UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Elastin as a kinetic elastomer : an analysis of its conformational, mechanical, and photoelastic properties Aaron, Ben-Meyer Benson 1980-12-31

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
UBC_1980_A6_7 A27.pdf [ 9.87MB ]
Metadata
JSON: 1.0094905.json
JSON-LD: 1.0094905+ld.json
RDF/XML (Pretty): 1.0094905.xml
RDF/JSON: 1.0094905+rdf.json
Turtle: 1.0094905+rdf-turtle.txt
N-Triples: 1.0094905+rdf-ntriples.txt
Original Record: 1.0094905 +original-record.json
Full Text
1.0094905.txt
Citation
1.0094905.ris

Full Text

cl  ELASTIN a s A KINETIC ELASTOMER: An A n a l y s i s of I t s C o n f o r m a t i o n a l , Mechanical, and P h o t o e l a s t i c Properties»  by BEN-MEYER BENSON AARCN B.Sc., The U n i v e r s i t y of B r i t i s h Columbia, 1976  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR A DEGREE OF MASTER OF SCIENCE  in THE FACULTY CF GRADUATE STUDIES Department of Zcclogy  We accept t h i s t h e s i s as conforming to the r e g u i r e d  standard  THE UNIVERSITY OF ERITISH COLUMBIA August 1980 ©  BEN-MEYER BENSON AARON, 1980 /  In  presenting  an  advanced  the I  degree  Library shall  further  for  this  agree  scholarly  by  his  of  this  written  thesis  in p a r t i a l  fulfilment  at  University  of  the  make  that  freely  permission  p u r p o s e s may  representatives. thesis  it  for  It  financial  is  of  The U n i v e r s i t y  of  B r i t i s h Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1W5  by  the  understood  permission.  Department  for  for extensive  gain  shall  the  requirements  B r i t i s h Columbia,  available  be g r a n t e d  of  copying Head o f  that  not  reference  be  of  I agree and this  or  allowed  without  that  study. thesis  my D e p a r t m e n t  copying  for  or  publication my  i  ABSTRACT The materials  elastic  tissue  t h a t are  characterized  mechanical  composite i s made up  properties..The  almost 80%  chemical  properties  of  indicates that e l a s t i n  other  20%.  The  the u n p u r i f i e d and  and  makes up  of bovine ligamgntum nachae with c o l l a g e n  mechanical  and  the  a n a l y s i s of purified  the  tissue  i s the dominant mechanical component at  s t r a i n s with c o l l a g e n  higher  different  p r o t e i n elastcmer e l a s t i n  matrix substances making up the  low  by  of a number of  contributing  significantly  at  the  extensions.  The  physical  s i n g l e , 5 to 8um  diameter,  water-swollen e l a s t i n f i b r e s were i n v e s t i g a t e d on a  micro-test  apparatus attached were analyzed by a n a l y s i s of the indicate  properties  to a p o l a r i z i n g microscope, and  using  the k i n e t i c theory  mechanical p r o p e r t i e s  that e l a s t i c  of  6000  to  chains  coeffecient,  C = 1 X 10-»m N-»  temperature  dependence  indicated  that  with i n c r e a s i n g for t h i s the  at extensions below  100%  of  X 10 Nm- , the s  and  at  2  2  the  24°C.  the  temperature. The  non-Gaussian  e x t e n s i o n s above 100%  order of  mechanical  1.6  and  suggest that the  approximately  10  in  Analysis  stress-optical  apparent  average the  stress-optical of  the  coeffecient  the p c l a r i z a b i l i t y of the random l i n k  process i s i n the  l i n k s contains  The  between c r o s s - l i n k s i s  7 100g/mol,  1  the r e s u l t s  relationships.  modulus, G = 4.1  molecular weight of the range  of  decreases  activation  energy  kcal/mole. A n a l y s i s optical chain  properties between  residues.  at  cross-  ' e f f e c t i v e ' random l i n k s ,  each l i n k c o n s i s t i n g of 7 to 8 amino a c i d  of  with  ii  The  e x p l i c i t assumption of a random network that i s made  by the k i n e t i c theory was t e s t e d by a 400  MHz  pmr  spectra  cf  resembled the spectra t h a t conformation, hydrolysate.  and  the  Polarized to  of  were p r e d i c t e d  spectra  f o r the  microscopy  studies  showed  Finally,  the parameters f o r the random chains i n  network  were  The c l o s e  published  values  GuHCl provided validating  random-coil  obtained f o r i t ' s amino a c i d  fibres  proteins.  techniques.  soluble alpha-elastin c l o s e l y  elastin  used  be  the  number  devoid of any c r y s t a l l i n e  intact  structures. the  elastin  to p r e d i c t the dimensions of other random correlation for  of  these  predictions  with  a s e r i e s of p r o t e i n s i n s o l u t i o n i n 6M  an independent t e s t c f the random conformation,  the use of  the  kinetic  analyze the macroscopic p r o p e r t i e s  theory  relationships  of e l a s t i n .  to  iii  TABLE OF CONTENTS  ABSTRACT  I  TABLE OF CONTENTS  i i i  LIST OF TABLES  X  LIST OF FIGURES  ,  .xi  ACKNO WL E EG EM EN TS  .xi V  ,  . 1  I. .INTRODUCTION  4  I I . GENERAL CHARACTERISTICS OF ELASTIC TISSOE  4  A. I n t r o d u c t i o n B. . E l a s t i n  Development  •  • 4  (a) Embryology (b) Morphogenesis  Of E l a s t i n  5  Fibres  7  (c) E l a s t i n Turnover C. E l a s t i n  7  Chemistry  (a) Amino Acid Composition  7  (b) E l a s t i n  9  Cross-links  (c) S o l u b l e E l a s t i n  . . 1 0 11  D. Composition Of E l a s t i c T i s s u e (a) Water Content (b) E l a s t i n  Content  (c) N e u t r a l Sugars (d) Mucopolysaccharides (e) C o l l a g e n  Content  E. O r g a n i z a t i o n Of E l a s t i c T i s s u e (a) Methodology  ..  12  . . . 1 2 .12 12 ...14 .14 . , . 14  i v  (b) O r g a n i z a t i o n Of ligament E l a s t i n  15  (c) O r g a n i z a t i o n  18  F. P u r i f i c a t i o n  Of A r t e r i a l E l a s t i n  Techniques  ......21  (a) Amino Acid Compositions  21  (b) Hexosamine And N e u t r a l Sugar  Content  24  (c) E v a l u a t i o n Ey Scanning E l e c t r o n Microscopy .....24 G. Mechanical P r o p e r t i e s Of E l a s t i n  Bundles  (a) Methods  23  (b) R e s u l t s  28  H. Mechanical P r o p e r t i e s Of S i n g l e E l a s t i n I.  24  Discussion  F i b r e s .....31 34  (a) V a r i a t i o n In E l a s t i n (b) The Composite  Biochemistry  Tissue  .....35  (c) E v a l u a t i o n Cf P u r i f i c a t i o n I I I . CONFORMATION OF ELASTIN: THE  ? - 34  Techniques ......... 36  CONTEOVEESY.  A. . I n t r o d u c t i o n  38 .....38  B. The Random Network Model  . 33  (a) E l a s t i n T h e r m c e l a s t i c i t y  .41  (b) D i f f e r e n t i a l Scanning C a l o r i m e t r y  43  (c) E.M.  43  And  X-ray D i f f r a c t i o n  Studies  (d) N.M.R. Evidence C. . L i q u i d Crop Model  44 .45  (a) P r e l i m i n a r y Evidence  45  (b) Thermodynamic Evidence  46  D. .Oiled C o i l Model  .47  E. F i b r i l l a r Models  .48  (a) E l e c t r o n Microscope Evidence  49  V  (b) F.  Nuclear  Magnetic  Discussion  ..  Two-phase Models  (b)  Evidence  For  (c) Hydrogen  G. IV.  Fibrillar  .  Secondary  Structures  Introduction  .....58  Models  ..  B.  The  C.  Preparation  ELASTIN: COACERV ATE •  Phenomenon Of Of  Removal Of  (c)  C h a r a c t e r i z a t i o n Of And  62  .  62 62 •  Relevant  (b)  Evaluation  (c)  A p p l i c a t i o n To  The  Alpha-elastin  65 .66  Eguation  Cf The  Studies  (a)  Viscosity  (b)  Calculation  Shape P a r a m e t e r  Soluble Of  66 ,  .69  Elastins  ..70  Alpha-Elastin  73  Measurements Cf  Partial  73 Specific  Volume  And  Hydration (c) F.  Results  Nuclear  3  65  Shape  The  6  .....64  SDS  (a)  Viscosity  STRUCTURE. ........  Alpha-Elastin  (b)  Viscosity  60  Coacervation  Enzyme H y d r o l y s i s  60  •  •  (a)  54 56  Conclusions  A.  E.  54  Ecnded S t r u c t u r e s  CONFORMATION OF  Do  .50  •  (a)  (d)  Resonance E v i d e n c e  76 And  Discussion  .76  M a g n e t i c Resonance  .........80  (a)  Theory  (b)  The  (c)  Relaxation  .80  Chemical S h i f t Processes  ..  .84 ••••  85  vi  G. N. Mo Ro S t u d i e s  Of A l p h a - E l a s t i n  ...87  (a)  Materials  And Method  .87  (b)  P r e d i c t i o n Cf E a n d o m - c o i l S p e c t r a  9 1  (c)  Results  92  And D i s c u s s i o n  H. C o n c l u s i o n s V. CON FO EM A H O N  .105  OF ELASTIN: BIREFRINGENCE PEOPEBTIES.  ...108  A. I n t r o d u c t i o n  ..108  B. P h e n o m e n o l o g i c a l E x p l a n a t i o n (a)  Retardation  (b)  Quantitating  Of Double R e f r a c t i o n  Of P o l a r i z e d L i g h t The R e t a r d a t i o n  .110 ,,  ,115  C. . Q u a l i f y i n g The T y p e s Of B i r e f r i n g e n c e (a)  Intrinsic  (b)  Form  {c)  Strain  E.  116 .........124  Birefringence  ....125  And Methods  Birefringence  -  Properties  (a)  Form  (b)  Intrinsic  (c)  Explanation  Of S i n g l e E l a s t i n  .127  Birefringence F o r The A p p a r e n t  132 Birefringence  Previous  (b)  The F i b r i l l a r  Studies  ...146  Models  .148  G. . C o n c l u s i o n s  MICROSCOPY A.  Introduction  B. Methods  ....136 ..146  (a)  CONFORMATION  126  F i b r e s .127  Birefringence  . F. D i s c u s s i o n  VI. ,  .....116  Birefringence  Eirefringence  D. M a t e r i a l s  .109  .150 OF  ELASTIN:  SCANNING  ELECTRON .152 152 152  vii  C. R e s u l t s  .  ..153  D. D i s c u s s i o n  156  VII. .ELASTIN AS A KINETIC ELASTOMER.  .....163  A. I n t r o d u c t i o n  .163  B. .Entropy Elastomers: The K i n e t i c  Theory  Of  Rubber  Elasticity  164  (a) Gaussian Chain S t a t i s t i c s And Entropy  164  (b) The E l a s t i c Network  .168  (c) Mechanical P r o p e r t i e s Of K i n e t i c Rubbers ...... 169 (d) P h o t o e l a s t i c i t y  171  (e) Non-Gaussian E f f e c t s And The E v a l u a t i o n (f) Reduction Cf E l a s t i n  Of S ..172  Data To The Unswollen Form 173  C. M a t e r i a l s  And Method  .174  (a) P u r i f i c a t i o n Of E l a s t i n  .174 i  (b) The Experimental Stage  .175 i  (c) P r e p a r a t i o n  Of Experimental Specimen  175  (d) Measurement Of S t r a i n  178  (e) Measurement Of Force  .178  (f) C a l c u l a t i o n Of C r o s s - s e c t i o n a l Area (g) Measurement Of B i r e f r i n g e n c e  .....179 ...180  (h) E r r o r s  180  D. P h y s i c a l P r o p e r t i e s Of S i n g l e E l a s t i n F i b r e s (a) General C h a r a c t e r i s t i c s (b) Mechanical P r o p e r t i e s  Temperature  r^  8  1  And The D e r i v a t i o n Of Mc 18 1  (c) P h o t o e l a s t i c i t y (d)  181  . Dependence  Of  185 The  Optical  viii  Anisotropy  188  (e) Non-Gaussian P r o p e r t i e s Of The E l a s t i n  Network  192  E. Conclusions  195  V I I I . .A PREDICTIVE TEST FOR ELASTIN CONFORMATION  197  A. . I n t r o d u c t i o n  197  B. C h a r a c t e r i z a t i o n Of Random P r o t e i n s  .197  (a) The Measurable Dimension  197  (b) Accounting For The N o n - i d e a l i t y  .20 1  C. R.M.S. Values From V i s c o s i t y (a)  202  V i s c o s i t y Of Random C o i l s  202  (b) C o r r e c t i o n Of V i s c o s i t y Values D. P r e d i c t i o n s From The E l a s t i n  203  Network  .204  (a) C a l c u l a t i o n Of S  .204  (b) C a l c u l a t i o n Of L  ..206  E. D i s c u s s i o n  And Conclusions  207  IX. CONCLUSIONS  211  APPENDIX.I: I h e r m o e l a s t i c i t y  .216  (A) Thermodynamic R e l a t i o n s h i p s  .216  (B)  The Thermodynamic Experiment  (C)  T h e r m o e l a s t i c i t y Of K i n e t i c Elastomers ...........218  APPENDIX.II:  PREPARATION  OF  SOLUBLE  217  ELASTIN  PHOTOLYSIS  BY ....... 220  A. I n t r o d u c t i o n  220  B. Methodology  .220  (a) R a t i o n a l e (b) Procedure C. R e s u l t s  And D i s c u s s i o n  .220 •  .224 ......225  ix  (a) Y i e l d  ..225  (b) C h a r a c t e r i z a t i o n Of The Soluble Peptides  ......226  A p p e n d i x . I l l : PREDICTIONS FOR TROPOELASTIN VISCOSITY ...,231 A. The Relevant Equation  231  5. A p p l i c a t i o n To T r o p o e l a s t i n  231  Appendix.IV: EVALUATION OF PROTEIN CONFORMATION Appendix.V:  DETERMINATION  COILS  OF  .236  SOLVENT EFFECTS ON RANDOM .........242  Appendix.VI: AMINO ACID COMPOSITION AND ELASTIN EVOLUTION 245 LITERATURE CITED  ..249  X  LIST OF TABLES  Table.2.1: Amino Acid Composition Of E l a s t i c P r o t e i n s .  .,  Table.2.2: Chemical Composition Of E l a s t i c T i s s u e Table.2.3:  Amino  Acid  Composition  Of  13 Elastin  P r e p a r a t i o n s . ........................................ Table. 4.1: Table.4.2:  25  V i s c o s i t y Of A l p h a - e l a s t i n Spectral  Parameters  Used  8  77 For  Bandom-coil  Predictions  .90  Table.4.3: Peak Assignments For A l p h a - e l a s t i n At 400  MHz. 95  Table.5.1: Form B i r e f r i n g e n c e  Of S i n g l e E l a s t i n F i b r e s .  .128  Table.7.1: K i n e t i c Theory Parameters For E l a s t i n .  ..186  Table.8.1: P r e d i c t i o n s For Random-coil P r o t e i n s .  ..205  T a i l e . A . 3 . 1 : P r e d i c t i o n For T r o p o e l a s t i n V i s c o s i t y . .....234 Table.A.6.1: D i f f e r e n c e Index For E l a s t i n Composition. .,246  xi  LIST OF FIGURES  F i g u r e . 2 . 1 : O r g a n i z a t i o n Of Ligament E l a s t i n . . .  .16  Figure.2.2: O r g a n i z a t i o n Of A r t e r i a l E l a s t i n .  .19  F i g u r e . 2 . 3 : The  E l a s t i c T i s s u e Composite..  Figure.2.4: E v a l u a t i o n Of P u r i f i c a t i o n  22  Technigues.  .26  Figure.2.5: Mechanical P r o p e r t i e s Of E l a s t i n Bundles. ... Figure.2.6:  Mechanical  Properties  Of  Single  29  Elastin  Fibres.  32  F i g u r e . 3 . 1 : P r o p o s a l s For The Conformation Of E l a s t i n ....39 Figure.3.2: Eeta-turns.  51  F i g u r e . 4 . 1 : C c a c e r v a t i o n P r o f i l e Of Figure.4.2:  Dependence  Of  The  Alpha-elastin.  Simha  Factor  On  ..67 Axial  Ratio Figure.4.3:  71 Dependence  Of  Elastin  Water  Content  On  Temperature  .74  F i g u r e . 4 . 4: V i s c o s i t y Of A l p h a - e l a s t i n  78  F i g u r e . 4 . 5 : P r e c e s s i o n Of A Proton In A Magnetic F i e l d . . 81 F i g u r e . 4 . 6 : The L o r e n t z i a n Line Shape Figure.4.7: 4C0  MHz  Nmr  Spectrum Of A l p h a - e l a s t i n . .......93  Figure.4.8: P r e d i c t e d Nmr F i g u r e . 4 . 9 : Nmr Figure.4. 10: Nmr Figure.5.1:  ..88  Spectra Fcr A l p h a - e l a s t i n  Spectrum Of A l p h a - e l a s t i n Hydrolysate.  ..100  Spectra Of Albumin.  Propagation  Of  Polarized  103 Light  Isotropic Material. Figure.5.2:  97  Propagation  Anisotropic Material  Through .....111  Of  Polarized  Light  Through .113  xii  FIGUfiE.5.3:  The  Birefringence  Figure.5.4:  The  Sign Of The  Figure.5.5:  Form B i r e f r i n g e n c e  Figure.5.6:  Form B i r e f r i n g e n c e  Of  Figure.5.7:  Form B i r e f r i n g e n c e  Of C o l l a g e n . . .  Figure.5.8:  Expected  117  Experiment  Birefringence.  . . . . . . . 1 2 0  .  122 Elastin  ,129  Fibres  Relationship  133  For  Intrinsic 137  Birefringence. Figure.5.9:  Expected  Birefringence  For  An  Anisotropic 140  Coating F i g u r e . 5 . 10:  Birefringence  Pattern  Of  Single  Elastin  . .  Fibres Figure.6.1:  S.E.M. Of  Figure.6.2:  Surface Texture Of  144  Unpurified  Ligament E l a s t i n  Autoclaved E l a s t i n  154  Fibre. Fibres.  157  Figure.6.3:  Fracture  Figure.7.1:  The  Random-coiled Chain  Figure.7.2:  The  Experimental Stage..  Figure.7.3:  P h y s i c a l P r o p e r t i e s Of  Surfaces Of E l a s t i n  Fibres.  ,159 166 .176  Single E l a s t i n  Fibres. 183  Figure.7.4: Figure.7.5:  Temperature Dependence Of P h o t o e l a s t i c i t y . . . 1 8 9 Non-Gaussian  Properties  Of  The  Elastin 193  Network. Figure.8.1:  The  Figure.8.2:  P r e d i c t i o n s For The  ....199  Randcm Walk.. Dimensions Of  208  Protein Figure.9.1:  Random-coil  Summary Figure  For The  F i g u r e . A. 2 . 1: P h o t o l y s i s Of E l a s t i n  Thesis.  213 222  xiii  Figure.A.2.2: Molecular Weight Of P h o t o l y s i s Peptides. Figure.A.3. 1 : V i s c o s i t y Of Eandom-coil  ..228  Proteins..  232  Figure.A.4. 1 : Badius Of Gyration For Various Shapes. ..... 237 Figure.A.4.2: E v a l u a t i o n Of P r o t e i n Conformation Figure.A.5. 1 :  Evaluation  Of  Solvent  Coils. F i g u r e . A. 6. 1 : E l a s t i n E v c l u t i c n .  239  E f f e c t s On Random 24 3 247  xiv  AKNOWLEDGEMENTS T h i s r e s e a r c h was support  cf  made  possible  through  procrastination  my  incompetence  and  the d i r e c t i n g  c f t h i s r e s e a r c h . I would  and  the  patience  and  many people. I would e s p e c i a l l y l i k e t o thank Dr.  J.M. G o s l i n e who s u f f e r e d  Karen  by  everpresent  without  bouts  losing interest i n  also  like  to  Martin f o r her help with the p r e p a r a t i o n of t h i s  f o r g i v i n g me the emotional  support  of  thank thesis  over these past years.  The occupants of the l a b and my other a s s o c i a t e s , Mark  Denny,  Tony Harmon, Bob Shadwick, and Kevin Bush have a l l c o n t r i b u t e d to  t h i s t h e s i s through  t h e i r many c r i t i c i s m s and d i s c u s s i o n s ,  and t h e i r i n p u t i s g r a t e f u l l y  acknowledged.  Lastly,  I  would  l i k e t o thank Dr. P.D. Burns from the Department of Chemistry, without the  whose c o - o p e r a t i o n and many hours of experimental  nmr  studies  would not have been p o s s i b l e , and T i n a Duke  from the Department of Computer Science who documentation of t h i s This  research  Foundation  work  was  helped  with  the  thesis. supported  by  grants from the B.C. Heart  and the Canadian N a t u r a l Sciences  C o u n c i l t o Dr. J.M. G o s l i n e .  and  Engineering  XV  This  thesis  is  Benson  and Seemah  Aaron,  whose  d e d i c a t e d t o my f a m i l y : my Aaron, support  c a t a l y s t f o r t h i s work.  and and  my  brother  advice  parents Solomon  provided  the  1  Chapter.I. INTRODUCTION,, We about  have a l l experienced i n s t a n c e s the  where  having  knowledge of an o b j e c t s f u n c t i o n have subsequently  been embarassed by our ignorance of the  'mode of  Proteins,  functions  present  which serve the  detergent  same  a vast number of  dilema.  commercials  certain  compounds.  does not  i n any  performs. character  way  I f one it  information  on  us about the  i s to comment on  the  protein,  necessary protein  the  protein's  manner  t h i s aspect to  obtain  i n question. can  in  e l a s t i c i t y assume d i f f e r e n t level  f o r the  material i n question.  that one  should  protein  in  an  some The  structural  same argument  at  knowledge  the  attempt to shed some l i g h t on the  elastin  was  to i n v e s t i g a t e the s t r u c t u r e of the e l a s t i n interpret  Hence the  it's  of  molecular  Hence, i t seems reasonable  of  to  a  of  theories  physical  this  validity  the wide spectrum of t h e o r i e s that c l a i m t c e x p l a i n the  then,  is  which i s the  i n v e s t i g a t e the molecular conformation of  elasticity.  it  of the enzyme's  Different  conformations  but  which  claim that e l a s t i n  state.  down  'function'  b a s i s of t h i s e l a s t i c i t y ,  conformational  nature,  break  t o p i c of i n t e r e s t , cannot be e l u c i d a t e d without the  in  enzymes  t e l l s us about t h e i r  inform  functioning',.  most of us know from watching  television,  This  becomes for  As  a p p l i e s t o e l a s t i n . Although one rubbery  boasted  of  basis  major focus of t h i s t h e s i s protein  and,  p r o p e r t i e s i n terms of a  t h e o r e t i c a l framework f o r rubber e l a s t i c i t y . The.techniques of c o n f o r m a t i o n a l i n v e s t i g a t i o n were s e l e c t e d on  analysis  t h e i r value  used  in  this  as s e n s i t i v e probes  2  of  structure  with  the  minimum amount of d i s t u r b a n c e to the  n a t i v e conformation i n terms of experimental studies  themselves  organization  were  starting  developed  with  the  at  techniques.  different  investigation  l e v e l s of  of  soluble  peptide p r o p e r t i e s and b u i l d i n g upto the i n t a c t e l a s t i c Nmr  and  v i s c o s i t y experiments  of  soluble  and  scanning e l e c t r o n microscopy  proteins  fibre.  were used to study the s t r u c t u r e  from e l a s t i n , with p o l a r i z e d  microscopy  p r o v i d i n g the t o o l s  a n a l y s i s of i n t a c t e l a s t i n f i b r e s . elastin  The  for  the  A l l of these s t u d i e s showed  to be a random network elastomer, and on the b a s i s of  this conclusion framework  I  then  proceeded  to  use  the  theoretical  provided by the k i n e t i c theory of rubber  to c h a r a c t e r i z e the macroscopic properties  of  this  protein.  network network conformation was dimensions  of  randcm-coil  mechanical  and  elasticity  photoelastic  F i n a l l y , a t e s t f o r the random conducted  proteins,  by  predicting  the  and comparing these t o  published values. The  t h e s i s i t s e l f i s organized i n the f o l l o w i n g manner, I  have s t a r t e d by d i s c u s s i n g the p r o p e r t i e s of the i n t a c t composite elastic  (chapter II) t o show the exact r e l a t i o n s h i p fibre  conformation  and  controversy  (chapter  investigations (chapter V) , and VI).  of  the  to the other components present i n the t i s s u e .  The f o l l o w i n g f o u r chapters d e a l with the q u e s t i o n of  (chapter  tissue  involves  the  III),  presentation the  nmr  elastin  of the c u r r e n t  and  viscosity  (chapter I V ) , the p o l a r i z e d microscopy s t u d i e s the  scanning  The next chapter  electron  microscopy  result-s  (chapter VIII) deals with the  3  e v a l u a t i o n of the e l a s t i n kinetic  theory  network p r o p e r t i e s i n terms  of  the  of rubber e l a s t i c i t y . The p r e d i c t i v e t e s t f o r  e l a s t i n conformation i s presented i n chapter IX..  4  Cha_)ter__II__  GENERAL CHARACTERISTICS OF ELASTIC TISSUE,,  A. . I n t r o d a c t i o n Given  a  macroscopic chemical  mechanically  functioning  material,  the  p r o p e r t i e s of t h i s t i s s u e w i l l depend on, (a) the  composition  of  the  tissue,  (b)  the  mechanical  p r o p e r t i e s of the i n d i v i d u a l components,  (c) the a r c h i t e c t u r a l  organization  (d) the e f f e c t of the  chemical  of  these  properties  of  p r o p e r t i e s of i t s e l f As  will  be  components, and one  component  and the other  shown  in  probably  justifiable  to  is  of e l a s t i c t i s s u e , the  with i t ' s  implicated  convenience  and i t  organization,  i n the pathology of the vascular activity  protein  chemistry of t h i s  determine i t ' s f u n c t i o n a l p r o p e r t i e s . . S i n c e  has been c o n s i d e r a b l e of  mechanical  components.  discuss  p a r t i c u l a r p r o t e i n which, along eventually  the  t h i s chapter the e l a s t i n  forms a major mechanical component is  on  in this field,  will  elastin  system, t h e r e  so f o r the  sake  (and l a c k of breath) I have j u s t o u t l i n e d the  major aspects of i t ' s b i o c h e m i s t r y .  A detailed  discussion  of  the chemistry of e l a s t i n has been presented by Sandberg (1976) and  Franzblau  (1971).  1  Elastin  Development  (a) Embryology All  elastin,  l i k e c o l l a g e n and other connective  a r i s e s from the t h i r d  germ  layer  commonly  referred  tissue, to  as  5  mesoderm.  The  particular  elastin  mesodermal  exact  type of mesoderm t h a t g i v e s r i s e t o  is  source  of the composition tissue  on  where  T h i s c o u l d account  that i s observed  (Kieth  for  the  the  et^a1_._. 1S79).  A  (the  from t h a t  variability  review  types of  of e l a s t i n  summarized  in  the  paragraph.  According of  occurs  between d i f f e r e n t  embryology i s given by Hass (1939) and i s following  it  of a r t e r i a l e l a s t i n i s d i f f e r e n t  of ligament or s k i n ) .  elastic  dependent  t o Hass, the v a s c u l a r system i s the f i r s t  body  to  demonstrated  be  to  supplied  with  elastin,  be present i n f o u r day  humans the e l a s t i n  i s f i r s t found  and  part  can  be  o l d c h i c k embryos. In  i n the t h i r d  or f o u r t h week.  In the embryo, the majority of the e l a s t i n i s concentrated the  aorta  with  the  exact  distribution  parturition.  A f t e r b i r t h , the r e l a t i v e  the  decreases while the r e l a t i v e  artery  increases.  A  development  similar of  the  alimentary  tract  recieve e l a s t i c  time  elastin  elastin in  skin is  course  Although fibre  of  in  after  elastin  in  amounts i n the v e i n s  can  be  found  for  the  i n the lungs, with the development of  lagging thought  by  about  three  months.  The  t o be one of the l a s t organs t o  tissue.  i t has been known is  a  two  for  component  a  fibres long  between  the  two  time  system,  amorphous core with a surrounding f i b r i l l a r relationship  changing  amount  Jbj_ Morphogenesis of e l a s t i n  elastin  any  has  i«.e..  coat,  only  that  the  an i n n e r  the  recently  exact been  6  e l u c i d a t e d . Ross and components  Eornstein  (1969) demonstrated that  of the e l a s t i n f i b r e are  the e x t e r n a l f i b r i l l a r  coat being  the amorphous core being  a  very d i s t i n c t polar  has  i n diameter, which c o u l d  reduced  with  an extremely hydrophobic p r o t e i n . been s t u d i e d  been shown to be composed of f i b r i l s , ranging  to 40 nm that  chemically,  glycoprotein  chemistry cf the m i c r o f i b r i l l a r component has it  these  be  extracted  d i - s u l p h i d e bonds (Robert e t . a l . 1971,  and  from  with  The  10  agents Anderson  1976) . In  order  glycoprotein involved  to  assign  fibrils  it  It  has  development, later  but  (which  been  proposed  has  a  to  that  they  p r o t e i n during  of e l a s t i n i n t h e i r e a r l y  elastin  tend to  positive  which,  the  the of  present.  p r o t e i n can  aggregate  are  it's  stages  between these m i c r o f i b r i l s  charge, may  net  these  shown t h a t e l a s t i c ligaments i n  stages of development the  negative  role  have a high amount of g l y c o p r o t e i n  to be i n t e r s p e r s e d  to t h e i r  has  been  embryo are almost devoid  seen  functional  with the a l i g n i n g of the e l a s t i n  secretion.  In the  a  be due  elastin  charge) around themselves  (Ross  et_.al o_ 1 97 7). Further microfibrils who  evidence  this  has been obtained  histologically  developing  for  type  of  a  by C o t t a - P e r e i r a  role  for  the  et.al..(1977),  demonstrated the presence of two  types of  e l a s t i n f i b r e s . Oxytalan f i b r e s , composed mainly of  glycoproteins,  and  microfibrils  and  observations  they  elaunin amorphous proposed  fibres elastin. a  which On  oxytalan--  consisted  the b a s i s of elaunin—  of their  mature  7  elastic  fibre  protein,  which i s s y n t h e s i z e d i n v i v o by f i b r o b l a s t and  muscle c e l l s  hierarchy  (Boucek 1959,  Jcj_ The  turnover of  characterized the l i f e by  for  by  a  and  Elastin  elastin  workers  alteration  in  increasing  with  is largely  due  that  the  normal  (Ayer  the  the  elastin smooth  1977).  turnover in  turnover  age and to  Pathrapamkel e t . a l .  elastic  tissue  h a l f - l i f e that i s approximately  span of the animal  some  the development of the  1969). .It has been  diseased  states  rate  the  of  is  egual to proposed  represent  elastic  an  tissue,  pathology. T h i s i n c r e a s e i n t u r n o v e r presence  of  degradative  processes  (Robert 1 977) . .  Elastin  _[a}_ Amino a c i d In  composition  order to c h a r a c t e r i z e the amino a c i d composition  p r o t e i n one be  Chemistry  has to f i r s t  considered  as  being  decide on the g u e s t i o n of what i s  to  the 'pure' p r o t e i n . . F o r t u n a t e l y the  case f o r e l a s t i n i s g u i t e c l e a r  cut,  have  been  still  a r r i v e at a p r o t e i n of constant  account  of a  in  that  investigators  able t o t r e a t e l a s t i c t i s s u e g u i t e d r a s t i c a l l y composition  which  and can  f o r the e l a s t i c i t y of the i n t a c t t i s s u e . T h i s r e s i d u e  that remains a f t e r treatment  i s termed e l a s t i n ,  d e f i n e d as such f o r the r e s t of t h i s Amino  acid  and  will  be  thesis.  