IV as to i t s p o s s i b l e nature a p p l i e s here a l s o . To p r e c i s , i t may be that both s e c u l a r d i p o l a r i n t e r a c t i o n s and slow r e o r i e n t a t i o n (slower than the average f o r the system as a whole) occur. I t i s i n t e r e s t i n g to note, i n passing, the s i m i l a r i t y between t h i s spectrum and that of l a b e l l e d wood (Figure V-4e), the l a t t e r i n the absence of paramagnetic i o n s ; the compar-i s o n serves to r e i n f o r c e the point that the r e s i d u a l s i g n a l represents a broader lineshape which has been s e l e c t e d from the envelope represented by the n a t i v e spectrum (Figure V-4b). 212 Figure V-6: N i c k e l ions as probes i n l a b e l l e d c e l l u l o s e s without and with spacer arms. Esr spectra of (a)(51)(aq.); (b)(51) + 2M N i S 0 4 ( a q . ) ; ( c ) ( 5 3 ) ( a q . ) ; (d)(53) + 2M N i S 0 4 ( a q . ) . Q(*1) a(*12) b(*12) F i g u r e V-7: S p e c t r a (a) and (b) from p r e v i o u s f i g u r e showing r e l a t i v e a m p l i f i c a t i o n s . ( a ) ( 5 1 ) ; ( b ) ( 5 1 ) + 2 M N i S 0 ( a q . ) -K3 h-1 214 Some d i s c u s s i o n of the nature of the broadening phenomenon i s approp-r i a t e here; the same p o i n t s are a l s o a p p l i c a b l e to nickel-broadening experiments described i n preceding chapters. Three mechanisms may be envisaged f o r the broadening of the n i t r o x i d e s i g n a l : Heisenberg exchange; s e c u l a r d i p o l a r i n t e r a c t i o n between surface-bound metal ions and n i t r o x i d e s ; and d i p o l e - d i p o l e s p i n - l a t t i c e r e l a x a t i o n . For t r a n s i t i o n metal ions and n i t r o x i d e s i n s o l u t i o n s of low v i s c o s i t y , exchange has always been found to dominate, even i n cases where the i o n i s present as a l a r g e complex (Chap-t e r I C ) . Where c o l l i s i o n s are p o s s i b l e between ions and surface-bound n i t r o x i d e s , exchange i s l i k e w i s e expected to p r e v a i l , though s t e r i c hindrance may make the ( l i n e a r ) c o n c e n t r a t i o n dependence shallower by reducing c o l l i -s i o n frequency. The same w i l l be true f o r metal ions bound at the surface (and such a phenomenon cannot be discounted i n c e l l u l o s e ) i n very c l o s e p r o x i m i t y to a n i t r o x i d e . Exchange, however, f a l l s o f f very r a p i d l y w i t h d i s t a n c e . Thus i t may be necessary to consider d i p o l a r e f f e c t s when n i t r o x i d e s are hidden i n pores at a s u r f a c e , s i n c e these i n t e r a c t i o n s gener-a l l y f a l l o f f l e s s r a p i d l y w i t h i n c r e a s i n g d i s t a n c e . Secular d i p o l a r broadening may be discounted s i n c e i t i s averaged by the r a p i d r e l a x a t i o n -12 -13 of the n i c k e l i o n (T^ - 10 - 10 s ) . D i p o l e - d i p o l e s p i n - l a t t i c e r e l a x a t i o n of the n i t r o x i d e by the metal i o n i s modulated e i t h e r f o r f r e e or bound metal by i t s T-i (not molecular tumbling, which i n e i t h e r case i s sl o w e r ) . This mechanism w i l l not, t h e r e f o r e , be p a r t i c u l a r l y e f f i c i e n t at —6 the resonant frequency; n e v e r t h e l e s s , because i t s (distance) dependence i s shallower than that f o r exchange, which drops o f f as the wavefunctions, i t may c o n t r i b u t e to the r e l a x a t i o n of i n a c c e s s i b l e n i t r o x i d e s at the surface where d e v i a t i o n s from l i n e a r metal c o n c e n t r a t i o n dependence are observed (Figure V-9). Q u a n t i t a t i o n of the r e l a t i v e c o n t r i b u t i o n s from d i f f e r e n t 215 mechanisms to e l e c t r o n r e l a x a t i o n i s a r a t h e r d i f f i c u l t task which w i l l not concern us here. C l e a r l y , however, the c o n c l u s i o n that the r e s i d u a l s i g n a l a r i s e s i n one way or another because of the r e l a t i v e i n a c c e s s i b i l i t y of some n i t r o x i d e s i s j u s t i f i e d . In the case of the spacer-conjugated m a t e r i a l line-broadening i s much more marked (Figure V-6c and d ) , the r e s i d u a l s i g n a l at 2.0M N i ^ being almost undetectable. Thus almost a l l the n i t r o x i d e p o p u l a t i o n appears, as a r e s u l t of extension from the surface by means of a spacer arm <_ ^  1.2 nm 46 i n l ength (as compared w i t h <_ ^  0.35 nm i n (51)), to have become a c c e s s i b l e to metal-induced r e l a x a t i o n from s o l u t i o n . In view of the g e n e r a l l y accepted n o t i o n of 'bound water' at the 22 c e l l u l o s e - s o l u t i o n i n t e r f a c e , a n a t u r a l question a r i s e s concerning the p a r t i c i p a t i o n , or l a c k o f , of t h i s water i n the f i r s t h y d r a t i o n sphere of the paramagnetic i o n ; or, as has p r e v i o u s l y been a l l u d e d t o , b i n d i n g of the i o n to surface hydroxyls. This would be expected to i n f l u e n c e the e f f i c i e n c y of metal-induced r e l a x a t i o n through i t s d i s t a n c e dependence. I t was t h e r e f o r e decided to i n v e s t i g a t e the e f f e c t of a paramagnetic i o n whose l i g a n d s do not exchange (chemically) w i t h s o l v e n t . F e r r i c y a n i d e was chosen; i t s e l e c t r o n s p i n - l a t t i c e r e l a x a t i o n i s s u f f i c i e n t l y r a p i d that no room temperature e l e c t r o n resonance i s detected, and i t i s known to undergo strong exchange w i t h n i t r o x i d e , about 20% of c o l l i s i o n s being s u c c e s s f u l The r e s u l t of a d d i t i o n of K 3Fe(CN) 5 to a c o n c e n t r a t i o n of 1.0M (an almost saturated s o l u t i o n ) to the two c e l l u l o s e d e r i v a t i v e s i s shown i n Figure V-8. I n t e r e s t i n g l y , almost no e f f e c t save f o r a very s l i g h t l o s s of r e s o l u t i o n i n the water features i s observed i n the d i r e c t l y l a b e l l e d system. Almost a l l the l a b e l s are unable to exchange w i t h f e r r i c y a n i d e . 216 Figure V-8: F e r r i c y a n i d e as a probe ion i n l a b e l l e d c e l l u l o s e s . Esr spectra of (a)(51)(aq.); (b)(51) + 1M K 3Fe(CN) 6(aq.); ( c ) ( 5 3 ) ( a q . ) ; (d)(53) + 1M K 3Fe(CN) 6(aq.). In c o n t r a s t , only a weak s i g n a l remains i n the spacer-conjugated system; i t i s th e r e f o r e c l e a r that extension from the surface by - 1.2 nm i s l a r g e l y s u f f i c i e n t to enable the n i t r o x i d e s to escape whatever s t e r i c hindrance occurs due to c e l l u l o s e molecules and bound water. Figures V-9 and V-10, i n which the increase i n peak-to-peak width of the centre l i n e i s p l o t t e d i n each case as a f u n c t i o n of metal i o n concen-t r a t i o n i n the two c e l l u l o s e s and i n a s o l u t i o n of n i t r o x i d e (2) i n water, throw more l i g h t on these experiments. C l e a r l y both immobilised l a b e l s are subject to hindrance at the sur f a c e , the spacer-conjugated n i t r o x i d e i n (53) l e s s so than the d i r e c t l y - c o n j u g a t e d one i n ( 5 1 ) , - t h e i r l i n e w i d t h s i n c r e a s i n g l e s s r a p i d l y w i t h c o n c e n t r a t i o n than f o r the s o l u t i o n case. Each system, however, appears to conta i n l a b e l s i n d i f f e r e n t s i t e s ; at low [ N i ^ ] q u i t e r a p i d broadening occurs i n both cellulose-bound l a b e l s w h i l e at higher c o n c e n t r a t i o n broadening i s slowed i n the spacer-conjugated sample (53) and e s s e n t i a l l y no f u r t h e r broadening i s observed i n the other. In the f e r r i c y a n i d e experiment i t i s again c l e a r that not only i s l i n e -broadening slower than i n s o l u t i o n , but i n the spacer-conjugated system (53) i t occurs over the e n t i r e c o n c e n t r a t i o n range. I t i s p o s s i b l e , t h e r e f o r e , to c h a r a c t e r i s e a c c e s s i b i l i t y changes i n terms of changes i n the gradient of the p l o t ; i t i s convenient to d e f i n e an ' a c c e s s i b i l i t y parameter', z^, as ^ _ gradient (immobilised species) [37] i gradient ( s o l u t i o n species) both gradients having dimensions of G .A.mole (these are nothing more than f i r s t - o r d e r r a t e constants). Table V - l shows z^-values c a l c u l a t e d from the data i n Figures V-9 and V-10. In the three cases where changes i n gradient occur as a f u n c t i o n of c o n c e n t r a t i o n , i n i t i a l ( Z Q ) a n d f i n a l 6 Figure V-9: Increase of n i t r o x i d e c e n t r e - f i e l d l i n e w i d t h AOJQCC) as a f u n c t i o n of n i c k e l ion c o n c e n t r a t i o n , ( a ) s p i n l a b e l (2) i n aqueous s o l u t i o n ; (b)(53)(with spacer); (c)(51)(no spacer) . 0 0 . 1 0 . 2 0 . 3 0 . 4 [K 3 Fe(CN) 6 ] (M) F i g u r e V - 1 0 : I n c r e a s e i n n i t r o x i d e c e n t r e - f i e l d l i n e w i d t h A w ( c ) a s a f u n c t i o n o f e r r i c y a n i d e i o n c o n c e n t r a t i o n . ( a ) l a b e l (2) i n a q u e o u s s o l u t i o n ; ( b ) (53) ( a q . ) ; 220 Table V - l : Low- (z ) and high- c o n c e n t r a t i o n (z ) a c c e s s i b i l i t y parameters o r f o r probe ions at l a b e l l e d c e l l u l o s e surfaces compared w i t h s o l u t i o n n i t r o x i d e . Number of p o i n t s used i n gradient measurements given i n parentheses. system and code number probe H2N-SL (2) ( s o l u t i o n ) Fe(CN) 6 c e l l u l o s e - S L (51) ce l l u l o s e - s p a c e r - S L (53) 3-H2N-SL (2) ( s o l u t i o n ) N i ( H 2 0 ) 6 c e l l u l o s e - S L (51) ce l l u l o s e - s p a c e r - S L (53) " 2+ z (number of p o i n t s ; 1 ^ 0 0.48 (8) 1 0.11 (4) 0.19 (4) z^ (number of po i n t s ) 1 % 0 0.07 (6) 1 ^ 0 0.03 (9) (z^) values are c a l c u l a t e d using l i n e a r r e gressions and the number of p o i n t s shown i n the t a b l e . Previous conclusions are r e s t a t e d i n the t a b l e : that d i r e c t l y -attached l a b e l s are i n a c c e s s i b l e to f e r r i c y a n i d e , and only p a r t i a l l y a c c e s s i b l e to n i c k e l o u s i o n s , w h i l e spacer-conjugated l a b e l s are found i n more and l e s s a c c e s s i b l e s i t e s as 'sensed' by both probes. z_^  = 0 i m p l i e s an exchange r a t e of zero, i . e . , t o t a l i n a c c e s s i b i l i t y . The d i v i s i o n i n t o i n i t i a l and f i n a l a c c e s s i b i l i t i e s i m p l i e s a two-s i t e model, but i n f a c t the data are not s u f f i c i e n t to d i s t i n g u i s h t h i s from one in v o k i n g a s e r i e s of s i t e s graded i n a c c e s s i b i l i t y . A 6- or 7-factor i n z^ separates the most from the l e a s t a c c e s s i b l e i n both experiments w i t h the spacer-conjugated n i t r o x i d e . This i s an important r e s u l t , because the q u a l i t a t i v e p i c t u r e i m p l i e d by the sp e c t r a i n Figures V-6 and V-8, where spacer-conjugated n i t r o x i d e s i g n a l s almost disappear, leaves open the p o s s i b i l i t y that i n r e a c t i n g w i t h a c t i v a t e d c e l l u l o s e , the spacer u n i t (6-amino c a p r o i c acid) 221 i t s e l f gains access only to unhindered s i t e s . However, i n extended conformations i t i s smaller i n c r o s s - s e c t i o n than l a b e l ( 2 ) , and the concentration-dependence data, though not proving that a l l the same s i t e s , are d e r i v a t i s e d i n the two l a b e l l e d c e l l u l o s e s , evidences a range of a c c e s s i b i l i t i e s i n both. Again, t h i s would not be suspected on the b a s i s of the unperturbed lineshape of the spacer-conjugated d e r i v a t i v e (53). One r a t h e r p u z z l i n g f e a t u r e remains to be explained i n Table V - l . This i s the f a c t that d e s p i t e the q u a l i t a t i v e s i m i l a r i t y of the r e s u l t s , both at high and low concentrations of probe the magnitude of the a c c e s s i -b i l i t y of the spacer-conjugated l a b e l to f e r r i c y a n i d e i s