a n a l y s i s of t h i s p r o t e i n shows e l a s t i n to be  8  'Table, 2. 1: A j i n c a c i d composit ion of e l a s t i c  proteins,  AMINO ACID COMPOSITION OF ELASTIC PROTEINS. ELASTIN  1  RESILIN  2  ABDUCTIN  3  *  CONNECTIN  4  OCTUPUS FIBRES  asx  6.4  102  69.9  92  90.6  thr  8.9  28  7.4  59  64.4  ser  9.9  80  36.4  60  71.8  9lx  15  47  19.4  128  121  pro  120  77  7.4  65  54.9  hyp  10.7  -  —  12  -  giy  324  385  620  104  85  ala  232  111  26.5  84  71  cy5  4.1  -  -  4  7.4  val  135  28  3.5  60  62  met  —  23  21  -  117  1le  25.5  17  4  52  60  leu  - 61.1  23  3  70  73.5  tyr  7.1  27  10.7  28  36.2  phe  30  26  51.3  29  42.3  his  0.6  9  0.3  15  21  lys  7.4  5  12.4  61  68.3  arg  5.4  35  9.8  56  45.8  * 1n residues/1000. ^ o s s and B o r n s t e i n 1969. We1s-Fogh 1961. K e l l y and Rice 1967. Maruyama e t . a l . 1976. Shadwick 1980.  i 3  4  5  5  9  one of the most  hydrophobic  nearly  the  60%  presented  of  proteins  residues  being  yet  discovered,  non-polar.  later, this characteristic,  more than any  been r e s p o n s i b l e f o r the m i s - i n t e r p r e t a t i o n of properties  of  this  acid  composition  of e l a s t i n  and i t ' s comparison to the (Anderson  1971) ,  (Maruyama ______ from octopus  other  Abductin  proline  known and  (Shadwick 1980)  elastin  is  chains  stress.  accomplished of  discovered  a  that the  polymer  the  case  Thomas  desmosine.  These  of  Resilin  elastomer  has  a  to  mechanical prevent  the t i s s u e from natural  (Flory  et.al.  this  rubber  structural  elastomer who  them  resonance  is  residues regard to  were  first  showed them t o be  desmosine  proposals  the  'flowing' this  1953).. With  (1963)  named  c o n c u r r e n t l y by nuclear magnetic and K a t r i t z k y  1969)  Rice 1967) , Connectin  that  up  of  and  The  cross-links  make  cross-links  by  has  i s shown i n t a b l e 2. 1. .  protein  chains  pyridinium derivatives  These  residues.  by forming c o v a l e n t bonds between carbon  adjacent  elastin,  In  be  physical  elastomers,  f u n c t i o n , i t has to be c r o s s - l i n k e d i n order  under  the  (Eoss and B o r n s t e i n  (Kelly  (b) E l a s t i n  polypeptide  other,  1976), and the r e c e n t l y d i s c o v e r e d  arteries  Since  will  elastomer. E l a s t i n a l s o has an u n u s u a l l y  l a r g e content of g l y c i n e , v a l i n e , and amino  As  with  and  were  iso-  confirmed  studies  (Bedford  1 963) .  same  authors  ( P a r t r i d g e ______  that these compounds c r o s s - l i n k e d two  1965)  polypeptide  l a t e r showed chains  and  10  proposed a p o s s i b l e route f o r the formation of these l i n k s . I t is  currently  thought  that the desmosines and  are formed by the condensation  of four l y s i n e s , three of which  have been o x i d i z e d by the enzyme l y s l There i s a l s o some (iso)desmosines,  evidence such  c r o s s - l i n k i n g agents  as in  iso-desmosines  that  oxidase  residues  (Sandberg 1976). other  than  lysinonorleucine, also the  elastin  network  the  serve  (Lent  as  et.al.  1969) .  Jc_ As  mentioned  cross-linking network. Due in  Soluble e l a s t i n  before, insoluble e l a s t i n  r e s u l t s from  of s o l u t l e e l a s t i n p r e c u r s o r s i n t o a  the  functional  to t h i s r e l a t i o n s h i p there has been some i n t e r e s t  the c h a r a c t e r i z a t i o n of these p r e c u r s o r p r o t e i n s . Although  elastin  are  (Partridge 1953), KOH  a  number  known,such and  of methods f o r the s o l u b i l i z a t i o n oxalic  acid  Adair 1955), e l a s t a s e (Hall 1961), urea  (Bowen  (Mcczar ______  as  digestion  1979). The  the moment f o r the i s c l a t i c n of an  only method a v a i l a b l e  formation involved requiring  method  is  at  unbranched p r e c u r s o r i s the  e x t r a c t i o n cf t r o p o e l a s t i n from l a t h y r i t i c This  with  of  animals.  based on the biochemistry  cf c r o s s - l i n k  which r e q u i r e s the o x i d a t i o n of the l y s i n e r e s i d u e s , i n the formation of  (iso)desmosines,  enzyme l y s l oxidase  by  copper  (Franzblau 1971). I n h i b i t i o n of  t h i s enzyme's a c t i v i t y , by r a i s i n g  animals  on copper d e f e c i e n t  d i e t s or by i n d u c i n g l a t h y r i s m using agents such propionitrile,  the  as  B-amino-  allows the e x t r a c t i o n of s o l u b l e p r o t e i n s from  11  the e l a s t i c t i s s u e of the The an  extraction  amino  acid  animal.  procedure r e s u l t s i n a p r o t e i n  composition t h a t  cross-links)  and  tropocollagen  scheme  currently  thought  tropoelastin,  that  weight of 72,000, r e p r e s e n t s  (Sandberg  the  1976).  which has  building  to  molecular weight s p e c i e s ,  procollagen,  presence  of  and  such  1977).. T h i s recent is  et^ali.  named  however, has  provides strong  that  the  having  et.al.  biosynthesis  precursor-product r e l a t i o n s h i p e l a s t i n has  a  1976,  to r e s t by  a  tropoelastin {Rosenbloom  elastin  (Narayanan and  (Sandberg e t ^ a L  proteins  Page 1976,  P a r t i a l primary seguence data f o r been published  between  tropoelastin  been demonstrated r e c e n t l y  v i t r o c r o s s - l i n k i n g of the s o l u b l e  also  comparable stated  been put  evidence  primary precursor i n e l a s t i n  fibrous  linked  have  (Foster  mature  there e x i s t s  proelastin,  140,000  is  1980).  The and  130,000 t o  that  the  It  of  which would be  publications  protein,  speculation,  paper t h a t  the  recent  a  molecular weight of  lack  a molecular  block  i n s o l u b l e e l a s t i n . There i s some s p e c u l a t i o n higher  t o the  i s termed t r o p o e l a s t i n i n analogy to  collagen-  a  has  i s . s i m i l a r to mature e l a s t i n  (with the e x c e p t i o n of a high l y s i n e content, due of  which  porcine  to  give  in  cross-  Smith e t . a l ^ .197 5).. tropoelastin  i n the l i t e r a t u r e on e l a s t i n  1977).  EU Composition of E l a s t i c  by the  Tissue  have  biochemistry  12  _[__ Unpurified elastin blotted  Water content samples  from  ligament  nachae  on paper towels to remove excess water and  weighed at  240C..The same samples were then d r i e d to constant an  oven  at  110<>C  ligament  elastin i s  similar  value  samples  of  (Harkness  and  were  weight  in  reweighed. The r e s u l t s i n d i c a t e t h a t  approximately  72%  water  by  weight..  70% has been r e p o r t e d f o r a r t e r i a l  A  elastin  ______.1957).  (b) E l a s t i n  _______  U n p u r i f i e d ligament e l a s t i n samples were d r i e d i n an oven to constant weight. autoclaving value  of  weight.  The samples were then p u r i f i e d by  (Partridge e t . a l . elastin  content  1974,  1955), d r i e d and reweighed. so  obtained was  Values f o r the e l a s t i n content  occur i n the range of 40% Harkness ______  repeated  elastin  of  The  about 80% by  thoracic  (w/dry weight)  dry  arteries  (Charm e t . a l .  1957)..  J c _ N e u t r a l sugars Neutral  sugar content of u n p u r i f i e d ligament e l a s t i n  evaluated using the p h e n o l - s u l p h u r i c a c i d assay of (1970),  using  glucose  It  is  almost  mucopolysaccharide  et_al.  (Sigma) as a standard. A value of  (w/dry weight of t i s s u e ) was  _d_  Lo  was  0.3%  obtained.  Mucopolysaccharides  impossible  to  determine  the  exact  content of e l a s t i c t i s s u e due t o the l a r g e  Itr  In  IP-  in>  CHEMICAL COMPOSITION OF ELASTIC TISSUE.  E  lO  Ligamentum nuchae Mucopolysaccharides:  2.3%  Aorta  II-  1.6%  1  hexosamlnes  (36%)  (26%)  uronic acids  (41%)  (34%)  sulphate  (15%)  (12%)  Collagen  20%  18%  Elastin  78%  18%  0.3%  ?  Neutral  Sugars  !/) HInIM-  IS t  14  variability given  in  arterial the  from sample to sample. A table  tissue  2.2  for  general  ligament  (Meyer  et.al.  (Kirk 1959). In the case of  mucopolysaccharides  composition 1956)  ligament  are seen t o make up about  is and  elastin  2.3%  of the  whole t i s s u e  (w/dry wt.  present  the form of hexosamines, 41% as u r o n i c a c i d s with  in  approximately  T i s s u e ) . Of t h i s approximately 36%  is  15% a c i d h y d r o l y z a b l e s u l p h a t e . .  Jej_ C o l l a g e n content A f t e r accounting f o r the v a r i o u s other components present i n ligament t i s s u e , the approximately  17.4%  a r t e r i a l samples  collagen  content  works  to  be  as compared to values of 18% r e p o r t e d f o r  (Harkness e t . a L . 1957) .  The summary f o r the chemical composition ligament  out  t i s s u e i s given i n t a b l e 2.2  of a r t e r i a l  and  .  E. O r g a n i z a t i o n of E l a s t i c Tissue Since the main f u n c t i o n of e l a s t i c t i s s u e i s a mechanical one, i t i s not s u r p r i s i n g that the o r g a n i z a t i o n of the e l a s t i n fibres  in  e l a s t i c t i s s u e v a r i e s with the d i r e c t i o n ( s )  s t r a i n that are imposed on the t i s s u e i n with  this  generally  p r e s e n t a t i o n of the extremes  .  In  keeping  accepted h y p o t h e s i s , the f o l l o w i n g i s a  microscopical  organization  of e l a s t i c t i s s u e as demonstrated  and a r t e r i a l  vivo  of the  elastin.  J a | Methodolc_g_y  of  the  two  by ligament nuchae  15  Histology, Samples cf u n p u r i f i e d p i g adventitial was  aorta  the  and  fixative  f o r 72 hours. They were then cut  ligament  and  samples  into  small  samples  was  cut  into  procedure,  which was  by  et_al_  Clark  pieces  5  to  a slight  both  rinsed  the  with  the  arterial Bouins  distilled  and embedded i n p a r a f f i n  7  After  embedding  um s e c t i o n s and  m o d i f i c a t i o n of  wax the  s t a i n e d . The  that  presented  (1973), c o n s i s t e d of s t a i n i n g s e c t i o n s with  o r c e i n f o l l o w e d by a c o u n t e r - s t a i n of napthol protocol  of  were t r e a t e d with  using standard h i s t o l o g i c a l t e c h n i q u e s . tissue  stripped  l a y e r and other adhering t i s s u e . Ligament e l a s t i n  a l s o cleaned of adhering t i s s u e ,  water,  were  results  in  red  elastin  fibres  green  B.  This  with the c o l l a g e n  f i b r e s appearing f a i n t green. . Scanning  electron  microscopy:  I n s t e a d of using f i x a t i v e s , the samples liguid onto  n i t r o g e n and stubs  examination Instrument chapter  and of  Company,  with  a  samples  fine  was  Stereoscan  microscope  expected,  ligament  elastin  of on  gold.  The  a Cambridge  (see  methods,  collagen  fibres  seem  elastin i n the l i g h t  microscope  i n the l o n g i t u d i n a l  direction,  which i s the d i r e c t i o n of the i n v i v o 1um  in  6 f o r more d e t a i l s ) .  shows a very d i s t i n c t alignment  The  layer  conducted  Jb_ O r g a n i z a t i o n of ________ As  frozen  d r i e d i n a vaccum. They were then mounted  coated the  were  tc  strain  (figure  2.1a)»  form a very f i n e network  16  F i g u r e . 2 . 1 : O r g a n i z a t i o n of ligament e l a s t i n . (a) light micrograph of sectioned, unpurified ligament nuchae . The e l a s t i n f i b r e s , E, are a l i g n e d in the d i r e c t i o n of the s t r a i n (arrow) with t h e c o l l a g e n (C) i n t e r s p e r s e d between the e l a s t i n . The bar r e p r e s e n t s 30um. . (b) s.e.m. of u n p u r i f i e d ligament showing the collagen. (c s.e.m. of u n p u r i f i e d ligament. The arrow i n d i c a t e s a branch point i n the e l a s t i n network. The s o l i d bar i n b, and c r e p r e s e n t s 10um.  17  Figure.2. 1.  18  around  (between) the e l a s t i n f i b r e s which have a  about  6  to  8 um.  presence of a  There was  collagen  diameter  no h i s t o l o g i c a l evidence f o r the  sheath,  cr  other  such  collagenous  s t r u c t u r e , a s s o c i a t e d with the i n d i v i d u a l e l a s t i n Scanning  electron  f i n d i n g s of the l i g h t clearer  microscopy  essentially  microscope study as  (figure  2.1b). There was  supported as  giving  J c _ Organization  2um  direction. organized  presence  thickness)  The  elastin  of  2.2c). The lamellae  in  change  running in  in  this  interlamellar collagen  in  a  elastic  seemed  dense  respect  to  the  any  in given  the  is  occur  (which  lamella  seem  lamellae  (figure also  evident  between  The  composite  collagen Glagov  of are  the  network  be  showing The  (figure elastin  network. Although i t was  (Wolinsky and  structural  to  2.2b).  not  alignment  l o n g i t u d i n a l d i r e c t i o n of the the  of  circumferential  the adjacent  fibres to  branching  lamellar organization  direction  fibrillar  have been r e p o r t s that  f o l l o w s a h e l i c a l path  network  cross-sections  p o s s i b l e to determine the d i r e c t i o n of the c o l l a g e n  there  a  elastin  ( f i g u r e 2.2a), with the l a m e l l a  u n i d i r e c t i o n a l l y , with  a successive  the  samples,  a r t e r i a l samples show a very d i s t i n c t  (with  the  ( f i g u r e 2.1c).  of a r t e r i a l  In c o n t r a s t t o the ligament  constituents  in  a l s o some evidence f o r the  of the i n d i v i d u a l e l a s t i n f i b r e s  about  well  fibres.  p i c t u r e of the f i n e d e t a i l s . I t showed a very d i f f u s e  network of c o l l a g e n f i b r e s d i s p e r s e d  it's  .of  artery) actually  1964).  organization  of ligament  and  19  Fig;ur;e__2, 2: O r g a n i z a t i o n of a r t e r i a l e l a s t i n . (a) s.e.m. shewing the l a m e l l a r (L) o r g a n i z a t i o n of arterial elastin. (b) Light micrograph of s e c t i o n e d a r t e r i a l media showing the r e l a t i v e organization of adjacent l a m e l l a e . The t a r r e p r e s e n t s 2Cum. (c) s.e.m. of a r t e r y showing the presence of the i n t e r l a m e l l a r f i b r e s (If) . The s o l i d bars i n a, and c r e p r e s e n t s 10um.  20  21  a r t e r i a l t i s s u e i s d e p i c t e d i n f i g u r e 2.3. analysis  of  the  s t r u c t u r e of e l a s t i c  a r t i c l e s by C o t t a - P e r e i r a e t . a l . and Kadar  it  more  t i s s u e can be found i n  Techniques  order to study the p r o p e r t i e s of the e l a s t i n  tissue  protein,  rest  of  the  with which i t occurs i n v i v o . T h i s can be done by  use of a number methodology  of  (Robert  purification and  et.al.  1971)  technigues  Hornebeck  e v a l u a t i o n of the r e s u l t i n g  elastin  procedure  and the repeated a u t o c l a v i n g method of  presented as chemical  and  exclusively. a  The  the  1962,  comparison  structural  of  these  of  the  chemical Grant  the  which  elastin  (Partridge e t ^ a l ^  19 55)  section i s therefore two  technigues  on  a  basis.  compositions  e v a l u a t i n g the amino a c i d composition on  which  the  (Lansing e t . a l . , 1 9 5 2 )  following  J § 1 Amino a c i d In  and  (Partridge  with the p h y s i c a l p r o p e r t i e s  p r o t e i n , the a l k a l i e x t r a c t i o n  used  1976)  for  has been w e l l documented. In t h i s t h e s i s ,  deals primarily  decide  (1977)  (1977) .  i s necessary t o i s o l a t e the p r o t e i n from the  were  compelete  ( 1 9 7 7 ) , Carnes e t . a l .  F«_. P u r i f i c a t i o n In  A  one has to  first  a standard a g a i n s t which t o compare the r e s u l t s of  the p u r i f i c a t i o n product. T h i s standard i s u s u a l l y taken to be the p r e c u r s o r molecule, amino  acid  composition  tropoelastin.  Table  2.3  lists  for t r o p o e l a s t i n , autoclaved  and a l k a l i e x t r a c t e d e l a s t i n  as  presented  by  Grant  the  elastin e t . al..  22  F i g u r e . 2 . 3 : The _______ t i s s u e __________. (A) The a r t e r i a l media showing the relative organization of the c o l l a g e n (C) and the e l a s t i n (E) . (B) A schematic diagram of ligamenturn ______ showing the o r g a n i z a t i o n of the e l a s t i n , c o l l a g e n , and the c o l l a g e n sheath (CS) .  23  Figure.2  24  (1971).  Both  procedures  are seen to give comparable e l a s t i n  p r e p a r a t i o n s with regard t o the amino a c i d  composition.  jb_l Hexosamine and n e u t r a l sucjar content The  data obtained by Grant e t . a l . .  alkali  extracted  elastin  contains  (1971) about  hexosamine as autoclaved samples (both are below The  n e u t r a l sugars  of Lo e t . a l . . (1970)  standard.  The  results  show  n e u t r a l sugar content of 0.02% sugars i n a l k a l i p u r i f i e d  using that  glucose  microscope  Both p u r i f i c a t i o n  elastin  2.4c). No  fibres  a  e l a s t i n had a  purified  elastin  were  with gold and  observed  as d e s c r i b e d b e f o r e .  procedures  scanning  and b ) . However, t h e r e was  as  microscopy  gave c l e a n  electron  preparations  microscope,  i n d i c a t i o n of c o l l a g e n or 'matrix' substances  the  w/w).  d r i e d i n a vaccum. The p i e c e s of  were mounted onto stubs, coated  the  0.04%  elastin.  f r o z e n i n l i g u i d n i t r o g e n and  in  much  as  (w/w). There were no d e t e c t a b l e  Samples of a u t o c l a v e d and a l k a l i  observed  half  (Sigma)  autoclaved  l£L E v a l u a t i o n by_ scanning e l e c t r o n  i n a Stereoscan  that  were g u a n t i t a t e d with the p h e n o l - s u l p h u r i c  a c i d assay  tissue  indicate  (figure  with 2.4,  as no a  some i n d i c a t i o n of a l k a l i a t t a c k of  prepared by 0.1N  such degradation was  observed  NaOH e x t r a c t i o n for autoclave  elastin.  G. Mechanical P r o p e r t i e s of E l a s t i n  Bundles  (figure purified  25-  TaLl_g.2.3:  _____o a c i d  composition  of e l a s t i c  AMINO ACID COMPOSITION OF ELASTIN PREPARATIONS* Tropoelastin  Autoclaved  . Alkcli  asx  3.3  6.4  5.4  thr  13.2  7.4  5.3  9.2  8.7  6.6  glx  15.8  15.7  11.9  pro  101.1  118.4  96.4  hyp  6.6  8.7  12.9  giy  333.4  310.5  316.2  ala  237  238.6  243.1  cys  —  —  —  val  125.4  143.9  154.3  met  —  —  --  ile  16.1  23.3  24.6  leu  47.5  59.5  63  tyr  14.1  10.7  13  phe  28.3  28.6  32.1  his  —  --  --  lys  45.1  3.1  3.1  4.3  7.7  2,7  ., ser  *from Grant e t . a l . 1971.  preparations.  26  fi^ureiJ,. E v a l u a t i o n o f £urification techniques. (a) s.e.m. of a l k a l i p u r i f i e d e l a s t i n . (b) s.e.m. of autoclave p u r i f i e d e l a s t i n . (c) Higher m a g n i f i c a t i o n of a l k a l i p u r i f i e d e l a s t i n showing the h y d r o l y t i c attack of the e l a s t i n f i b r e . The s o l i d bars represent 10um.  27  28  (a) Methods Samples of ligament e l a s t i n were were  embedded  i n t o threaded  were then hydrated days  under  s t r a i n p r o p e r t i e s of Instron 1.5  tensile  unstrained  f o l l o w s . The was was  and  with  of  using  a  (Gosline  with  an  (of about  area  was  measured  as  the  of  L°,  extension  used  ratio  for and  the  the  subsequent  unstrained cross-  mechanical  test,  the  cut at the anchor p o i n t s , d r i e d t o c o n s t a n t weight  weight  p r o t e i n . The  stress-  c a l i p e r s before the s t a r t of a t e s t . T h i s  weighed. The volume of the  this  determined  seven  a t a r a t e of 1mm/min.  s e c t i o n a l a r e a . Immediately a f t e r was  were  cross-sectional  taken to be the value  sample  ends  length of the sample between the anchoring p o i n t s  measured  calculation  of  t e s t i n g . The  machine, with the samples  cm length) being extended The  before  elastin  testing  their  water over a p e r i o d  conditions the  and  s t e e l cups with epoxy glue. They  in distilled  sterile  dried  and  a  value of  protein  was  fraction  1978) , which was  of  from  1. 33g/cc f o r the d e n s i t y of the  volume of the hydrated sample  volume  calculated  0.65  fcr  the temperature  was  clculated  the p r o t e i n at 24°C at which  the  tests  were conducted.  The ncminal c r o s s - s e c t i o n a l area could then  calculated  dividing  by  by  the value f o r the hydrated  be  volume by  LO.  _(b) R e s u l t s Figure  2.5  shows  the  results  of  the  stress-strain  29  Figure.2.5;  ________  Mechanical  __________  of  elastin  P l o t s of nominal s t r e s s versus s t r a i n f o r : (a) autoclaved ligament elastin bundles. r e p r e s e n t s the f a i l u r e s t r a i n . (b) u n p u r i f i e d ligament e l a s t i n bundles.  (x)  30-  figure.2.5.  50  100 Extension %  150  31  experiments elastin.  cn  unpurified  and  autoclave  The u n p u r i f i e d samples show a b i p h a s i c curve  i n i t i a l t e n s i l e modulus of 6.87 X 10 of  3.8 X 10  by about  purified  6  to  Nm  -2  their  initial  length  before  modulus  of  the  extension. samples  These  are  values  in  Mukerjee e t . a l . Although purified  unpurified  close  this  sectional  as  bundles  for  in  the  same r e g i o n o f  f o r autoclaved  elastin  is  to  higher  modulus  the  unpurified  to 50%  component the  of  f o r the tissue,  the u n p u r i f i e d  mechanical  extension.  Since  properties f o r the  cross-  c a l c u l a t e d f o r the whole t i s s u e , of which  only 80% i s e l a s t i n , the nominal due  a  g l a n c e , i t can be e x p l a i n e d i f one  elastin  contributing  area  of  compared  approaching  underestimate  value  agreement with the values a t t a i n e d by  result,  assumes t h a t only the  elongations  at about  (1976)..  ligament  is  In  which i s higher than the  -2  obtained  appears r i d i c u l o u s a t f i r s t  samples  Nm  modulus  failure.  50% e x t e n s i o n , and upto f a i l u r e e x h i b i t e d a s i n g l e s  an  c o u l d be extended  t h i s , autoclaved e l a s t i n samples f a i l e d  the t e n s i l e modulus c f 8.6 X 10  with  and a f i n a l  -2  Nm ..These u n p u r i f i e d bundles  150% c f  contrast  s  ligament  to  stress  the overestimate  c r o s s - s e c t i o n a l by approximately  values  will  be  an  of the ' c o n t r i b u t i n g '  20%. T h i s i s borne out by the  absolute magnitude of the two m o d u l i i with the  ratio  of the  u n p u r i f i e d m o d u l u s / p u r i f i e d modulus being about 0.75 ..  H_.Mechanical P r o p e r t i e s of ______ The  first  studies  on  the  Elastin Fibres  mechanical  properties  of  32  Fi^are_. 2_. 6j_ Mechanical p r o p e r t i e s of s i n g l e elastin fibres., j?lots of nominal f o r c e versus e x t e n s i o n f o r : (a) u n p u r i f i e d f i b r e s {Carton e t ^ a l . . 1962). (b) a u t o c l a v e d f i b r e s .  50  100  Extension %  150  34  unpurified single et.al..  (1960,  1962).  the mechanical fibres in  biphasic  in  by  Carton  along  for purified single  figure  obtained  s t r e s s - s t r a i n curve bundles  2.6  of  by  (the  100%  Carton  similar  elastin.  a u t o c l a v e d e l a s t i n f i b r e s show a upto approximately  reported  with  elastin  methodology  to In  also exhibited a  that  obtained  comparison  fairly  linear  for  to  this,  relationship  e x t e n s i o n , beyond which they a l s o tend  to d e v i a t e upwards, but by an amount that i s much smaller that  shown  by  the  a u t o c l a v e d bundles, to  is  7).  relationship  unpurified  were  Their r e s u l t s are presented  study  i n chapter  The  fibres  p r o p e r t i e s obtained  this  discussed  elastin  unpurified purified  fibres.  In  the i n i t i a l e x t e n s i o n  c o n t r a s t t o the  single f i b r e s could  120-150% extension before f a i l u r e . The region,  a u t o c l a v e d s i n g l e f i b r e s , had  of  both  than  be  extended  Young's modulus f o r the  unpurified  a value of about 1 X 10  6  and  Nm . -2  I_. D i s c u s s i o n  Ja_ Variation i n e l a s t i n Although" composition few  is  a  general  concensus  and/or primary  differences  exist  sequence of  elastin  in  the amino a c i d from  different  t h e r e have been r e p o r t s on the e l a s t i n  cartilaqe,  about  of e l a s t i n , there i s a l s o some i n d i c a t i o n  distinct  Recently  there  biochemistry  that  the a  profiles sources.  from  auricular  which have shown t h i s p r o t e i n to have an  unusually  l a r g e content  of p o l a r r e s i d u e s  (twice  as  much  as  arterial  35  elastin)  with a 20%  (Field e t ^ a l i The  reduction i n the amount of v a l i n e r e s i d u e s  1 9 78) .  sequential  Al._  (1979), who  of  elastin  variability  was  analyzed the v a l i n e - p r o l i n e  from  different  tissue.  p a r t i c u l a r seguence occurs about aortic  pointed out by K i e t h et..  elastin  and  only  e l a s t i n . On the b a s i s  41  seguence  They  showed  times/1000  content that t h i s  residues  in  9 times/1000 r e s i d u e s i n a u r i c u l a r  of  these  e x i s t e n c e of more than one  results  they  favoured  gene f o r the e l a s t i n  the  protein.  This aspect of e l a s t i n b i o c h e m i s t r y i s worrisome s i n c e i t r a i s e s doubts about the use of e l a s t i n from ligament nuchae t o make  g e n e r a l i z a t i o n s about  e l a s t i n from  a r t e r i a l elastin.These differences would  be  very  (Urry  et^al..  determinant  crucial, 1S77a) ,  of e l a s t i n  the  as  as suggested  val-pro  composite  by s e v e r a l authors is  a  and  major  tissue  composite  o r g a n i z a t i o n i s seen to vary from  t i s s u e to  in  vivo  tissue  occurs .  Its  depending  exact f u n c t i o n a l s t a t e . In g e n e r a l , i t seems e v i d e n t  that the e l a s t i n p r o t e i n i s probably the component  seguence  t i s s u e , l i k e most other b i o l o g i c a l t i s s u e ,  chemical  it's  primary  seguence  mechanical  on  a  the  e.g.,  structure.  Jb}_ The Elastic  if  in  other sources  at  low  dominant  mechanical  s t r a i n s with the c o l l a g e n c o n t r i b u t i n g  the  more s i g n i f i c a n t component at higher e l o n g a t i o n s . Comparison of unpurified  elastin,  single seems  fibre to  results show  for  purified  t h a t another  and  mechanical  36  component i s i n t i m a t e l y a s s o c i a t e d fibre.  There  is  seme  with  speculation  which i s present  individual  f i b r e s ( F i n l a y and  as a sheath  F r a c a s s i n i et_ A l . 1977). This aspect which  is  based  on  mechanical  supported by the r e s u l t s electron  of  the  mechanical  the  a  around  of e l a s t i n  the  Serafini-  organization  properties,  could  histological  and  not  be  scanning  report.  ground substance i s u n l i k e l y to be a major  component  contributes  elastin component  Steven 1973,  microscope s t u d i e s conducted f o r t h i s  Although  single  t h a t t h i s other  c o n s i s t s of c o l l a g e n elastin  the  in . elastic  tissue  small amount as shown by  (it  Banga and  probably  Balo 1960),  i t ' s chemical p r o p e r t i e s are probably important i n determining the environment within these  environmental  the  elastic  properties  would  mechanical p r o p e r t i e s of the e l a s t i c Recent s t u d i e s cn the dynamic elastin  (Gosline and  properties  of  the  French 1979) elastin  slight  degrees of dehydration.  role,  of  tissue.  Alterations  i n e v i t a b l y e f f e c t the  component. mechanical  properties  of  i n d i c a t e that the f u n c t i o n a l  p r o t e i n are adversely In view  of  this  a l t e r e d by  the  crucial  ' c o n t r o l l i n g the e n v i r o n m e n t , which i s performed 1  the embedding matrix i n vivo cannot be  _c_ Evaluation  procedure  one  has  tc f i r s t consider  any  techniques given  alkali  purification  purification  the experiment that i s to  be conducted on the p u r i f i e d sample. I t i s evident that  by  ignored.  of _ u r i f i c a t i o n  In comparing the f e a s i b i l i t y of  study  in  yields  from  this  a more pure e l a s t i n  37  (chemically  closer  procedure.  