greater than the magnitude of i t s a c c e s s i b i l i t y to n i c k e l o u s i o n ; t h i s d e s p i t e the observa-t i o n that i n the d i r e c t l y l a b e l l e d m a t e r i a l the s i t u a t i o n i s reversed. Consistency i s observed i n that z (Fe(CN)i~) z f(Fe(CN)e") : ~-7 -'- 2.3 and ^-r— - 2.5. Z o ( N i ( H 2 0 ) 6 ) z f ( N i ( H 2 0 ) 6 ) One can only speculate as to the reasons. Factors independent of the b a s i c s t r u c t u r e of c e l l u l o s e may play a p a r t ; one p o s s i b l e v a r i a b l e i s charge. Reaction w i t h CNBr i s l i k e l y to have introduced a net p o s i t i v e charge to the matrix. This may r e s u l t i n an increased gradient i n the c o n c e n t r a t i o n dependence of broadening by a negative i o n , w h i l e decreasing that of a p o s i t i v e s p e c ies. That charge i s i n f l u e n t i a l i n determining a c c e s s i b i l i t y i s supported by r e s u l t s i n the next s e c t i o n , ( i i i ) Reduction Reduction of surface-bound n i t r o x i d e by agents present i n s o l u t i o n represents a conceptually simpler v a r i a n t on the exchange-broadening experiment. This type of approach has been used to monitor f l i p - f l o p 48 r a t e s of s p i n l a b e l l e d l i p i d molecules i n liposomes and c e l l s . Those 222 n i t r o x i d e s which are a c c e s s i b l e are reduced to diamagnetic species such as the N-hydroxy analogue, w i t h concomitant l o s s of the esr s i g n a l . Three reducing species were chosen f o r such experiments i n the l a b e l l e d c e l l u l o s e systems: sodium d i t h i o n i t e (^28204) and ferrous sulphate (FeSO^). In each case the exact course of the r e d u c t i o n i s unknown; a l l three give paramagnetic products, the f i r s t two w i t h sharp s i g n a l s at g = 2 which do not i n t e r f e r e w i t h d e t e c t i o n of outer l i n e s i n the n i t r o x i d e spectrum. F e r r i c i o n s , which are produced by o x i d a t i o n of ferrous ions by n i t r o x i d e , have a broad resonance which overlaps that of the l a t t e r and must be leached out of the c e l l u l o s e p r i o r to re c o r d i n g s p e c t r a . Both 0.1M sodium ascorbate, pH 7.0, and 0.2M sodium d i t h i o n i t e , pH 7.0 when added to e i t h e r of the two l a b e l l e d c e l l u l o s e d e r i v a t i v e s caused immediate ( i . e . , w i t h i n - 30 s, the time r e q u i r e d to perform the measurement) disappearance of the n i t r o x i d e s i g n a l . No r e s i d u a l s i g n a l whatever could be observed. However, p l a c i n g a 1.0M s o l u t i o n of FeSO^ i n t o contact w i t h the wet, l a b e l l e d c e l l u l o s e , followed by s e v e r a l renewals of the fe r r o u s s o l u t i o n caused only p a r t i a l r e d u c t i o n of the d i r e c t l y conjugated s p i n l a b e l s (Figure V - l l c ) ; the r e s i d u a l spectrum d i s p l a y s a lineshape not u n l i k e that of the 'nati v e ' l a b e l l e d m a t e r i a l (51) at lower s i g n a l - t o - n o i s e . In t h i s case the r e a c t i o n took = 2 h to completion at which point a d d i t i o n of f r e s h f e r r o u s CH20H sodium ascorbate CHOH Ho* "0 OH 223 Figure V - l l : The p a r t i a l reducing a c t i o n of ferrous ions on l a b e l l e d c e l l u l o s e (51). (The spacer-conjugated n i t r o x i d e s i n (53) were a l l reduced.) Esr spectra of (a)(51)(aq.); (b)(51) + 2M N i S 0 4 ( a q . ) ; (c) (51) , reduced exh a u s t i v e l y with aqueous ferrous sulphate s o l u t i o n s ; (d) p a r t i a l l y reduced (51) from c + 2M NiSO. (aq.). 224 s o l u t i o n s r e s u l t e d "in no f u r t h e r r e d u c t i o n . The spacer-conjugated c e l l u l o s e d e r i v a t i v e was immediately and completely reduced by f e r r o u s i o n s . These l a s t experiments provide e x t r a c o n f i r m a t i o n that a c c e s s i b i l i t y d i f f e r e n c e s e x i s t at the s u r f a c e ; as the lineshape of p a r t i a l l y reduced n i t r o x i d e - l a b e l l e d c e l l u l o s e i s s i m i l a r to that of the n a t i v e m a t e r i a l (51) i t would seem that r a t e of tumbling i s not a p a r t i c u l a r l y s e n s i t i v e index of a c c e s s i b i l i t y . The use of three reducing agents a l s o emphasises the c o n c l u s i o n which was h i n t e d at i n the previous s e c t i o n , that a c c e s s i b i l i t y i s not only dependent on s i z e . I f ascorbate (unhydrated r a d i u s ^ 0.40 nm) 2+ can reduce a l l surface-bound n i t r o x i d e s , why cannot Fe(H20)g (radius ^ 0.45 nm) do the same? Again one must conclude that n e g a t i v e l y charged species are at an advantage i n approaching the s u r f a c e , which at n e u t r a l pH i s l i k e l y to have a net p o s i t i v e charge a f t e r m o d i f i c a t i o n . I t should be remarked here that a d i r e c t comparison between r e d u c t i o n and exchange i s dangerous because w h i l e the former may r e q u i r e only one or a few encounters, the l a t t e r must occur r a p i d l y and continuously to i n f l u -ence the l i n e w i d t h . The microscopic d i s t a n c e and o r i e n t a t i o n dependencies of the two processes may a l s o d i f f e r . Nevertheless, i t i s of i n t e r e s t to compare the exchange-broadened s i g n a l observed a f t e r p a r t i a l r e d u c t i o n of d i r e c t l y - b o u n d n i t r o x i d e s w i t h ferrous ions w i t h that observed i n the n a t i v e m a t e r i a l , each i n the presence of 2.0M n i c k e l o u s i o n . To t h i s end hexaquo n i c k e l o u s ions were added to a c o n c e n t r a t i o n of 2.0M to the p a r t i -a l l y reduced sample. The r e s u l t i n g spectrum i s shown i n Figure V - l l d . The f i r s t p o i n t t o be made i s that only a s m a l l (- 1.5x) r e d u c t i o n i n s i g n a l - t o - n o i s e has occurred. The change i n lineshape ( g i v i n g an appear-ance s i m i l a r to that of the unreduced, d i r e c t l y - l a b e l l e d c e l l u l o s e i n the presence of N i ^ , Figure V - l l b ) shows that some of the r e s i d u a l spins are 225 nevertheless a c c e s s i b l e to exchange; so that although the populations i n f l u e n c e d by the two s i m i l a r d i v a l e n t ions are s i m i l a r (compare the much greater r e d u c t i o n i n s i g n a l i n t e n s i t y from Figure V - l l a to b ) , they are not the same. This c o n c l u s i o n i s supported by the f a c t that the r e s i d u a l spectrum of the unreduced m a t e r i a l i n the presence of n i c k e l ( I I ) has higher s i g n a l - t o - n o i s e (7x) than that a f t e r r e d u c t i o n . VC: Triazine-Mediated L a b e l l i n g The a l t e r n a t i v e l a b e l l i n g method used i n t h i s group of experiments was as f o l l o w s : I-OH + iJyl V - i o n ^ (49) C l (8) (54) (55) I t was u s e f u l i n two r e s p e c t s . In the f i r s t p l a c e , the somewhat curious o b s e r v a t i o n that n i t r o x i d e s a f f i x e d t o c e l l u l o s e under aqueous c o n d i t i o n s are not always a v a i l a b l e to aqueous reagents of s i m i l a r s i z e i n s o l u t i o n r e q u i r e d that the dependence of t h i s e f f e c t on attachment chemistry, and the s i z e of the l a b e l s p e c i e s , be enquired i n t o . In the second p l a c e , i t was p o s s i b l e to perform the above chemistry i n aqueous (50% aqueous acetone c o n t a i n i n g 4% NaOH) and nonaqueous ( r e f l u x i n g hexane) c o n d i t i o n s , under which the surface pore s t r u c t u r e would be expected to d i f f e r . Powdered 226 c e l l u l o s e s (49) l a b e l l e d w i t h the t r i a z i n e - n i t r o x i d e reagent (8) under these two types of c o n d i t i o n were obtained from Dr. M. J . Adam, w i t h whose a s s i s -tance the f o l l o w i n g experiments were performed. Figure V-12 shows the s p e c t r a of the two l a b e l l e d m a t e r i a l s together w i t h t h e i r s p e c t r a i n the presence of 2.0M aqueous n i c k e l sulphate. As was found w i t h the cyanogen bromide-labelled c e l l u l o s e (Figure V-5), m o b i l i t y i n c r e a s e s , i n the sample l a b e l l e d i n hexane, on passing from i t to water. In both s p e c t r a (Figure V-12a and b) a t r a c e of adsorbed l a b e l appears to be present. This was not amenable to l e a c h i n g under c o n d i t i o n s p r e v i o u s l y described. The s p l i t t i n g s (2T) between the outer features i n Figures V-12a and V-5f are 64 G and 65 G r e s p e c t i v e l y ; the i m p l i e d l a c k of spacer-dependence of c o r r e l a t i o n time again suggests a t r a p p i n g or f o l d i n g back to the matrix. In water, the two samples show s i m i l a r l i n e s h a p e s , both contain-ing evidence of r o t a t i o n a l a n i s o t r o p y , which, given the nature of the l i n k i n g u n i t , i s expected to be more pronounced than i n the cyanogen bromide-l a b e l l e d m a t e r i a l , s i n c e r o t a t i o n of the aromatic r i n g i s l i k e l y to be much slower than r o t a t i o n of the n i t r o x i d e about the two bonds j o i n i n g i t to the r i n g : slower f a s t e r Cl NH / \ Q (54) (51) 227 Figure V-12: Esr spectra of t r i a z i n e - l a b e l l e d c e l l u l o s e s (54). (a)(54)(synthesized i n n-hexane) i n n-hexane; (b)(54)(synthesized i n n-hexane) i n water; (c)as b with added NiS0 4(aq.)(2M); (d)(54) (synthesized i n aqueous acetone) i n water; (e)sample from d with added NiS0 4(aq.)(2M). 228 (Note a l s o the p o s s i b i l i t y that both c h l o r i n e s may be d i s p l a c e d from the t r i a z i n e d e r i v a t i v e to give (55) i n which r o t a t i o n may only occur about two bonds.) Otherwise the s p e c t r a of d e r i v a t i v e s (54) and (51) represent n i t r o x i d e s i n q u i t e s i m i l a r motional regimes, though the t r i a z i n e - l a b e l l e d samples have c o n s i d e r a b l y reduced s i g n a l - t o - n o i s e . 2+ In the presence of 2.0M Ni (^0)5 both t r i a z i n e - l a b e l l e d m a t e r i a l s show a r e s i d u a l s i g n a l w i t h a broader lineshape than the o r i g i n a l aqueous samples (Figures V-12c and e ) . This lineshape i s s i m i l a r i n nature both to that observed i n Figure V-12a and to the r e s i d u a l s i g n a l seen i n the c r o s s -l a b e l l e d c e l l u l o s e i n the presence of n i c k e l i o n s . I n t e r e s t i n g l y , however, though both are weak, the m a t e r i a l l a b e l l e d i n hexane shows a considerably (3x) smaller r e s i d u a l p o p u l a t i o n . This i s as expected s i n c e fewer surface hydroxyls (only the r e l a t i v e l y unhindered, a c c e s s i b l e ones) should be a v a i l -a ble f o r l a b e l l i n g i n hexane than i n aqueous acetone w i t h sodium hydroxide owing to the s w e l l i n g (hydrogen bond-disrupting) p r o p e r t i e s of the l a t t e r . However, there i s no f i r m evidence i n the unperturbed sp e c t r a f o r s i t e d i f -f e r e n t i a t i o n (e.g. , on the b a s i s of m o b i l i t y ) . A more q u a n t i t a t i v e study of these e f f e c t s r e q u i r e s enhanced s i g n a l - t o - n o i s e . However, i t i s i n t e r -e s t i n g to note that a r e s i d u a l s i g n a l i s observed at a l l ; molecular models show that l i k e the cyanogen bromide-labelled product (51), the t r i a z i n e -conjugated n i t r o x i d e i s able to f o l d back to hydrogen bond to a f l a t s u r f a c e , and t h i s may provide one 'masking' mechanism. At 77 K the s i g n a l - t o - n o i s e r a t i o was too poor to a c c u r a t e l y determine d^/d i n e i t h e r case, but i n d i c a -t i o n s were that spins were too widely spaced to enable d i p o l a r i n t e r a c t i o n to be measured. 229 VD: Surface Area Most of the methods which have been used to measure the surface area of c e l l u l o s e i n v o l v e the adsorption of gaseous s p e c i e s , and many d i f f e r e n t numbers have been obtained, w i t h a p a r t i c u l a r l y marked dependence on hydro-gen bonding a b i l i t y of the adsorbed molecule. Hence the idea of ' s w e l l i n g ' . Such methods are by nature i n d i r e c t i n the sense that the adsorbed species are not a c t u a l l y observed i n t h e i r surface l o c a t i o n . One p o t e n t i a l l y important idea which developed during the course of the present s p i n l a b e l -l i n g experiments on c e l l u l o s e concerned the a b i l i t y of the technique to measure surface areas through d i r e c t observation of i n t e r a c t i o n s between surface-bound species. The p r i n c i p l e s of the measurement are as f o l l o w s . F i r s t l y , by double i n t e g r a t i o n and comparison w i t h standard s o l u t i o n s i t i s p o s s i b l e to d e r i v e , as before, the number of n i t r o x i d e s per gram of polys a c c h a r i d e . Secondly, p r o v i d i n g spins are s u f f i c i e n t l y c l o s e l y spaced at the su r f a c e , the magnitude of t h e i r d i p o l a r i n t e r a c t i o n i n the absence of motional averaging ( i . e . , at 77 K) can be measured, g i v i n g by comparison w i t h Figure 1-11 a mean distance between nearest neighbour spins which may be converted to an average number of spins per u n i t area. Then i n t e g r a t e d i n t e n s i t y (spins.g "S r ,2 -ls roon  a 1—^—^ °-—_2 = surface area (m .g ) [38] surface d e n s i t y of spins ( p ) (spins.m ) I t w i l l be noted that the success of t h i s method does not depend upon p p r o v i d i n g that i t f a l l s w i t h i n c e r t a i n bounds which d e f i n e the l i m i t s w i t h i n which the d i p o l a r i n t e r a c t i o n may be measured (see Chapter I C ) . In p r a c t i c e the observed distance between l a b e l s on c e l l u l o s e f a l l s towards the outer l i m i t of these bounds, where d j / d i s l e s s s e n s i t i v e to changes i n d i s t a n c e ; the method could t h e r e f o r e be improved by using a s l i g h t l y more e f f i c i e n t l a b e l l i n g method. I t should a l s o be noted that the method measures 230 a through-space i n t e r a c t i o n and hence i t must be assumed that the d i s t r i -b u t i o n of l a b e l s i s two dimensional. A t y p i c a l sample of d i r e c t l y l a b e l l e d c e l l u l o s e (using the CNBr method, compound (51)) was found to c o n t a i n , a f t e r c o r r e c t i n g the weight on the b a s i s of an estimated (using elemental a n a l y s i s ) 5% bound water, one s p i n per 169 glucose r e s i d u e s , or 2.2 x 1 0 1 9 spins.g ^. The mean nearest-neighbour distance ( r ) , found from the d^/d parameter i n the esr spectrum measured at 77 K, was 1.83 nm; the d e n s i t y i n two dimensions, p , i s r e l a t e d - - -2 to r by p = (2r) (equation [A2], see Appendix 2) which gives the value of -2 -2 p of 7.47 x 10 s p i n nm . X-ray d i f f r a c t i o n s t u d i e s i n d i c a t e that at an ordered ( m i c r o c r y s t a l l i n e ) s u r f a c e , roughly f i v e glucose residues might o -2 -1 occupy an area of 1 nm . Then 5 x (7.47 x 10 ) - 67 residues are present at the surface f o r each l a b e l ; very roughly, (67/169) x 100 = 40% of g l u -cose residues might be supposed to be a c c e s s i b l e to n i t r o x i d e . The c a l c u l a t i o n then proceeds as f o l l o w s : This f i g u r e i s r a t h e r l a r g e r than previous measurements taken from the 25 l i t e r a t u r e ; using water vapour, Klenkova and I v a s k i n found a surface 9 -1 area of 137 m .g , w h i l s t adsorption of non-hydrogen bonding species l i k e ? -1 N 2 gives f i g u r e s of the order of 0.7 m .g . I n c r e a s i n g the s i z e of the 28 29 s o l u t e has been shown to reduce the ' a v a i l a b l e s o l v e n t ' ' ; the present r e s u l t , even i n the l i g h t of the r a t h e r l a r g e e r r o r l i m i t , i s s u f f i c i e n t l y l a r g e to suggest that the assumptions i n v o l v e d i n i t s d e r i v a t i o n may be questionable, s i n c e a c o n s i d e r a b l y greater surface area appears to be a v a i l a b l e to the n i t r o x i d e than to water. S i m i l a r conclusions can be 27 drawn on c o n s i d e r a t i o n of the reports of Rowlands , who found that surface area = 2.2 x 1 01 9 7.47 x 1 0 1 8 x 10' 231 N , N - d i e t h y l a z i r i d i n i u m c h l o r i d e (molecular weight 136) gained access to about 40% of the hydroxyls a v a i l a b l e to water (as D 20), w h i l e f o r Diphenyl Fast Red 5BL (molecular weight 676) the f i g u r e was 5%. The molecular weight of the n i t r o x i d e i s 171. The same sample of c e l l u l o s e , l a b e l l e d i n aqueous cyanogen bromide, was then d r i e d i n vacuo overnight at 393 K, a f t e r which i t s esr spectrum at 77 K was recorded. In t h i s case the same procedure u t i l i s i n g equation [38] p -1 -gave a surface area of 332 m .g , r i n c r e a s i n g to 1.94 nm. F i r s t i t should be noted that t h i s f i g u r e does not represent the surface area of dry c e l l u -l o s e ; to o b t a i n t h i s i t would be necessary to perform the l a b e l l i n g e x p e r i -ment under dry c o n d i t i o n s . Second, i t i s rewarding that the d i p o l a r i n t e r a c t i o n i s s e n s i t i v e to changes i n the s t r u c t u r e of the surface occur-r i n g during d r y i n g , and gives room f o r optimism about the general u t i l i t y of the method to t e x t i l e chemists. T h i r d l y , however, one can only specu-l a t e at present as to why the magnitude of the i n t e r a c t i o n s should decrease, g i v i n g a greater c a l c u l a t e d 'surface area' (given equal l o a d i n g ) . Although an o v e r a l l e r r o r of 50% has been estimated f o r t h e i r absolute v a l u e s , the f i g u r e s f o r wet and dry c e l l u l o s e are c o r r e c t r e l a t i v e to one another to about 5% (the measurement e r r o r i n d^/d). I t seems l i k e l y that t h i s must be the r e s u l t of a s i g n i f i c a n t p r o p o r t i o n of the i n t e r a c t i o n s being between n i t r o x i d e s a f f i x e d to d i f f e r e n t .polyglucose chains, which are p u l l e d away from each other as s t r a i n i s created by the removal of hydrogen-bonding solvent molecules. Increased s e p a r a t i o n may conceivably occur between strands i n amorphous r e g i o n s , or between surfaces i n regions of type B i n Figure V-2c. I t i s c l e a r l y a phenomenon worthy of f u r t h e r i n v e s t i g a t i o n . The a c t u a l magnitude of the surface area of wet c e l l u l o s e might be expected to be l a r g e r than the present measurement i f i n t e r a c t i o n s i n three, r a t h e r 232 than two, dimensions were important; however, the decrease i n the d i s t a n c e between l a b e l s seen upon dryi n g suggests that a three dimensional micro-s c o p i c model may be more v a l i d . VE: Summary and Prospects The r e s u l t s of the present chapter may conveniently be summarised w i t h i n the framework of an e m p i r i c a l pore model as shown s c h e m a t i c a l l y i n Figure V-13. Pores are presumed to e x i s t i n a v a r i e t y of s i z e s which other experiments have suggested may range downwards from > 10 nm i n d i a -meter. They may then i n theory be a s s o c i a t e d w i t h d i f f e r e n t l e v e l s , types or magnitudes of s t r u c t u r e i n the m i c r o f i b r i l : surfaces of m i c r o c r y s t a l -l i t e s , amorphous re g i o n s , and so on. Solvent molecules at the surface may, i n t h i s s i m p l i f i e d model, be d i v i d e d i n t o t h r e e : those trapped w i t h i n pores and t h e r e f o r e able to exchange only s l o w l y w i t h bulk s o l v e n t ; those at r e a d i l y a c c e s s i b l e surfaces and hence i n r a p i d exchange w i t h bulk solvent (not shown i n Figure V-13); and the bulk solvent i t s e l f . In the same way two types of d i r e c t l y attached n i t r o x i d e may be d i s t i n g u i s h e d at the s u r f a c e : those at a c c e s s i b l e s u r f a c e s , and t h e r e f o r e s o l v a t e d e i t h e r by bulk solvent or by rapidly-exchanging s o l v e n t ; and those i n s i d e pores. I t i s tempting to suggest that i n the l a t t e r case m o b i l i t y might be r e s t r i c t e d by s t e r i c hindrance at the w a l l s of s m a l l pores, l e a d i n g to a decrease of conformational entropy and a tendency to hydrogen bond to groups at the surface. The l a t t e r could be accomplished i n more than one p o s i t i o n : the n i t r o x i d e oxygen as w e l l as the imino- and amino-nitrogens i n the spacer u n i t . This should be detectable i n two ways: an increase i n c o r r e l a t i o n time f o r those l a b e l s i n small pores ( i n p r a c t i c e t h i s probably means a d i s t r i b u t i o n of c o r r e l a t i o n times, assuming a d i s t r i b u t i o n of pore s i z e s ) and a decrease i n a c c e s s i b i l i t y to s o l u t i o n reagents. Neither phenomenon of course d i s t i n g u i s h e s between Figure V-13: Schematic p i c t u r e of a simple generalized h y d r o x y l i c surface c o n t a i n i n g pores. Labels are attached v i a short or long spacer arms and may be found w i t h i n or without pores. Escape from w i t h i n a pore depends on spacer length. The probable a b i l i t y of hydrated ions to coordinate to surface s u b s t i t u e n t s i s shown. 235 'masking' by s t e r i c hindrance and by hydrogen bonding. I t can be s a i d that on the b a s i s of the r e s u l t s presented the d i r e c t l y attached l a b e l (at <_ 0.35 nm from the surface) i s subject to masking of one s o r t or another; not a l l 2+ 2+ of i t i s a c c e s s i b l e to Ni(H20)g nor Fe(H 20)g ( i o n i c r a d i i - 0.45 nm) and 3-almost none i s a c c e s s i b l e to Fe(CN)g ( i o n i c r a d i u s - 0.60 nm). In c o n t r a s t the spacer-conjugated l a b e l s at _< 1.2 nm from the surface are rendered a c c e s s i b l e to a l l three reagents and the evidence from the broadening e x p e r i -ments suggests that t h e i r attachment s i t e s are not a l l u n iformly unhindered. However, though a considerable increase i n r a t e of tumbling occurs as a r e s u l t of the presence of a spacer as expected i f the s t e r i c hindrance experienced by the d i r e c t l y bound l a b e l i s t r u l y a surface phenomenon, co n s i d e r a t i o n s based on T alone do not enable anything to be s a i d about the u n i f o r m i t y of the spacer-conjugated p o p u l a t i o n . I t i s p o s s i b l e t h a t , enabling the n i t r o x i d e moiety to escape from the pore (Figure V-13), the spacer arm renders i t s motion i n s e n s i t i v e to s i t e of f i x a t i o n , or simply that the spectrum contains species whose c o r r e l a t i o n times d i f f e r i n s u f f i c i -e n t l y to enable s p e c t r a l r e s o l u t i o n to occur. On the b a s i s of the r e s u l t s obtained using these two d e r i v a t i v e s , a c l u e t o . the d i s t a n c e dependence of masking can be obtained: most i n t e r a c t i o n s are s u b s t a n t i a l l y reduced at 1.2 nm from the su r f a c e , though s t i l l very c l e a r l y measurable i n terms of the a c c e s s i b i l i t y paramaters z^. I t i s c l e a r that the study of a l a r g e r s e r i e s of e e l l u l o s e - n i t r o x i d e d e r i v a t i v e s w i t h d i f f e r e n t spacer lengths would provide u s e f u l e l u c i d a t i o n of the d i s t a n c e dependence of surface e f f e c t s and the d i s t r i b u t i o n of pore s i z e s , and furthermore that the same ideas may be a p p l i e d to the study of o t h e r , n o n - c e l l u l o s i c s u r f a c e s . I t i s important i n p a r t i c u l a r to know at what distance from the surface an immobilised reagent best resembles i t s s o l u t i o n analogue. C o r r e l a t i o n time was found i n 236 Chapter IV to be a u s e f u l i n d i c a t o r of a c c e s s i b i l i t y f o r a f f i n i t y chromato-graphy on Sepharose 4B; the present r e s u l t s show that more s u b t l e e