On  showed t h a t , results  to  the  tropoelastin)  other  hand, scanning  structurally,  in  than  both  the  autoclaving  e l e c t r o n microscopy  preparations  gave  similar  g r c s s o b s e r v a t i o n . However, c l o s e r examination of  the p r e p a r a t i o n s  evidence  of  h y d r o l y t i c attack of the e l a s t i n f i b r e i n the samples t h a t  had  been  purified  revealed t h a t  there  was  some  by a l k a l i treatment. In c o n t r a s t , there was  i n d i c a t i o n of such unwanted s i d e  effects  in  the  no  autoclaved  preparations. This  brings  me  experiment t h a t one then  it  would  afforded  by  experiment  had  be  the  back  to  the  first  i n mind  was  biochemical  advisable alkali  involves  the  statement. I f the  to use the c l e a n e r  technique.  an  autoclaved  nature,  preparation  Alternately,  if  the  measurement of p h y s i c a l p r o p e r t i e s ,  such as s t r e s s - s t r a i n r e l a t i o n s h i p s , i t i s more use  in  feasible  to  sample which, though l e s s pure c h e m i c a l l y ,  i s probably  more a p p r o p r i a t e  no evidence  of e l a s t i n  f o r such purposes s i n c e there  degradation.  is  38  C h a p t e r ^ I I I i . CONFOJiMATION OF ELASTIN^ THE CONTROVERSY..  Introduction Several  models  years. These range triple  helix  the other similar  from  of  other  elastin the  structure,  extreme to  for  a  have been proposed  extreme  of  the  the  collagen-like  proposed  by Ramachandran  totally  amorphous  k i n e t i c elastomers  over  (1963), t o  random  (Hoeve and  network  Flory  1958).  The remaining p o s s i b i l i t i e s l i e somewhere i n between these  two  extremes,  such as  and  Anderson  ( 1970) ,  the  Liguid-drop  the  Oiled-coil  model  of  Weis-Fogh  model of Gray e t . a l .  and the r e c e n t l y p u b l i s h e d c r o s s - B e t a s p i r a l of Urry These models f o r e l a s t i n This  chapter  (1973)  (1976a) .  s t r u c t u r e are d e p i c t e d i n f i g u r e  3.1i  w i l l be d i r e c t e d towards p r e s e n t i n g the v a r i o u s  p r o p o s a l s f o r e l a s t i n conformation, and w i l l t r y  to  evaluate  the evidence on which they are based. .  The Random Network Model The  random  e x t e n s i o n from of  hydrocarbon  network model f o r e l a s t i n was  the work of polymer chemists on the elastomers.  mechanical behaviour standard new  theories  Since  these m a t e r i a l s e x h i b i t e d  that could not be accounted cf s o l i d e l a s t i c i t y  The  of Rubber E l a s t i c i t y , was theory  was  random conformation  an  properties  for  by  the  present a t the time, a  t h e o r e t i c a l framework, commonly r e f e r r e d t o as the  Theory ).  essentially  developed  (Guth  Kinetic  et.al^  1946  based on Gaussian s t a t i s t i c s r e g u i r i n g a f o r the m a t e r i a l i n g u e s t i o n .  39  F i g u r e . 3 . 1: Proposals f o r the conformation of elastin (A) C o l l a g e n - l i k e t r i p l e - h e l i x (Samachandran 1 9 6 3 ) . (B) B e t a - s p i r a l s t r u c t u r e (Urry 1 9 7 8 b ) . (C) O i l e d - c o i l model (Gray e t ^ a l ^ 1 S 7 3 ) . (D) L i q u i d - d r o p model (Weis-Fogh and Anderson 1979). (E) Random network conformation (Hoeve and Flory 1 9 5 8) .  4 0  F i g u r e . 3.1.  B  rigid t B-spiral I  | dynamic j_-spiral  c  41  This K i n e t i c theory was  a l r e a d y i n e x i s t e n c e when  s t a r t e d to study the p r o t e i n rubbers, and i t i s not that  the  initial  these p r o t e i n kinetic hinge  elastomers  theory  involved  Abductin  rubber  the  T h i s was  (Alexander  Resilin  application  1966)  (Weis-Fogh  kinetic  theory.  Since  network s t r u c t u r e , i t was conformation random  at  of  these  the  conformation  and  a n a l y s i s of  the  1961a).  The elastin,  elastin  using 1),  associated  work  the  on a random  idea  properties  of  1936,  length  that  et.al.  these i n v e s t i g a t o r s f a i l e d dependent  swelling  of  a  according  and  Fiery  on the to  the  p r o p e r t i e s of  experimental  l a r g e energy mechanism  technique  component  which  was  to  in  (Meyer and  l a t e r shown that  account  the  elastin,  (1958)  was  1943). I t was  which  component a s s o c i a t e d with i t (Hoeve and Hoeve  the  random  a l s o based  thermoelastic  elastic  Wohlisch  to  a  c o n t r a d i c t i o n to the n o t i o n o f an entropy elastomer Ferri  by  s t a t e t h a t they were  p r o t e i n was  the  constant  the  predicted  thermoelasticity  on  indicated  with  The  were  elasticity.  Elastin  initial  (appendix  level.  i t ' s macroscopic  _a_  and  the  insect  Both  t h i s theory i s based  elastomers  k i n e t i c theory of rubber  the  l o g i c a l t o then e x t r a p o l a t e  molecular  for  of  done f o r the b i v a l v e  c o n v i n c i n g l y shown t c be e n t r o p i c elastomers as the  surprising  to c h a r a c t e r i z e the p r o p e r i e s of  relationships.  ligament  protein  attempts  people  had  for has Flory gotten  a  temperature large  energy  1974). around  this  42  experimental  difficulty,  of  temperature-deswelling,  studying the t h e r m o e l a s t i c i t y of e l a s t i n i n system,  of  30:70  e l a s t i n was showed  mixed  diluent  e t h y l e n e - g l y c o l : w a t e r , where the volume of  independent  that,  a  by  under  of  temperature.  these  Their  conditions,  elatin  experiments behaved as a  t y p i c a l k i n e t i c rubber with the energy  component  to  supported by V o l p i n  zero.  Cifferi  This  conclusion  (1970) who  temperature  although  temperature  types  the  approach  (1960) ,  solvent It  basic  i.e.  (1968)  who  circumvented  in  the  pointed  the problem  did  not  was  test  out of a  for  a  the changes i n the d i s t r i b u t i o n of  molecules  bound  as  a  function  of  was t h e r e f o r e necessary to make some changes theoretical  relationships  to  explain  the  behaviour of open systems such as e l a s t i n . T h i s  accomplished by Oplatka e t . a l . ,  Smith  experiment  temperature.  by Oplatka _t_al__£ this  of  thermoelastic was  of  change  temperature. in  similar  dependent volume change i t  composition the  a  and  mixed d i l u e n t systems u t i l i z e d by Hoeve and F l o r y  later criticized that  conducted  later  close  range of 50°-70°C, where the volume of e l a s t i n i s  a l s o independent The  was  being  for  (1960),  and  Bashaw  and  pclymer systems of highly swollen rubbers.  The s p e c i f i c a p p l i c a t i o n of these r e l a t i o n s h i p s t o the e l a s t i n p r o t e i n was  provided by M i s t r a l i _______  Hence with regard to rate,  elastin  (1971).  thermoelasticity,  any  i t seems f a i r l y s a f e t o conclude t h a t the r e s u l t s p o i n t  t o e l a s t i n being a t y p i c a l k i n e t i c elastomer as the  at  described  by  k i n e t i c theory cf rubber e l a s t i c i t y . Since t h i s theory i s  43  based  on a random network conformation, i t seems reasonable t o  expect e l a s t i n to be random i n i t ' s conformation as well,  Jb_ Kakivaya  and  differential elastin. second  D i f f e r e n t i a l scanning Hoeve  order  to  the  of s t a b l e secondary  that  elastin  second  undergoes  transitions  d i d not see any  be a s s o c i a t e d  structures.  temperatures  evidence  The  extensible  found t h a t depressed  polymer  diluents, the  glass  such  (Gotte as  well  first  for  with  the the  glass  observed  characterized  regarding  transition  the  use  of  ______  ethylene  by  glycol  a  the  diluent  thermodynamic s t u d i e s of e l a s t i n . They a l s o argued results  were c o n s i s t e n t  a random network model  consisting  of  a  single  And  X-ray d i f f r a c t i o n  studies  on a  Oplatka  system f o r that  with a random network model, and  phase.  _ c _ _____  of  also  water,  equally,  objection  mixed  and  a  glass  1965). I t was  temperature  volume b a s i s , which argues a g a i n s t (1960)  are  with the 'melting'  values  correlated  for  sudden drop i n the modulus as i t goes frcm being a r i g i d an  a  range that depends  order  mechanical behaviour of e l a s t i n which i s  to  of  g l a s s t r a n s i t i o n s that are observed f o r most  t r a n s i t i o n s that might  transition  technique  to study the g l a s s p o i n t of  show  content.. Such  amorphous polymers..They order  the  t r a n s i t i o n , i n a temperature  on i t ' s water cf  used  scanning c a l o r i m e t r y  They were able  typical  (1975)  calprimetry  these only  homogeneous  44  Early in  examination  of n e g a t i v e l y s t a i n e d e l a s t i n  the e l e c t r o n microscope, showed e l a s t i n to be an  p r o t e i n with  no d e t e c t a b l e s t r u c t u r a l f e a t u r e s  1962,  and  Eoss  Bornstein  however was not investigators  an at  unanimous that  s t r u c t u r e s w i t h i n the Gross  1949).  studies,  filamentous  these  also  elastin  as  reported  fibre  structures observations  been  form  the  model of e l a s t i n , t h a t w i l l  Little  1961)..This  various seeing  (Lansing  have  amorphous  (Cox and  Karrer and Cox  conclusion  time  Similar  and  1969,  fibres,  other  fibrillar  et.al.  1952,  seen i n r e c e n t basis  for  a  be d i s c u s s e d l a t e r on  i n t h i s chapter. . The X-ray d i f f r a c t i o n s t u d i e s that  time  elastin reported did  were a l s o i n support  (Astbury diffuse  not change  typical  of  crystalline  Cox  upon  Little  conducted  1962).. Both  polymers Again  This and  the  type  of  indicated  1 935,  papers  Bear  1942  pattern  is  a  of  interpretations  some r e p o r t s o f s t r o n g d i f f r a c t i o n  (Kolpack  at  haloes a t 4.6A0 and 7.8A° which  stretching.  structure.  elastin  of a random network model f o r  and  diffraction  amorphous  unanimous with elastin  1940,  of  and  r e s u l t s were l a t e r shown to be a r t i f a c t s  lack were  not  patterns f o r  1944)..All of, these caused  by  collagen  contamination.  J d l N.M. B.  Evidence  The r a t i o n a l e f o r t e s t i n g the random netwcwrk model using nuclear  magnetic  t h i s technigue  resonance  lies  i n the high s e n s i t i v i t y o f  i n measuring the t h e m o b i l i t y of tihe components  45  i n v o l v e d . T o r c h i a and of  ligament  correlation backbone kinetic  elastin  (1973) obtained  and  they  l3  C-nmr  able  to show t h a t  the  indicators  of  are  taken  movements  (with  the  s m a l l times r e p r e s e n t i n g  indicating a  high  had  from  carbons  in  for  ( l y e r l a and  were proposed to  the  values  mobility  be  to  spectrum  which  resonances,  backbone  were  a  times,  motion), as obtained  various  percent  Piez  be  line the  about  Torchia  widths  of  the  10 nanosecond range 80  percent  1975). The  involved  fast  in  the  of  the  remaining  20  cross-linking  region which would be expected to have lower m o b i l i t i e s . These data a l s o support  the random network conformation.  C_. L i q u i d Drop Model  (a) P r e l i m i n a r y The seeded  initial by  Partridge  corpuscular filtration  idea  experiments  f i b r e s . He  columns  could  molecular  were c o n s i s t e n t with that  contained  was  seperate  weights,  a two-phase model f o r e l a s t i n  (1967a,  structure  elastin  of  for  1967b) elastin  conducted  who on  the  basis  packed  the  basis  of  their  and t h a t these s e p a r a t i o n c h a r a c t e r i s t i c s the matrix  (elastin)  being  a  material  pores of about 32A° i n diameter, or a sytem of a length of  16A°.  Partridge  n o t i c e d that t h i s system would adsorb a l c o h o l s , and  t h i s adsorption  gel  able to show t h a t these e l a s t i n on  columns  of  a  with  molecules  on  hypothesized  was  packed  randomly d i s t r i b u t e d rods having also  evidence  of  alcohols  was  directly  related  to  that the  46  hydrocarbon  chain  l e n g t h . I t t h e r e f o r e seemed reasonable, at  the time, to think of  elastin  discrete  hydrophobic  regions  of  as  a  two-phase  clusters  system  and i n t e r s p e r s e d  s o l v e n t , e s p e c i a l l y s i n c e these s t r u c t u r e s were electron A  microscope similar  Poullain  and  degradation  evidence  type Robert  showed  that  of  model  was  using  elastin.  eluded  Jhey  addition  of  from  proposed  elastin  solvent  hydrophobicity  a l c o h o l s to the  reaction  elastin.  They  dispersion  of  hydrophobic  by the o r g a n i c s o l v e n t f o l l o w e d by a l k a l i n e a t t a c k of  the p e p t i d e m o i e t i e s . Although there  alkali  a s e r i e s of steps i n the degradation process of  which i n v o l v e d the i n i t i a l  regions  alkali  g u a n t i t a t e d the  mixture g r e a t l y f a c i l i t a t e d the degradation of then  by  t o by K o r n f e l d -  evidence  as a f u n c t i o n c f  the  supported  ( P a r t r i d g e 1 968).  (1968)  studies  d i g e s t i o n of e l a s t i n and  of  with  is  an  implicit  i t is  assumption  not  stated  explicitly,  i n t h i s type of degradation  process of a two-phase system.  Jb_  Thermodynamic  evidence  Using the constant temperature (appendix  1)  Weis-Fogh  during e l o n g a t i o n t h a t was extension.  This  with the random entropy  based  was  and  Anderson  ( 1970)  observed  viewed as a r e s u l t that was  network  experiment a heat  many times l a r g e r than the work  model  for  elastin  of  inconsistent  since,  for  an  e l a s t i c i t y , the heat r e l e a s e d during e x t e n s i o n  should be egual to the mechanical sample  thermoelastic  (see appendix  1). I t was  work done  a l s o observed  to  elongate  that t h i s  the  effect  47  of  'excess  heat'  cculd  be  reduced  by  hydrocarbon molecules l i k e a l c o h o l s . On results  they  proposed  s t r u c t u r e , which hydrophobic Extension  liguid  consisted  of  drop  chain  of  these  basis  model  corpuscular  for  units  elastin having  of t h i s network would f o r c e p a r t s of the  1976)  model was  and  the r e t r a c t i v e f o r c e was  obtained  from n.m.r. Data  by G o s l i n e §tj,al. (1975) who  a n a l y s i s to confirm  this reversible  groups,  corpuscular  from  hydrophillic  the  hypothesized  solvent during  The  o i l e d c o i l model was  is  based  these  interspersed  Model  tropoelastin  (1973),  (Foster  the  category  were assigned  to  the  flexible  the a l a n i n e and and  regions.  valine  data  cross  link  It which  lysine  residues  the  To  rich being  i s thought that align  by  et..al..  et^al... 1971, 1 972) . They d e l i n e a t e d t h e i r  a l a n i n e r e s i d u e s form a l p h a - h e l i c e s residues  the  hydrophobic  proposed by Gray e t ^ a l ^  with the g l y c i n e , p r o l i n e  assigned  to  of  probe  (b) r e s i d u e s i n v o l v e d i n the e x t e n s i b l e r e g i o n s .  first  areas,  exposure  c a t g o r i e s : (a) r e s i d u e s i n v o l v e d i n  r e g i o n and  Packer  on the amino a c i d seguence data accumulated  Sandberg  i n t o two  ( E l l i s and  extension.  i n v e s t i g a t o r s f o r porcine  1973,  support  used f l o u r e s c e n c e  units,  p. O i l e d C o i l  and  a  hydrophobic  a r i s e from t h i s i n t e r f a c i a l enercjy. e f f e c t . F u r t h e r  for this  the  the  long  core with the h y d r o p h i l l i c groups on the s u r f a c e .  core to the s u r f a c e and to  a  adding  the  lysine  i n t o a p o s i t i o n that favours t h e i r c r o s s l i n k i n g i n t o  desmosine and isodesmosine r e s i d u e s .  48  The link  o v e r a l l model envisaged a system having h e l i c a l  regions  with  o i l e d - c o i l . I t was would  occupy  exposed  the  solvent.  the  with  the  buried  inside  lies  in  these c l u s t e r s to be to  be  of  the  proposal  globules,  fibrillar.  by Weis-Fogh and  at  the  model  for  with a very taken  to  an  Santhanam elastin.  low  shape  only  of  the  presume  whereas the o i l e d - c o i l  proposes  identically,  the c a l o r i m e t r i c data  (1970) was  of the f i b r i l l a r  when  classified  conceptually  (1967). The  the  also  cited  obtained  as  evidence  Models models f o r e l a s t i n was  i n the c o l l a g e n investigators  f i e l d t h a t was started  s t r u c t u r e . These workers approached i t from a which  is  the  model.  activity  time  other  seems to  Ii F i b r i l l a r  the i n t e n s e  solvent  and  Both of them would behave  Anderson  f o r the o i l e d - c o i l  tone  residues  the c o i l s away from  model of P a r t r i d g e  i n the thermodynamic sense, and  The  valine,  l i g u i d drop model of P a r t r i d g e  hydrophobic c l u s t e r s . The  them  proline,  As i s obvious, t h i s type  difference  forming a broad  e x t e r i o r of these o i l e d - c o i l s i n a  residues  s i m i l a r to the  regions  f u r t h e r proposed that the g l y c i n e  position,  hydrophobic  extensible  cross  i t as being  melting extreme  (1957) who  a  study point  (Astbury  by  prevelant elastin of  view  part of the c o l l a g e n f a m i l y ,  temperature in  to  set  1940).  It  but was  p u b l i c a t i o n by Samachandran  and  proposed a c o l l a g e n - l i k e t r i p l e h e l i x f o r  This idea of wanting to f i t e l a s t i n i n t o a s t r u c t u r a l  hierarchy,  as borrowed  from  the  collagen  group,  is  still  49  prevelant  today and  forms the  i n t e r e s t i n the f i b r i l l a r  major impetus f o r the  models.  (a) E l e c t r o n microscope The  fibrillar  microscope was elastin  appearance  first  f i b r e was  of  evidence  elastin  noted by Gross  in  the  electron  stated that  the  a composite of a twisted rope s t r u c t u r e s of  Other s t u d i e s a l s o reported  the e l a s t i n  in  (1949) who  80A° diameter, which were cemented together matrix.  increased  fibre  via  an  amorphous  such f i b r i l l a r  components  (Lansing e t ^ a l ^ . 1952,  Ehodin and  Dalhamn  1955). A flood  of papers on e l a s t i n  t h i s theme of a f i b r i l l a r These  investigators  s o n i c a t i o n and arrays  of  model f o r e l a s t i n  have u t i l i z e d  heavy  metal  fibrils,  30A°  in  in  1973,  et.al.  Gotte  1976,1978). The 40-50AO  ligament with  the  diameter  visualize (Gotte  1974,  l a s t authors  periodicity  a  recent  of  an  components,  fibre  as  ordered  et.al..1965,  the  existence  (Quintarelli  et^al.  Serafini-Fracassini  have a l s o reported  of  et.al.  a  reflection  i n the X-ray d i f f r a c t i o n  p a t t e r n s of  been s t r e t c h e d by  This  60%..  (Pasquali-Eonchetti  f r e e z e f r a c t u r e and  presence  structural  study  such  1966). Recent p u b l i c a t i o n s  elastin  e l a s t i n t h a t had  utilizing the  to  up  some s u c c e s s .  microscope s t u d i e s have a l s o confirmed  of s u b - s t r u c t u r e  of  with  d r a s t i c technigues  staining  S e r a f i n i - F r a c a s s i n i and Tristam electron  u l t r a s t r u c t u r e have taken  et^al^  etching technigues,  extension present  related strong  along  which  alignment evidence  1979),  of for  show the the  50  fibrillar In  model. general,  the  electron  microscope evidence  seems t o  support a f i b r i l l a r  model which when taken t o an extreme,  be  a t w i s t e d rope array of two 15A° f i l a m e n t s  visualized  as  that,  as a u n i t , are v i s u a l i z e d i n the e l c t r o n  30A°  fibrils  (Gotte ______ 30A°  scanning  1976). There i s a l s o some  these  are  150 t o  clustered 20Cnm  in  e l e c t r o n microscope  Nuclear  evidence  that  together i n t o a higher order of diameter,  as  (Hart ______  magnetic resonance  observed  i n . the  1978).  ________  has been pointed out i n seme recent p u b l i c a t i o n s t h a t  the primary the  seguence cf t r o p o e l a s t i n , which i s thought  precursor  of  the  fibrous  protein,  to  Based on these primary  sequence data Urry and  1972).  h i s co-wprkers  s y n t h e s i z e d a number of repeat peptides corresponding t o  the seguences i n t r o p o e l a s t i n , and have attempted t o the  be  contains recurring  sequences of c e r t a i n amino a c i d s (Sandberg _ t _ a l . , 1 9 7 1 ,  have  as  fibre  J__ It  microscope  running along the long a x i s of the e l a s t i n  fibrils  sub-fibre,  can  conformation  of  the  elastin  protein  by  s y n t h e t i c peptides as models. T h e i r e f f o r t s have on three such  decipher  using  these  concentrated  model peptides:  1. A t e t r a p e p t i d e (Val-Pro-Gly-Gly) n 2. A pentapeptide 3. A hexapeptide For  the  sake  of  (Val-Pro-Gly-Val-Gly) n (Ala-Pro-Gly-Val-Gly-Val) n  simplicity  tetrapeptide studies follows.  a  short  discussion  of the  51  £ i a ^ r e _ . 3^2.2 Beta-turns. (A) The t e t r a p e p t i d e ( v a l - p r c - g l y - g l y ) showing the results of nmr studies: (•) solvent shielded moieties, (o) solvent exposed moieties (from U r r y a n d L o n g 1976a) . (B) Beta-turn proposal based on these results. Dashed line i n d i c a t e s t h e n y d r o g e n bond (from U r r y and Long 1977b).  52-  F i g u r e . 3.2.  53  Figure summary data  3.2 (a  of  the  obtained  S  b ) , shows  the  tetrapeptide  proton and carbon nuclear  from  solvent  Triflouroethanol),  which  titration  involves  the  magnetic resonance technigues  (DMSO-  monitoring  of the  resonances as a f u n c t i o n of s o l v e n t composition 1976a). I t was found that some moities dependence  of  relatively  their  chemical  uneffected.  (Urry and Long  showed a marked  shifts,  These u n e f f e c t e d  and the  while  solvent  ethers  residues  were  were proposed  to p a r t i c i p a t e i n hydrogen bonds. In  the  case  of  the t e t r a p e p t i d e ,  proton resonace data  showed t h e GlyU NH to be a s o l v e n t s h i e l d e d moiety involved  in a  hydrogen bond with the carbonyl  which  was  oxygen of the  Val1 r e s i d u e . In comparison t o t h i s , the Gly3 and the Val1 NH were  found  to  be  solvent  p a r t i c i p a t e i n hydrogen supported  exposed, and were not thought t o  bonds.  Carbon  Long  proposed  1976a) . .. On a  Beta-turn  tetrapeptide  also  the  s o l v e n t exposed  b a s i s of these f i n d i n g s they have  stucture  (figure  3.2b)  of the high  for  polymers o f t h i s t e t r a p e p t i d e  i n d i c a t e d t h a t , above 50oc, i n a d d i t i o n to the 12,  (Urry  this  (Urry and Long 1977b).  Studies  Gly8,  data  these r e s u l t s , showing that the Val1 CO was i n f a c t  s o l v e n t s h i e l d e d with the Gly4 CO being and  resonance  and  16  NH*s  also  Gly4  behaved i n a s o l v e n t  repeat  NH,  the  shielded  manner. .Furthermore, the Gly4 CO behaved as though i t too was involved  in  a hydrcgen bond. To account f o r these a d d i t i o n a l  findings a Beta-spiral structure e x i s t above 50oc  ( f i g u r e 3.3) was proposed  (Urry e j ^ a l . . . 197 7a) . .  to  54  Farther  support  for  these  obtained through c o n f o r m a t i o n a l et.al_,1976)  Beta-turn  energy  and n u c l e a r overhauser  calculations  enhancement  of  the  Val1  (CH )  two  turn)  residues  (Urry e t . a l .  and  the  as  a  the  side  the  poly-pentapeptide  poly-hexapeptide  chains  preferred  (rigid)  (Urry  (Urry e t . a l .  of  1974,  ______ 1978a,b)  were a l s o i n v o l v e d i n the  conformation.  A  Beta-  number of a d d i t i o n a l  hydrogen bonds are present i n the hexapeptide more s t a b l e  This  1977a) .  i n d i c a t e d that these polymers turn  protons r e s u l t e d i n  a f t e r r i n g c l o s u r e (formation of a Beta-  S i m i l a r s t u d i e s cn 1976b)  which  proton resonances.  i n d i c a t e d the c l o s e a s s o c i a t i o n between these  was  (Khaled  enhancement s t u d i e s ,  showed t h a t the i r r a d i a t i o n of the £ro2 CH an  structures  which make i t  s t r u c t u r e than the ether two  a  repeats (Urry  1978a) . Assimilating the e l e c t r o n a  microscope  hierarchial  3.1.  this  model  nuclear  magnetic  evidence, Urry has proceded for elastin  occuring  regions in  condensation  the  with  the  cross-link  data to  with  define  which i s d e p i c t e d i n f i g u r e  T h i s s t r u c t u r e c o n s i s t s of dynamic  extensible  resonance  rigid  Beta-spirals  hexapeptide  regions,  in  Beta-spirals  facilitating  of the l y s i n e r e s i d u e s i n t o the  the  the  (iso) desmosines.  _. D i s c u s s i o n  J a _ _________ Under  this  models  c l a s i f i c a t i o n I have grouped  the  liquid-drop  55  model  (Partridge  oiled-coil propose  1967, Weis-Fogh and Anderson  model  (Gray  et.al.  1973)  1970)  since  and  both  the  of these  d i s t i n c t r e g i o n s of hydrophobic c l u s t e r s surrounded by  solvent. The the  major support f o r these two-phase models i s based  thermodynamic  evidence  of Weis-Fogh and Anderson  on  (1970)  and i t i s cn t h i s very evidence that i t has been r e f u t e d . In a communication by Grut and McCrum  (1S74)  that  Weis-Fogh  the  data  obtained  by  p r e d i c t a b l e from the k i n e t i c theory of (which  supports  i t was  of  energy  of  term  process.  water  onto  represented  This  out  Anderson  rubber  was  elasticity  a random c o n f o r m a t i o n ) . They argued t h a t the  excess heat observed during e l o n g a t i o n was adsorption  and  pointed  also  a  result  of the  the non-polar groups and that the the  heat  explained  of  the  dilution  decrease  from  this  i n the energy  component with the a d d i t i o n of o r g a n i c s o l v e n t s t o the d i l u e n t (which would have a lower heat of elaborated  d i l u t i o n ) . . The  b y Dorrington ______  further  the same c o n c l u s i o n s . T h i s a b s o r p t i o n elastin  network during extension  a l s o e x p l a i n the r e s u l t s of the (Gcsline e t . a l .  the  water  flcurescence  by  the  1974) c o u l d  probe  analysis  1975) which c o u l d , i n r e t r o s p e c t , be p r e d i c t e d  two-phase  model  would  g l a s s t r a n s i t i o n temperature (1 975) .  was  (1975) who a r i v e d at  (Hoeve and F l o r y  by the k i n e t i c theory r e l a t i o n s h i p s A  of  point  (Mark  1976).  a l s o be i n c o n s i s t e n t with the  studies  of  Kakivaya  and  Hoeve  These authors e x p l a i n e d t h a t i f t h i s g l a s s t r a n s i t i o n  occured i n s i d e these  •hydrophobic  g l o b u l e s ' , then i t would  be  56  hard  to  account  temperature on these  for  dependence  the amount of water t h a t  of  the  was  transition  present  outside  globules.. It  has  a l s o been pointed  temperature-swelling the  the  Flory-Behner  out i n recent  behaviour of e l a s t i n i s  in  guestion  (Gosline  magnetic resonance  studies  indicated  rapid  a  consistent  model f o r network s w e l l i n g  based on an amorphous, s i n g l e phase network (elastin)  studies that  very  1977,  (Torchia back-bone  for  the  Piez  motion  for these  models  elastin  struture,  protein  have  reduced  a  since  the  mobility  nuclear  the  i s a l s o hard to r e c o n c i l e with  expected to  polymer  1973),  protein.This for  with  which, a g a i n , i s  1978) . Carbon  and  the  inside  have  elastin  two  phase  would  such  be  compact  structures.  IhL  Evidence f o r secondary  Investigators have confirmed residues  in  that  Piez  e t . a L . 1 973,  location  of  are  1973,  is  twenty in  and  present  at  per-cent  secondary  Torchia  et.a. JU . 1977, the  technigues  1975,  of  of e l a s t i n  e x t e n s i b l e p a r t s of the polymer and  his  or  are  the  structures Starcher  Marami efj.aU . 