f f e c t s , such as that of non-uniform surface topography, may best be approached i n other ways—most notably by determination of the dependence of on spacer le n g t h . I t i s i n t e r e s t i n g , f o r example, that the p o p u l a t i o n of n i t r o x i d e s unreduced by f e r r o u s i o n i s not s u b s t a n t i a l l y l e s s mobile than the average i n the d i r e c t l y l a b e l l e d c e l l u l o s e . • This p o i n t emphasises the need f o r more experiments to s o r t out s e v e r a l unexplained observations. I t i s p o s s i b l e that the d i f f e r e n c e between the e f f e c t s of f e r r i c y a n i d e and hexaquo n i c k e l (and i r o n ) ions on the ' d i r e c t l y ' attached n i t r o x i d e may not be based so much on d i f f e r e n c e s i n s i z e , which are s m a l l , as upon the a b i l i t y of the hydrated ions to penetrate the s o l v a -t i o n l a y e r of the surface (Figure V-13), i n c o r p o r a t i n g bound water or even surface f u n c t i o n a l i t i e s i n t h e i r f i r s t c o o r d i n a t i o n sphere. Perhaps d i t h i o n i t e and ascorbate can do the same, yet the s i z e of the l a t t e r ( l a r g e r than unhydrated N i ^ ) suggests that other e f f e c t s may be at p l a y . Charge and hydrogen bonding character are the two most prominent of these; the nature of t h e i r counterions i s another p e r t u r b a t i o n . I t i s c e r t a i n that a f t e r r e a c t i o n w i t h cyanogen bromide a net p o s i t i v e charge r e s i d e s on the m a t r i x at n e u t r a l pH (see Chapter IV) which would be expected to grant n e g a t i v e l y charged species a greater apparent a c c e s s i b i l i t y . F o r t u n a t e l y the observed a c c e s s i b i l i t i e s are not s o l e l y the r e s u l t of Coulombic a t t r a c -_2 t i o n ( q i q 2 r , where q's are charges) s i n c e f o r f e r r i c y a n i d e , on a p o i n t charge b a s i s , t h i s should be three times as great as f o r ascorbate and twice as great as d i t h i o n i t e . The e f f i c a c y of ascorbate r e d u c t i o n should perhaps be seen i n the l i g h t of the p o s s i b i l i t y that the atom t r a n s f e r i n v o l v e d i n n i t r o x i d e r e d u c t i o n may be able to occur over longer distances than exchange, 237 perhaps through intermediates w i t h n i t r o g e n - c o n t a i n i n g f u n c t i o n a l groups introduced during the a c t i v a t i o n procedure. (The i n t r i g u i n g p o s s i b i l i t y a r i s e s of using present methods to modify and study e l e c t r o d e surfaces!) As f a r as u n r a v e l l i n g the s t r u c t u r e of the c e l l u l o s e surface goes, however, i t i s c l e a r that two improvements to present methods are d e s i r a b l e : the use of l a b e l l i n g chemistry that does not introduce charge at the s u r f a c e , and, conversely, the use of uncharged reducing (or, indeed, o x i d i s i n g ) agents and probe ions. I t has been found i n previous s o l u t e p e n e t r a t i o n experiments that the most c o n s i s t e n t r e s u l t s have been obtained using n e u t r a l s o l u t e s whose chemical nature i s c l o s e s t to that of c e l l u l o s e : 19 glucose o l i g o s a c c h a r i d e s , dextrans and polyethylene g l y c o l s . I d e a l l y , then, a s e r i e s of n e u t r a l h y d r o p h i l i c reducing agents or r a d i c a l probes of i n c r e a s i n g s i z e would be u t i l i z e d ; d e r i v a t i v e s of hydrazine, benzyl r a d i -c a l s and n e u t r a l t r a n s i t i o n metal complexes represent three p o s s i b i l i t i e s f o r c o n s i d e r a t i o n . The t r i a z i n e - n i t r o x i d e l a b e l l i n g chemistry suggested here f u l f i l l s the requirement of n e u t r a l i t y but introduces a bulky and hydro-phobic group to the s u r f a c e . Another i n t e r e s t i n g avenue yet to be explored concerns the use of o r g a n i c - s o l u b l e probes or reducing agents to i n v e s t i g a t e changes i n surface topography r e s u l t i n g from exposure to non-polar s o l v e n t s . P r e l i m i n a r y r e s u l t s from d i s t a n c e and surface area measurements s i m i l a r l y suggest a l a r g e number of f u t u r e experiments which time d i d not a l l o w the author to c a r r y out, and again the prospects f o r a p p l i c a t i o n to other surfaces are good. In c e l l u l o s e , t h i s may prove to be the method of choice f o r determining the nature of the d i f f e r e n t c l a s s e s of a c c e s s i b l e 42 43 surface seen by previous i n v e s t i g a t o r s ' using n i t r o g e n l a b e l l i n g and p a r t i a l a c i d h y d r o l y s i s . Are t h e i r s urface areas the same? Is the m o b i l -i t y and a c c e s s i b i l i t y of a l a b e l s e n s i t i v e to t h i s type of surface 238 o r g a n i s a t i o n ? The i n s t a b i l i t y of the n i t r o x i d e group under a c i d i c condi-t i o n s remains a stumbling block. Nevertheless, the r a t h e r u n s a t i s f a c t o r y nature of the gen e r a l i s e d pore model i n e x p l a i n i n g the observed decrease i n n i t r o x i d e - n i t r o x i d e i n t e r a c t i o n s upon dryi n g l a b e l l e d c e l l u l o s e — t h e more r e a l i s t i c d e s c r i p t i o n of the surface i n terms of i n d i v i d u a l chains or bundles thereof being more conveni e n t — s u g g e s t s that the s p i n l a b e l l i n g method i s capable of p r o v i d i n g a sorely-needed bridge between the pore concept and the more complete models shown i n Figure V-2, and, u l t i m a t e l y , new i n s i g h t i n t o d i f f e r e n t regenerated and n a t u r a l l y o c c u r r i n g forms of c e l l u l o s e . 239 References 1. F. Hoyle, A. H. Olaveson, and N. C. Wickramasinghe, Nature, 271, 229-231 (1978). 2. D. A. Rees, 'Polysaccharide Shapes,' Chapman and H a l l , London, 1977. 3. J . F. Si a u , 'Flow i n Wood,' Syracuse U n i v e r s i t y P r e s s , Syracuse, New York, 1971. 4. R. L. W h i s t l e r , Ed., 'Methods i n Carbohydrate Chemistry,' V o l . I l l , Academic, New York, 1963. 5. 0. H. Northcote, Essays Biochem., _8, 89-137 (1972). 6. A. H. Nissan, Macromolecules, 10, 660-662 (1977). 7. A. F. Turbak, R. B. Hammer, R. E. Davies, and N. A. Portnoy, ACS Symp. Ser., 58., 12-24 (1977). 8. A. D. French, Carbohydr. Res., 61, 67-80 (1978). 9. R. H. Marchessault, and A. Sarko, Adv. Carbohydr. Chem., J22, 421-482 (1967). 10. A. J . S t i p a n o v i c , and A. Sarko, Macromolecules, J3, 851-857 (1976). 11. A. Sarko, J . Southwick, and J . Hayashi, i b i d . , % 857-863 (1976). 12. H. T. Lokhande, J . Appl. Polym. S c i . , 20, 2313-2319 (1976). 13. A. H. Nissan, G. K. Hunger, and S. S. S t e r n s t e i n , E n c y c l . Polym. S c i . Technol., 3^ , 131-226 (1965). 14. R. D. Preston, and J . Cronshaw, Nature, 181, 248-250 (1958). 15. A. Frey-Wyssling, Science, 119, 80-82 (1954). 16. P. K. Chidambareswaran, N. B. P a t i l , and V. Sunderam, J . Appl. Polym. S c i . , 20, 2297-2298 (1976). 17. K. Hess, H. Mah, and E. Gutter, K o l l o i d z e i t s c h r i f t , 155, 1-9 (1957). 18. G. B. RSnby i n 'Handbuch der P f l a n z e n p h y s i o l o g i e , ' Ed. R. W. B e r l i n , V o l . 6, 268-304, Spr i n g e r - V e r l a g , 1958. 19. S. P. Rowland, Text. Chem. C o l o u r i s t , 4_, 204-210 (1972). 20. G. MacLachlan, Trends Biochem. S c i . , 18, 226-228 (1977). 21. J . R. C o l v i n , Tappi, 60, 59-62 (1977). 240 22. S. P. Rowland, ACS Symp. Ser., 49, 20-45 (1976). 23. C. M. Hunt, R. L. B l a i n e , and J . Rowan, Text. Res. J . , 20, 43-50 (1950). 24. F. H. F o r z i a t i , R. M. Brownell, and C. M. Hunt, J . Res. Nat. Bur. Stand., 50, 139-145 (1953). 25. N. I. Klenkova, and G. P. I v a s k i n , Zh. P r i k l , Khim, 36^ , 398-408 (1963). 26. B. R. P o r t e r , and M. L. R o l l i n s , J . Appl. Polym. S c i . , 16, 217-236 (1972). 27. S. P. Rowland, E n c y c l . Polym. S c i . Technol., Suppl. 1, 146-175 (1976). 28. J . E. Stone, and A. M. S c a l l a n , C e l l . Chem. Technol., 2, 343-358 (1968). 29. S. P. Rowland, and N. R. B e r t o n i e r , Text. Res. J.,'46, 770-775 (1976). 30. J'. Tankard, J . Text. I n s t . , 28, T263-T266 (1937). 31. E. Heymann, and G. C. M c K i l l o p , J . Phys. Chem.,45, 195-199 (1941). 32. D. V. Bien, and A. B. Lindenberg, Compt. Rend. Acad. S c i . , 257, 2283-2286 (1963). 33. C. Heinegard, T. Lindstrom, and C. Soremark, C o l l o i d I n t e r f a c e S c i . , (Proc. 50th I n t . Conf.), 4, 139-155 (1976). 34. R. Meredith i n 'Moisture i n T e x t i l e s , ' Ed. R. Meredith, and J . W. S. Hearle, 141-159, W i l e y - I n t e r s c i e n c e , New York, 1960. 35. W. H. Rees, i b i d . , 51-58. 36. F. C. Magne, and E. L. Skau, Text. Res. J . , _22, 748-756 (1952). 37. D. A. I. Goring, Pulp Pap. Mag. Can., 67, T519-T524 (1966). 38. Y. Ogiwara, M. Kobute, S. Hayoshi, and N. Mitomo, J . Appl. Polym. S c i . , 13, 1689-1695 (1969). 39. T. F. C h i l d , Polymer, 13, 259-264 (1972). 40. M. F. F r i o x , and R. Nelson, Macromolecules, 8, 726-730 (1975). 41. W. Drost-Hansen, Ind. Eng. Chem., 61, 10-47 (1969). 42. E. J . Roberts, J . L. Bose, and S. P. Rowland, Text. Res. J . , ^ 2 , 217-221 (1972). 43. S. P. Rowland, and E. J . Roberts, J . Polym. S c i . , A-1, 10, 2447-2461 (1972). R. M. Marupov, P. Kh. Bobodzhanov, N. V. K o s t i n a , and A. B. Shapiro, B i o f i z i k a , 21, 825-828 (1976). M. F r o i x , Chem. Scr., 10, 190-192 (1976). 'CRC Handbook of Chemistry and P h y s i c s , ' 58th E d i t i o n , F-215, 1977. A. V. K u l i k o v , and G. I. L i k h t e n s h t e i n , Adv. Mol. Relaxn. I n t . P r o c , 10, 47-79 (1977). L. J . B e r l i n e r , Ed., 'Spin L a b e l i n g . Theory and A p p l i c a t i o n s , ' Academic, New York, 1976. CHAPTER VI GLYCOPROTEINS VIA: I n t r o d u c t i o n In t h i s chapter a modest beginning i s made towards answering the questions of how carbohydrates attached to p r o t e i n r e o r i e n t i n s o l u t i o n s , i n gels and at membranes; to what extent conformations are r e s t r i c t e d by hydrogen bonding and s t e r i c b a r r i e r s (one example i s already known of an o l i g o s a c c h a r i d e appearing to ' f o l d back' i n t o a b i n d i n g s i t e presumed to be i n the p r o t e i n part of the molecule''") ; how t h i s a f f e c t s saccharides at non-reducing t e r m i n i of short or long p r o s t h e t i c groups; and, u l t i m a t e l y , what relevance t h i s has to t h e i r f u n c t i o n . I f spe c t r o s c o p i c l a b e l s are to be of use i n p r o v i d i n g answers to these and many other r e l a t e d questions i t i s c l e a r that new chemical methods that are s p e c i f i c , m i l d and g e n e r a l l y a p p l i c a b l e must be devised. The present chapter i s addressed to t h i s problem. The chemistry described i s a p p l i e d to s p i n l a b e l s , but may prove e q u a l l y u s e f u l i n flu o r e s c e n c e , nmr or other forms of spectroscopy. E q u a l l y , chemical m o d i f i c a t i o n has proved to be important i n i t s own r i g h t (Chapter I F ) ; i t may be a n t i c i p a t e d that, i n f u t u r e , s p e c i f i c methods to change the r e s i d e n t charge, h y d r o p h i l i c i t y , or a v a i l a b l e f u n c t i o n a l groups i n carbohydrates w i l l be used to a l t e r the p r o p e r t i e s of complex systems such as the c e l l . For the purposes of t e s t i n g the methods described, two very d i f f e r e n t , commercially a v a i l a b l e g l y c o p r o t e i n s were c h o s e n — c a l f f e t u i n (56), a serum 242 243 g l y c o p r o t e i n , and bovine submaxillary mucin (57) , which i s assumed to p l a y a part i n l u b r i c a t i n g and p r o t e c t i n g the cow's mouth. The experiments are a l s o extended to l a b e l l i n g of the outer surface of human ery t h r o c y t e s and of carbohydrates present i n t i s s u e s e c t i o n s from human colon. I t i s proposed a f t e r i n t r o d u c i n g the m a t e r i a l s used, to di s c u s s the chemistry and s e l e c t