1970) . . The moment  involves  these secondary s t r u c t u r e s : do they occur i n  c r o s s - l i n k regions  Urry  spectroscopic  involved  Lyerla  Tamburro that  different  approximately  elastin  (Torchia and  controversy  using  structures  they  present  in  the the the  chains.  co-workers  have  p u b l i c a t i o n s that these ordered r e g i o n s  argued are an  in  numerous  integral  part  57  of  the  elastin  discussed  chain  in  Beta-turn  structures. ______  glycine On  (1980),  resonance technigues, residues i n e l a s t i n In  extensible  judging  the who  residues other  also  between  these  two  (b)  can  In answer to the f i r s t occur  as  would occur  their  secondary  turns  resonance Hoeve  of  magnetic  has to ask  the  amino  a  acid  which form the b a s i s of  in  20%  the e l a s t i n that  of  the  primary although  the s t r u c t u r e s proposed  as a major conformation  assuming t h a t still  the  retain  This assumption, however, i s not  s i n c e even a r e l a t i v e l y  conservative  (val--pro) r e s u l t s i n the d i s r u p t i o n of the  Beta-  1977a). tc  the second q u e s t i o n , i t i s p o s s i b l e to  thermodynamic results  (1S75) using  calorimetry,  estimate,  structure.  reference  assimilate  one  could t o l e r a t e s u b s t i t u t i o n s and  (Urry ______ In  views  1973). Urry has argued  borne out e x p e r i m e n t a l l y substitution  by  possibilities?  peptides  t h i s i s i n f a c t a reasonable  peptides  nuclear  g u e s t i o n , only about  repeat  (Foster et . a l .  repeat  in  r e s u l t s from other methods be used t c  d i f f e r e n t i a t e between he two  residues  involved  hand, a recent study  (a) what percentage  Urry's  him  as  have shown t h a t almost a l l of the v a l i n e  as the repeat p e p t i d e s ,  work?  are  used  r e s i d u e s occur  by  and  are c h a r a c t e r i z e d by r a p i d movements.  number of q u e s t i o n s :  sequence  regions,  p r e v i o u s l y , these i n v e s t i g a t o r s b e l i e v e t h a t most of  the v a l i n e , p r o l i n e , and  Fleming  the  failed  tc  data  obtain  the to  with a  technigue  the  nuclear  magnetic  c l e a r e r s t o r y . Kakivaya of  observe any  differential first  order  and  scanning  transitions.  58  arguing a g a i n s t the presence would  be  expected  transition high  of B e t a - s p i r a l s t r u c t u r e s ,  which  to give a sharp peak i n the r e g i o n of the  (from the s p i r a l s to the random c o i l as induced  temperatures).  Their  results  by  would tend to favour the  view t h a t e l a s t i n i s e s s e n t i a l l y an amorphous p r o t e i n . I t must a l s o be kept i n mind t h a t most of the proposals f o r the structures fragments,  are  based  which may  on  studies  involving  not be s a t i s f a c t o r y  small  models f o r  Betapeptide  insoluble  elastin.  Jc]_ It  Hydrogen bonded s t r u c t u r e s  i s evident t h a t the Beta-turn and the B e t a - s p i r a l  structures  that  stability,  and  as pointed out b e f o r e , the thermodynamic data  of  and  Hoeve  Kakivaya  interpretations  depend  of  on  hydrogen  (1975)  elastin  are  energy  to  observe  for  inconsistent  a  for  with  the  failure  these  of  f i r s t order t r a n s i t i o n :  these (a) the  change a s s o c i a t e d with the t r a n s i t i o n i s very small  (b) these s t r u c t u r e s are extremely range of the experiment This  again  s t a b l e over the  conformation  or  temperature  (0O-200OC) .  creates  a  dilema.  If  the  energy  t r a n s i t i o n from the B e t a - s p i r a l s t r u c t u r e t o the  f o r the  random  ceil  i s very s m a l l , then there i s no reason to assume  that the p e p t i d e - p e p t i d e turns)  their  s t r u c t u r e . There are a number of  p o s s i b i l i t i e s t h a t could account investigators  bonding  are  should  i n t e r a c t i o n s . The  be  hydrogen  favoured  system  is  then  bonds over very  (needed the  for  Beta-  peptide-solvent  close  to  being  an  59  amorphous,  single-phase  movements. This  network  with  view i s a l s o c o n s i s t e n t  rapid  back-bone  the  Beta-spiral  with  s t r u c t u r e s , i f they are present, being very dynamic ones. On  the  other hand, i f these s t r u c t u r e s  then p r o t e i n - p r o t e i n favoured  over  interactions  protein-solvent  can  be  are very  stable,  considered  to  i n t e r a c t i o n s . That t h i s  could  occur i s net s u r p r i s i n g since they are known t o be present other  fibrous  proteins  such as c o l l a g e n ,  silk,  which have hydrogen bended o r g a n i z a t o n s l i k e and  beta-sheet  Any  hydrogen  structures. bonded  peptide-peptide  be  in  and k e r a t i n ,  the  alpha-helix  This i s where the dilema a r i s e s .  system  that  interactions  would  has  conseguence, e x h i b i t r e l a t i v e l y s t i f f  so  overly  t o , as  an  favour  inevitable  mechanical p r o p e r t i e s as  do the above mentioned group of p r o t e i n s . E l a s t i n a l s o e x h i b i t s such p r o p e r t i e s , The  dry s t a t e  (or low h y d r a t i o n  conditions)  to be analogous to a system where, due plasticising to a p o i n t glass.  function  lack  of the  the  bonding  peptide  functional elastic  tissue.  In view of the high i t must  be  behaves  as  a  c f water i n systems such as these i s t o  the  structures  the  water, the p e p t i d e - p e p t i d e i n t e r a c t i o n s dominate  with  •dissolving'  elastin,  to  i t i s dry.  can be c o n s i d e r e d  where the normally rubbery m a t e r i a l  The  compete  when  interactions,  back-bone  extensibility concluded  that  and a l l o w i n g  essentially i t t o be a  and  low  modulus  the  hydrogen  of  bonded  cannot be very s t a b l e ones, and i t seems reasonable  to expect e l a s t i n t o be an amorphous protein..The  possibility  60  for  the s t a b i l i z a t i o n of the Beta-type s t r u c t u r e s i n d i s e a s e d  s t a t e s i s a more p l a u s i b l e i d e a , which at the an open  moment  remains  guestion.  (d) As  was  pointed  Fibrillar  models  cut before,  the f i b r i l l a r  model as based  on the e l e c t r o n microscope s t u d i e s , i s thought t o c o n s i s t o f 3 to 5 nm f i l a m e n t s  organized along the long a x i s of the e l a s t i n  f i b r e . . Since t h i s type of o r g a n i z a t i o n of  discrete  regions  presented a g a i n s t model  also  this objection  here.  The only  and  the  liquid  drop  p o s s i b l e way to get around  that  the  protein  making  up the  i s i n a random conformation. T h i s type of s t r u c t u r e  would be c o n s i s t e n t  with a l l t h e evidence pesented i n  of the k i n e t i c theory e x p l a n a t i o n s is  presence  i s t o propose that the f i l a m e n t s a r e themselves  an i s o t r o p i c system L e ^ filaments  the  c f p r o t e i n and water, a l l the arguments  the o i l e d - c o i l model  apply  involves  support  forelastin elasticity..But  t h i s a reasonable statement? At the moment there e x i s t s no  evidence to support t h i s assumption. On an i n t u i t i v e b a s i s , i t i s hard t o v i s u a l i z e the 3 t o 5nm f i b r i l s accomodating  as being capable  random c o i l s of p r o t e i n s , e s p e c i a l l y  the s i z e of i n d i v i d u a l amino a c i d  of  considering  residues.  G. Conclusions Examination of the different  methods  cf  various study  s t r u c t u r e can be a s s i m i l a t e d  structural  seems  models  to i n d i c a t e that  most s a t i s f a c t o r i l y  and the elastin  i n terms of a  61  conformation random  that i s very c l o s e to the  network  structure  that  theory of rubber e l a s t i c i t y . The further . delineate  is  v i a b l e d e s c r i p t i o n of e l a s t i n  demanded  following  (confuse, d i s g u i s e )  here, and to t e s t the v a l i d i t y of  kinetically  the  by the  chapters  the c o n f l i c t s random  conformation.  agitated, kinetic try  to  presented  network  as  a  62  Chapter^IV..  CO N FOB MAT ION  Ai The  OF ELASTINl COACEBV ATE  IHt^oduction  a n a l a y s i s of the c o n f o r m a t i o n a l s t a t e of the  p r o t e i n i s g e n e r a l l y confined to the i n s o l u b l e , of t h i s protein,.Another of  approach i s t o study  the s o l u b l e p r o t e i n s , and  final  product. Although  The  fibrous  the  form  conformation  t h i s i s an i n d i r e c t  approach  to  the  results.  Phenomenon of Coa eery a t i o n  operational  Bungenberg de Jong  elastin  to e x t r a p o l a t e from these to the  problem i t c o u l d provide some i n t e r e s t i n g  An  STB UCTUB E^  definition  cf c o a c e r v a t i o n was given  by  (1949) as:  " I f one s t a r t s from a s o l , that i s a s o l u t i o n of c o l l o i d i n an a p p r o p r i a t e s o l v e n t , then according to the nature cf the colloid, v a r i o u s changes (temperature, pH, a d d i t i o n of a substance) can b r i n g about a r e d u c t i o n of the s o l u b i l i t y as a r e s u l t of which a l a r g e r part of the c o l l o i d seperates out i n a new phase. The o r i g i n a l one-phase system-the s o l thus d i v i d e s i n t o two phases, one of which i s r i c h i n c o l l o i d , the ether p o o r . . . . . . . . . . . . Macroscopic or microscopic investigation allows one to d i s t i n g u i s h between c r y s t a l l i z a t i o n when o b v i o u s l y c r y s t a l l i n e i n d i v i d u a l s are formed and c o a c e r v a t i o n , when amorphous l i g u i d drops are formed, which l a t e r coalesce more or l e s s readily into one clear homogeneous c o l l o i d - r i c h liguid l a y e r , c a l l e d the coacervate l a y e r " . S o l u b l e e l a s t i n s d i s p l a y t h i s phenomenon of  coacervation..Both  t r o p o e l a s t i n and a l p h a - e l a s t i n are f r e e l y s o l u b l e i n water room  temperature,  but  upon r a i s i n g the temperature t o about  37oc, they both show c o a c e r v a t i o n p r o p e r t i e s . . I f l e f t f o r about 20-24  hours,  at  this  coacervate  becomes  standing  insoluble,  63  presumably due t o the entanglement c f the p r o t e i n chains  (Wood  1958) . E l e c t r o n microscope peptides  and  fibrillar  appearance  (Volpin  ______  s t u d i e s of the coacervates of s o l u b l e  synthetic  peptides  when  1976,  of  visualized a,  b,  coacervate  organization,  with  c).  c o n t r o v e r s y , F i r s t , are these f i b r i l s the  elastin,  or,  This  be  capable  of  are  accomodating  w i t h i n such a r e s t r i c t e d In seems  one  elastin  raises  another  they  random  these  of the sha_e  types  and  coacervates... In  present chapter deals resonance  stains  a  result  do e x i s t ,  of would  c o i l s of p r o t e i n s  of  questions,  should choose experimental methods that  allow an examination soluble  negative  domain?  attempting t c answer that  a very  a t r u e r e p r e s e n t a t i o n of  p r o c e d u r a l a r t i f a c t s ? Second, i f these f i b r i l s they  show  with  the  view  viscosity  ________  of  of t h i s purpose, and  nuclear  s t u d i e s which can, i n p r i n c i p l e , t e s t f o r shape  elastin,  m.w.  will the the  magnetic  m o b i l i t y , r e s p e c t i v e l y . The d i s c u s s i o n w i l l be l i m i t e d to 66,000  it  and the  peptide fragment commonly r e f e r r e d to as alpha-  but the arguments  presented  here  are  apply to the p r e c u r s o r p r o t e i n , t r o p o e l a s t i n , as  expected  to  well.  C_ P r e p a r a t i o n Of A l p h a - E l a s t i n Instead  of  using  the usual method of producing s o l u b l e  e l a s t i n s by o x a l i c a c i d d i g e s t ( P a r t r i d g e  et.al.  alpha-elastin  prepared  used  in  this  study  was  h y d r o l y s i s of i n s o l u b l e e l a s t i n as o u t l i n e d by H a l l  1955),  the  by enzyme (1976)  and  64  H a l l and Czerkawski is  advantageous  compared  to  attained  (1961). The enzyme method  since  buffer:  of  mixture  that  with  10ml of  borate/chloride  ph8.5  and  ionic  0.2M  NaOH,  and  s o l u t i o n was prepared  strength  beef  of e l a s t i n :  t i s s u e was  finely  boiling d i s t i l l e d tissue  was  of 0.384 was  3 °3 B  a n <  ^  0.2M  t o 250ml..The  dissolving  12.369g  water..  Ligamentum ______  obtained from mature  c a t t l e was s t r i p p e d of f r e e adhering t i s s u e and p u r i f i e d  by repeated a u t o c l a v i n g  SDS  H  diluting by  H-^BO- and 14.911 KCl i n 1L of d i s t i l l e d Preparation  otherwise  hydrolysis  prepared by mixing 50ml of a mixture of 0.2M KCl  is  digests.  J a _ ______ Borate  preparation  the product i s a monomeric peptide as  the pclydisperse  through a c i d  of  was  (Partridge _t_a1_  minced,  washed  with  1955). The r e s u l t a n t several  water and d r i e d . About 5g o f t h i s  litres  autoclaved  suspended i n 380ml of the borate b u f f e r , 0.5,g o f  was added and the mixture was s t i r r e d a t 37°C f o r 1hr. then  contained  of  chilled  to  4<>C  5mg of e l a s t a s e  and  20ml of borate b u f f e r ,  It  which  (Sigma chemical company), was added.  The  r e a c t i o n v e s s e l was put i n t o a shaking, water-bath at 370C  and  the r e a c t i o n was allowed to proceed u n t i l a l l the  had  been  period  dissolved  the s o l u t i o n  reaction.  The  elastin  ( u s u a l l y about 5 h r s ) . At t h e end of t h i s was  stop  the  e n t i r e sample was f r e e z e - d r i e d and stored  at -  70°c u n t i l f u r t h e r use.  brought  tc a  boil  to  65  Jb_ The  SDS was removed from the peptide by a m o d i f i c a t i o n o f  the i o n - p a i r e x t r a c t i o n (1979).  About  exhaustively peptide  method proposed  against  distilled  water  (5ml:5ml:5ml) and s t i r r e d  acetone was g r a d u a l l y precipitation  extracted  and  Henderson  powder was d i a l y s e d freeze-dried..  at room  This  temperature  the  for  to 4"C and 85ml of anhydrous  added t o the s o l u t i o n . This  of  ______  i n a s o l u t i o n of t r i e t h y l a m i n e : a c e t i c  The mixture was then cooled  the  by  100mg of the a l p h a - e l a s t i n  was r e d i s s o l v e d  acid:water 1hr.  Removal o f SDS  protein  while  results  the  SDS  salt i s  i n the acetone. The p r e c i p i t a t e was r e d i s s o l v e d  •washing' s o l u t i o n , which c o n s i s t e d  in  in a  of 5ml of water, and  95ml  the acetone was added t o p r e c i p i t a t e cut the p r o t e i n .  This  •washing' was repeated 4 t o 5 times and the f i n a l product  was  of  blown  dry  i n an oven to remove a l l t r a c e s of acetone. N.m.r.  Spectroscopy showed t h i s p r e p a r a t i o n  t o be t o t a l l y  f r e e of any  SDS.  J_L The  Characterization  r e s u l t a n t peptide  66,000  M.W.,  electrophoresis 1971).  It  also  as  of the a l p h a - e l a s t i n  was  shown  a  monomeric  by  preparation  SDS-polyacrylimide  (Weber and Osborn 1969, Chrambach displayed  the  property  and  as  a  function  of  temperature  Robard  at  as  380nm  ( f i g u r e 4.1). The amino a c i d  composition  was shown to be s i m i l a r to  comparing  the  n.m.r.  gel  of c o a c e r v a t i o n  monitored by the absorbance o f the peptide s o l u t i o n  of  spectrum  insoluble of  the  elastin  by  alpha-elastin  66  hydrolysate The 6M  with a spectrum of ligament  h y d r c l y s a t e s were prepared HC1  at 1150C  f o r 24hrs.  i§l  larger  the  The  relevant  Shape  equation  adds a number of s o l i d  particles,  than the s o l v e n t molecules,  n, one  observes an i n c r e a s e i n the  s o l u t i o n due  induced  assuming  prevent  to a s o l v e n t of macroscopic  that  The  n  solution  and  as  are  this  lines,  was  to  the v i s c o s i t i e s of the s o l v e n t and  the  1961) :  with  4.1  V  s  and  e f f e c t of the s o l u t e on the  The experimental  representing.  as the s p e c i f i c  manipulation  v i s c o s i t y of the viscosity,  the  solvent  n":  4.2 for  non-ideal  molecules,  to  f o r the c o n c e n t r a t i o n e f f e c t s , i n v o l v e s the e m p i r i c a l  e v a l u a t i o n of the reduced v i s c o s i t y , n"/c,  c-*0,  for  by  n"= (n'-n) /n  the  of  volume f r a c t i o n of the s o l u t e .  i s u s u a l l y expressed  account  viscosity,  given  (Tanford  respectively,  'geometry' and The  n'  much  viscosity  relationship  flow  n'=n (1+v>0) where  are  the p a r t i c l e s are f a r enough apart  o v e r l a p of the d i s t o r t e d  E i n s t e i n i n 1906  which  to the d i s t o r t i o n of flow p a t t e r n s , which i s  by the s o l u t e p a r t i c l e s .  effect,  hydrolysate.  by a c i d h y d r o l y s i s i n vacuo i n  D_i V i s c o s i t y and  I f one  elastin  c o n c e n t r a t i o n , c. The  as  a  g r a p h i c a l p l o t c f n"/c  g i v e s a value termed the  intrinsic  function  of  versus  as  v i s c o s i t y [n] :  c,  67  ___________ Coaceryat ion p r o f i l e of a l p h a - e l a s t i n . Plot of normalized absorbance at 360nra versus temperature °C, f o r a s o l u t i o n of a l p h a - e l a s t i n at a concentration cf 6.8nag/ml, ph 7 . This sample was subsequently used f o r the nmr experiments.  69  [n]=n"/c, as c-»0 S u b s t i t u t i n g eguation 4.3  4.3  i n t o eguation  [n]=¥^/c s i n c e the volume f r a c t i o n  4.1  gives:  4.4  of the s c l u t e can be represented  jzS=Nv c/molecular weight of the s o l u t e 4.5 h where N i s Avogadro's number, c i s the c o n c e n t r a t i o n i n and  v  i s the hydrodynamic h Tanford as (196 1) : v, = (m. w./N) n  volume,  defined  ( v+SvO)  by:  g/ml,  according to  4.6  where v i s the p a r t i a l s p e c i f i c volume of the s o l u t e molecule, vO i s the p a r t i a l s p e c i f i c volume of the s o l v e n t molecule, & i s the h y d r a t i o n expressed eguations 4.6,  4.5,  and  4.4,  as g s o l v e n t / g s o l u t e . Combining gives:  [ n]=v(v+kvO) In  4.7  the case of water, v« i s egual t c u n i t y , and  reduces  eguation  4.7  to: £n] = v(v+ S )  IhL The  varying  symbol,  axial  v-,  i n eguation  4.7  r e p r e s e n t s the shape of  and has been e v a l u a t e d f o r  r a t i o by Simha  p r o l a t e e l l i p s o i d s of (Tanford  4.8  E v a l u a t i o n of the shape parameter  the s o l u t e molecule  axial  ellipsoids  of  (1940). For the s p e c i f i c case of ratio,  J,  it  takes  the  form  1961): v=J2/15 (ln2J-3/2)  Since  and  v> can be c a l c u l a t e d  4.9  i f the values of [ n ] , v,  , and  v°  70  are known. I h i s method can be used t o e m p i r i c a l l y geometry of the s o l u t e molecules. shape of the s o l u t e molecule,  Jc_ If  the  The dependence of \r, on the  i s shown i n f i g u r e 4.2.  A p p l i c a t i o n to s o l u b l e e l a s t i n s  process  of  coacervation  does  change of shape, t o a more a n i s o t r o p i c the  value  of  v-, as  given  of  v- w i l l  tropoelastin,  as  probably  by the r a t i o  the  calculated  other  hand,  f i l a m e n t o u s systems, then dramatically  at  the  form,  then  (v+ ^>)/[n], should range.  The  exact  l i e i n the range of 12.18 f o r from  eguation  and  the  i f these p e p t i d e s do i n f a c t  form  sedimentation data of Schein e t _ a l _ On  not r e s u l t i n a  fibrillar  remain constant over the e n t i r e temperature value  evaluate the  the  (1977).  value  critical  4.9  of  v- should  temperature  at  increase which  the  t r a n s i t i o n takes place. The a b s o l u t e magnitude of T, a f t e r the t r a n s i t i o n , can be p r e d i c t e d assuming  that  the  fibrillar  s t r u c t u r e s as suggested According to Urry to  have  a  f o r the  diameter  dimensions  850  by Urry and Long  (1977b).  (1976a), the B e t a - s p i r a l s are cf  15nm  residues  with  f o r the  1976) ,  which  has  the  expected  length . and  diameter grossly  r e p r e s e n t a t i v e of t h i s shape, the a x i a l r a t i o of 41.3  amounts  a  Simha  factor,  a  tropoelastin  as being  to  Assuming  of  expected  a t r a n s l a t i o n length of  (Sandberg  are 62nm and 1.5nm  respectively.  molecule  f o n t i s made up of B e t a - s p i r a l  0.44nm/6aa r e s i d u e s . In the case approximately  tropoelastin  V, s  prolate e l l i p s o i d  of  39.3  (eguation 4.9).  A similar  71  _ i a J J £ _ _ i i _ 2 i 3___jn^__ce of the Simha f a c t o r on axial _a_i°*. Evaluation of the Simha f a c t o r , v> as a functxon of axial ratio f c r prolate (a) and ofclate (b) e l l i p s o i d s , according to equation 4 . 9 .  Simha factor, \r Jvj UT  - —  >  X  cr  cn  - *  o  j—  N>  i  o  73  prediction branched  alpha-elastin  nature,  increase in  for  but  Hi  microscope  Viscosity  _[a]_ All  the  conducted  an  times f o r  transit  times  corrections. viscosity  fibrillar  studies  Viscosity  due  water  type  in  should  structures  excess  of the  w i t h an O s t w a l d  here  evaluated water,  a t each  using  300sec. need type  physical  f o r kinematic viscometer  The  and  by  apparatus  and t i s t h e  constant,  calibration  viscosity  260mg  of  water.  of protein  purified  values  with from  clear  This solution  supernatant  B, was  distilled standard  dissolving  about  250ml o f  a t 12,000X  for  m a t t e r and l a r g e a g g r e g a t e s , and  was u s e d  of the protein  in  was c e n t r i f u g e d  f o r t h e subseguent  e x p e r i m e n t s . The e x a c t c o n c e n t r a t i o n aligucts  was made up by  alpha-elastin  15min., t o remove p a r t i c u l a t e  1ml  the  tables..  distilled  the  long  4. 10  temperature  A standard solution about  These  n', i s g i v e n b y :  seconds.  density  were  viscometer, with  where B i s an a p p a r a t u s c o n s t a n t , p i s t h e d e n s i t y in  as r e p o r t e d  described  capillary  obviate  of the s o l u t i o n ,  time  i t ' s  measurements  n'=Bpt  transit  to  (Cox e t . a l . .1973).  experiments  Ostwald  In dealing  made  S t u d i e s Of A l p h a - E i a s t i n  viscosity  with  be  i t i s a l s o e x p e c t e d t o show a d r a s t i c  i n r i f i t t o o forms  the electron  transit  cannot  was measured  solution,  drying  viscosity  by t a k i n g  a  them i n an oven,  74  ____________  __________ of e l a s t i n water content 211 temperature. P l o t of r e l a t i v e volume (taken t o be 1 at 30OC) versus temperature °C f o r ligament e l a s t i n (from G o s l i n e 1978).  76  and  measuring the  solution  was  residue  weight. . 10ml  pippetted  into  the  of  the  alpha-elastin  visccmeter and  the t r a n s i t  times were measured with an e l e c t r o n i c stop-watch The  viscosity  p l o t t e d i n the  for alpha-elastin  was  partial  the  each  (see eguation 4. 3) .  intrinsic  viscosity,  s p e c i f i c volume and  method  of  and  which Edsall  amino-acid composition f o r a l p h a - e l a s t i n (1973), had  a value of 0.739. The  temperature dependence of hydration Gosline  Ceccorulli  [n],  hydration  Cohn  a l p h a - e l a s t i n , at each temperature, was  given by  temperature  s p e c i f i c volume of the a l p h a - e l a s t i n ,  by S t a r c h e r e t . a l . of the  at  versus c  of the  c a l c u l a t e d according t o the  (1943) from the  data  at each temperature.  C a l c u l a t i o n of p a r t i a l The  the  form of n"/c  This allowed the e v a l u a t i o n  Jb_  sec).  measurements were repeated on s e r i a l l y d i l u t e d  s o l u t i o n s of a l p h a - e l a s t i n and were  (±0.1  (1978)  ______  ( f i g u r e 4.3).  (1977)  have  given  hydration  calculated  from  for insoluble e l a s t i n  T h i s seems  shown  that the  valid two  since  types of  e l a s t i n e x h i b i t s i m i l a r temperature dependence f o r t h e i r water contents.  Jc_ The  Besults  r e s u l t s of the  summarized i n t a b l e 4.1 intrinsic  is a slight  discussion  v i s c o s i t y studies and  v i s c o s i t y , Xn3,  unchanged  and  f i g u r e 4.4.  on a l p h a - e a l s t i n  I t i s evident  of a l p h a - e l a s t i n remains  with temperature  that  are the  relatively  ( f i g u r e 4.4,a). I f anything, t h e r e  decrease i n [ n ] with i n c r e a s i n g temperature, which  VISCOSITY OF ALPHA-ELASTIN.  TC U  n  sp^  slope  c  v  e  r  s  u  s  c  Pl°  t s  I  n  ]  correlation coeffecient, r  12  0.67  0.99  3.7  22  1.30  0.84  4.1  36  1.20  0.97  3.1  41  1.40  0.84  3.6  78  F igurei4..4j. V i s c c s i t y of a l p h a - e l a s t i n ^ (A) Plot of i n t r i n s i c viscosity, £n], versus temperature °C. The clashed l i n e represents the expected r e l a t i o n s h i p c a l c u l a t e d from e q u a t i o n 4.9, assuming no change of shape, and the h y d r a t i o n values from f i g u r e 4. 3. (B) P l o t of the Simha f a c t o r , v, versus temperature oc calculated according t o equation 4.8 using the needed values from table 4 . 1 and f i g u r e 4.3.  S i m h a factor, to  <*>  r  (n] (cc/g)  tn "i  ro i  <*> i  *^ i  O  ro o u> o o  o  o tn o  o o  tn o axial r a t i o . o ' b  /•  o o  O  /  rO  O  9!  / / /  tn i  80  i s expected peptide line).  from  the decrease  (figure  i n the  water  content  4.3)  and  eguation 4.8 ( f i g u r e  of  the  Simha  Evaluation  factor,  v,  eguation 4.8 i n d i c a t e s t h a t t h e a l p h a - e l a s t i n or  less  spherical,  temperature  and  that  there  4.4, a dashed according  molecule  i s no  ( f i g u r e 4.4,b). I n c o r p o r a t i n g the  change  values  with  of the  weight,  possible  to  molecule,  assuming a s p h e r i c a l shape, t o be i n t h e order  calculate  to  i s more  molecular  in  partial  of the  s p e c i f i c volume, and h y d r a t i o n i t i s the  dimensions  of the a l p h a - e l a s t i n 20nm  diameter. Interestingly,  however, a glance at t a b l e 4.1 shows t h a t  the s l o p e s of the n"/c versus c p l o t s tend increasing  temperature.  This  can  be  to  interpreted  f o l l o w i n g way. I f the p a r t i c l e s i n s o l u t i o n with  each  other,  should be zero  then  general,  not  i n the interact  (Tanford 1961, Huggins 1942). The f a c t t h a t the  of aggregation processes  seem  do  with  the s l o p e of the n"/c versus c p l o t s  s l o p e s f o r the a l p h a - e l a s t i n s o l u t i o n s are the presence  increase  to  be  favoured  positive  supports  (Tanford 196 1) which, i n  with i n c r e a s i n g  temperatures  ( t a b l e 4. 1) .  F_ Nuclear Magnetic  Resonance  (a) Theory The particle, spinning  p o s s e s s i o n of both such  as  a  spin  proton,  and a  charge  magnetic  confers moment.  on  a  Thus, a  proton can be regarded as a bar-magnet along the a x i s  81  _ i _ u r j _ _ _ _ _ _ Precession of a _ r o t o n in a magnetic field. A prcton of magnetic moment, u, i s p l a c e d i n an e x t e r n a l magnetic f i e l d , H , causing i t to precess around the z axis at an an angle 0 . H, i s the o r t h o g o n a l magnetic f i e l d that i s used t o determine the frequency c f p r e c e s s i o n . Q  82  Figure.U.5  83  of  spin,  and the s t r e n g t h c f t h i s magnetic  as the n u c l e a r magnetic magnetic  field,  magnetic itself  H ,  moment, u. I f placed the  0  d i p o l e i s expressed in  an  external  i n t e r a c t i o n of t h i s f i e l d  with the  moment of the proton w i l l i n f l u e n c e i t t o in  the  d i r e c t i o n of the f i e l d ,  t u t the e f f e c t o f the  s p i n c r e a t e s a torque which causes the i t to the  z  axis  at  orientate  precess  around  a frequency, v° , and angle 0 (Stothers 1973,  f i g u r e 4.5). The anqular v e l o c i t y of t h i s p r e c e s s i o n , co , i s a  given by : = % H  where  &  4. 11  o  i s the gyromagnetic  r a t i o d e f i n e d as:  * =2u /h  4.12  r  where u^ i s the p r o j e c t i o n of the v e c t o r u i n the d i r e c t i o n of the  field  H , Q  determined protons  and  h  is  Planck's c o n s t a n t . The angle 0 i s  by the s p i n number of the  nucleus,  I,  180O-54.9O  energy,  for  has the value of 1/2. The angle of the p r e c e s s i o n f o r  t h i s s p i n number i s r e s t r i c t e d t o two o r i e n t a t i o n s , and  which  respectively  (Metcalfe  E, of the i n t e r a c t i o n of tihe  at  54.9°  1970). The p o t e n t i a l  magnetic  moment  u^ i s  given by: E — U^H .....o 4. 1c =  e  and  the  separation  between  the  two  energy  l e v e l s f o r the  proton i s : AE=2u^H If H  0  a small r o t a t i n g f i e l d  (figure  both H  (  4.5),  ,.4.14  0  H  (  i s generated  to  u would e x p e r i e n c e the combined e f f e c t s of  and H . The nucleus would a l s o r b energy c  orthogonal  from Hj i f the  84  angular  frequency of H  electromagnetic is  given  is  (  radiation  egual which  to will  only  orientation (Metcalfe  to  come i n t o  guanta  another  energy  possible  the  resonance  absorption,  when  H,  is  (chemical s h i f t )  The c h e m i c a l  identical  frguencies  derived  from  resonance frequency  on t h e d e t e c t i o n  spins  of  electrical  and  magnetic  1973).  The n u c l e i  field  by  as  the field the  each  proton  extra-nuclear  field  however,  (Walton  from  the  electrons,  could  sensitive  applied  e  be the  toi t ' s  and B l a c k w e l l  hence,  e x p e r i e n c e d by t h e n u c l e u s , H applied  system  resonance at  information  is  environment  c a n be s h i e l d e d  the  shift  technique.. Fortunately, of  w i t h v°  1  w i t h H| .  no c o n f o r m a t i o n a l  this  or  f r o m one  i n "resonance  a l l t h e p r o t o n s were t o u n d e r g o m a g n e t i c  magnetic  The  transitions  causes the nucleus t o f l i p  when t h e n u c l e a r  Jill  same  such  1 9 7 0 ) . N.m.r. S p e c t r o s c o p y i s b a s e d  the absorbed  If  effect  8.  4. 15  two v a l u e s o f 8 a r e  emmision, of energy  of  changing  by: hv°=AE=2u_H_  Since  v°,  ,  magnetic  the e f f e c t i v e i s  and c a n be e x p r e s s e d a s  not  the  (Metcalfe  1970) : H  where  =H (1-rj-)  e  CT i s t h e s h i e l d i n g  electron It  distribution is  distribution  factor  around  therefore around  4.16  0  which  of  the  the observed proton.  expected  the  i s a reflection  nuclei,  that  changes  i.e.  in  electron  conformational  or  85  bonding  changes, would e f f e c t the system because of s h i e l d i n g  effects,  and  with  characteristic  a  equivalent are  one  would expect  to observe  chemical  million  /H^,  S  5  and  The  chemical  number expressed  ) X 106.  group  of  shifts, S  ,  as p a r t s per  As s t a t e d b e f o r e , protons magnetic  field  are r e - o r i e n t a t e d i n an a p p l i e d  from  . The  however, have an egual p r o b a b i l i t y were  and  processes  by the a b s o r p t i o n of e l e c t r o m a g n e t i c  the resonance freguency  there  4.17  respectively.  JcJL B e l a x a t i o n  if  each  are the resonance f i e l d s f o r the sample  r e f e r e n c e compound  of  for  signal,  (ppm) , and i s d e f i n e d as: £> = (H -H^  where H  seperate  shift  protons i n the molecule.  measured i n a dimensionless  a  induced  radiation  transitions,  i n e i t h e r d i r e c t i o n so t h a t  egual p o p u l a t i o n s c f n u c l e i i n the two  l e v e l s , there would be no net a b s o r b t i o n of energy.  energy  In  fact,  however, there i s a s m a l l excess of n u c l e i i n the lower  energy  s t a t e which i s determined two  levels  are given  (equation 4.13)  by the energy d i f f e r e n c e between the and  the r e l a t i v e p o p u l a t i o n s  by: n'/n=exp (- A E/BT)  where lower  n'  and  n  are  l e v e l s r e s p e c t i v e l y . The  suffecient  to  give  4.18  the number c f protons i n the upper  the order of 7ppm i n a f i e l d is  (n'/n)  excess  of 10kG  of protons  is  only  (Metcalfe 1970), but  and in this  a net a b s o r p t i o n of energy s i n c e the  number of upward t r a n s i t i o n s are s l i g h t l y g r e a t e r than i n  the  86  other d i r e c t i o n . These t r a n s i t i o n s that of the e x c i t e d  determine the l i f e t i m e  n u c l e i are termed r e l a x a t i o n  Spin-lattice  relaxation  processes.  (longitudinal  relaxation), T j ,  r e s u l t s i n the d i s s i p a t i o n of the absorbed energy energy  to  the  collectively directly  other  nuclei  and  r e f e r r e d to as the  to  electrons  lattice.  as  thermal  i n the sample,  This  process  acts  maintain an excess of n u c l e i i n the lower energy  state. T, 2  the other process o f r e l a x a t i o n ,  of an e x c i t e d a  spin r e l a x a t i o n the  lower  (transverse  re-distribution  contribute  out  .  For  energy excess  and  spin-  results does  nuclei  not  i n the  processes determine the  processes  example,  are  influenced  by  molecular  the r a p i d thermal motions i n l i q u i d s  spin-lattice relaxation  times (Metcalfe  e f f e c t i v e f i e l d experienced by the n u c l e i i s a l s o  1970). averaged  by the r a p i d i s o t r o p i c movements, which r e s u l t s i n narrow  line-widths that  absorbed  process  with  line_widths.  leads to l a r g e The  of  to the maintainance of the  Beth r e l a x a t i o n motion  This i s termed  r e l a x a t i o n ) . This  s t a t e . Both of these r e l a x a t i o n  spectral  lifetime  nucleus by a mutual exchange of o r i e n t a t i o n  neighbouring nucleus of the same kind.  in  l i m i t s the  and l a r g e  the a b i l i t y  directly  on  o t h e r . This  values f o r T_. The reason  for this  of two n u c l e i to exchange t h e i r spins  depends  how long t h e i r motions remain i n phase with each  time during which r e l a t i v e o r i e n t a t i o n  c a l l e d the c o r r e l a t i o n time, characterizes  is  > -nd  i s the  persists i s  parameter  the molecular motions. Thus n u c l e i that  that  exhibit  87  fast,  isotropic  correlation n.m.r.  motions  times and  spectra  can  will  be  characterized  narrow l i n e - w i d t h s . The therefore  be  used  to  small  a n a l y s i s of deduce  the  protein  mobility.  G_ ______ In result  comparison  Studies Of  to  the  i s an e v a l u a t i o n  magnetic  resonance  molecules m o b i l i t y  of  Alpha,Elastin  viscosity a  molecule's  experiment  allows  and  n.m.r.  line-widths,  spectra  (conformation)  if of  it  a  nuclear  the e v a l u a t i o n of  and  of i t ' s component  should  be  the  composition  the  constituent  A l t e r n a t e l y , i f the ccmpcsition conformation,  shape,  (Dwek 1 9 7 3 ) F u r t h e r m o r e , s i n c e any  spectrum i s a ccmposite of the sum shifts  experiment whose main  predict  assumption, which i s then  is  a  and  chemical  the  mobility  molecules  known  by  n.m.r.  possible to predict  one  spectrum  tested  the  is  can  based  on  obtaining  known.  assume  a  such  an  experimental  values.  _a_ M a t e r i a l s and The  purified  alpha-elastin  method was  dissolved  concentration  of approximately 6mg/ml. Proton nmr  obtained  a  University  cn of  Bruker  to  75°C,  s p e c t r a were the  B r i t i s h Columbia, Department of Chemistry  with  spectrometer  of Dr. . A. G. .. M a r s h a l l and  Buns were made at 10°C 15°C  FT-NME  at a  at  the kind c o - o p e r a t i o n  WD-400  i n D_0  which  Dr. .P. D. . Bur ns.  i n t e r v a l s over a temperature spans  the  region  of  range  of  alpha-elastin  88  gigjjg! s.i*.i6l The L o r e n t z i a n l i n e shape.. The L o r e n t z i a n l i n e , as d e t e r m i n e d by e q u a t i o n 4.19, where 1/t i s the h a l f - w i d t h at h a l f - h e i g h t . w" i s t h e r e s o n a n t p r e c e s s i o n f r e q u e n c y o f the nucleus.. 5  89  Figure. U. 6  :  1/t  w.  90  Table..__2:_  S p e c t r a l  p a r a m e t e r s  PARAMETERS FOR THE  used  f o r  i___B.'g-»8!feBft  SPECTRA AT 400MH1. FROM OSS. CHEMICAL SHIFT Hz PPM ala  CH  3  arg «-CM  2  LINE-HI DTH  NUMBER OF PROTONS IN: Albumin^ a.a Clattlnj  1.34  534  Hz 25  3  783  138  1.75  700  35  2  10  46  1.67  667  32  3.11  1240  21  2 2  10 10  46 46  asn O-CHj  2.69  1074  28  2  "  20  asp a-CH  2.73  1092  28  2  9  76  30  2  43  32  J-CH  2  2  1.96  780  <-CH  2.43  971  43  2  43  32  glu »-CH i-CH  1.95  780  30  2  118  942  35  2  -  2.35  gin 8-CH  2  2  2  2  118  3.92  1569  18  2  610  30  his C2-H C4-H H-CH  8.54 7.09 3.05  3414 2835 1222  18 18 23  1 1 2  ** "  17 17 34  ile a-CH l-CH  1.59 1.59 0.81  634  39  2  34  28  634  39 30  2 6  34 103  28 84  1.59  634 634 322  39  2  107  122  39 30  1 6  54 322  61 366  gly*-CH  2  2  2  2  (CH ) 3  leu fl-CH  2  2  l-CH (CH ) 3  1.59 0.84  2  322  1.70  40  2  11  2  1. 39  680 554  118  2  30  2  11  S-CH  1.62  647  30  2  11  118 118  2  S-CH  2.92  1167  25  2  11  118  2  net «-CH  870 1040  24  2  8  31  2  -  <-CH  2.17 2.60  S-CHj  2.02  807  36  3  lys 3-CH ir-CH  2  2  pre t(C« )  --  --  8 12  7.20  2880  19  5  147  130  o-CM  2.93  1172  10  2  59  52  NCH o-CH  3.56 1.92  1423 769  59 36  2 2  234 234  56  2  l-CH  2  2.00  796  36  2  234  56  ser 3-CH  3.81  1520  36  2  22  56  tf.r  1.14  454  21  3  46  102  4.13  1652  10  1  15  34  7.03 7.00 6.70 6.66 2.90  2811 2798 2678 2654 1160  14 15 15 14 17  .5 1.5 1.5 .5 2  10 30 30 10 41  10 29 29 10 38  2.20 0.87  878 347  40 32  2  232 696  72 216  2 2  2  pro  2  2  CH P-CH  3  tyr i»(CH ) 2  2  0-CH  2  val Q-CH (CM ) 2  3  2  6  ^amlno add composition from Starcher et.al. 1973. ^amino acid composition from Dayhoff 1976.  56  r a n d o m - c o i l  91  coacervation were: 4.6  16k  sec.  (see  f . i . d data delay  resonance  figure  with quadrature  s e t , 1.4  between  saturation.  4.1).Typical sec.  spectral  d e t e c t i o n and  parameters  a c g u i s i t i o n time with a  successive  A  spectral  acquisitions  to  avoid  width of 6000 Hz was  phase  alteration  used  sequence  and  e x p o n e n t i a l a p c d i z a t i c n e q u i v a l e n t to 2 Hz l i n e broadening, enhance  the  to  s i g n a l to noise r a t i o . A l l assignments were made  from the r e s i d u a l H 0 peak taken to be at 4.68ppm from DSS  at  2  250C.  Jb_  P r e d i c t i o n of randc m-coil s p e c t r a  In approaching route  which  obtained  utilizes  1979,  various  parameters  The  shape  of  the  curves  figure  Where A(w)  fitted  and  coil  to  the  (Marshall  4.19  r e p r e s e n t s the a b s o r p t i o n at the freguency  resonance  been  4.6):  freguency  (i.e.  the  chemical  w,  shift  w° i s of  a  t d e s c r i b e s the l i n e - w i d t h such t h a t :  1/t=1/2 (width at h a l f - h e i g h t ) The  have  f o r a L o r e n t z i a n l i n e , which takes the form  and  residue)  that  was  A (w) =t/ (1* (w°-w) z _ 2  the  f o r an e m p i r i c a l  f c r p r o t e i n s which are known to be i n the random  conformation. eguation  t h i s problem I have opted  values used f o r the p r e d i c t i o n s  composition,  the  chemical  parameters frcm Bradbury and spectra  shift  4.20  involve data,  R a t t l e (1972)  The  predicted  4.19  on a D i g i t a l Equipment pdp11  the and  (see  amino  line-width table  were c a l c u l a t e d according to computer,  using  acid  a  4.2).  eguation Fortran  92  program.  J S l J e s u i t s and d i s c u s s i o n Figure elastin  4.7  in D 0 z  shews  the  400HHZ proton s p e c t r a f o r a l p h a -  at 25°C. The assignments  (see legend t a b l e  were made on the b a s i s of the p r e d i c t e d s p e c t r a and  other  et. a l . the  published  spectra  1 980) . Examination  main  (Egmond  (figure  et.al.  of f i g u r e s 4.7  and  a b s o r p t i o n peaks i n the n.m.r.  4.3)  1979, 4.8  4.8a) Cozzone  shows  spectrum  that  correspond  to the v a l i n e , a l a n i n e , and g l y c i n e r e s i d u e s . . T h i s  aspect  of  the r e s u l t i s v a l u a b l e because the d i s t r i b u t i o n of these amino acids  is  almost  exclusively in  glycine  very  and  distinct,  i n t h a t the a l a n i n e r e s i d u e s  the  cross-link  v a l i n e s are r e s t r i c t e d  the e l a s t i n chains  (Gray  et_^aJU_.  regions,  occur  whereas  the  to the e x t e n s i b l e p a r t s of 1973,  Anwar  1977).  These  r e s i d u e s can t h e r e f o r e serve as probes f o r the conformation the  different  similarity coil  regions  and  supports  the presence  elastin.  However, in  satisfactorily confidence.  the  e l a s t i n network. The  between the p r e d i c t e d s p e c t r a  conformation)  differences  of  the  of  the a  random are spectra  cenformation also  some  which  before the above statement  The  in  results alphadistinct  explained  can be accepted  two  restricted  to three types of r e s i d u e s :  the  (1.3ppm),  the  residues  be  general random-  for  very  must  the  (broad envelope  differences  glycine  a  e x p e r i m e n t a l l y observed  there two  (assuming  (3.9ppm)  in  with  s p e c t r a seem to be alanine and  at 3ppm and the peaks between 6.7  the and  residues aromatics 7.2ppm).  93  F i g u r e . 4 . 7 : 400 MHz nmr spectrum of a l p h a - e l a s t i n . Pmr spectrum of alpha-elstin in D0 at a concentration c f 6.8 mg/ml, ph 6.8, a t 25°C (see t a b l e 4.3 f o r peak assignments) .. The l a r g e peak at 4.68ppm i s from the r e s i d u a l H,0. 2  >3  laJUj§.i J*.*i;i  P^i.k a s s i g n m e n t s  for alph^-giastin  PEAK ASSIGNMENTS FOR ALPHA-ELASTIN. 1.  lie.,  2.  thr  : CH  l>s  : V-CH  3.  ala  : CH  4.,  ile  , leu  5.  srn  leu, val  :  (CH ) 3  3  2  3  : <3-CH ,  tf-CHj  2  lys  : S - C H _ , Jf-CH_ : ? - C H , g-CH^  pro  : P -CH , *-CH  glu  : P-CH  7.  val  :P-CH  8.  glu  :  tf-f.H  9.  asp  :  P-CH  10.  phe : (i  6.  2  2  2  2  2  2  2  2  -CH  2  lys  : « -CH  tyr  : |3 - C H  2  11  arg  :  S-CH  2  12.  pro  :  N-CH  2  2  2  13.  ser  : P"CH  I** •  gl y  :-<-CH  15.  envelope of alpha-carbon  2  16.  tyr ring : H 3 , 5  17.  tyr ring : H 2 , 6  lfi.  phe  :c?<CH ) 2  2  19.  i 30-desmosine  20.  iso-desmosine  and desmosine  a t 4 0 0 MHz^  96  I n t e r e s t i n g l y , a l l the d e v i a t i o n s seem to be of one  type, i . e .  the observed l i n e - w i d t h s are broader than the p r e d i c t e d ones. As  mentioned b e f o r e , the a l a n i n e r e s i d u e s i n the  network  are  localized  i n the c r o s s - l i n k r e g i o n s . The  of seguence s t u d i e s i n d i c a t e that these a l a n i n e s i n s e g u e n t i a l runs of 4-5 that  these  elastin results  are  grouped  r e s i d u e s , and t h e r e i s some  evidence  a l a n i n e s form a <x-helix  1976).  The  presence of h e l i c a l s t r u c t u r e s would be expected to r e s u l t  in  restriction  of  the  molecular  r e g i o n s , as r e f l e c t e d i n the Since the the  aromatics  (Foster e t . a l .  motions  in  the  cross-link  broadening of the s p e c t r a l l i n e s .  are  also  concentrated  around  the  c r o s s - l i n k r e g i o n s a s i m i l a r e x p l a n a t i o n , as the one  forwarded  for  the  the  alanine  residues,  broadening of these glycines?  There  can  residues  are  two  be  as  invoked  well.  Eut  alternatives  for what  in  secondary  broadening due  structures  does not  shewn  visualized  in  glycines  to r e s t r i c t e d motions. T h i s i s probably  seguence  (Sandberg e t . a l . any  presence  the  narrow  the the are  which could r e s u l t i n l i n e  case s i n c e the g l y c i n e r e s i d u e s occur elastin  about  t h a t can e x p l a i n  broadening of t h i s peak. I t i s p o s s i b l e t h a t the invoved  line  of  not  with the v a l i n e s i n  1977)  and  secondary  line-widths  the the  t h i s l a t e r group structures, (figure  as  4.7).  A l t e r n a t e l y , the observed g l y c i n e peak could be a composite of a broad d i s t r i b u t i o n number  of d i f f e r e n t  of  narrow  resonances  arising  glycine populations. This i s a  statement s i n c e the g l y c i n e protons alpha-carbon of the r e s i d u e and  are d i r e c t l y  would  therefore  from  reasonable  bonded to be  a  the  markedly  97  Figure.4.8: P r e d i c t e d nmr s p e c t r a f o r a l p h a - e l a s t i n . (A) P r e d i c t e d randcm-coil s p e c t r a f o r a l p h a - e l a s t i n using the s p e c t r a l parameters from table 4.2, and the amino a c i d composition from Starcher e t . a l . 1 973. (B) The same spectrum with the phe, t y r , and ala residues broadened to 50 Hz t o simulate their p a r t i c i p a t i o n i n secondary s t r u c t u r e s . The gly peak has been broadened to 30 Hz.  98  99 a f f e c t e d by the type of r e s i d u e that occurred arcund i t i n the amino  acid  sequence.  Hence d i f f e r e n t primary sequences,  with  regard t o the g l y c i n e r e s i d u e , would be expected t o r e s u l t different  chemical  in  s h i f t s f o r the g l y c i n e protons, and,since  there i s no reason to b e l i e v e that any cne type of amino  acid  always  this  occurs  with  a  g l y c i n e i n the primary seguence,  e x p l a n a t i o n f o r the l i n e broadening of the g l y c i n e peak acceptable. the  When the e x p l a n a t i o n s f o r the d i f f e r e n c e s between  randcm-coil  spectra  (figure  spectrum 4.7)  (figure  4.8a)  are i n c o r p o r a t e d  and  the  4.9 shows the spectrum  As  expected  t h e alpha-protons  been  broadened  (3 t o 4.5ppm)  a f f e c t e d the most by the h y d r o l y s i s of the p e p t i d e bonds, upfield  and  obtained f o r a s o l u t i o n of  h y d r o l y z e d a l p h a - e a l s t i n where the peaks have 30hz.  theory  ( f i g u r e 4.8b and l e g e n d ) .  Figure  to  observed  i n t o the p r e d i c t i v e  process, one observes a c l o s e r agreement betweeen experiment  seems  r e g i o n of t h i s spectrum,  seen t o be i d e n t i c a l with alpha-elastin  (figure  are The  f c r the free amino a c i d s , i s  the spectra  obtained  for intact  4. 7)., T h i s again supports the presence  of a r a n d c m - c o i l conformation f o r t h i s e l a s t i n p e p t i d e . The p o s s i b i l i t y  e x i s t s , however, that the f a s t motions o f  the protons deduced from the narrow l i n e - w i d t h s , i s a of  a  rapid  tumbling  result  of the whole a l p h a - e l a s t i n molecule i n  s o l u t i o n r a t h e r than being a r e f l e c t i o n of a k i n e t i c a l l y protein  chains.  To  ensure  against  this  s p e c t r a was obtained f o r bovine serum albumin Company). Albumin  free  a r t i f a c t a proton (Sigma  has a s i m i l a r molecular weight  Chemical  and i n t r i n s i c  100  F i g u r e . 4.9:. Nmr spectrum of alpha-glastin iil__c 400 MHz spectrum of a l p h a - e l a s t i n hydrolyzed i n vacuo i n 6M HCl at 115<>C f o r 24hrs. The spectrum has been broadened to 30 Hz.  101  Figure. U  102  viscosity  (Tanford  1961)  as  alpha-elastin  s e c t i o n of t h i s chapter) and, hence, frictional to  contain  stable  elastin,  however,  secondary  albumin  in  broad  spectrum  the  n.m.r.  resonance  with  the  and t h i s  of  resonances  envelopes,  secondary used  reasonable  to  studies of t h i s this  of  state for  protein  structures.  Broadening  1976)  t o 100hz r e s u l t s  the that  the  the  (amino a c i d  in  a  better  (figure 4.10,c).  results the  all  presented here i t seems  conclusicn  alpha-elastin  molecule  of  a  random  based  on the  a n a l y s i s i s not a r e s u l t of experimental a r t i f a c t it  is  conformational  is  demonstrates  i n the p r e d i c t i o n of the s p e c t r a  basis  conformation  a  true  state.  reflection Furthermore,  observable temperature dependence of that  mass  as compared to the p r e d i c t e d  approximation of the observed r e s u l t s the  be  motion of t h i s molecule does not mask the  composition from Dayhoff  rather,  to  should  a t 25°C. The  1967), more i m p o r t a n t l y ,  tumbling  presence  n.m.r.  shown  spectrum. F i g u r e 4.10,a, shows the  the r e s u l t s of C D .  (Timasheff e t ^ a l ^  On  been  f o r the random-coil conformation ( f i g u r e 4.10,b),  consistent  that  similar  s t r u c t u r e s which are mostly a l p h a -  400MHz spectrum obtained f o r albumin i n D^O of  display  has  i n nature (Timasheff e t . a l . . 1967)  reflected  viscosity  p r o p e r t i e s and tumbling p r o p e r t i e s i n s o l u t i o n . In  contrast  helical  should  (see  of since  the  this their  spectra,  but,  proteins was it  seems  the coacervate of a l p h a - e l a s t i n i s a l s o c h a r a c t e r i z e d  a random conformation.  no  by  103  F i g u r e . 4.10: Nmr s p e c t r a of albumin.. (A) 400 MHz spectrum of bovine serum albumin at 250C. . (B) P r e d i c t e d random-coil s p e c t r a f o r albumin using spectral parameters from t a b l e 4.2 and amino a c i d composition from Dayhoff 1976. . (C) Same as spectrum B, but with a l l the resonances broadened to 100 Hz t o simulate the presence of s t a b l e secondary s t r u c u t r e s .  104  Figure. 4.10_  A  n  105  !!•. Conclusions Because of the insoluble  difficulties  materials  such  as  involved  fibrous  r e s e a r c h e r s have chosen to look at the of  the  soluble  elastins:  in  a number o f  structural  properties  alpha-elastin  (Partridge  content,  elastin and  elastins  with  exhibit  temperatures  has  since  interactions  a  a  there  very is  (Partridge  the  high hydrophobic an  temperature  (370C).  phase  increase  and  (Tanford  phenomenon  of  for  molecules  1973),  and  coacervation  at  S e v e r a l workers have shown exhibit  high  that  a fibrillar  precursor  both  tropoelastin  similar  as  and  ones  Cliff  observed  e l a s t i n preparations  (Gotte e t . a l . . 1976).  workers  have  supported  presence  of these f i b r i l s  Urry  1978),  with  f o r insoluble and  h i s co-  t h i s view by demonstrating  i n the c c a c e r v a t e s of the  (Rapaka and Urry  and  s t r u c t u r e i n the coacervate  1974, C l e a r y the  the  crucial  (urry 1978c).  s t a t e . (Cox e t ^ a l ^ . 1 9 7 3 , periodicities  t o be a  cross-linking  i n t o the f i b r o u s t i s s u e  repeat p e p t i d e s  the s o l u b l e  of the s o l u t i o n i n t o a p r o t e i n dense  concentrating  also  hydrophobic  This c o a c e r v a t i o n , which i s c h a r a c t e r i z e d  separation  alpha-elastin  amino a c i d  in  phase and an e g u i l i b r i u m s o l u t i o n , i s thought step  et.al.  1 979) .  Since  by  with  elastin,  1955), and the precursor p r o t e i n , t r o p o e l a s t i n Whiting  working  1978, V o l p i n e t . a l .  the  synthetic 1976a, b,  c) . Although very  obvious,  the the  concentrating 'aligning'  effect, aspect  of c o a c e r v a t i o n , i s  implies  an  ordered  106  (fibrillar)  structure  elastins  i n the  c o a c e r v a t e states..Taken a step f u r t h e r , the presence  of the  filaments  argues  f o r these  against  an  soluble  entropic  elasticity  e l a s t i n p r o t e i n , and t h i s forms the b a s i s of currently  surrounding  the  s t r u c t u r e of e l a s t i n  This controversy i s further fueled obtained by  the  various  the  f o r the  controversy coacervates.  by the c o n f l i c t i n g  workers  who  are  evidence  addressing  this  question. The  n.m.r.  data  is  r e p o r t e d by Urry and long i n d i c a t e an Lyerla  increase  ______  The a n a l y s i s chapter  in  (1977b), order  of  the  viscosity that  with  f o r the  formation  of  the t h e C-n.rn.r 13  elastic 1976)  light  a  whereas, elastin.  presented  process  of  aggregation  in  this  alpha-elastin  processes  with  nc  of p a r a l l e l i s o t r o p i c f i l a m e n t s microscope.  This  the r e s u l t s i s i n e x c e l l e n t agreement with r e s u l t s of  Lyerla  e t . a l . . (1 975),  s c a t t e r r i n g experiments  and sedimentation  support  studies  for fibrous  which a r e seen i n the t r a n s m i s s i o n e l e c t r o n interpretation  that  temperature,  studies  the  c o a c e r v a t i o n i s dominated by  in  f o r the s y n t h e t i c p e p t i d e s ,  (1975) r e p o r t the converse  indicates  evidence  contradictory  random  data  (Jamieson  (Schein ______  conformation  quasi-  e t . a l . 1972,  1977) , which  f o r the  soluble  also  elastin  coacervates. That there are some secondary is  clearly  ______  demonstrated  by  19 73, Tamburro ______  unresolved  guestion  deals  a  structures i n number  1977, F o s t e r with  the  elastin  of s t u d i e s (Starcher et.al.  whether  these  1976). areas  The of  107  secondary s t r u c t u r e s are l o c a l i z e d i n the c r o s s - l i n k  area  whether  of the e x t e n s i b l e  regions.  Since the a n a l y s i s of the e l a s t i n as a k i n e t i c rubber  assumes  they  that there that n.m.r.  this  are  characteristic  are no s t a b l e secondary s t r u c t u r e s , i t i s important conflict  be  resolved.  r e s u l t s presented i n t h i s  secondary  or  The r e s u l t s of the proton chapter  suggest  that  the  s t r u c t u r e s i n the e l a s t i n network are r e s t r i c t e d t o  the c r o s s - l i n k r e g i o n s , are c h a r a c t e r i z e d  and t h a t the majority  of the  by random, i s o t r o p i c motions.  residues  108  Chapter^Vo. C 0 NFO EM ATICN OF  EL ASTIN ± BIBEFBINGENCE PEOPEETIES^  Introduction As  pointed  out  microscope s t u d i e s coacervates  of  earlier,  several  of n e g a t i v e l y s t a i n e d f i b r o u s  soluble  elastin,  reveal  a n i s o t r o p i c s t r u c t u r e c o n s i s t i n g of 3 t c are  presumed  to run  fibre  (Quintarelli  1976  and  Cliff  1978). The  can  be  1 978,  1977,  Gosline  explained  random  network  highly  5nm  Cox  Volpin  this  ordered,  filaments  filamentous  that  elastin et.al.  et.a lg_. 1 974,  Cleary  of  however,  elastin,  Dorrington  and  theory  structure  and  and  by the K i n e t i c Theory of Bubber  F l o r y 1958,  1978,  1980), and  elastin  Serafini-Fracassini  mechanical p r o p e r t i e s  Gosline  anisotropic,  1973,  Gotte ejUal.. 1974,  (Hoeve and  a  electron  p a r a l l e l to the long a x i s of the  et.al.  accurately  Elasticity  recent  Cifferri  is  that  based  is  systems  et,al..  very seen  1970, on  an  1975  and  Aaron  and  isotropic,  d i f f e r e n t from in  the  the  electron  microscope. Given these two one  should  be  p o s s i b l e types of s t r u c t u r e s f o r e l a s t i n ,  able t o d i s t i n g u i s h between them by  the b i r e f r i n g e n c e of s i n g l e e l a s t i n f i b r e s i n microscope.  This  approach  does not  r e q u i r e any  reducing  the chance of any  technique  is  very  s t r u c t u r e at two level  was  procedural  of the  p r o t e i n , thus  artifacts..Further,  allowing  l e v e l s of o r g a n i z a t i o n :  as r e f l e c t e d by the  polarizing  chosen because the technique  p h y s i c a l disturbance  versatile,  the  observing  the  evaluation  (a) at the  the of  molecular  i n t r i n s i c b i r e f r i n g e n c e , and  (b)  at  109  the sub-microscopic l e v e l 5nm  ( i _ e _ , at the l e v e l of  f i l a m e n t s ) as i n d i c a t e d by the form If  elastin  is  should be r e f l e c t e d  as  when  this  observed  the other hand, i f e l a s t i n i s i n  implied  by  the  elctron  microscope  evidence, then t h i s should be manifested as an observable b i r e f r i n g e n c e . S i m i l a r s t u d i e s on c o l l a g e n ( P f e i f f e r Chitin  (Diehl and I t e r s o n 1935)  this  type  of  birefringence analysis  analysis.  a t t e s t to the This  chapter  p r o p e r t i e s of s i n g l e  in  to  birefringence.  as a lack c f b i r e f r i n g e n c e  filamentous,  3  i s o t r o p i c i n i t s o r g a n i z a t i o n , then  between c r c s e d p o l a r i z e r s . On fact  the  terms  of  the  elastin  form  1943)  and  reliability  of  deals  with  fibres  implications  and  for  the it's  elastin  conformation.  