i v i t y of two r e a c t i o n s before r e t u r n i n g to d i s c u s s the s i g n i f -icance of the e.s.r. data. VIB: BSM and F e t u i n 2 Bovine su b m a x i l l a r y mucin (BSM) (57) i s a g l y c o p r o t e i n of molecular weight 4 x 10 6 (lower values have been repo r t e d , probably owing to p a r t i a l degradation) which behaves as a h i g h l y hydrated r i g i d rod upon the surface of which carbohydrate i s evenly and densely d i s t r i b u t e d . Two f r a c t i o n s , "major" and "minor" have been d i s t i n g u i s h e d , and Immunoelectrophoresis shows two arcs f o r the major and three f o r the minor component. The m a j o r i t y of 2 6 the major component, i s present as a d i s a c c h a r i d e , NANA——GalNAc, O - g l y c o s i d i c a l l y bound to s e r i n e or threonine; other, l a r g e r o l i g o s a c c h a r -ides are probably a l s o present i n small q u a n t i t i e s . The s i a l i c a c i d occurs as N - a c e t y l , N - g l y c o l y l , N - a c e t y l - 7 - 0 - a c e t y l , N - a c e t y l - 8 - 0 - a c e t y l , N - a c e t y l - 7 , 8 - d i - 0 - a c e t y l and N - a c e t y l - t r i - O - a c e t y l ( p o s i t i o n s of O-acetyl s u b s t i t u e n t s unknown) d e r i v a t i v e s . I t i s hot known how these s u b s t i t u t i o n p a t t e r n s and, indeed, the d i f f e r e n t o l i g o s a c c h a r i d e s are r e l a t e d to the v a r i o u s f r a c t i o n s d i s t i n g u i s h e d by Immunoelectrophoresis. The p r i n c i p a l aminoacids are a l a n i n e , g l y c i n e , p r o l i n e , s e r i n e , and threonine. I t i s thought that the m a j o r i t y of the p r o t e i n comprises 3 repeating u n i t s of about 28 aminoacids , though some homologous s u b s t i t u -t i o n probably occurs. During the course of s t u d i e s on the aminoacid make-up of BSM, Pigman and co-workers, l a t t e r l y the p r i n c i p a l researchers 244 i n the f i e l d , found that the enzymic or a c i d - c a t a l y s e d removal of s i a l i c a c i d enabled enzymic p r o t e o l y s i s to proceed to a con s i d e r a b l y g r e a t e r 3 extent , p r o v i d i n g f u r t h e r evidence of the p r o t e c t i v e r o l e of carbohydrate a l l u d e d to i n Chapter IF. Fe t u i n (56), an a - g l o b u l i n , d i s p l a y s many of the feat u r e s common to 4 gl o b u l a r p r o t e i n s . I t can comprise up to 45% of the t o t a l p r o t e i n i n f o e t a l c a l f serum at concentrations up to 10 mg/ml, so i s r e l a t i v e l y e a s i l y obtained, i n p r a c t i c a l q u a n t i t i e s , u s u a l l y by ammonium sulphate p r e c i p i t a -t i o n . I t s molecular weight i s about 4.8 x 10^ and i t s a c i d i c p r o p e r t i e s are almost e n t i r e l y due to i t s s i a l i c a c i d content, which a l s o seems to be re s p o n s i b l e f o r a c e r t a i n microheterogeneity (though no 0- a c e t y l s u b s t i t u -ents are p r e s e n t ) , and, perhaps, the apparent dependence of the molecular shape on i o n i c s t r e n g t h ^ . Most common aminoacids are present, i n c l u d i n g c y s t i n e but not c y s t e i n e , i n a s i n g l e polypeptide chain. About 20% of the dry weight i s 5 6 carbohydrate, present as three d i f f e r e n t o l i g o s a c c h a r i d e s ' . Two of these are 0- l i n k e d to s e r i n e and threonine: NANA: 2 3 •Gal-1 3 -GalNAc-6| 1 -Ser, Thr a a 2|a NANA 2 3 1 3 1 NANA^-^Gal-^Ga lNAc- -Ser, Thr and the other N - g l y c o s i d i c a l l y l i n k e d to asparagine: 245 NANA a|2 3 Gal [ l I* GlcNAc 1 NANA—^GalNAc-—GlcNAc-—Man 3 a l l 1 4 1 4 1 Mart=—^GlcNAc^— 1 GlcNAc-—Asn N A N A 2 - A ; a l N A c ^ - A ; i cNAc^-^Man Each f e t u i n molecule contains three O - g l y c o s i d i c a l l y l i n k e d and three N - g l y c o s i d i c a l l y l i n k e d o l i g o s a c c h a r i d e s . The i n v i v o r o l e of f e t u i n i s s t i l l unknown (although i t has been suggested^ that i t may play a part i n r e g u l a t i n g the immune response), but i n v i t r o a c t i v i t i e s and uses have g been found: f e t u i n s t i m u l a t e s lymphocytes , i n h i b i t s e r y t h r o c y t e agglu-9 t i n a t i o n by i n f l u e n z a v i r u s , has been used as f e t u i n - c e l l u l o s e as an a f f i n i t y m a t r i x f o r neuraminidase"^, and as f e r r i t i n - f e t u i n to i d e n t i f y the l o c a t i o n of neuraminidase a c t i v i t y under the e l e c t r o n microscope"'""'". A s i a l o f e t u i n g l y c o p e p t i d e s , conjugated to other p r o t e i n s , have been shown 12 to cause r a p i d removal of the l a t t e r from c i r c u l a t i o n by the l i v e r , and indeed most of the abovementioned a c t i v i t i e s are i n some measure a s s o c i -13 ated w i t h s i a l i c a c i d , of which i t has a l s o been used as a source, though now superseded by Chinese b i r d ' s nest and the s i a l i c a c i d polymers found i n b a c t e r i a l c e l l w a l l s . 246 H 0. C0 2 H (13) s i a l i c a c i d s (13a) R' = Ac, R = H : N - a c e t y l neuraminic a c i d (NANA) R N H / I O R H OH OR H VIC: Carbodiimide Coupling Amongst the saccharides present i n t y p i c a l g l y c o p r o t e i n s , two f u n c t i o n a l groups suggest themselves as p o s s i b l e s i t e s f o r s p e c i f i c modi-f i c a t i o n : amine and carboxylate. The presence of too many groups of s i m i l a r r e a c t i v i t y moderates against the use of h y d r o x y l , at l e a s t w i t h p r e s e n t l y a v a i l a b l e aqueous methods. Unfortunately most or a l l of the amine f u n c t i o n a l groups present i n glycoconjugates are a c e t y l a t e d , g r e a t l y reducing the n u c l e o p h i l i c i t y of the n i t r o g e n ; a f u r t h e r o b j e c t i o n to a t t a c k at C-2 n i t r o g e n i n hexosamines i s that these are r a t h e r too l i b e r -a l l y s c a t t e r e d as N-acetyl glucosamine and galactosamine. The most obvious s i t e f o r m o d i f i c a t i o n i s at C - l of s i a l i c a c i d , which i s the only carbohydrate c a r b o x y l a t e , i s expected to be a c c e s s i b l e from s o l u t i o n , and, furthermore, places the l a b e l at a s i t e known to be a s s o c i a t e d w i t h essen-13 t i a l b i o l o g i c a l f u n c t i o n s . One important disadvantage accrues: the t o t a l charge of the molecule w i l l be a l t e r e d as a n i o n i c carboxylates are covered. However, t h i s can be turned to advantage by the a s s e r t i o n that a means of n e u t r a l i s i n g C - l without e f f e c t i n g other changes would enable determination of the r61e of the negative charge i n b i o l o g i c a l a c t i v i t y . Carbodiimides, p a r t i c u l a r l y d i c y c l o h e x y l c a r b o d i i m i d e (DCC),have been wid e l y used f o r peptide s y n t h e s i s i n organic media, and more r e c e n t l y i o n i c analogues such as N-ethyl-N'-dimethyl-aminopropyl carbodiimide hydro-c h l o r i d e (EDC) (20) and N-cyclohexyl-N-(2-morpholinoethyl) carbodiimide 247 p-toluenesulphonate (CMC) (21) have been introduced to promote the forma-t i o n of amide bonds i n aqueous media. An a d d i t i o n a l advantage CMC (21) H CH 3 CH 2 N = C = N ( C H 2 ) 3 N(CH 3) 2 Cl" EDC (20) N = C=N DCC accompanying t h e i r use i s the w a t e r - s o l u b i l i t y of the urea byproduct that i s otherwise o f t e n d i f f i c u l t to separate from the d e s i r e d product. Although r e a c t i o n s between amines (RNH2) and c a r b o x y l i c a c i d s (R'C02H) i n 14 the presence of DCC are thought to progress through an ' a c t i v a t e d e s t e r ' of the form recent work has shown that aqueous r e a c t i o n s a s s i s t e d by EDC, which experiences r i n g - c h a i n tautomerism"''^' ^ , i n s t e a d proceed at a c i d pH as f o l l o w s : 0 II OCR HN R NR 248 The optimum pH f o r the r e a c t i o n has been found to l i e between 4.5 and 6.0. Recent use has been made of EDC i n preparing s y n t h e t i c g l y c o p r o t e i n s u s i n g 1-amino-l-deoxy"'"^ and l-amino-2-acetamido.'''^ sugars coupled to peptide c a r b o x y l groups, as w e l l as a l d o n i c a c i d s coupled to amine f u n c t i o n a l i t i e s of protexns The general s t r a t e g y adopted i n v o l v e s comparing r e s u l t s obtained using the two n a t i v e g l y c o p r o t e i n s w i t h those of c o n t r o l experiments us i n g m a t e r i a l s that have been d e s i a l y l a t e d using e i t h e r a c i d or neuraminidase. S i a l i c a c i d was checked at each stage using a combination of two c o l o u r -i m e t r i c assay procedures: the t h i o b a r b i t u r i c a c i d (TBA) method f o r f r e e s i a l i c a c i d o n l y , and the p e r i o d a t e - r e s o r c i n o l method, which can be adapted e i t h e r f o r k e t o s i d i c a l l y bound s i a l i c a c i d only (PRBA), or f o r both f r e e and bound m a t e r i a l (PRT). Where i t was necessary to separate macromolecular m a t e r i a l from f r e e s i a l i c a c i d , t h i s was achieved us i n g f i l t r a t i o n through microporous membranes, g e l f i l t r a t i o n or d i a l y s i s . 249 VID: Carbodiimide-Mediated L a b e l l i n g ( i ) BSM BSM (57) was f i r s t de-O-acetylated (the presence of O-acetyl sub-21 s t i t u e n t s i n s i a l i c a c i d may i n h i b i t neuraminidase-catalysed cleavage ) by treatment w i t h 0.1M potassium hydroxide followed by n e u t r a l i s a t i o n w i t h 0.05M s u l p h u r i c a c i d . This m a t e r i a l was found to c o n t a i n about 8.5% s i a l i c a c i d . I t s spectrum a f t e r l a b e l l i n g u s i n g a mixture of EDC (20) and 4-amino-2,2,6,6-tetramethylpiperidine-l-oxyl (2) at pH 5.0 i s shown i n Figure V l - l a . A d d i t i o n of calcium ions d i d not a l t e r the lineshape. S i g n a l - t o - n o i s e was found to be optimised by using l a r g e (100-fold) excesses of l a b e l and c o u p l i n g reagent. The r e a c t i o n was complete a f t e r 3 hours gentle shaking at room temperature, during which a brown granular p r e c i p -i t a t e appeared on occasion. This could be removed by c e n t r i f u g a t i o n using a bench c e n t r i f u g e , and, as i t contained no s i a l i c a c i d , was thought to be an i m p u r i t y . L a b e l l i n g at C - l would be expected to render a s i a l i c a c i d 22 residue r e s i s t a n t to V i b r i o c h o l e r a neuraminidase , and i t was found that the spectrum remained unchanged a f t e r treatment of the l a b e l l e d BSM w i t h the enzyme and removal of products of low molecular weight. Figure V l - l b shows the spectrum of BSM f o l l o w i n g d e s i a l y l a t i o n w i t h a c i d and l a b e l l i n g under the same c o n d i t i o n s , i n d i c a t i n g a r a t h e r s m a l l amount of l a b e l l i n g at n o n - s i a l i c c a r b o x y l a t e s . Another c o n t r o l experiment was performed: the de-O-acetylated BSM was reacted w i t h EDC i n the absence of l a b e l but under otherwise i d e n t i c a l c o n d i t i o n s . The main purpose of t h i s was to t e s t the r e l i a b i l i t y of the c o l o u r i m e t r i c assays i n the presence of unknown proporti o n s of chemically modified s i a l i c a c i d s . Figure VI-2 shows the r e a c t i o n scheme and assay r e s u l t s using the PRT method ( t o t a l s i a l i c a c i d ) . Aside from the experimental e r r o r of ± ^ 10%, anomalies are evident; i t 250 Figure VI-1: Esr spectra of f e t u i n and BSM l a b e l l e d using the EDC coupling procedure, (a)BSM; (b)asialo-BSM; ( c ) f e t u i n ; ( d ) a s i a l o f e t u i n ; ( e ) f e t u i n l a b e l l e d at p r o t e i n amine groups using EDC with l a b e l ( 4 ) (a-d a l l use l a b e l ( 2 ) ) . BSM(EDC) neuraminidase > BSM(EDC)(-sa) 4.79 EDC BSM — - — ^ d e - O - a c e t y l a t e d BSM• 8.46 BSM(-sa) 9.52 7.48 f i l t r a t e 6.37 re t e n t a t e 0.43 EDC,SLNH„ . _„.. C I neuraminidase - 2—^ BSM-SL 2.94 » BSM-SL(-sa) 5.44 f i l t r a t e 7.59 re t e n t a t e 0.14 f i l t r a t e 3.63 r e t e n t a