Mi  Phengmengloqical  e_p_.anat.ion of double  __________  The b a s i s f o r the phenomenon cf b i r e f r i n g e n c e , use  as  an  indicator  of  and  the  wave  nature  light  mediums,  then,  by  electric  of  e x p l a i n and a c c o u n t , f o r the observed two  it's  molecular c c n f o r m a t i o n , e v e n t u a l l y  r e s t s on the i n t e r a c t i o n between the particles  and  properties  l i g h t . Hence i f one interaction  between  of can the  comparing the nature of the i n c i d e n t  to the r e s u l t i n g r a d i a t i o n , one can make some deductions  as to the s p a t i a l arrangement of the i n t e r a c t i n g seems f i t t i n g processes  of  then, that something molecular  phenomenon of double  should be  interactions  refraction.  that  particles. It  said  about  the  g i v e r i s e t o the  110  (a) R e t a r d a t i o n The  retardation  concept  of  that chemical  of l i n e a r l y  axis  is  along  up  the  fast  Consider  of  ncn-retarded  axis a  of  5.IC).  of  a  beam  of  of  the  this  is  to  vector  normal to i t  velocity.  propagated elliptically by  and  ( i_e_ can  in  object,  the  the  be  represented  the  zy  as  plane  (figure  light  would  be  Since there i s no  (which  is  at  90° by  viewed  analyzer.  However,  for  a  positively  birefringent,  o b j e c t , o r i e n t a t e d along the z a x i s , the zy  a  ( f i g u r e 5.IB),  to the p o l a r i z e r ) , none of i t w i l l be t r a n s m i t t e d  through the  of  t o pass through a  t h a t depicted i n f i g u r e 5. 1A. axis  direction  out of the plane  resulting  analyzer  it's  the a n a l y z e r . Hence the specimen w i l l appear dark when  i n the  vector  p o l a r i z e d l i g h t with i t ' s  p o l a r i z e d l i g h t was  component p a r a l l e l to the relative  parallel  passed  vector l y i n g i n the xy plane  plane  non-birefringent identical  (which to give  which  linearly  along the y a x i s  vector l y i n g If  the  1971).  propagation  the  vector  transmission)  component  the paper). T h i s e l e c t r i c  and  on  to as the slow a x i s cf t r a n s m i s s i o n .  vector V as shown i n f i g u r e 5.1&,  resultant  based  the b i r e f r i n g e n t o b j e c t the r e t a r d e d  with the  (Wilkes  electric  is  'retarding' i t ' s transmission  referred  polarized l i g h t , analyzer  light  p o l a r i z e d l i g h t whose e l e c t r i c vector i s  Upon emerging frcm w i l l add  polarized  bonds w i l l i n t e r a c t with t h a t component  to the bond d i r e c t i o n , This  of p o l a r i z e d l i g h t  plane  will  be  parallel  to  cylindrical  plane p o l a r i z e d l i g h t the  slow  axis  of  111  ____________ Propagation of _________ light through i_:otro^ic material^ (A) Looking down the a x i s of propagation, showing the r e l a t i v e p o s i t i o n s o f the p o l a r i z e r (pol and the a n a l y z e r (an) . (B) The e l e c t r i c v e c t o r i n the xy plane. (C) The e l e c t r i c v e c t o r i n the zy plane. .  112  F i g u r e . 5,J.  113  Figure.5.2: Propagation of p o l a r i z e d l i g h t through anisotropic material. (A) The e l e c t r i c vector i n the xy plane, normal to the slow a x i s . (B) The e l c t r i c vector i n the zy plane is parallel to the slow a x i s and i s r e t a r d e d by an amount 0 r e l a t i v e t o the vector i n the xy plane. (C) The r e s u l t a n t l i g h t i s e l l i p t i c a l l y polarized with a ccmponent (V) that i s t r a n s m i t t e d by the a n a l y z e r (an) .  11"  115  transmission  (figure  i t ' s transmission the  xy plane  and  will  5.2B),  v e l o c i t y . On the other hand, t h e  vector  frcm  the  unaltered  birefringent  (figure  5.2A).  object the retarded  Upon  vector,  which propagated along the slow a x i s , w i l l be out of phase an  amount  ^  with the unaltered one. As s t a t e d before,  two v e c t o r s w i l l add up to g i v e e l l i p t i c a l l y which  has a component V  the a n a l y z e r maximum  parallel  will  at  given  the analyzer  Jpl  (  at  90° of  to the  45°  with  Q u a n t i t a t i n g the r e t a r d a t i o n  r e t a r d a t i o n of the e l e c t r i c  calibrated  light  1950).  vector propagating  the slow a x i s c f t r a n s m i s s i o n can be g u a n t i t a t e d by a  these  r e t a r d a t i o n , the  occur at a specimen o r i e n t a t i o n  r e s p e c t to the p o l a r i z e r (Bennett  The  polarized  by  t o the t r a n s m i s s i o n a x i s of  ( f i g u r e 5.2C). For any  transmission  polarizer)  in  w i l l be normal to t h i s slow a x i s o f t r a n s m i s s i o n  t h e r e f o r e , remain  emerging  r e s u l t i n g i n the r e t a r d a t i o n o f  compensator between  along  inserting  the b i r e f r i n g e n t o b j e c t and  the a n a l y s e r . This compensator, as the name i m p l i e s , f u n c t i o n s by r e v e r s i n g the e f f e c t s  of  changes  the  linearly  polarized light,  to  elliptically  the  birefringent  polarized  emergent  with i t ' s e l e c t r i c  specimen. light  vector  It  back t o parallel  t h a t of the p o l a r i z e r . Since t h e analyzer w i l l not pass any  component  that  experimental  i s at  90°  manipulation  extinction  (cf the  'reverse'  retardation  to  i t ' s t r a n s m i s s i o n a x i s , the  i n v o l v e s r o t a t i n g the compensator t o  bright of  specimen), the  at  compensator,  which  point  the  i s egual t o the  116  i n i t i a l r e t a r d a t i o n cf the specimen. The r e t a r d a t i o n can  then be t a b u l a t e d  equations f o r any This  value  length,  of  compensator and  retardation  is  a value f o r the  Figure  5.3  is  p o s i t i o n s of the birefringence the  wavelength  divided  summary  be  ( 1953)  Wilkes  of  by the o p t i c a l path  molecular  the  specimen,  showing the  relative of  the  involved i n polarized l i g h t Bennett  The  Types of  (1950),  Frey-  Birefringence  birefringence  i n t r i n s i c or c r y s t a l l i n e b i r e f r i n g e n c e ,  which i s thought t o be the r e f l e c t i o n of the the  light.  (1971).  J _ l Intrinsic concept  of  A more d e t a i l e d account  found in a r t i c l e s by and  the  appropriate  components used i n the study  methodology  C_ __________  The  figure  p r o p e r t i e s of m a t e r i a l s .  microscopy can Wyssling  a  and  of  b i r e f r i n g e n c e , which i s u n i t l e s s .  various  theory  value  u t i l i z i n g the  d, u s u a l l y equated to the t h i c k n e s s of  to y i e l d  of  given  by  absolute  level,  is  best  introduced  organization  at  by c o n s i d e r i n g  an  i s o t r o p i c m a t e r i a l . In such a m a t e r i a l the chemical bonds distributed linearly  egually  over a l l angles,  p o l a r i z e d l i g h t . As  distribution  light  will  vectors,  have  r e l a t i v e t o the i n c i d e n t  result  of bond angles there  of the component e l e c t r i c polarized  a  of  this  homogeneous  i s no s e l e c t i v e r e t a r d a t i o n and  the  emergent  i t ' s e l e c t r i c vector  that of the i n c i d e n t beam. Hence the i s o t r o p i c appear dark when viewed between  are  crossed-polars.  plane  parallel  material  to  will  117  Ficj.ure._5_.j:. The b i r e f r i n gence experiment. Pol: polarizer. S: specimen. D: path l e n g t h . C: compensator. An: a n a l y z e r . Ob: observer. The arrow shows the d i r e c t i o n c f propagation.  118  _  Z  _  _  _  _  _  _  _  _  j  o  119  This  system  however,  cannot  between  an  motion  of  segments (such as the k i n e t i c elastomers) and  the  i s o t r o p y that i s a r e s u l t of anisotropic  the  differentiate random  thermal  i s o t r o p y that a r i s e s from a homogeneous d i s t r i b u t i o n of segments  (such  as  glass).  Fortunately,  molecular c h a r a c t e r i z a t i o n can the  mechanical  properties  the  materials  K i n e t i c elastomers are c h a r a c t e r i z e d the order have  of  10  modulus  6  Now  that  than the rubbery  object  bond  light  will  of  study.  modulus  in  three t c f o u r orders  of  materials.  a m a t e r i a l whose molecular s t r u c t u r e shows a  light, will result polarized  basis  under  by a Young's  are atout  predominant d i r e c t i o n a l i t y of i t ' s of  the  2  consider  orientation  of  Newtcns/m , whereas g l a s s y substances u s u a l l y  values  magnitude higher  t h i s aspect  be e l u c i d a t e d on the  of  rigid  angles,  in to  appear  the  units.  Such  with reference  transformation  elliptically  polarized  bright  examined  when  a  preferred  to the i n c i d e n t  of  the light,  between  linearly and  the  crossed  polars. The length,  amount is  an  organization.  of  indicator The  the d i r e c t i o n of the conventionally parallel  to  retardation of  a  f u n c t i o n of the  extent  of  path  molecular  s i g n of the b i r e f r i n g e n c e , which r e f e r s to slow  axis  'given  f a s t a x i s p a r a l l e l to the  i s s a i d to be n e g a t i v e l y  of  transmission,  has  been  p o s i t i v e when the slow a x i s i s  dimentional  a x i s of a c y l i n d r i c a l o b j e c t . it's  the  d e f i n e d as being a  as  feature'  Conversely, i f 'given  such as the the  dimentional  b i r e f r i n g e n t (figure  object  long has  feature', i t  5.4).  120  F igure,_J5_. 4^ The s i g n of t h e b i r e f r i n g e n c e . (A) Negatively birefringent specimen: fast p a r a l l e l to the ' d i m e n s i o n a l feature . (B) Positively birefringent material: slow p a r a l l e l to the ' d i m e n s i o n a l f e a t u r e ' .  axis  1  axis  122  Z i g u r e ^ S i S . : Form b i r e f r i n g e n c e . (A) A composite of f i b r e s with r e f r a c t i v e index n^- , i n a matrix of r e f r a c t i v e index n . (B) Evaluation of the form b i r e f r i n g e n c e according to eguation 5.2: (a) f o r f i b r e s with positive birefringence (b) f o r i s o t r c p i c fibres (c) f o r n e g a t i v e l y b i r e f r i n g e n t f i b r e s . The i n s e t shows the r e l a t i v e dependence of the form b i r e f r i n g e n c e on the volume f r a c t i o n cf the f i b r e s , \ . m  123  F i g u r e . 5. 5.  R  124  It) Unlike  Form b i r e f r i n g e n c e  intrinsic  birefringence  which  a n i s o t r o p y at the molecular l e v e l , form from  an  anisotropy  of  a  scale  light.  The  limit  to  their  possess t r u e phase boundaries Consider  the  refractive form  index  n^ ,  birefringence  1950).  The  first  of  parallel,  surrounded  of  theoretical  the  is  set  by  isotropic  by  as  this  will  differing  refractive  i n 19 12, and i t has formed  form  n 2-n_2= ( n ^ 2 - n  parallel  m 2 ) 2  f  birefringence  ( 1  _  f )/  A  f  2  (  1  _  f  )  the  basis  s t u d i e s t o date. For a  «. _ n  to and normal t o the dominant with  2( 1 + f  _  5  .  (Bear  r  a  axis  of  the  specimen  f r e p r e s e n t i n g the volume f r a c t i o n of the  f i b r e s . Another r e l a t i o n s h i p f o r a on  index  and n_ represent the r e f r a c t i v e index of the system  respectively,  based  of  r e l a t i o n s h i p f o r such composites  19 37) :  u  5.5A..  exhibit  ______  n  to  an i s o t r o p i c medium of  of r o d s , the t h e o r e t i c a l e x p r e s s i o n has the form  where.  the  fibres  system  u  of  a r i s i n g from the p r e f e r r e d o r i e n t a t i o n of  was presented by Weiner most  wavelength  (Frey-Wyssling 1953).  index n^. A s t r u c t u r e such  (Bennett  the  than the  u n i t s should be b i g enough  a s s y m e t r i c bodies i n a medium of  of  than  larger  an  arises  s t r u c t u r a l composite shown i n f i g u r e  T h i s r e p r e s e n t s a system refractive  is  'smallness*  requirement t h a t the s t r u c t u r a l  from  birefringence  that  dimensions of molecules but s m a l l e r  arises  modification  of  the  similar  type  of  system,  Weiner e q u a t i o n , has been  125  proposed by Bear e t . a l . .  (1937).  I f n_c and n,^ a r e n e a r l y e g u a l , be s i m p l i f i e d t o an experimentally  the above r e l a t i o n s h i p can more u s e f u l one of the form  (Stokes 1 963) : n, -n _= ( n n ) 2 f ( 1 - f ) / i i v  J  A graphical representation figure  5.5B,  curve  b.  (form and i n t r i n s i c ) birefringence  of  of t h i s eguation i s  shown  system  be  additive,  can be c h a r a c t e r i z e d  the  total  as shown i n  f i g u r e 5.5B, curve a, f o r rods of p o s i t i v e b i r e f r i n g e n c e , curve all the  cases, the form b i r e f r i n g e n c e birefringence  f o r any  egual to 0.5  cf  the  goes t o zero when n =n _, and +  system  eguals  given  occurs at a volume f r a c t i o n of  fibres,  birefringence  along  one  of  object  i t ' s axis,  i s subjected t o  i t will  exhibit  which i s commonly l a b e l l e d a c c i d e n t a l or  angles,  d i c r e c t i o n a l i t y on in  the  originally  the  distribution  isotropic  subsequently r e f l e c t e d as an o p t i c a l The  molecular changes r e s p o n s i b l e  material,  a  strain  b i r e f r i n g e n c e . . The imposed s t r a i n has the e f f e c t of i n d u c i n g preferred  f,  ( f i g u r e 5.5 i n s e t ) .  When an i s o t r o p i c n o n - b i r e f r i n g e n t  birefringence  intrinsic  r e f r a c t i v e index d i f f e r e n c e the maximum  _[£L Strain  strain  the  n  of the f i b r e s . I t a l s o f o l l o w s from eguation 5. 2  form b i r e f r i n g e n c e  a  and  c , f o r rods having negative i n t r i n s i c b i r e f r i n g e n c e . In  birefringence that  in  Since the two types of b i r e f r i n g e n c e  are thought t o  a  5.2  m  r  a  of the bond which  is  anisctropy.. f o r the observed  strain  126  birefringence optical  depends on the type of m a t e r i a l  anisoptropy  t h a t r e s u l t s when c r y s t a l l i n e s o l i d s are  s t r a i n e d , a r i s e s from the deformation of the  material,  which  distances  the  birefringence  (Frey-Wyssling of  t h e o r e t i c a l b a s i s has t o  arising  from  birefringence  1953). When  rubbery  different  the  electron  the  chains  displacement  be of  of  however,  invoked.  Rather  electrons,  the  a  than strain  of these m a t e r i a l s i s thought t o r e s u l t from the the  kinetically  of the polymer network. The o p t i c a l p r o p e r t i e s o f  l i n k s themselves .remain The  in  considering  polymers  alignment of the 'random l i n k s ' t h a t make up free  orbits  i n t u r n i s a conseguence of the a l t e r e d  inter-atomic strain  i n guestion. The  uneffected.  t h e o r e t i c a l eguations f o r the  rubbery  polymers  birefrinigence  w i l l be more a p p r o p r i a t e l y  chapter 7. For the moment i t qualitative  strain  differences  suffices  between  the  to  discussed i n  simply  two  state  types  of  the  strain  birefringence.  D_ M a t e r i a l s  and Methods  S i n g l e , 6 t o 8 un, e l a s t i n f i b r e s from 2i3§i™en_u_  f i n e forceps  viewed between microscope, using was  a Zeiss  provided  bovine  nuchae and from ligament t h a t had been p u r i f i e d by  repeated a u t c c l a v i n g with  untreated  (Partridge e t _ ___  were  isolated  on a d i s s e c t i n g micrcsccpe. The f i b r e s were  crossed  and  1955)  the  polars  on  retardations  a  Wild  were  M-21  determined at 546nm  1/30 wavelength r o t a r y compensator. by a 100 watt  quartz  lamp  polarizing  which  Illumination was  used  in  127  conjunction  with  an  interference  filter  to  give  the  monochromatic green l i g h t .  Birefringence  was  dividing  at  of the f i b r e by the  fibre  the  retardation  the center  calculated  by  diameter. The  form  measurements collagen  birefringence on  single  curves  elastin  f i b r e s that had been  fibres  swollen  r e f r a c t i v e index. These are l i s t e d The  had  in  liguids  circulaticn connected  chanelled  of to  water.  a  out  The  thermostated  to  an  allow  that  had  been  outlets  from  Unless otherwise s t a t e d , a l l  known  i n the l a b t o  aluminium  the  circulating  calibrated  of  piece  an i n t e r n a l c l o s e d stage  water  temperature a t the sample was monitored with bead  tendon  i n t a b l e 5.1..  microscope. I t c o n s i s t e d of  been  from  and r a t t a i l  temperature c o n t r o l l e d stage was b u i l t  f i t the Wild that  were . obtained  were  bath.  GB32  The  thermistor  with a mercury thermometer.  experiments  were  conducted  at  240C.  Ii  Birefringence  P r o p e r t i e s of S i n g l e E l a s t i n  Fibres  iJLL Form b i r e f r i n g e n c e Single  elastin  f i b r e s were e i t h e r swollen  known r e f r a c t i v e index air  dried  refractive and  and  (water, ethylene  immersed  in  organic  index. In a l l cases the  unpurified) ,  gave  a constant  glycol, solvents  elastin  i n l i g u i d s of glycerol)  or  of d i f f e r e n t  fibres  (purified  low b i r e f r i n g e n c e value of  about 2 X 10-* (table 5.1, f i g u r e 5.6 curve a ) . There  was  no  128  Iable.5.1;  Form b i t e f r i n qsncQ of s i n g l e e l a s t i n  0}  •«- i <t-  «3-  —  01 —  u >>  rr  fibres.  129  Figure.5.6: Form _____________ of e l a s t i n f i b r e s . (a) For s i n g l e e l a s t i n f i b r e s i n (x) water n=1.33 (+) ethylene g l y c o l n=1.43 and g l y c e r o l n=1.48„ {•) f o r o r g a n i c s o l v e n t of d i f f e r e n t r e f r a c t i v e index.. (b) Theoretical birefringence calculated for i s o t r o p i c f i b r e s a c c o r d i n g to eguation 5.2. (c) R e s u l t s obtained f o r s i n g l e elastin f i b r e s by S e r a f i n i - F r a c a s s i n i e t . a l . 1976. The inset shows the temperature dependence f o r the b i r e f r i n g e n c e of s i n g l e e l a s t i n f i b r e s .  13 1  indication  of form  b i r e f r i n g e n c e , which i s u s u a l l y  as a U-shaped curve f c r refractive  index  characteristic structure,  of  of  the  graph  of  visualized  birefringence  versus  imbibing l i g u i d . T h i s U-shaped curve i s  fibrous  proteins  with  filamentous  sub-  such as c o l l a g e n , which shows a very d i s t i n c t  b i r e f r i n g e n c e curve  (figure  5.7  results  chitin,  and k e r a t i n  for  silk,  curve  a  ),  and  form  reported  (Frey-Wyssling  1948,  1953) . If elastin electron then  was  i n f a c t f i l a m e n t o u s , as suggested  microscope  one  should  by  studies (Serafini-Fracassini et.al. expect  a  result  the  1976),  t h a t c l o s e l y f o l l o w s the  t h e o r e t i c a l p r e d i c t i o n a f f o r d e d by eguation 5.2,  as  figure  calculated  5.6  curve b. T h i s t h e o r e t i c a l curve was  assuming the f i l a m e n t s ( i f present) a value of 1.55 protein. 0.65  The  for  in  refractive  It  should  these  reflection  be  index  condition  are  cf  the  mentioned  that  elastin  experimental  results  obtained  considered to be a more a c c u r a t e as  compared  the data p o i n t s which were obtained f o r dry e l a s t i n  in  the  encouraging,  and  prcbably  showed i d e n t i c a l  results.  Lastly,  should  obtained  in  it  these  be  using  elastin  to  organic  by  glycerol retains i t ' s  of e l a s t i n ' s f u n c t i o n a l conformation,  different  in  taken to have a value of  water, ethylene g l y c o l , and  e l a s t i c p r o p e r t i e s , hence the under  to be i s o t r o p i c , and  volume f r a c t i o n , f , was  (Gosline 1978).  swollen  the  shown  liquids. relevant,  pointed  experiments  are  Nevertheless,  fibres it  is  that both s e t s of data  out  that  the  inconsistent  results with  the  132  reports  of  Fracassini  positive  form  birefringence  e t . a l . . 1976),  figure  5.6  curves  curve  c, and a l a t e r  report  of a negative form  Gotte  1977) f o r s i n g l e e l a s t i n f i b r e s . A p o s s i b l e  for  these  conflicting  birefringence  results  will  curve  be  (Serafini-  (Bairati  and  explanation  presented  i n the  d i s c u s s i o n s e c t i o n of t h i s chapter.  Jb_ Since there reasonable represents  tc  Intrinsic  birefringence  i s no i n d i c a t i o n of form b i r e f r i n g e n c e , assume  that  the i n t r i n s i c  the  constant  it is  value of 2 X 10-*  component of the b i r e f r i n g e n c e .  This  value i s seen t o be temperature independent f o r e l a s t i n f i b r e s in  water  (figure  5.6  inset),  and  compared to other known c r y s t a l l i n e It  is  therefore  tempting  tc  is  extremely  proteins  conclude  s m a l l as  (Schmitt  that  1950).  e l a s t i n i s an  amorphous p r o t e i n . One might argue however, that intrinsic  birefringence  t o t a l birefringence form  birefringence,  birefringence magnitude so)  is  this  actually  low  value  an a r t i f a c t . Since the  c f a system i s a sum of the i n t r i n s i c i t is  counteract  each other when  and have opposite  the  birefringence  acid f i x e d collagen function  fibres  and  p o s s i b l e that these two types o f they  are  s i g n s , to y i e l d a zero  t o t a l b i r e f r i n g e n c e . This i s demonstrated i n  where  f o r the  of  egual  (or n e a r l y  figure  5.7,  of d r i e d c o l l a g e n f i b r e s and t a n n i c (Pfeiffer  1943)  is  plotted  as  of the r e f r a c t i v e index of the immersion medium.  Collagen  f i b r e s , which a r e known t o be a n i s o t r o p i c at the  133  F i g u r e .5.7: Form b i r e f r i n g e n c e of collagen.. (a) u n p u r i f i e d c o l l a g e n f i b r e s . (b) e l a s t i n f i b r e s . (c) Tannic a c i d fixed collagen fibres (Pfeiffer 1 943) .  134  135  molecular form and a).  and  the sub-microscopic  positive intrinsic birefringence  Tannic  acid  b i r e f r i n g e n c e , and collagen n  m  appear  reverses  the  as a r e s u l t isotropic  egual to 1.40  of  l e v e l s , show both  and  1.67  of  sign this,  {figure of  positive  5.7  the  tannic  curve  intrinsic acid  fixed  (show zero t o t a l b i r e f r i n g e n c e ) at  (figure 5.7  curve c) . That the  lack  b i r e f r i n g e n c e i n s i n g l e e l a s t i n f i b r e s does not occur as a  r e s u l t of t h i s phenomenon i s supported  by the  following.  In order f o r the t h e o r e t i c a l form b i r e f r i n g e n c e curve intersect  the  observed  has to e i t h e r positive)  (a)  to  data point f o r e l a s t i n i n water,  assign  an  unusally  large  one  negative  i n t r i n s i c b i r e f r i n g e n c e to e l a s t i n , or  (or  (b) assume a  r e f r a c t i v e index f o r the p r o t e i n t h a t i s much s m a l l e r than value of are  1.55  used i n t h i s study. Both of these  unreasonable.  indicate  a  supported  by the X-ray  second  very  A  choice,  high  intrinsic  crystalline  and  that  of  is  indeces f o r a  wide and  i s precluded by the  fact  section,  fibres  that,  as  show  presented a  very  b i r e f r i n g e n c e over a bread range of r e f r a c t i v e 5.6).  This  is  taken  in low, index  X  10-*,  which  is  taken  an  variety  artifact the  to be the value of the  last  constant liguids  to mean that there i s no  b i r e f r i n g e n c e component a s s o c i a t e d with the observed 2  The  1.55.  i m p o r t a n t l y however, the p o s s i b i l i t y of an  elastin  not  a low r e f r a c t i v e index, would be  of p r o t e i n s , with values that l i e between 1.50  (figure  this  would  d i f f r a c t i o n r e p o r t s (Astbury 1940).  e x c e p t i o n to the known r e f r a c t i v e  Most  possibilities  birefringence  structure  the  value  form of  apparent  136  intrinsic  b i r e f r i n g e n c e . The  •apparent' i s d i s c u s s e d  _[cl E x p l a n a t i o n Although  the  timely i n t e r j e c t i o n  source of t h i s  i s faced  with three  the  word  i n the f o l l o w i n g s e c t i o n .  f o r the apparent b i r e f r i n g e n c e  magnitude of the r e s i d u a l b i r e f r i n g e n c e i s  extremely s m a l l , i t might be i n t e r e s t i n g the  of  to  speculate  about  ' i n t r i n s i c ' b i r e f r i n g e n c e . In doing so  one  possibilities:  1. The b i r e f r i n g e n c e i s an i n h e r e n t property of the fibre. 2. That the birefringence i s a result of an a n i s o t r o p i c r e s i d u e around the f i b r e . 3. That i t i s due to the r e f r a c t i v e index d i f f e r e n c e at the i n t e r f a c e of the swollen protein and the surrounding water.  Inherent  birefringence:  Since  the  r e t a r d a t i o n i s p r o p o r t i o n a l t o the  path  length  through the f i b r e , then, i f the b i r e f r i n g e n c e  was  the  at the center  f i b r e one  would expect i t to be  greatest  inherent  the f i b r e where the path l e n g t h i s at i t ' s maximum value.. a  circular  cross-section  r e t a r d a t i o n s across  one  the f i b r e of r a d i u s , r , i n  the  relative  the case of the u n p u r i f i e d f i b r e s i t i s p o s s i b l e  that  according  to:  This r e l a t i o n s h i p i s depicted  lirefringent  terms  For  the  p= 2 (cos6r)  there  predict  of  of  path length,p,  In  can  to  5.3  in figure  5.8..  coating:  e x i s t s a sheath of b i r e f r i n g e n t m a t e r i a l  (such  as  the  137  ___________  ^i^ef£i£3snce_  Expected  relationship  for  intrinsic  G r a p h i c a l r e p r e s e n t a t i o n of the r e l a t i v e i n t e n s i t y versus d i s t a n c e across f i b r e c a l c u l a t e d according to eguation 5.3. M i s the middle of the f i b r e .  138  Figure.5.8.  2  139  glycoprotein  micro-fibrils)  of the f i b r e .  S i m i l a r l y , i n the case of the autoclaved  one  around the c e n t r a l amorphous core  might expect t h a t a r e s i d u e c o u l d  f i b r e s , which i f c r y s t a l l i n e ,  be  left  elastin  covering  would give r i s e t o an  the  observable  b i r e f r i n g e n c e . Again assuming a c i r c u l a r c r o s s - s e c t i o n f o r the elastin  fibre,  i t should  be p o s s i b l e t o p r e d i c t the r e l a t i v e  r e t a r d a t i o n s through the a n a l y s i s of the the  length  across  fibre. Consider  a beam of p o l a r i z e d l i g h t  c y l i n d e r of r a d i u s r ' , c o n t a i n i n g radius  r " , with both having  away from the center  propagating  within  common c e n t e r s  t o t a l path l e n g t h , p, t r a n s v e r s e d  it a  through a  cylinder  (figure  5.9A).  of .The  by the ray a t a d i s t a n c e , x,  i s given by:  p=2(r« -x2) V 2  The  path  5.4  2  component, p , of t h i s t o t a l path l e n g t h which l i e s i n s i d e 1  the i n n e r c y l i n d e r of r a d i u s , r , at a d i s t a n c e , x, away M  the c e n t e r ,  i s given by: p  Since  only  from  i-2(r"2-x2) V  the c o a t i n g matrix  5.5  2  i s b i r e f r i n g e n t , the e f f e c t i v e  path l e n g t h , p", which i s the path length c o n t r i b u t i n g to the r e t a r d a t i o n , at a d i s t a n c e , x, away from the c e n t e r , f o r x<r", i s given by: p"=p-p« For  values  of  5.6  x>r", p' goes to zero, and the above  eguation  reduces t o : p"=p These equations were evaluated  5.7 f o r various  ratios  of  r':r".  140  F iqure.5.9: Expected _____________ for an a _ _ _ _ t _ _ _ i _ coating. (A) I s o t r o p i c m a t e r i a l of r a d i u s r " surrounded by a birefringent c o a t i n g of r a d i u s r * . M r e p r e s e n t s the middle of the f i b r e . (B) Graphical representation of the relative intensity versus d i s t a n c e across f i b r e c a l c u l a t e d a c c o r d i n g to eguations 5.6 and 5.7, for different r a t i o s of r / r ' : (a) 0.75 (bj 0.90 (c) 0.95. n  142  and  the  r e s u l t s are  system  predicts  shown i n f i g u r e 5. 9 E . This a n a l y s i s of  t h a t the b i r e f r i n g e n c e  the edges, f a l l i n g to a f i n i t e  should be  value towards the  the  greatest  at  middle.  I n t e r f a c i a l effects.: In t h i s case, the source of the to be the  birefringence  i n t e r f a c e at the border of the  surrounding  water.  