t e 1.13 Figure VI-2: Experimental scheme f o r BSM spin l a b e l l i n g and assay using the PRT method f o r t o t a l s i a l i c acid(sa)(quoted as % dry weight of g l y c o p r o t e i n ) . i s not c l e a r , f o r example, why the apparent s i a l i c a c i d content ( f r e e + enzymically released) increased i n both the l a b e l l e d BSM and the EDC-tr e a t e d c o n t r o l a f t e r treatment w i t h neuraminidase. The EDC treatment tended to reduce the apparent content, and treatment w i t h both EDC and n i t r o x i d e diminished i t s t i l l f u r t h e r . However, these e f f e c t s , being aside from the main purpose of the experiments,were not f u r t h e r i n v e s t i -gated. Instead, the f o l l o w i n g computations were considered to be j u s t i -f i a b l e : by s u b t r a c t i n g the p r o p o r t i o n of t o t a l s i a l i c a c i d hydrolysed enzymically from the l a b e l l e d m a t e r i a l ( . * ,^  x 100 = 66.7%) from the w • 44 J p r o p o r t i o n hydrolysed from the EDC-treated .control ( ^ * x 100 = 85.2%) (0.43 and 1.13 were too small to be used), a maximum extent of l a b e l l i n g of 18.5% can be estimated; that i s , assuming the enzyme to be f u l l y e f f i c i e n t i n the absence of C - l s u b s t i t u e n t s on s i a l i c a c i d , about one f i f t h of the s i a l i c acids present were l a b e l l e d . That a greater propor-t i o n of these were a c t u a l l y modified (probably forming the s t a b l e N-acyl 14 urea ) could be shown by e l e c t r o p h o r e s i s , s i n c e most of the negative charges of BSM are due to s i a l i c a c i d . The l a b e l l e d BSM upon e l e c t r o -phoresis on c e l l u l o s e acetate s t r i p s f a i l e d to migrate from the o r i g i n ; a standard BSM sample migrated towards the anode (+). S t a i n i n g i n t e n s i t y (by the c a t i o n i c dye a l c i a n blue) was a l s o g r e a t l y reduced, a f u r t h e r i n d i c a t i o n that a m a j o r i t y of c a r b o x y l f u n c t i o n a l i t i e s had reacted, ( i i ) F e t u i n U n l i k e BSM, f e t u i n contains no O-acetylated s i a l i c a c i d s , and was used as purchased. I t was found to c o n t a i n a, 8.0% s i a l i c a c i d . The spectrum of the l a b e l l e d m a t e r i a l i s shown i n Figure V I - l c , and that of a s i a l o f e t u i n , t r e a t e d s i m i l a r l y , i n Figure V l - l d . Both lineshapes were 2+ i n s e n s i t i v e to the presence of Ca . Reaction c o n d i t i o n s were as f o r 253 BSM. In t h i s case a very s i g n i f i c a n t background s i g n a l i s present, so a r a t h e r more complex matrix of experiments and c o n t r o l experiments was attempted. Instead of se p a r a t i n g high molecular weight c o n s t i t u e n t s from t h e i r l i g h t e r counterparts f o r the purposes of assaying s i a l i c a c i d , d i f f e r e n t assays were used upon a l i q u o t s from the same s o l u t i o n s to detect f r e e , bound or t o t a l q u a n t i t i e s . The experiments are summarised i n Figure VI-3, along w i t h the r e s u l t s of the assays. Both enzymic and a c i d i c d e s i a l y l a t i o n were used i n case the l a t t e r should be causing the exposure of new carboxylate groups, as by l i b e r a t i n g a s p a r t i c a c i d from asparagine; the r e s u l t s , however, were i d e n t i c a l i n a l l r e s p e c t s . A neuraminidase-catalysed h y d r o l y s i s was a l s o performed i h the presence of f r e e n i t r o x i d e l e s t the l a t t e r i n t e r f e r e w i t h the former, but t h i s d i d not occur e i t h e r . As w i t h BSM, l a b e l l i n g caused a decrease i n apparent s i a l i c a c i d content; treatment w i t h neuraminidase caused the measured value ( f r e e + bound) to increase again, though not to i t s o r i g i n a l value. In t h i s case assays were coupled w i t h esr i n t e g r a t i o n of l a b e l l e d f e t u i n and a s i a l o f e t u i n , which i n d i c a t e d that i n the case of the n a t i v e m a t e r i a l , the s i g n a l could be accounted f o r i f ^  18% of the s i a l i c a c i d r esidues had been l a b e l l e d . However, < 1% of the s i g n a l i n t e n s i t y of the a s i a -l o f e t u i n could be accounted f o r by i t s r e s i d u a l s i a l i c a c i d (about 3% of that present i n the n a t i v e m a t e r i a l ) . I t s s i g n a l i s h a l f as intense as that of the n a t i v e m a t e r i a l ! Thus i t i s l i k e l y that the s i g n a l due to n a t i v e f e t u i n does not a r i s e e x c l u s i v e l y from n i t r o x i d e s i a l a m i d e s . The 'background' s i g n a l may best be accounted f o r by p r o t e i n c a r b o x y l a t e s i d e chains; about twice as many a s p a r t i c and glutamic a c i d residues per weight p r o t e i n occur i n f e t u i n (17%) as i n BSM (9%) (some present, however, as amides) and i n a d d i t i o n the former has fewer s i a l i c a c i d r e s i d u e s . I t i n t e g r a t i o n 7.46 H?Q8l Figure VI-3: Experimental scheme f o r f e t u i n s p i n l a b e l l i n g and assay, using the PRT method • f o r t o t a l s i a l i c acid(sa) (ODi > t h e P R B A method f o r bound s i a l i c acid(|—[x]) , and the TBA method f o r f r e e s i a l i c acid(unenclosed f i g u r e s ) . Figures represent % dry weight of g l y c o p r o t e i n . 255 was hoped that s i n c e the pK & of s i a l i c a c i d s l i e s at about 2.5, w h i l e those of a s p a r t i c and glutamic a c i d s i d e chain carboxylates are higher (3.8 and 4.2 r e s p e c t i v e l y ) reducing the r e a c t i o n pH might enhance i t s s e l e c t i v i t y . At pH 3, however, the y i e l d was too low to be u s e f u l , and below t h i s pH there i s a danger that s i a l i c a c i d w i l l be l o s t . Reducing the pr o p o r t i o n s of c o u p l i n g agent and/or l a b e l achieved nothing more than a reduced y i e l d ; use of CMC (21) i n s t e a d of EDC (20) as c o u p l i n g reagent gave s i m i l a r r e s u l t s . These f a c t o r s , however, are i n s u f f i c i e n t to s a t i s f a c t o r i l y r a t i o n a l i s e the d i f f e r e n c e i n s e l e c t i v i t y of the method i n the two g l y c o -p r o t e i n s , and i t may be that we are observing another m a n i f e s t a t i o n of the p r o t e c t i v e f u n c t i o n that has been a s c r i b e d to carbohydrate i n mucous g l y c o p r o t e i n s ; that i s , access to aminoacid s i d e chains by reagents i n s o l u t i o n may be s t e r i c a l l y r e s t r i c t e d by the dense carbohydrate c o a t i n g , even i n the absence of s i a l i c a c i d . In f e t u i n , t h i s i s u n l i k e l y to be the case, as only 6 o l i g o s a c c h a r i d e s . a r e present per p r o t e i n monomer. As a s i d e l i n e , f e t u i n was s u c c e s s f u l l y l a b e l l e d at p r o t e i n amine groups by a r e v e r s a l of the above method; under the same experimental c o n d i t i o n s 4 - c a r b o x y l - 2 , 2 , 6 , 6 - t e t r a m e t h y l p i p e r i d i n e - l - o x y l (4) was reacted w i t h f e t u i n i n the presence of EDC. The spectrum of the product, a f t e r removal of unreacted l a b e l by exhaustive d i a l y s i s , i s shown i n Figure V l - l e . Used at h i g h d i l u t i o n of p r o t e i n (to avoid c r o s s - l i n k i n g ) t h i s may be a l a b e l l i n g method worthy of f u r t h e r i n v e s t i g a t i o n , ( i i i ) Non-glycoprotein C o n t r o l M a t e r i a l s In the l i g h t of these r e s u l t s , s i m i l a r experiments were performed on m a t e r i a l s known to c o n t a i n a n i o n i c groups of non-carbohydrate o r i g i n : bovine and human serum albumins (BSA and HSA) (carboxyls) and c a l f thymus DNA (deoxyribonucleic acid) (phosphates). A l l of these m a t e r i a l s , 256 t r e a t e d to the same l a b e l l i n g procedure, d i s p l a y e d s u b s t a n t i a l s i g n a l s , confirming that the method as employed f a i l e d to d i s t i n g u i s h between phos-phates, aminoacid s i d e - c h a i n c a r b o x y l a t e s , and s i a l i c a c i d c a r b o x y l a t e s . I-carrageenan, however, which contains sulphate s u b s t i t u e n t s , was not l a b e l l e d under the same c o n d i t i o n s , i n d i c a t i n g that h a l f - s u l p h a t e e s t e r s present i n mucus or connective t i s s u e polysaccharide would not c o n t r i b u t e a background s i g n a l . ( i v ) Human Tissue Sections and Eryth r o c y t e s The method was found to be s u f f i c i e n t l y m i l d to be a p p l i c a b l e to both t i s s u e s e c t i o n s and e r y t h r o c y t e s . The spectrum of a formaldehyde-f i x e d 5y l a t e r a l s e c t i o n of human co l o n , mounted on a 9 * 29 mm cover s l i p and de-O-acetylated using 0.5% potassium hydroxide i n 70% ethanol f o r 30 minutes at room temperature, i s shown i n Figure VI-4a, and VI-4b shows an a c i d - d e s i a l y t e d s e c t i o n from the same colon l a b e l l e d under the same con-d i t i o n s . Unreacted reagents and products of sm a l l molecular weight are e a s i l y removed by washing. The background s i g n a l i n t h i s case could have a r i s e n from uronic a c i d s i n connective t i s s u e as w e l l as aminoacid carboxy-l a t e s , and phosphates. The s i g n a l could be completely abolished by p r i o r . methylation of the s e c t i o n w i t h methanol i n HC1. Other types of t i s s u e (Brunner's gland, bowel) were a l s o t r i e d , but poorer s i g n a l i n t e n s i t y r e s u l t e d . I t i s hoped that t h i s type of chemistry can be a p p l i e d to h i s t o c h e m i c a l s t a i n i n g procedures, where methods which d i s t i n g u i s h , f o r example, phosphates from carboxylates are i n great demand. A s i n g l e experiment using the amine-containing dye t h i o n i n e 257 Figure VI-4: Esr spectra of l a b e l l e d t i s s u e s e c t i o n s and ery t h r o c y t e s . (a)5u s e c t i o n of human colon; ( b l d e s i a l v l a t s e c t i o n ; ( c ) e r y t h r o c y t e s ; ( d ) d e s i a l y l a t e d e r y t h r o c y t e s 258 w i t h human colon and the same c o n d i t i o n s as f o r s p i n l a b e l l i n g i n d i c a t e d c o n s i d e r a b l e p o t e n t i a l , as n u c l e i c a c i d phosphates were, r a t h e r s u r p r i s i n g l y , s t a i n e d much more i n t e n s e l y than were mucin ca r b o x y l a t e s . Unfixed human ery t h r o c y t e s were reacted under somewhat m i l d e r con-d i t i o n s , at pH 6.0 f o r 1 hour i n 0.154M ( p h y s i o l o g i c a l ) s a l i n e on a 'rocker', r e s i d u a l l a b e l and coupling reagent being removed by repeated c e n t r i f u g a -t i o n and resuspension i n s a l i n e . Removal of s i a l i c a c i d was accomplished using neuraminidase. The s p e c t r a of the packed c e l l s and t h e i r d e s i a l y -l a t e d c o n t r o l are shown i n Figures VI-4c and d. Again a r a t h e r substan-t i a l background s i g n a l i s i n evidence owing presumably to l i p i d head group phosphates and the aminoacid carboxylates of membrane p r o t e i n s . C e l l l y s i s was minimal. A d d i t i o n of potassium f e r r i c y a n i d e to a c o n c e n t r a t i o n of 0.3M caused the s i g n a l to be broadened beyond the d e t e c t i o n l i m i t , i n d i c a t i n g that l a b e l s were present e x c l u s i v e l y on the outer surface of the c e l l s ; when the membranes were separated by c e l l l y s i s and c e n t r i -f u g a t i o n , the s i g n a l was found t h e r e i n , and none was present i n the super-natant. I t has been shown that e r y t h r o c y t e s t r e a t e d w i t h CMC (21) a f t e r 23 three hours have zero surface charge ; the net negative charge of the 24 normal e r y t h r o c y t e i s a s c r i b a b l e to s i a l i c a c i d . In a d d i t i o n , EDC has 25 been shown to i n t e r f e r e w i t h o x i d a t i v e phosphorylation , which renders i t s use u n d e s i r a b l e i n c e r t a i n cases. VIE: P e r i o d a t e O x i d a t i o n — R e d u c t i v e Amination In view of the l a c k of s p e c i f i c i t y of the carbodiimide c o u p l i n g method, a p r e l i m i n a r y study was c a r r i e d out* using an a t t r a c t i v e a l t e r n a t i v e procedure. Period a t e o x i d a t i o n , which r e l i e s on the presence of a 1 , 2 - d i o l , *In c o l l a b o r a t i o n w i t h M. A. B e r n s t e i n . 