The  is  p r o t e i n f i b r e and  hydrated p r o t e i n would be  pure p r o t e i n , probably around  water the  r e f r a c t i v e index of the hydrated p r o t e i n i s i n c o n s e g u e n t i a l  as  as i t d i f f e r e n t from the  (which i t o b v i o u s l y derive it  was  a  is).  The  pure  to  exact value f o r  long  1.5.  the  expected  have a r e f r a c t i v e index somewhere between that of and  thought  value of the surrounding  Although  it  was  not  liquid  possible  t h e o r e t i c a l r e l a t i o n s h i p f o r a system such as  p o s s i b l e to make some p r e d i c t i o n s  based  on  to  this,  empirical  observations. Towards birefringence it  was  at the to  this  purpose,  patterns  observations  at o i l - w a t e r  were  i n t e r f a c e s . In  p o s s i b l e t o observe a very evident i n t e r f a c e . Unfortunately  extract  s i n c e the  any  useful  information  by chance, i n the case of a few trapped  bubbles  represent  distinct  birefringence,  pattern  of  this  a  three an  structure.  on  all  not  from these  the  cases  birefringence  however, i t was  i n t e r f a c e s were e s s e n t i a l l y two  of a i r bubbles were  made  zone  possible  observations  dimentional.  Totally  of these experiments, a number  in  the  o i l . Since  dimentional effort The  was  system, made  results  to  showed  these and  air  show a  study  the  that  the  143  birefringence  was  greatest  at the edges , f a l l i n g  off  towards  the c e n t e r cf the bubble. In  analogy  to  this,  one might  expect t h a t the e l a s t i n  f i b r e surrounded by water should behave i n a Furthermore,  within  the  resolution  similar  of  the  methods, i t seems that the b i r e f r i n g e n c e  should  theoretical  which  amorphous  core  described  5.10A  elastin  for  surrounded  in figure  Figure an  prediction  a  system  by  an  elastin  shows the b i r e f r i n g e n c e  f i b r e i n water.  birefringence  distinguish experiment liguid  c o n s i s t s of an sheath,  as  pattern  observed f o r  I t i s c l e a r that the r e t a r d a t i o n ,  is  with l a s t two  between  an  at the edges of the  possibility  inherent  that  the  of  the  property  them,  without  if  altering  i s caused by  the the  sheath, then i t should remain birefringence be  uniformity approaches For hydrating  choices, one  i t would be p o s s i b l e t o could  manipulate  the  by changing the r e f r a c t i v e index of the surrounding  birefringence  should  the  protein.  In d e a l i n g  the  resemble  5.9..  This c o n c l u s i v e l y r u l e s out the  •apparent'  experimental  anisotropic  as i n d i c a t e d by the i n t e n s i t y , i s g r e a t e s t fibre.  manner.  hydrated presence  protein. of  an  If  the  anisotropic  unchanged. Cn the other hand, i f  i s a r e s u l t of i n t e r f a c i a l e f f e c t s , then i t  considerably  reduced  as  the  system  approaches  (as the r e f r a c t i v e index of the surrounding l i g u i d that of the p r o t e i n ) .  p r a c t i c a l purposes, i t i s p o s s i b l e t o achieve t h i s by the e l a s t i n f i b r e i n the vapour phase over a  dilute  144  £i_u££i5__0_  _______  Birefringence  pattern  of ______  elastin  (A) The b i r e f r i n g e n c e pattern of s i n g l e e l a s t i n f i b r e s n=1.55, i n water n=1.33, between crossed polars. The b i r e f r i n g e n c e i s seen t o be highest at the edges. The bar represents 10uni. (B) Densitometer t r a c i n g s f o r negatives of s i n g l e elastin fibres i n : (1) water, n=1„33. (2) immersion o i l n=1.52. (3) c o n t r o l t r a c i n g of blank f i e l d .  145  DISTANCE ACROSS  FIBER  146  salt  s o l u t i o n , and  which w i l l not value  then c o v e r i n g  were then scanned on  elastin  (1.52) that  Elastin fibres  mounted between crossed  Figure  f i b r e with immersion o i l  i n t e r a c t with the hydrated p r o t e i n , but  of r e f r a c t i v e index  swollen f i b r e .  the  i s c l o s e to that of  treated  p c l a r s and  in  5.10B,  tracing  were  negatives  curve 1, i s a densitometer t r a c i n g f o r the  previous observation  of  the  same  the  fibre.  interface,  above.  the  retardations  observed, that  Curve  f i b r e after reduction the  is  the  manner  r e f r a c t i v e index d i f f e r e n c e a t It  a  a Joyce-Loebel scanning densitometer.  f i b r e i n water, confirming  a  this  photographed. The  the h i g h e r i n t e n s i t i e s at the edges of shows  has  as  of  an of 2, the  described are  reduced  d r a s t i c a l l y , and  i s i n d i s t i n g u i s h a b l e from the c o n t r o l t r a c i n g  of a blank f i e l d  (curve  the  residual  'apparent'  3). Hence i t seems s a f e to s t a t e birefringence  r e f r a c t i v e index d i f f e r e n c e a t the that the  a c t u a l value f o r the  is  a  fibre-water  r e s u l t of interface,  that the and  b i r e f r i n g e n c e of e l a s t i n i s very  c l o s e to zero, as expected f o r a random p r o t e i n .  F.  Discussion  (a) Previous There  are  studies  a number of s t u d i e s  that d e a l with the  birefringence  properties  i n t e r p r e t a t i o n of these p r o p e r t i e s structure  of  Eomhanyi 1958,  this Gotte  protein 1965,  present i n the  Bairati  of e l a s t i n and  i n terms of  (Schmidt and  1939, Gotte  literature  the  molecular  Dempsey 1977,  the  1952,  Serafini-  147  Fracassini  ______  1976,  F i s c h e r 1979). .Most of these s t u d i e s  are i n agreement with r e f e r e n c e to the elastin  (with  the  exception  of  low  birefringence  Serafini-Fracassin  1976), r e p o r t i n g values around 2 X 10-*  as  study.. A l l  have  of  these  other  studies  obtained  et.al. in  on  the  •visualize  basis the  1  (Eomhanyi 1958) reaction  cf  structure  assumption utilizing  manipulations  1979). . Using  they  models  favoured  result  in  this  results  study  has  the i n v i v o rubbery  mentioned  in  the  water,  a  on  Gctte  elastin  which  of  these  the a n a l y s i s of  condition  that  before the r e s u l t s obtained i n t h i s  by  most  state.  with r e g a r d to the form b i r e f r i n g e n c e , are  B a i r a t i and  these ordered  occurence  concentrated  c l o s e l y resembles  reported  from  blue  p r o t e i n , which a l s o occur i n  fibres  those  reaction  anisotropic,  on u n p u r i f i e d  purified elastin  As  the  the  t i s s u e . In order to prevent  artifacts  phenol  to  a r t i f a c t s from the chemical i n t e r a c t i o n s of  components other than the e l a s t i n elastic  the  proceeded  f o r e l a s t i n . I t should be pointed out t h a t  the above s t u d i e s were conducted could  have  low  fibre,  and the p e r m a n g a n a t e - b i s u l f i t e - t o l u i d i n e  (Fischer  structural  this  this  assumed t h i s  b i r e f r i n g e n c e to be an i n h e r e n t property of the e l a s t i n and  of  Serafini-Fracassini  in  study,  conflict  et.al.  with  (1976)  and  (1S77). This study f a i l e d t o r e v e a l any  form  b i r e f r i n g e n c e f o r e l a s t i n , whereas S e r a f i n i - F r a c a s s i n i and h i s co-workers obtained a very d i s t i n c t i v e curve as w e l l as a high i n t r i n s i c b i r e f r i n g e n c e of 1 X 1 0 , -z  being i n favour of the f i b r i l l a r  which they i n t e r p r e t e d  models. In t r y i n g to  as  explain  148  this  conflict  a  clue  is  afforded  b i r e f r i n g e n c e curve obtained curve  c)  and  by  by g l a n c i n g  at the  authors  (figure  these  comparing i t with the r e s u l t s obtained  study f o r the form b i r e f r i n g e n c e of c o l l a g e n a).  The  resemblance i s g u i t e marked and  one  5.6  in this  ( f i g u r e 5.7 might  form  curve  speculate  that these i n v e s t i g a t o r s were a c t u a l l y d e a l i n g with a c o l l a g e n fibre. This  objection  f a c t t h a t these protein, high  and  is  authcrs  too f a r f e t c h e d c o n s i d e r i n g  used  collagenase  that t h i s technigue has  1977).  Furthermore, both B a i r a t i and  Serafinin-Fracassini contained  t h i s i t could artifacts,  (1976),  more  than one  be argued  caused  used  that  to  purify  purified Gotte  experimental  e l a s t i n f i b r e . On these  Jb_  authors  tissue  (1977)  The  of the f i b r i l l a r  the b a s i s  were  1. E l a s t i n i s  and  that  made  of  reporting be  fibres.  fibrillar  models in  the  models f o r e l a s t i n one  i s faced  with  up  parallel  possibilities:  filaments  and  preparations  In t r y i n g to i n t e r p r e t the b i r e f r i n g e n c e r e s u l t s  two  their  by the i n t e r f a c i a l e f f e c t s , which would  very l a r g e f o r ccnglcmorations of  context  the  been shown to r e s u l t i n  l e v e l s of c o l l a g e n contamination i n the  (Kadar  that  not  a l i g n e d i n the  of  an  array  of  d i r e c t i o n of the long a x i s ,  these f i l a m e n t s are themselves i s o t r o p i c ,  p o s s i b l y accomodating random c o i l s of p r o t e i n . 2. That e l a s t i n i s filamentous  in i t ' s organization,  149  as mentioned above, and i n a d d i t i o n filaments  contain  to  this  these  secondary s t r u c t u r e s such as the  Beta-turns. In the f i r s t case, that of i s o t r o p i c  f i l a m e n t s , one would  expect e l a s t i n  to e x h i b i t a marked  form  would  to  refractive  reduce  zero  when  the  birefringence  immersion medium e q u a l l e d  t h a t of the  protein  the  would  expect  second  case,  birefringence  cne  arising  also  from  the  arrangement of the f i l a m e n t s along  well  index  which of  the  filaments.  In  a  marked  defined  spatial  the long a x i s of the f i b r e .  But i n a d d i t i o n , t o t h i s form b i r e f r i n g e n c e , one should intrinsic  birefringence  form  associated  with  the  see an  crystalline  s t r u c t u r e of the f i l a m e n t s themselves. In the s p e c i f i c case o f the occurence of the Beta-turns i n the f i l a m e n t s one  would p r e d i c t a negative  peptide  back-bone i n v o l v e d  intrinsic  (Drry  1978c) ,  b i r e f r i n g e n c e s i n c e the  i n t h i s type of s t r u c t u r e would  be  running normal to the long a x i s of the f i l a m e n t s . As elastin seem  is  guite  evident  from the previous  f i b r e s do not show any form b i r e f r i n g e n c e nor do  to have any i n t r i n s i c b i r e f r i n g e n c e . These r e s u l t s  against  the presence c f any f i l a m e n t o u s  occurence  of  stable  secondary  p r o t e i n . Furthermore, the f a c t any  section, single  temperature  against  any  conformation workers  structures  in  that e l a s t i n does  argue  or  the  the  elastin  not  display  dependence o f i t ' s b i r e f r i n g e n c e a l s o argues  drastic, to  organization,  they  a  temperature, more  ordered  induced  change  of i t ' s  s t a t e as suggested by some  {Urry 1976a). F i n a l l y , s i n c e the s y n t h e t i c  fibres  of  150  elastin  (Orry  birefringent the  use  and  (Long  Long  1979)  1977b)  one  of these m a t e r i a l s  are  might be  known  to  be  justified in  as models f o r e l a s t i n  highly  questioning  structure.  G_.Con e l u s i o n s In view cf the evidence presented i n t h i s chapter, probably  justifiable  to  random network s t r u c t u r e kinetic  which  that  is  not  only  able  deal with the  to  typical  cf  other  known  d i s t r i b u t i o n of s t a t i c  u n i t s and  light  'average conformation',  distinguish  between  the  and  homogeneous  the random k i n e t i c  movements  of mobile a n i s o t r o p i c u n i t s . Hence i t cannot argue against presence of as  disregard of  dynamic  the  (in r a p i d  the  presence  One  s t r u c t u r e s , and  the  chains  nmr  organization  drying  T h i s could along  studies the  were  receding  electron  elastin  of the heavy-metal s a l t s used f o r negative  by  which  analysis  v i s u a l i z e d i n the  well be the r e s u l t of  microscope  the  4.  filamentous  high e l o n g a t i o n s process.  probably  s t r u c t u r e on  I t i s a l s o p l a u s i b l e that the samples of e l a s t i n electron  can  p l i a n t mechanical p r o p e r t i e s of e l a s t i n ,  presented i n chapter  microscope may  movements).  presence of s t a t i c , c r y s t a l l i n e  are i n c o n s i s t e n t with g l a s s y  The  the  of secondary s t r u c t u r e s as long as these are thought  being  basis  is  e l a s t i n possesses a  elastomers. However, the technique of p o l a r i z e d  microscopy can is  conclude  it  in  the  staining.  prepared  for  i n a d v e r t e n t l y extended to water  during  r e s u l t i n the alignment of the  the a x i s of the f i b r e ,  the  drying  polypeptide  which as a r e s u l t of  the  151  •static' would  view a f f o r d e d  be  a  by the e l e c t r o n  mis-interpretation  kinetically  free  filamentous  system  structure,  as  of  microscope the  being  technique,  normally a  agitated,  highly  organized  that i s i r r e l e v a n t t c the i n vivo rubbery  condition. T h i s p o i n t i s supported by e l e c t r o n which,  using  gradual  d r y i n g down of  filamentous 200%  the  freeze-etching the  technigues  tissue,  could  is  also  1978)  my  opinion  avoid  the  demonstrate  are  large  idea o f e l a s t i n being  et.al..1979).  that the 200nm s u b - f i b r e s  enough  to  random network s t r u c t u r e , and are not  of e l a s t i n  only  (Pasquali-Eonchetti  have been observed i n the scanning e l e c t r o n et.al.  to  studies,  s t r u c t u r e i n samples t h a t had been extended 150 t o  of t h e i r i n i t i a l length It  microscope  microscope  (Hart  accomodate an i s o t r o p i c in conflict  with  a t y p i c a l k i n e t i c elastomer. This  organization  which  i s examined i n the next  chapter.  the  aspect  152  Chapter^VI^  CONFORMATION OF ELASTINj. SCANNING ELECTRON MICROSCOPY^  A. I n t r o d u c t i o n In  the  microscopic  previous  and  p r o t e i n . . Having  the  chapter  I  mclecular  have  examined  structure  of  the sub-  the  elastin  done so i t seems a p p r o p r i a t e a t t h i s time t c  e v a l u a t e the o r g a n i z a t i o n of e l a s t i n a t a l e v e l of order approaches  the  range  of  scanning  electron  that  miscroscope  t e c h n i q u e s , which should allow an  examination  sub-fibres  present. T h i s i s a f e a s i b l e  i f they  are i n f a c t  p r o p o s i t i o n s i n c e the procedures use  o f quick f r e e z i n g technigues  (water)  that c l o s e l y resembles  procedural artifacts. technigue  aspects The of  should  utilized in this  the 200nm  study  i n the presence  protect lies  electron  against in  the  make  of a s o l v e n t  the i n v i v c environment.  disadvantage  scanning  of  These  organizational fact  microscopy  that  only  the  allows  v i s u a l i z a t i o n of the surface t e x t u r e , which can e a s i l y  a  lead t o  m i s - i n t e r p r e t a t i o n of the images. .  Ii Elastin,  from  Methods  unpurified  ligament,  alkali  extracted  ligament,  and autoclaved ligament, was c u t with a sharp r a z o r  i n t o cubes  ( l a r g e s t dimension  distilled The by  less  than  1mm)  and  left  in  water at 4°c o v e r n i g h t . p r e p a r a t i o n f o r scanning e l e c t r o n microscopy  was done  dropping the hydrated p i e c e s o f t i s s u e i n t o a butanol  bath  153  that was cooled fractured  in liguid  while  freeze dryer  frozen.  A l l samples  were  samples  were  dehydrated i n a  (operating a t -50°C) and mounted onto stubs  conducting  silver  in  ,  vacuo  n i t r o g e n . Some of the  p a i n t . These stubs  with  were subsequently  a t h i n l a y e r of gold approximately  with  coated, 100A° i n  thickness. The  specimens  Company, voltage film,  were  Stereoscan  viewed  microscope,  of 50kv. The images and  the  in  negatives  at  were were  a  Cambridge  Instrument  a filament accelerating  recorded  on  preserved  Polaroid  655  by treatment  with  sodium s u l p h i t e f o r one hour.  C_ R e s u l t s Preparations indication  of  of u n p u r i f i e d ligament e l a s t i n  a  'fibrillar'  fibre  that i s thought t o occur  failed  indicating  i t i s probably  that  the p e r i p h e r y  the the c o l l a g e n  around the i n d i v i d u a l e l a s t i n  the e l a s t i n  support  such  of  sub-structure  the s u r f a c e as a granular  an  organization,  a f e a t u r e t h a t i s confined t o  fibres. f i b r e s r e s u l t e d i n the removal o f  'surface c o a t i n g ' r e v e a l i n g what appears  indication  for  to  of the e l a s t i n  Autoclaving  at  which  ( F i n l a y and Steven 1973). The f r a c t u r e s u r f a c e s of these  f i b r e s , however,  the  structures,  diameters of 80 t c 100nm, could represent  sheath  some  arrangement when viewed i n the  l o n g i t u d i n a l d i r e c t i o n ( f i g u r e 6.1)..These had  showed  to  be  a  clear  ( f i g u r e 6.2), t h a t i s v i s u a l i z e d appearance with r i d g e s thrown  in  good measure. But again, c r o s s - s e c t i o n a l views of the same  154  F i g u r e . 6._1i S.E.M. Of unpurified fibre_ (E) E l a s t i n . (CS) Collagen sheath. The bar r e p r e s e n t s 10um,  ligament  elastin  155  156  preparations  f a i l e d t c show any s u b - s t r u c t u r e .  A l k a l i extracted structure,  with  200nm apart,  specimens showed a very  40nm  fibres  surface  t h a t were spaced approximately  s i m i l a r t c these observed f o r a r t e r i a l e l a s t i n by  Carnes e t ^ a l . .  (1977)  ( f i g u r e 6.3a).  Higher  the f r a c t u r e s u r f a c e s showed a very any  distinct  magnification  of  smooth appearance, without  i n d i c a t i o n of i n t e r n a l s t r u c t u r e  ( f i g u r e 6.3b).  M>. D i s c u s s i o n Almost dealing  a l l of  the  s t u d i e s reported i n the l i t e r a t u r e ,  with the scanning e l e c t r o n u i c r c s c o p y  supported the 200nm protein.  In  sub-fibre  objection  to  arrangement  this  of e l a s t i n , f o r the  have  elastin  p o i n t of view, i t should have  looked  be  stated that  most of these i n v e s t i g a t o r s  at the  appearance  of the f i b r e s i n the l o n g i t u d i n a l d i r e c t i o n . As i s  shown by t h i s study, t h i s approach to the problem can be leading and  since  inherent  i t cannot d i s t i n g u i s h between surface  organization..The  the c r o s s - s e c t i o n a l view of  mis-  features  few papers that have d e a l t with the  elastin  fibres  (Minns  and  Stevens 1974) are i n agreement with the f i n d i n g s of t h i s study that  there  is  nc  indication  i n t e r e s t i n g t o note that observed  in  the  these  of  sub-fibres  transmission  electron  f o l l o w i n g are a few mere aspects of argue a g a i n s t Since  sub-structure.  the  have  It i s  never  microscope.  results  which  been The also  the presence of these 200nm s u b - f i b r e s .  the  fracture  propagation of a crack  of  a  substance  through the  material,  results and  from the since  the  157  ___________ _______  Surface  texture  of  autoclaved  elastin  There is seme indication of s u b s t r u c t u r e as r e f l e c t e d i n the s u r f a c e t e x t u r e of these fibres. The bar represents 2um.  158  ___________  159  F iqure.6.3: F r a c t u r e s u r f a c e s of e l a s t i n f i b r e s . Alkali purified f i b x e s showing the 200nm spaces on the e l a s t i n f i b r e s . ..The f r a c t u r e surfaces, howver, are smooth and do not i n d i c a t e the presence of 200nm s u b - f i b r e s . The t a r represents 2um.  160  Figure.6.  16 1  path  of  crack  propagation f o l l o w s a route  l e a s t expenditure of energy, one properties  of  a  wculd  that reguires  expect  the  fracture  homogeneous substance, such as g l a s s , to  n o t i c e a b l y d i f f e r e n t from a composite m a t e r i a l , such as g l a s s which i s made up of g l a s s f i b r e s embedded  in  the  be  fibre-  an  epoxy  matrix. Applying elastin, 90°C)  one  to  this  analogy  to  the  fracture  properties  would expect a •homogeneous' e l a s t i n f i b r e  fracture  in  a  smooth  manner  similar  to  m a t e r i a l s . I f , however, these s u b - f i b r e s do e x i s t the could  be represented  protein  (2C0nm)  of t h i s m a t e r i a l  •splayed' As  fracture  (at -90°C) should  give r e s u l t s s i m i l a r to  these  200nm  some  of  sub-fibres  appearance cf the f r a c t u r e  fibrils.  It  is  embedding matrix m a t e r i a l has  the  the  cracks  resulting in a  surface.  mentioned before, the f r a c t u r e s u r f a c e s  identical  material  embedded i n a cementing matrix. The  between  'splayed'  glassy  elastin  f i b r e s were always smooth, with no evidence f o r of  (at  as a composite c o n s i s t i n g of the  f r a c t u r e of f i b r e - g l a s s m a t e r i a l s , with propagating  of  possible  of the the  elastin presence  though,  that  the  mechanical p r o p e r t i e s  that  are  to the p r c t e i n f i b r e s as well as being s t r u c t u r a l l y  continuous with these s u b - f i b r e s . In which case, the  material  would f r a c t u r e i n a homogeneous manner. This  leaves  me  with one  presence of these 200nm surface The state  r e p o r t s that have  that  they  last  p o i n t : how  to e x p l a i n  the  texture.  observed  these  sub-fibres  often  are only seen a f t e r the p u r i f i c a t i o n of  the  162  protein  {Hart ______  1978). On the b a s i s of t h i s comment i t i s  i n t e r e s t i n g to speculate-that artifact during would  caused  by  the  the  200nm  structures  removal c f the g l y c o p r o t e i n  p u r i f i c a t i o n , and a c t u a l l y represent  the  r e s u l t from t h e i r removal. T h i s e x p l a n a t i o n  because  the  glycoproteins  do  microscope  Having  (larenbach  presented  the s u b - f i b r e s , I would proper  perspective  the  being  still  not  a typical  enough  to  however, properties  ______  i s plausible  transmission  1966).  arguments a g a i n s t the presence o f  l i k e to put the controversy  into i t ' s  by s t a t i n g that i t i s g u i t e i r r e l e v a n t . I f  be  elastin  fibre,  they  i n c o n t r a d i c t i o n t o the idea of e l a s t i n  k i n e t i c elastomer, s i n c e t h e i r s i z e  is  large  accomodate random c o i l s of p r o t e i n . T h e i r presence  would of  alter  the  elastic  interpretations tissue  since  of  they  i n t r o d u c t i o n of another a r c h i t e c t u r a l f a c t o r composite.  that  i n f a c t occur i n 100 t o 200nm  they are i n f a c t r e a l a t t r i b u t e s of the would  fibrils  spaces  bundles around the e l a s t i n f i b r e , as seen i n the electron  are an  in  the would the  failure be  an  network  163  Chapter.VII. ELASTIN AS  A_o_ In  the  previous  Introduction  c h a p t e r s I have attempted t o  examine the c o n f o r m a t i o n a l reason  for  use of the basis  a  the  conditions But  (in f u t i l i t y ? )  elastomer,  do these t e s t s on  a  number  single  fibre  properties using  are  studies.  The  more  has  fibres.  convenient  'architecture'  This study was the  from  architectural  interpretation  which was  the  interference  cf  attempted  relationships.  all  fibres?  off  Hence  the  the  Aside there  been based on this  form  experiment,  a  using  is  sample  purpose of by  studying  various  it's sub-  properties material.  getting  elastin fibres.  the  without  with  p r o p e r t i e s of the  the p r o p e r t i e s at the by  experiments  Although of  the  elastomeric  to d i s t i n g u i s h the  undertaken with the  p r o p e r t i e s of s i n g l e , 6 to 8um, an  that  elastin  analysis  s t r u c t u r e w i l l make i t d i f f i c u l t the  elastin  met.  disadvantage l i e s i n the f a c t that using  of  of  This  p h i l o s o p h i c a l arguments,  of e l a s t i n upto now,  the  adeguate  of p r a c t i c a l reasons f o r s u f f e r i n g through  bundles of e l a s t i n  guestion  assuming  single  from i r r e l e v a n t e x i s t e n t i a l and are  The  t o v a l i d a t e the  with the e v a l u a t i o n  of the k i n e t i c theory  why  protein.  p r o p e r t i e s of t h i s p r o t e i n .  expressly  kinetic  was  rigourously  of rubber e l a s t i c i t y as an  elastomeric  then, deals typical  s t a t e of the e l a s t i n  exercise  k i n e t i c theory  for  chapter, as  that  A KINETIC ELASTOMER.  the  around physical  This  allowed  molecular  level,  kinetic  theory  the major product of t h i s study was  the  164  evaluation apply  of the  various k i n e t i c theory  to the e l a s t i n Furthermore,  parameters,  elastin  s i n c e the f i b r e s i n the e l a s t i n bundles  extension).  is s t i l l  one  be  •Gaussian  extension,  and  1  shown  in  at low this  strain chapter,  r e g i o n of i t ' s macroscopic  t h e r e f o r e , bundle s t u d i e s  a n a l y s i s of the non-Gaussian p r o p e r t i e s of  the  network. i f one  It  is  these  information  do is  polymer  properties  i s to e x t r a p o l a t e  that  need to  be  from the e l a s t i n system to  p r o t e i n s i n the random conformation. L a s t l y , the p h o t c - e l a s t i c  experimental  setup  utilize  experiments single  e f f e c t s , that  masks  the  properties  demand  of  t o the  the  The  K i n e t i c ______  J i l l Gaussian chain In  trying  to  the  Elasticity  entropy  s t a t i s t i c a l mechanics of  random c o i l e d molecules i t i s convenient to represent the  chain,  c h a r a c t e r i z e the quantity  termed  a,  to  be  fixed  at  the  origin,  'random walk' to the other end, the end-to-end d i s t a n c e  p r o b a b i l i t y density  fibres,  impossible.  of ______  s t a t i s t i c s a_d  understand  the  interfacial  single  making the e v a l u a t i o n of the bundle p r o p e r t i e s  _ _ t r o a _ ___________  that  f i b r e s , s i n c e bundles of  f i b r e s have a large form b i r e f r i n g e n c e , due  of  are  that  by the  other  will  to examine the a d d i t i o n a l  realized  evaluated,  As  i n the  p r o p e r t i e s , at 50% not allow  they  protein.  not c o n t i n u o u s , p u r i f i e d bundles of e l a s t i n f a i l (about 50%  as  and  end to  via  the  r, (figure 7 . 1 ) .  The  f u n c t i o n , of f i n d i n g b i n the  b,  one  vicinity  of  165  point be  P  w i t h i n the  represented  values)  forms the  The  placing  the  the  of the  of the  of  chain  of  end,  function i s described 3  TT3/2)  where p [ x , y , z ] r e p r e s e n t s end  b a distance, r  b i s defined  the  the  2  2  eguation is  7*1,  similar  saying the  Since  (Treloar  maximum  when  constant.  is As  If  then  7. 1 of f i n d i n g a.  the  chain  The  term  7.2  the  links The  f u n c t i o n has  b,  entropy  i n the  important a  of  the  being  at  the  the  and  chain  1 is  result  maximum a t r=0,  most  of  chain  1975)  the  entropy  of  which  conformations origin  are  (no  pun  i s proportional  number o f c o n f o r m a t i o n s a v a i l a b l e t o  whose p o i n t s  c f the  are  c h a i n , S,  i s a l s o s e e n t o be  an  arbitrary  i s evident  from  seperated  i s given 2  c  egually probable,  2  2  link.  S=c-kb r where  the  to the  at  a  r=0.  a chain  entropy  allow  volume e l e m e n t .  2  that  end,  of the  system  the  random  i s t h a t the  with  logarithm  For  the  ( T r e c l a r 1975): 2  number o f random  each  tc  intended). the  the  of  consistent  that  exp ( - b r )  2  r  by:  is  length  by  of  d e p e n d s on  ( r = x + y + z ) , away f r o m e n d ,  2  s  a given  probability  b = (3/2)sl where  probabilities  within  also  theory r e l a t i o n s h i p s .  