259 26 has been widely a p p l i e d to polysaccharide sequencing and d e r i v a t i s a t i o n ( I t was avoided i n the s p i n l a b e l l i n g experiments w i t h polysaccharides because i n t r a c y c l i c cleavage i s expected to a l t e r the motional p r o p e r t i e s of polysaccharide chains.) More r e c e n t l y i t has been used to modify c e l l 27 surface carbohydrates . The present a p p l i c a t i o n r e l i e s on the f o l l o w i n g two-step r e a c t i o n : | " 0 H -IPJ FC = 0 R N « 2 f-CHNHR L - , C - ° H l>C = 0 B H j C N ~ | _ ^ In complex carbohydrates, more than one unsubstitued v i c i n a l d i o l i s 28 l i k e l y to be present. However, i t i s e s t a b l i s h e d that owing to the intermediacy of a c y c l i c periodate adduct i n the o x i d a t i o n step, r e a c t i v -i t i e s vary i n the order e x t r a c y c l i c > i n t r a c y c l i c c i s - > i n t r a c y c l i c t r a n s -In g l y c o p r o t e i n s , t h e r e f o r e , the e x t r a c y c l i c t r i o l of s i a l i c a c i d i s most s u s c e p t i b l e to a t t a c k ( c r e a t i n g a C7 or C8 aldehyde); the i n t r a c y c l i c c i s - d i o l s of mannose, galactose and fucose are next most r e a c t i v e . There i s evidence that i t i s p o s s i b l e , using very mi l d c o n d i t i o n s , to s e l e c t i v e l y 29-39 o x i d i s e s i a l i c a c i d . On the b a s i s of these previous s t u d i e s , the f o l l o w i n g r e a c t i o n c o n d i t i o n s were chosen: 5 moles of sodium metaperio-date per mole of s i a l i c a c i d at 273 K f o r 35 minutes. The periodate can subsequently be destroyed using potassium i o d i d e and sodium t h i o s u l p h a t e . A f t e r d e s a l t i n g , the r e d u c t i v e amination step proceeds using a l a r g e excess of l a b e l (2) w i t h sodium cyanoborohydride at pH 8 f o r 2 hours at room temperature. The spectrum of f e t u i n l a b e l l e d i n t h i s f a s h i o n i s shown 260 together w i t h that of i t s unoxidised counterpart i n Figure VT-5a and b. Assays and double i n t e g r a t i o n i n d i c a t e d that 20 - 30% of s i a l i c a c i d l a b e l l e d . I t i s appropriate to mention here that s i n c e the i n c e p t i o n of these experiments two other groups have reported the use of s i m i l a r methods to 34 s p i n l a b e l the carbohydrate component of immunoglobulins. In one case o x i d a t i o n was allowed to proceed f o r 8 hours at room temperature; i n the 40 41 other ' f o r 16 - 24 hours at room temperature or i n the c o l d . In a d d i t i o n one group had d i f f i c u l t i e s w i t h adsorption of l a b e l and r e v i s e d r e s u l t s had to be published. The experiments described i n t h i s s e c t i o n together w i t h other l i t e r a t u r e r e p o r t s i n d i c a t e that under the c o n d i t i o n s described i n these two r e p o r t s residues other than s i a l i c a c i d would have been o x i d i s e d . One group e x p l i c i t l y s t a t e d that only about 60% of the l a b e l was attached to s i a l i c a c i d . This more s p e c i f i c chemistry has been extended i n s e v e r a l d i r e c -42 43 .tions ' : red blood c e l l s have been s i m i l a r l y d e r i v a t i s e d w i t h greater s e l e c t i v i t y than i n the case of the EDC amidation. In a d d i t i o n , p e r i o -date has been shown to be r a t h e r more v e r s a t i l e , s i n c e a f t e r removal of s i a l i c a c i d , f o r example, i n f e t u i n , exposure to periodate f o r s l i g h t l y longer periods enables galactose residues newly exposed at the non-reducing t e r m i n i to be o x i d i s e d and r e d u c t i v e l y aminated. F i n a l l y , these may be yet more s e l e c t i v e l y d e r i v a t i s e d by o x i d a t i o n i n the presence of the enzyme galactose oxidase, which e f f e c t s the transformations 261 Figure VI-5: Esr spectra of ( a ) f e t u i n l a b e l l e d by periodate o x i d a t i o n followed by r e d u c t i v e amination; (b)unoxidised n a t i v e f e t u i n perfused with cyanoborohydride and l a b e l ( 2 ) ; ( c ) f e t u i n as a, immobilised on Sepharose 4B. The outer parts of the spectrum are a l s o a m p l i f i e d 10 x showing a broader underlying s i g n a l . 262 H HO(NHAc) H HO (NHAc) again followed by r e d u c t i v e amination. I t i s l i k e l y that enzymic methods of t h i s k i n d , because of t h e i r mildness and s p e c i f i c i t y , w i l l f i n d i n c r e a s -i n g f u t u r e use i n chemical m o d i f i c a t i o n of complex mixtures. VIF: The M o b i l i t y of Sugars i n Glycoproteins C o r r e l a t i o n times c a l c u l a t e d f o r the v a r i o u s experiments discussed i n t h i s chapter are c o l l e c t e d together i n Table VI-1. The f i r s t p o i n t to be made about the sp e c t r a concerns t h e i r s i m i l a r i t y where d i f f e r e n t chemical methods and even d i f f e r e n t systems have been used. No superimposition of spe c t r a w i t h d i f f e r e n t l i n e w i d t h s can be detected i n the EDC-amidated samples d e s p i t e the known heterogeneity of l a b e l l e d s i t e s ; the removal of s i a l i c a c i d and l a b e l l i n g at the penultimate sugar using the galactose oxidase or periodate o x i d a t i o n followed by r e d u c t i v e amination makes l i t t l e d i f f e r e n c e to the r a t e of tumbling of the l a b e l (and can even in c r e a s e i t ) ; indeed a l l the macromolecular samples whether p r o t e i n s l a b e l l e d at aminoacid carboxylates (HSA, BSA), f e t u i n or BSM l a b e l l e d at s i a l i c a c i d or ga l a c t o s e , or f e t u i n l a b e l l e d at l y s i n e E-NH2, show remark-ably s i m i l a r T values. The value of T c a l c u l a t e d f o r 'whole-molecule' 263 Table VI-1: C o r r e l a t i o n times of n i t r o x i d e s attached to g l y c o p r o t e i n s . Except where i n d i c a t e d , l a b e l (2) was used. m a t e r i a l method x ( e x p e r i -mental)* T (ns) (Stokes l a w ) * * (ns) f e t u i n EDC coupling of l a b e l (4) to p r o t e i n -NH2 EDC coupling to -CO2H periodate o x i d a t i o n / r e d u c t i v e amination 1.00 1.55 0.75 4 3 14.4 a s i a l o f e t u i n galactose oxidase/reductive amination periodate o x i d a t i o n / r e d u c t i v e amination EDC coupling to -C0 2H 0.52 4 2 43 1.20 1,38 BSM EDC cou p l i n g to -C0 2H periodate o x i d a t i o n / r e d u c t i v e amination 1.83 0.35 4 3 •>300 asialo-BSM galactose oxidase/reductive amination periodate o x i d a t i o n / r e d u c t i v e amination 43 0.49 0.35 4 3 human eryt h r o c y t e s EDC coupling to -C0 2H periodate o x i d a t i o n / r e d u c t i v e amination 0.52 i . o 4 2 BSA re d u c t i v e amination of l a b e l ( 1 ) to p r o t e i n - NH 2 EDC coupling to -C0 2H 1.50 4 2 2.98 20.0 HSA EDC coupling to -C0 2H 2.90 20.0 *C a l c u l a t e d using equation [20] ** C a l c u l a t e d assuming r i g i d unhydrated s p h e r i c a l molecules using the method given i n reference 44. 264 r o t a t i o n using Stokes' law i s l a r g e r than the experimental value i n a l l cases and more p a r t i c u l a r l y so f o r the l a r g e r species l a b e l l e d . Although the spec t r a r e f l e c t a l e s s r a p i d r e o r i e n t a t i o n than that found i n s o l u t i o n s of small n i t r o x i d e s , i t i s s u f f i c i e n t l y r a p i d to suggest that i n accordance w i t h present views of g l y c o p r o t e i n s t r u c t u r e the l a b e l s are, i n a l l cases, present at 'outer', s o l u t i o n - a c c e s s i b l e s i t e s , r a t h e r than i n i n t e r i o r pockets of the p r o t e i n s where s t e r i c hindrance might r e t a r d r e o r i e n t a t i o n ; i n the case of the e r y t h r o c y t e s , the l a b e l l e d carbohydrate appears to be moving f r e e l y i n the e x t r a c e l l u l a r s o l u t i o n though l a t e r a l m o b i l i t y of l a b e l l e d components ( e s p e c i a l l y s i a l o g l y c o -45 l i p i d s ) may a l s o c o n t r i b u t e to r e l a x a t i o n . These r e s u l t s may g a i n f u l l y be compared w i t h recent work on the g l o b u l a r membrane p r o t e i n s p e c t r i n , 46 which a s s o c i a t e s w i t h the cytoplasmic surface of the ery t h r o c y t e . This was l a b e l l e d using maleimide d e r i v a t i v e s of a n i t r o x i d e , which were found to be s t r o n g l y immobilised, and the more so when allowed to r e a s s o c i a t e w i t h the membrane. The l a b e l s were thought to r e s i d e at t h i o l f u n c t i o n a l groups w i t h i n the p r o t e i n . The l a c k of a s u b s t a n t i a l d i f f e r e n t i a l between l a b e l l e d BSM and f e t u i n suggests that the length of o l i g o s a c c h a r i d e connecting the l a b e l to the p r o t e i n i s unimportant. S i m i l a r c o r r e l a t i o n times have r e c e n t l y been 42 obtained i n t h i s l a b o r a t o r y using deuterium n.m.r. This could e i t h e r be because the l a b e l r e o r i e n t s independently of the saccharide, that i s , about s i n g l e bonds which connect the two, or because a d i s a c c h a r i d e u n i t i s j u s t as co n f o r m a t i o n a l l y mobile as a l a r g e r o l i g o s a c c h a r i d e , a con c l u -s i o n which i s not supported by model-building, though the l a r g e number of p o s s i b l e i n t e r a c t i o n s that could s t a b i l i s e an i n d i v i d u a l conformation means that the l a t t e r i s not very r e l i a b l e . The s i m i l a r i t y of T values 265 f o r species l a b e l l e d at a c c e s s i b l e p r o t e i n side-chains seems to suggest that the former—independent r o t a t i o n of the l a b e l — m a y dominate i n each case. The co n c l u s i o n that l a b e l s attached to carbohydrate on the outer surface of the c e l l membrane r e o r i e n t r a t h e r r a p i d l y i s i n agreement w i t h 47 previous r e p o r t s concerning randomly-labelled g a n g l i o s i d e s ; calcium-induced l a t e r a l phase separations and changes i n membrane f l u i d i t y were not detected as m o b i l i t y changes i n the l a b e l l e d carbohydrate on the outer surface of the membrane, where r e o r i e n t a t i o n was always r a p i d (T- - 10 s ) . This i n t e r p r e t a t i o n i s not rendered l e s s l i k e l y by the observation that some of the spec t r a (Figure VI-5a) suggest the presence of r o t a t i o n a l a n i s o t r o p y , which can be i d e n t i f i e d from the equal heights of the centre and l o w - f i e l d peaks; i n an i s o t r o p i c spectrum the l a t t e r i s smaller than the former except at the l i m i t of s m a l l x. This could a r i s e from r a p i d r o t a t i o n about the bonds connecting the l a b e l to sugar or aminoacid, r o t a t i o n normal to t h i s being more r e s t r i c t e d . To be more systematic, we may f a c t o r i s e the o v e r a l l r a t e of r e o r i e n -t a t i o n i n t o s e v e r a l c o n t r i b u t i n g processes as f o l l o w s : x = x ^ (whole-molecule r o t a t i o n ) + x (segmental motion i n p r o t e i n ) + x "'"(glycosyl aminoacid l i n k a g e ) + x ^ ( g l y c o s i d i c bonds i n p r o s t h e t i c group) + x (label-carbohydrate bonds) [39] These may, of course, be f u r t h e r decomposed i f d e s i r e d . The r e s u l t s w i t h BSM show that at l e a s t i n that case, whole-molecule r o t a t i o n makes a n e g l i g i b l e c o n t r i b u t i o n to x. Experiments can be envisaged i n which each of these c o n t r i b u t i o n s may be assessed. Thus by m u l t i - s i t e i m m o b i l i s a t i o n of whole g l y c o p r o t e i n , whole-molecule r o t a t i o n a l c o n t r i b u t i o n s are e l i m i n -ated. The f i r s t two f a c t o r s are e l i m i n a t e d by i m m o b i l i s i n g s u i t a b l e 266 glycopeptides, obtained by p r o t e o l y t i c d i g e s t i o n . The l a s t two may s i m i l a r l y be i s o l a t e d by i m m o b i l i s a t i o n of the l a b e l l e d o l i g o s a c c h a r i d e a f t e r s eparation and p u r i f i c a t i o n . Such experiments suggest an i n t e r e s t -i n g common denominator between t h i s chapter and the preceding ones; u n f o r t u n a t e l y , i t was only p o s s i b l e f o r the present i n v e s t i g a t o r to pursue one of these avenues, that of i m m o b i l i s i n g whole g l y c o p r o t e i n . Figure VI-5c shows the spectrum of f e t u i n , l a b e l l e d at s i a l i c a c i d by the p e r i o d a t e - r e d u c t i v e amination procedure, and subsequently coupled to Sepharose 4B by means of cyanogen bromide a c t i v a t i o n . x f o r t h i s l a b e l -9 i s 0.85 x 10 s. C l e a r l y i m m o b i l i s a t i o n has had l i t t l e e f f e c t upon i t s m o b i l i t y , w i t h the exception of a small component which i s q u i t e broad and whose o r i g i n i s not c l e a r (trapping between g l y c o p r o t e i n and m a t r i x , or neighbouring g l y c o p r o t e i n s i s one p o s s i b i l i t y ; f u r t h e r i n v e s t i g a t i o n i s c a l l e d f o r ) . Two other r e p o r t s have appeared concerning the i m m o b i l i s a t i o n 47 48 of l a b e l l e d p r o t e i n s , one d e a l i n g w i t h RNAse and the other w i t h t r y p s i n , both l a b e l l e d at the a c t i v e s i t e ; i n both cases greater increases i n x were observed upon i m m o b i l i s i n g the p r o t e i n . U n f o r t u n a t e l y , however, the only p o s i t i v e c o n c l u s i o n that i t i s p o s s i b l e to draw at t h i s stage from the present work i s that l a b e l s present at carbohydrate p r o s t h e t i c groups are not so i n f l u e n c e d , e i t h e r because of unhindered r o t a t i o n r e l a t i v e to the l a b e l l e d s p e c i e s , or because of the m o b i l i t y of the p r o s t h e t i c group i t s e l f . This u nderlines the weakness of the s p i n l a b e l l i n g technique. The r e s u l t may however be seen as l e s s s u r p r i s i n g i n the context of Chapter IV, s i n c e the f e t u i n molecule e s s e n t i a l l y c o n s t i t u t e s an extremely l a r g e spacer arm! The f i n a l p o i n t f o r d i s c u s s i o n i n t h i s chapter i s one which a l s o demands f u r t h e r i n v e s t i g a t i o n , namely, the reason f o r the r a t h e r broader 267 spectrum e x h i b i t e d by the l a b e l l e d t i s s u e s e c t i o n (Figure VI-4a; a x-value i s not quoted here as the spectrum f a l l s i n the intermediate motional' regime and shows some a n i s o t r o p y ) • I t may be that a good part of t h i s s i g n a l a r i s e s from uronic a c i d carboxylates present i n connective t i s s u e , s i n c e a f t e r d e s i a l y l a t i o n a considerable background s i g n a l , w i t h lineshape of similar-':form, i s v i s i b l e (Figure VI-4b). Such an i n t e r p r e -t a t i o n i s r e i n f o r c e d by the r e s u l t s of experiments using the p e r i o d a t e -42 r e d u c t i v e amination method , which gave r a t h e r weaker s i g n a l s than the EDC amidation. Other than these uronic a c i d s , the components of the s e c t i o n are g e n e r a l l y s i m i l a r to those l a b e l l e d s e p a r a t e l y : c e l l surface carbohydrates ( i n r a t h e r small q u a n t i t i e s ) together w i t h mucins and plasma g l y c o p r o t e i n s . The r e s u l t s d i s c u s s e d - i n the present chapter would sug-gest that c r o s s - l i n k i n g processes thought to be i n v o l v e d i n formaldehyde f i x a t i o n should not i n f l u e n c e the: m o b i l i t y of l a b e l s attached to carbohy-drate c o n s t i t u e n t s of these molecules, nor indeed to aminoacid s i d e chains. I t i s i n t e r e s t i n g to note i n c o n c l u s i o n that s c i e n t i s t s attempting to c h e m i c a l l y modify complex b i o l o g i c a l systems are i n constant need of means to q u a n t i t a t e t h e i r e f f o r t s . This has not been at a l l d i f f i c u l t i n the present work owing to the s e n s i t i v i t y and r a p i d i t y of the esr double i n t e g r a t i o n procedure, which i s t h e r e f o r e presented as a u s e f u l a l t e r n a -t i v e i n cases where d i f f i c u l t i e s a r i s e w i t h more commonly u t i l i s e d r a d i o -chemical procedures; any motional i n f o r m a t i o n a f f o r d e d can i n t h i s case be viewed as a bonus. 268 References 1.. R. J . S t o c k e r t , A. G. M o r e l l , and I. H. Scheinberg, Science, 197, 667-668 (1977). 2. A. Gottschalk, A. S. Bhargava, and V. L. N. Murty, i n 'The Glyco-p r o t e i n s , ' Ed. A. Gottschalk, BBA L i b r a r y , V o l . 5, Chapter 7, Sec-t i o n 4, E l s e v i e r , Amsterdam, 1972. 3. W. Pigman, J . Moschera, M. Weiss, and G. Tettamanti, Eur. J . Biochem., 32, 148-154 (1973). 4. E. R. B. Graham i n 'The G l y c o p r o t e i n s , ' Ed. A. Got t s c h a l k , BBA L i b r a r y , V o l . 5, Chapter 6, Section 5, E l s e v i e r , Amsterdam, 1972. 5. R. G. S p i r o , and V. D. Bhoyroo, J . B i o l . Chem., 249, 5704-5717 (1974). 6. B. N i l s s o n , N. E. Norden, and S. Svensson, i b i d . , i n press. 7. S. Yachnin, J . Exp. Med., 141, 242-256 (1975). 8. C C S . Hsu, W. I . Waithe, P. Hathaway, and K. Hirschhorn, C l i n . Exp. Immunol., 15, 427-434: (1973). 9. E. R. B. Graham, A u s t r a l i a n J . S c i . , 24, 140-146 (1961). 10. J . L. Winkelhake, and G. L. N i c o l s o n , Anal. Biochem. , 7_1_, 281-289 (1976). 11. H. A. Blough, V i r o l o g y , 31, 514-522 (1967). 12. J . C. Rogers, and S. K o r n f e l d , Biochem. Biophys. Res. Commn., 45, 622-629 (1971). 13. A. Rosenberg, and C.-L. Schengrund, Ed ., ' B i o l o g i c a l Roles of S i a l i c A c i d , ' Plenum, New York, 1976. 14. A. F. Hegarty, M. T. McCormack, G. Ferguson, and P. J . Roberts, J . Am. Chem. S o c , 99, 2015-2016 (1977). 15. I. T. Ibrahim, and A. W i l l i a m s , i b i d . , 100, 7420-7421 (1978). 16. J . C. Sheehan, P. A. Cruickshank, and G. L. Boshart, J . Org. Chem., 26, 2525-2528 (1961). 17. T. Tenforde, R. A. Fawwaz, N. K. Freeman, and N. C a s t a g n o l i , i b i d . , 37., 3372-3374 (1972). 18. E. Moczar, and G. Vass, Carbohydr. Res., 50, 133-141 (1976). 19. E. Moczar, and J . Leboul, F.E.B.S. L e t t . , 80, 300-302 (1975). 269 20. J . Lonngren, I. J . G o l d s t e i n , and J . E. Niederhuber, Arch. Biochem. Biophys.,175, 661-669 (1976). 21. H. F a i l l a r d , C. F. DuAmaral, and M. Blohm, Hoppe-Seyl. Z. P h y s i o l . Chem.,350, 792-802 (1969). 22. R. Brossmer, and L. Holmquist, i b i d . , 352, 1715-1719 (1971). 23. T. S. Tenforde, Adv. B i o l . Med. Phys., 13, 43-105 (1970). 24. G. M. W. Cook, D. Heard, and G. V. F. Seaman, Nature, 188, 1011-1012 (1961). 25. Y. Kagawa, and E. Racker, J . B i o l . Chem., 246, 5477-5487 (1971). 26. B. Lindberg, J . Lonngren, and S. Svensson, Adv. Carbohydr. Chem. Biochem., 31, 185-240 (1975). 27. C. G. Gahmberg, I . V i r t a n e n , and J . Wartiovaara, Biochem. J . , 171, 683-686 (1978). 28. J . M. B o b b i t t , Adv. Carbohydr. Chem.,11, 1-41 (1956). 29. L. Van Lenten, and G. Ashwell, J . B i o l . Chem., 246, 1889-1894 (1971). 30. R. L. McLean, M. S u t t a j i t , B. B e i d l e r , and R. J . W i n z l e r , i b i d . , 246, 803-809 (1971). 31. M. S u l t a j i t , Ph.D. Thesis, State U n i v e r s i t y of New York, B u f f a l o , 1972. 32. 0. 0. Blumenfeld, P. M. Ga l l o p , and T.-H. L i a o , Biochem. Biophys. Res. Commn., 48, 242-250 (1972). 33. T.-H. L i a o , P. M. Ga l l o p , and 0. 0. Blumenfeld, J . B i o l . Chem., 248, 8247-8253 (1973). 34. K. J . W i l l a n , B. Golding, D. G i v o l , and R. A. Dwek, F.E.B.S. L e t t . , 80, 133-136 (1977). 35. P. Weber, and L. Hof, Biochem. Biophys. Res. Commn., b5_, 1293-1302 (1975). 37. G. P. Roberts, Histochem. J . , % 97-101 (1977). 38. R. G. S p i r o , J . B i o l . Chem., 239, 567-573 (1964). 39. E. H. Ey.lar, and R. W. Jean l o z , J . B i o l . Chem., 237, 1021-1025 (1962). 40. R. S. N e z l i n , V. P. Timofeev, Yu. K. Sykulev, and S. E. Zurabyan, Immunochem., 15, 143-144 (1978). 270 41. V. P. Timofeev, I. V. Dudich, Yu. K. Sykulev, and R. S. N e z l i n , F.E.B.S. L e t t . , 89, 191-195 (1978). 42. M. A. B e r n s t e i n , unpublished r e s u l t s . 43. J . D. A p l i n , M. A. B e r n s t e i n , C. F. A. C u l l i n g , L. D. H a l l , and P. E. Reid, submitted f o r p u b l i c a t i o n . 44. A. G. M a r s h a l l , ' B i o p h y s i c a l Chemistry,' Wiley, 1978. 45. P. Devaux, and H. M. McConnell, J . Am. Chem. S o c , 94, 4475-4481 (1972). 46. R. Cassoly, D. Daveloose, C. Wolf, and F. L e t e r r i e r , Compt. Rend. Acad. S c i . , 286, s e r i e s D, 1009-1012 (1978). 47. F. J . Sharom, and C. W. M. Grant, Biochem. Biophys. Res. Commn., 74, 1039-1045 (1977). 48. R. Reiner, and H.-U. Siebeneick, Enzyme Eng., Pap. Res. Rep. Eng. Found. 2nd Conf., 179-180 (1974). 49. L. J . B e r l i n e r , S. T. M i l l e r , R. Uy, and G. P. Royer, Biochim. Biophys. A c t a , 315, 195-199 (1973). CHAPTER V I I SUMMARY Aqueous s o l u t i o n s and gels c o n t a i n i n g macromolecular carbohydrates have been observed using n i t r o x i d e e s r , from two d i f f e r e n t p e r s p e c t i v e s : that of a probe (Chapter I I ) and that of a l a b e l (Chapters I I I and VI) . Perhaps not s u r p r i s i n g l y , the probe rela y e d i n f o r m a t i o n not about the j£ 5% carbohydrate present, but about the main c o n s t i t u e n t , water, whose p r o p e r t i e s were found to be s i m i l a r to those i n the pure l i q u i d . This d i s c o v e r y i s i n agreement w i t h previous r e p o r t s of n i t r o x i d e probes i n s y n t h e t i c polymer s o l u t i o n s . N i c k e l ions used as probes i n s o l u t i o n s of n i t r o x i d e - l a b e l l e d agarose, however, were found to undergo r a p i d e l e c t r o n exchange w i t h the n i t r o x i d e , causing l i n e - b r o a d e n i n g , thus i n d i c a t i n g an a b i l i t y to approach the v i c i n i t y of the polysaccharide. The two r e s u l t s are by no means i n c o n s i s t e n t ; only a s m a l l f r a c t i o n of the metal i o n s — those c l o s e at a given i n s t a n t to the polymer—need be i n v o l v e d i n c o l l i -s ions w i t h n i t r o x i d e s to create the observed e f f e c t . Labels attached i n v a r i o u s ways to agarose, a l g i n a t e , f e t u i n and BSM i n s o l u t i o n were found to r e o r i e n t q u i t e r a p i d l y (Table V I I - 1 ) , though l e s s so than the probe molecules (T ^ 10 s ) , and i n a l l cases f a s t e r than would be expected f o r the o v e r a l l r e o r i e n t a t i o n of the macromolecule ( c h a r a c t e r i s e d by a time x ). This h i g h l i g h t s a.problem common to many s t u d i e s using s p i n l a b e l s and other e x t r i n s i c r e p o r t e r molecules c o v a l e n t l y l i n k e d to the system of i n t e r e s t , namely that r e o r i e n t a t i o n may occur about the bonds i n the 271 272 Table V I I - 1 : C o r r e l a t i o n times (10 y x, s) f o r l a b e l l e d g l y c o p r o t e i n s and polysaccharides i n s o l u t i o n BSM (EDC method) (300 K) 1.83. F e t u i n (EDC method) (300 K) 1.55 Sodium A l g i n a t e (CNBr method) (300 K) 0.6 Agarose (chloroacetimide method) (307.5 K) 0.35 Sodium carboxymethyl c e l l u l o s e (CNBr method) (300 K) (see Appendix) 0.78 li n k a g e i n such a way that the m o b i l i t y of the l a b e l i s greater than that of the macromolecule (and there i s a r e s u l t i n g need f o r new s y n t h e t i c i n i t i a t i v e s to circumvent t h i s problem). Indeed i t was shown by immobil-i s a t i o n of l a b e l l e d f e t u i n on Sepharose 4B that x-values f o r n i