a l l conformations are  p [ x , y , z ]= ( b /  function  conformations  b,  (which can  density  kinetic  these  number  assumes t h a t density  probability  b a s i s of t h e  derivation  evaluation  one  as  volume e l e m e n t [ d x , d y , d z ]  2  by  by  a distance,  (Treolar  r,  1975):  7. 3  constant, eguation  and 7.3,  k i s the the  Boltzmann  imposition  of  166  F i g u r e _ 7 . 1 : The r a n d o m - c o i l e d c h a i n The random c h a i n w i t h end a a t t h e o r i g i n and end b at a point P(x,y,z) within a volume element _dx,dy,dz2.  167  F i g u r e . 7.1.  168  s t r a i n on t h i s system of  the  w i l l i n c r e a s e r, r e s u l t i n g  c c n f i g u r a t i o n a l entropy of the system. T h i s decrease,  i n e n t r o p y , forms kinetic  the  basis  for  the  retractive  elastomers. The i n t e r n a l energy  and does not c o n t r i b u t e to the e l a s t i c  Jb_ The e l a s t i c Any on  i n a decrease  force  term remains  of  constant  properties.  _______  molecular i n t e r p r e t a t i o n of a rubber  polymer,  based  the k i n e t i c theory of rubber e l a s t i c i t y , assumes a network  made up of a  three  dimensional  array  of  idealized  random  c h a i n s . The c h a i n s are c r o s s - l i n k e d at various p o i n t s ( t h i s i s necessary  if  the  network i s to maintain i t ' s s t r u c t u r a l  mechanical i n t e g r i t y characterize  the  when  chain  strained) between Mc,  and  one  cross-links  and c o n s i s t i n g  can  having  an  s  number  of  molecular weight  random  l i n k s each of l e n g t h 1. In the case of r e a l  are  restricted  by  valence  s t e r i c h i n d r a n c e s . One  of  cccur s i n c e bond  angles,  therefore  as  average  however, i d e a l random l i n k s cannot  and  molecules, movements  potential barriers,  and  t h e r e f o r e has to invoke the concept  of  a ' f u n c t i o n a l ' random l i n k . T h i s l i n k w i l l c o n s i s t of a number of  bonds,  which as a u n i t , appear to s a t i s f y the  reguirements  cf the  ' i d e a l ' random  As s t a t e d b e f o r e , imposing decrease  the  configurational  link.  a s t r a i n on t h i s network entropy,  r e t r a c t i v e f o r c e . The b a s i c assumptions which are d i s c u s s e d i n d e t a i l  statistical  by  giving  rise  will t o the  of the k i n e t i c t h e o r y ,  Treolar  (1975)  (1 953), are as f e l l o w s : 1. The network c o n t a i n s N chains per u n i t  and volume.  Flory  169  These c h a i n s , which were d e f i n e d above, are held i n a network by a few s t a b l e c r o s s - l i n k s . 2. The mean square end-to-end d i s t a n c e f o r the whole assembly of chains in the u n s t r a i n e d s t a t e i s the same as f o r a corresponding set of f r e e c h a i n s . 3. Deformation does not lead to a change i n volume. 4. The components of length of each chain change i n the same r a t i c as the corresponding dimensions of the bulk rubber. 5. The entropy of the network i s the sum of the e n t r o p i e s of the i n d i v i d u a l c h a i n s .  J c _ Mechanical p r o p e r t i e s of k i n e t i c Assuming  an  ideal  Gaussian  rubbers  system, the k i n e t i c  theory  makes i t p o s s i b l e t c c h a r a c t e r i z e the t e n s i o n a l e l a s t i c f o r c e , r e s u l t i n g from the s t r a i n induced  f r e e energy change (egual t o  the entropy  rubber),  eguations  change f o r an  ideal  by  the  following  ( T r e o l a r 1975): f=NkT ( > - > ~ 2 )  7.4  T=NkT( > 2 - >-*)  7.5  and,  where  f  is  the  nominal s t r e s s  (force/unit unstrained  s e c t i o n a l area),  i s the t r u e  stress  cross  area),  is  sectional  (1.38  X 10~  the  number  and  >  to  the  erg K - ) , 1  the  T i s the a b s o l u t e  of random chains per  i s the  unstrained In  16  k  extension  ratio  cross  (force/unit  strained  Boltzmann  constant  temperature,  N  is  u n i t volume of the m a t e r i a l , expressed  in  units  of  the  length.  the case of swollen prctein  elastomers,  rubbers, a  a state that i s relevant  volume  fraction  i n c o r p o r a t e d to g i v e : f=NkTv_-/ ( > - > ) 3  -2  7.6  term  is  170  and, 0- = N k T v V { X 3  7.7  2  2  where  v  is  z  the  unswollen  r e f e r to the f o r c e per area, and  f o r c e per  volume/swollen volume.  u n i t swollen u n s t r a i n e d  f and  cr  cross-sectional  unit swollen s t r a i n e d c r o s s - s e c t i o n a l  area  these  that  respectively. All  four  characterizes  of the  eguations  stiffness  of  c a l l e d the e l a s t i c modulus, G,  the  assumption linking  5),  and  density,  attaching  which  #c  seems  intuitively  is  molecular  N is itself  for  the  the  weight  M  number  N  (based  r e l a t e d to the of  one  on  cross-  cross-link  7. S  of c r o s s - l i n k s per  possible between  (1/2)#c),  to  obtain  cross-links  from the  This i s r e a l i z e d i n the  i s the  following  polymer  measure  (which  of  for  the  a  to the number of  given cross-  relationship: 7.10  density,  (0.082 X 10- m atm m o l - i K 3  a  unit volume. I t  value cf the e l a s t i c modulus G,  G=Nkt= f ET/Mc  constant  to:  case  composition i s i n v e r s e l y p r o p o r t i o n a l  p  tested,  four chains i s approximately egual t c :  where  where  being  number of chains,  N=2 (#c)  links,  term  7.8  to the  since  a  material  according  G=NkT Since G i s p r o p o r t i o n a l  define  3  - 1  B  is  the  ),  and  Mc  universal i s the  gas  molecular  weight between c r o s s - l i n k s . Water equilibrium  swollen with  elastin, a  being  hydrophillic  a hydrophobic p r o t e i n i n solvent,  presents  some  17 1  problems when one  t r i e s to i n t e r p r e t i t ' s p r o p e r t i e s  above r e l a t i o n s h i p s since contrary  to the  function  cf  Hoeve and  Flory  Smith  i t ' s eguilibrium  assumption of the  both  using  degree of  the  swelling,  k i n e t i c theory, changes as a  temperature  and  elongation  1976). Oplatka e t . a l .  (1960)  (Gosline  and  1978,  Bashaw  and  (1968) extended the .kinetic theory to account f o r these  c o m p o s i t i o n a l changes that take place Mistrali  e t^al....  (1971) p r o v i d i n g  i t ' s a p p l i c a t i o n to they are  elastin.  measurements  critical  appraisal  were  the  These  mentioned here, were not  physical  in  systems,  preliminary  although  rigourously  accurate  with  support f o r  relationships,  tested  not  open  since  the  enough to allow a  of such r e f i n e d d i s t i n c t i o n s .  (d) Photo e l a s t i c i t y The  k i n e t i c theory a l s o a f f o r d s a  strain-birefringence  where  and  parallel  solvent  (fi2 + 2)22T  L  to  are  H_«<,-o<J  the  (Treolar v^/  3  (\  the  1975):  2 -  >-i)/ii45  p o l a r i z a b i l i t i e s of  i t ' s length  and  7.11  the  random  normal to i t , r e s p e c t i v e l y ,  the average r e f r a c t i v e index of the as  for  behaviour of rubbery polymers swollen i n  an o p t i c a l l y n e u t r a l n„-n =  relationship  p r o t e i n , and  N is  link n is  defined  before. The  assumption  anisotropy  of the  alignment  of  remains  made by  t h i s theory that the  polymer when i t i s s t r a i n e d  arises  the random l i n k s , whose inherent  unchanged,  birefringence  is  is  in not  the  direction  thought  to  of  from  an  polarizability  the  represent  optical  strain. an  The  anisotropy  172  a r i s i n g from the displacement  of e l e c t r o n o r b i t a l s  (due to /the  a l t e r a t i o n of the i n t e r a t o m i c d i s t a n c e s ) , as i s the case  with  c r y s t a l l i n e or g l a s s y polymers. The  relationship  for  the  s t r e s s - b i r e f r i n g e n c e can  d e r i v e d from the above eguation by 7.7, and  in  equation  which d e s c r i b e s the r e l a t i o n s h i p between the true s t r e s s the e x t e n s i o n , i n t o equation 7.11 n  Since  n ~ _. n  CT(n +2) 22 TT  =  to  This  and n are presumed to incorporate  constant, C ,  to g i v e :  /n45kT = CTC  2  (ot\ - °_)  possible  all  7.12  remain  constant,  it  the parameters i n t o an  which i s termed the s t r e s s - o p t i c a l  should be i n v e r s e l y (<*|-°_)»  coeffecient.  link,  is  of the random l i n k , and  p r o p o r t i o n a l t o the temperature.  which  represents  theoretically  the  is  overall  c o e f f e c i e n t depends only on the mean r e f r a c t i v e index  the polymer and the p c l a r i z a b i l i t y  of  substituting  be  The  of it  value  a n i s o t r o p y of the random  independent  of  strain,  Hence i t ' s constancy  swelling,  s t r e s s , and  temperature.  over a number of  parameters  could be used as an i n d i c a t o r of the peptide back-  bone s t a b i l i t y .  Je_ Non-Gaussian e f f e c t s and the e v a l u a t i o n of s Since  the  mechanical  and  i s the r e s u l t l i n k s , and intuitively  non-Gaussian  effects  cbserved  for  p h o t o e l a s t i c p r o p e r t i e s of the e l a s t i c  the  network  of the f i n i t e l e n g t h of the c h a i n between c r o s s -  hence a f i n i t e obvious  number of random l i n k s , s. I t  that  one  •backwards' and e v a l u a t e the value  should 's' by  be  able  analysis  to of  seems work these  173  non-Gaussian p r o p e r t i e s . The  Gaussian  relationships  that the end-to-end d i s t a n c e , r , fully  mentioned is  upto now,  small  extended l e n g t h of the chains  compared  (r/sl«1).  assume to  the  T h i s assumption  r e s t r i c t s t h e i r a p p l i c a t i o n t o small s t r a i n s , and n e c e s s i t a t e s the  use  higher  of  the  more accurate non-Gaussian r e l a t i o n s h i p s at  l e v e l s of e x t e n s i o n . For the  case  of  the  mechanical  data, the r e l a t i o n s h i p takes the form of a s e r i e s expansion which the f i r s t  f i v e terms are  ( T r e o l a r 1975) :  f=Nkt ( X - X ~ ) [ 1+ (3/25s) (3 > 2  2  + 4 / X ) + (297/6125s ) (5 X * + 2  8 % + 8/ X ) + (123 12/2 20 5000s ) (3 5 X 2  8 +  5  ] data,  u  (p P ) -  j  Z  i s d e f i n e d according t o : 2  2  2  +  ( V 1 5 0 s ) (6 X * + 2 X - 8 / x ) + 2  (1/35Cs ) (10 X + 6 X " 1 6 / x 6  2  3  Comparison of the e x p e r i m e n t a l l y and  phctoelastic  r e l a t i o n s h i p s should  and  the  )  7.15  obtained curves  properties)  (for the  with  data to the unswollen  p r o t e i n elastomers  E l a s t i n , exhibit  3  these  allow an e v a l u a t i o n of s.  l i L I n d u c t i o n of e l a s t i n Since  non-Gaussian  7. 14  z  (P, - P ) = N ( * . - * ) [ (1/5) X - ( 1 / X )  mechanical  the  ( T r e o l a r 1975):  o -n^= (r\?+2) *4-rr (P, -P )/3fi where  2  7.13  stress-optical  r e l a t i o n s h i p takes the form  3  3  1 1 2 0 X + 1 440 > + ( 1 5 3 6 / x ) +  128C/ X *) + the  + 6 0 X + 7 2+6 4/ x ) +  6  3  (1261 1 7/693 (673750)s* (630 X  For  of  their  such as E e s i l i n ,  elastomeric  behaviour  form Abductin, only  when  174  they are swollen by a d i l u e n t little  irrelevant  to  (such as water), i t might seem a  attempt  the  obtained f o r the s w o l l e n form to an  reduction  of  the  'unswollen' s t a t e .  data  This i s  n e c e s s a r y , however, i f cne i s make a v a l i d comparison  between  the  systems  theoretical  mentioned  relationships  i n the l a s t  section  fcr (which  unswollen network) and the e l a s t i n The  data  for  elastin  the non-Gaussian derived  for  the  according  to  the  data.  was  r e l a t i o n s h i p s provided by F l o r y  are  reduced  (1953):  f o = f / v / / 3 . ... . . 7. 16 s  X °= > V v V  3  7.17  q-°=g-Vv_2/3  7. 18  2  ( ^ - n j o=(n -n )/v|/3 ||  where the s u p e r s c r i p t s swollen  forms  of  0  7.19  1  and s r e f e r t c  the  'unswollen'  the designated terms, which  are d e f i n e d  and as  before.  C. M a t e r i a l s and Method  Jaj_ P u r i f i c a t i o n Bovine obtained  ligamentum  of  mature  beef  cattle  was  from a l o c a l s l a u g h t e r house and p u r i f i e d by repated  autoclaving i n d i s t i l l e d Partridge  et__al_,  per  water  (1S55).  •cooking' the e l a s t i n pounds  nuchae  of e l a s t i n  i n an  according  Briefly,  this  autoclave  to  the  method  procedure  operating  at  of  involves fifteen  sguare i n c h f o r a p e r i o d of an hour. T h i s removes  the a s s o c i a t e d p r o t e i n a c e o u s m a t e r i a l , such  as  the  collagen  175  and  the  matrix  substances,  elastin  p r o t e i n . The  fresh  changes  preparation. indefinite  behind  the  insoluble  procedure i s repeated 6 to 8 times,  of  distilled  Tissue period  leaving  thus of  water,  prepared  time  with  to  can  be  ensure  a  stored  with clean  for  an  o c c a s i o n a l s t e r i l z a t i o n by  a u t o c l a v i n g . The e v a l u a t i o n of t h i s p u r i f i c a t i o n  procedure  has  been presented i n chapter 2.  _b_ The along  major piece of  with  rod,  the  experiment,  p o s i t i o n s of the v a r i o u s components, was  assembled  from  a  5mm  microforce  diameter  transducer,  was  Corning high vaccum grease was slide  and  the  during the experiment desired the one  p c i n t . The  stage  obtained.  a p p l i e d between the movable  t o smooth out the  manipulations  temperature  c o n t r o l system  was  at  the  the same as  5.  in  of  dried dimension)  purified was  elastin  mounted  (approximately  directly  onto  experimental stage by anchoring one end t o the f l e x i b l e rod  by  P r e p a r a t i o n of e x p e r i c e n t a l specimen  strip  X 5mm  made  and to keep the e x t e n s i o n s f i x e d  d e s c r i b e d i n chapter  Jc_ A  glass  s o l i d . g l a s s rod ever a bunsen burner  u n t i l the d e s i r e d diameter of 80 to lOOum was.  glass  2mm  in  glued together with epoxy adhesive. The f l e x i b l e g l a s s  pulling  Dow  used  7.2..The stage  which served as a  flame  apparatus  the r e l a t i v e  i s shown i n f i g u r e slides  The experimental stacje  and the other end to the movable g l a s s s l i d e  the glass  using rubber  176  F D e f s m e f d f  i i x i l e l i e i  g u r e . 7 . 2 : The e x p e r i m e n t a l ______ agram o f t h e e x p e r i m e n t a l s t a g e used f o t e n s i o n , a n d r e t a r d a t i o n measurements. The b r e , E, was extended by m a n i p u l a t i n g t h e i d e , S, a n d t h e e x t e n s i o n was m o n i t o a s u r i n g t h e d i s t a n c e between two landmarks a s t i n f i b r e . The f o r c e e x p e r i e n c e d by t h e b r e was c a l c u l a t e d from t h e measurement f l e c t i o n , d, o f t h e g l a s s r o d , G, r e l a t i x e d marker, M.  r f o r c e , e l a s t i n movable r e d by on t h e e l a s t i n o f t h e v e t o a  177  178  cement. D i s t i l l e d  water was  the e l a s t i n  f i b r e s teased  with  forceps,  fine  then added to the p r e p a r a t i o n ,  away, under a d i s s e c t i n g  u n t i l a s i n g l e connecting  i n t a c t between the g l a s s rod and  the  microscope  f i b r e was  movable  and  slide  left  (figure  7.2).. The  p r e p a r a t i o n was  on a Wild  M21  conducted  then mounted between crossed  p o l a r i z i n g microscope, and  by the manipulation  m a g n i f i c a t i o n . The shifting  the  extension,  f i b r e was  movable  the  experiment  was  of the mounted specimen at 300X  strained in a  glass  polars,  slide,  random  and  steps,  by  the values f o r the  f o r c e , and r e t a r d a t i o n , were obtained  as  described  noted i n the  unstrained  below.  (d) Measurement cf s t r a i n Two  distinguishing  s t a t e and Filar  extension  represented the i n i t i a l  step,  moving  the  elastin  fibre  fixed  marker  separating and  the  calibrated  d i s t a n c e was  elongation  using  a  evaluated  at  could  then  be  ratio  of  done  by  f o r c e on  the  length.  manipulation  deflection  was  as an absolute v a l u e , c r as an e x t e n s i o n  _e_ The  were  the d i s t a n c e between them  micrometer.. The  each  marks  glass  Measurement of f o r c e of the e l a s t i n f i b r e , which was  slide,  which c c u l d  of  the  exerted be  flexible  ( f i g u r e 7.2),  an extending  guantitated  by  measuring  g l a s s rod with r e f e r e n c e to  using  a  Filar  the the  micrometer.. T h i s  179  value  could  utilizing  then  be  converted  t o an absolute f o r c e , F, by  the equation f o r the bending of a beam: F=d (3EI/13)  7.20  Where d i s the d e f l e c t i o n of t h e g l a s s fibre  the  the  second moment of area, which f o r a rod of c i r c u l a r of r a d i u s ,  (usually around  1 i s the  of  section  glass  fibre,  value  obtained rod,  of  E,  1.3 t o 1.5 cm), and I i s  7.21  which i s the Young's modulus c f g l a s s , was  by measuring t h e d e f l e c t i o n of  when e x p e r i e n c i n g a bending f o r c e  weights. force  Substituting  i n t o eguation  solving  cross-  r , takes t h e form: I=TTr*/4  The  length  these  values  a  macroscopic  from s e v e r a l  glass  calibrated  f o r the d e f l e c t i o n and  7.20 along with the  dimension  data,  and  f o r E gave a value of 6.2 X 10*ONm- f o r the Young's 2  modulus of g l a s s . The  experimentally  represented unstrained  this  the  the  force nominal  area)  or  strained cross-sectional  could  then  stress the  area).  presence of erroneous values r e s u l t i n g  be  (force/unit true  stress  To i n s u r e from  the  against use o f  method, one of the m i c r o - f o r c e t r a n s d u c e r g l a s s rods was  calibrated the  either  cross-sectional  (force/unit the  as  obtained  by hanging known weights on i t ' s end and  deflections. deflections  These  predicted  Jf1 Calculation The  unstrained  measuring  d e f l e c t i o n s were always within  2% of  by use o f eguations 7.20 and 7.21..  cf c r o s s - s e c t i o n a l  diameters were  measured  area with  the  Filar  180  micrometer  and  the  c r o s s - s e c t i o n a l areas were c a l c u l a t e d  assuming a c i r c u l a r c r o s s - s e c t i o n . The for  the  length  strained  change by  fibres  were  c a l c u l a t e d from the  assuming  that  the  remained c o n s t a n t with  specimen on the  between crossed it's  long  polars  which along with  monochromatic l i g h t The extension  measured  the  fibres  birefringence  ( p o l a r i z e r at 0°, analyzer  orientated  at 90°)  with  p o l a r i z e r ( p o s i t i v e angles being  I l l u m i n a t i o n was an  of  p o l a r i z i n g microscope was  a x i s at +45° to the  counter-clockwise).  volume  areas  extension.  JLal Measurement of The  cress-sectional  by  interference  by a 100watt quartz lamp, filter  provided  a  green  (546nm) source.  retardations  were  measured,  measurement, at the center  after  each  of the f i b r e using  forcea  1/30  wavelength Z e i s s r o t a r y compensator. This value of  retardation  was  to  d i v i d e d by the  diameter  at  each  optical  path  extension  length  step)  to  (taken give  the  be  the  value f o r  birefringence.  Jh_ Since i t i s hard dealing  accordingly,  the  data  evaluate  with a system such as the  a statistical  with the  to  Errors  approach was the r e l e v a n t  the  one  errors  utilized  thought to be  more  involved  in  i n t h i s study, feasible,  and  s t a t i s t i c a l parameters are presented  r e s u l t s . In g e n e r a l , and  the  minimum  the  e x c e l l e n t r e p r o d u c i b i l i t y of  scatter,  is  taken to be a good  181  i n d i c a t o r of the systems  P h y s i c a l P r o p e r t i e s of S J J _ _ l e E l a s t i n F i b r e s  D_  Ja_ The  reliability.  mechanical  observed  to  be  General  characteristics  behaviour  of  completely  single  elastin  elastic  with  h y s t e r e s i s or change with r e p e t i t i v e c y c l i n g  fibres  no of  was  observable the  strain.  T y p i c a l l y , the f i b r e s c o u l d be elongated to 100%-200% of t h e i r initial  length  ( X = 2 t c 3) before breaking. The cause of the  f a i l u r e c o u l d not be determined s t a t e d that failure.  there  The  failure  breakage of the breaking  was  no  away  of  the  observable  itself  strained  with c e r t a i n t y . I t can only be  under  from  the  a  r e s i d u a l bundles  of  preparation.  The  tensile  s t r e n g t h i s probably i n the range of 10&Nm- .  il§chanica_ p r o p e r t i e s and the d e r i v a t i o n of Mc  mechanical  7.6  i n d i c a t e s t h a t i f the data obtained f o r the  p r o p e r t i e s of s i n g l e e l a s t i n  of nominal line  s t r e s s versus of  F i g u r e 7.3,a, shows elastin  the  2  Eguation  straight  anchoring  or  which were used  graph  before  observation,  elastin  IhL  for  flew  occured e i t h e r as a r e s u l t of a  portion fibre  plastic  (X-X  zero i n t e r c e p t the  and  mechanical  fibres in d i s t i l l e d  - 2  f i b r e s i s p l o t t e d as a  ),  it  should  yield  a slope egual to Gv^/ . 3  data  for  eight  (an  single  water at 24°C. I t i s evident t h a t  the r e l a t i o n s h i p obeys the p r e d i c t i o n of a s t r a i g h t l i n e }\ =2  a  upto  extension of 100%) .. Beyond t h i s e x t e n s i o n , however,  182  the  experimental p o i n t s deviate from the s t r a i g h t  as an upturn i n the graph. T h i s d e v i a t i o n non-Gaussian p r o p e r t i e s in  detail  later.  restricted  to  seen  i s the r e s u l t of the  of the network, which w i l l be analyzed  Fcr  the  line,  the  linear  moment,  the  portion  of  analysis the  will  be  stress-strain  relationship. The the  elastic  data  according  4.1 X 10 Nm- . 5  2  linear portion v^O.65 the  modulus, to  This  G, obtained from the treatment of  eguation  was  calculated  of the p l o t  (Gosline  from  modulus, and  has  from  a  value  the  of  slope of the  ( f i g u r e 7.3,a), using  a  value  of  1978). . S i m i l a r l y , the value of Mc, which i s  molecular weight c f the  evaluated  7.8,  eguation  ^ = 1.33.  chain  7.10,  between the  .This gave  a  cross-links,  value  value  of  of  the  7,100  was  elastic f o r the  molecular weight of the chain between c r o s s - l i n k s . This  use  of  eguation  7.10  assumes i d e a l t e t r a - f u n c t i o n a l an o v e r s i m p l i f i c a t i o n loose  ends  possible  etc.  cross-links. This i s  be  present  by  utilizing  eguation 7.10, according t c ( T r e l o a r G= f RT (1-2MC/M) /Mc  M  in  the  network. I t i s  f o r these i m p e r f e c t i o n s , assuming randomly  distributed cross-linking,  Where  obviously  s i n c e , i n r e a l i t y , i m p e r f e c t i o n s such as  will  tc correct  f o r the c a l c u l a t i o n of Mc  a  modification  1975): 7.22  i s the molecular weight c f the polymer before  cross-  linking.  T h i s r e l a t i o n s h i p cannot, i n the s t r i c t e s t sense,  applied  to  elastin  precursor protein,  since  the  of  cross-linking  t r o p o e l a s t i n , are not randomly  sites  be  on the  distributed  183  r_±2ii£!__Z___ P h y s i c a l __o__rtj.es of s i n g l e e l a s t i n _______. ,(A) Graph of the f o r c e e x t e n s i o n data f o r s i n g l e e l a s t i n f i b r e s plotted according to equation 7.6. The l i n e a r r e g r e s s i o n , through the f i r s t 30 p o i n t s , has a c o r r e l a t i o n c o e f f e c i e n t r=0.91.. (B) Graph of the B i r e f r i n g e n c e - s t r e s s data f o r single e l a s t i n f i b r e s p l o t t e d according to eguation 7.12. The l i n e a r r e g r e s s i o n , through the f i r s t 30 p o i n t s , has a c o r r e l a t i o n c o e f f e c i e n t r=0.88.  1814  185  but,  rather,  molecule  cccur i n d i s t i n c t l o c a t i o n s on the t r o p o e l a s t i n  (Sandberg  eguation  7.22  et^al..  should  1972).  Nevertheless,  give an estimate of the lower  Mc. U t i l i z i n g the value obtained above, of 7100 eguation  use  of  l i m i t of  f o r Mc,  and  7.22, along with a value of 72,000 f o r the molecular  weight of the p r e c u r s o r t r o p o e l a s t i n corrected  molecular  weight  of  (Sandberg  6000  1976), g i v e s  range  of  7100  to  6000  agreement with the expected biochemical  composition  insoluble elastin  According t o  value  in  g/mcle. T h i s r e s u l t i s i n good  the  (Gray e t . a l .  lc)  Mc  values c a l c u l a t e d from  and  a  f o r the chain between  c r o s s l i n k s . Hence the data i s c o n s i s t e n t with a the  the  the  cross-linking  known  p r o f i l e s of  1973) .  Photoelasticity  eguation  7. 12,  a  plot  of b i r e f r i n g e n c e  versus s t r e s s , should give a s t r a i g h t l i n e of slope C .  Figure  7.3,b, r e p r e s e n t s the data f o r e i g h t s i n g l e e l a s t i c f i b r e s . As with  the  relationship deviates  can  relationship,  the  i s followed t o a c e r t a i n p o i n t ,  towards  approaching theory  stress-strain  the  stress  i t ' s ultimate again  be  axis,  value.  with  This  theoretical  after  which i t  the b i r e f r i n g e n c e  departure  from  the  e x p l a i n e d i n terms of the non-Gaussian  p r o p e r t i e s of the e l a s t i n network: the f i n i t e  length  of  the  c h a i n between c r o s s - l i n k s i s r e s p o n s i b l e f c r t h i s non-Gaussian shift,  with  the  assymptotic  l i m i t r e p r e s e n t i n g the s t a t e of  's' l i n k s i n f u l l alignment.  The. e x i s t e n c e  causal  by the o b s e r v a t i o n that both the  element, i s supported  of  this  common  186  T a b l e . 7 . 1 : K i n e t i c theory parameters f o r e-lasti  cn  o o  n  CM  E  o  o  r-H  o o  CO  o  E  O  n i—i  X  X  o —t  cvj  CO  I  t-  ID  c  —- U —-  o  4O  0  E CU  4-  o  sU  c  01 + J QJ -C 0) S- O ) 2 CU •— 4->  ft3 U •— +-» •M C C L CU O -  to  M  to  >—  ra  CU 4CU  s- o  *J  cu» x:  o o  f—  187  stress-strain  graph  birefringence region  graph  (figure  7.3,a) 7.3 b)  7. 3,b),  and  enter  r  the  stress-  the non-Gaussian  (X=2).  at the same l e v e l cf e x t e n s i o n  Concentrating  of  (figure  on the l i n e a r p o r t i o n of t h i s graph  the slope  (figure  of t h i s r e l a t i o n s h i p y i e l d s a value  1.0 X lO-^mZN- . U t i l i z i n g t h i s value of C 1  f o r C'  and a value o f  n=1.55, i t i s p o s s i b l e to s o l v e f o r the p o l a r i z a b i l i t y of the random  link  i^r^z) °  Doing  this  one  2.84 x 1 0 - O m 3 f o r the p o l a r i z a b i l i t y  obtains  of the  3  a  value of  random  link  at  24°C. The value c a l c u l a t e d f o r the s t r e s s - o p t i c a l c o e f f e c i e n t , C , i s comparable to the r e s u l t s obtained  by Weis-Fogh  for  (1.0 X 10- m N  1  the  i n v e r t e b r a t e elastomer, r e s i l i n  e l a s t i n as compared to 1.1 X 10- m N 9  not  surprising  since  they  2  _l  link.  _l  for  This  is  9  for resilin).  2  are both p r o t e i n elastomers and  would, t h e r e f o r e , be expected t o have s i m i l a r values average  (196 1b)  r e f r a c t i v e index and the p o l a r i z a b i l i t y  The k i n e t i c theory parameters d e r i v e d  f o r the  of the random  f o r elastin  are  summarized i n f i g u r e 7. 1. . It  should  be  mentioned  birefringence extrapolate stress.  This  unstrained  has  birefringence  of  of  been  d i f f e r e n c e at  from z e r o  with  single elastin fibres,  birefringence  the  the data  for elastin  to a positive birefringence  i s consistent  residual birefringence  index  that  t h e r e s u l t s obtained f o r  2.4 X 10~*. T h i s to  the unstressed 1980)..  residual  a r i s e from the r e f r a c t i v e  water/fibre  (Aaron and Gosline  zero  which appear t o have a s m a l l  about  shown  at  interface.  The  "true"  fibre i s indistinguishable  138  Jdj_ Temperature dependence of the o p t i c a l Eguation  7.12  coeffecient, absolute index  C,  alsc  states  should  be  that  and  the  polarizability  unchanged. F i g u r e results  7.4,a,  f o r the  the  inversely  temperature, T, assuming  shows  the  the  the  are  random  t o the  refractive link  remain obtained  temperature dependence of C*. I t seems t h a t  followed  (figure  qualitatively  but  7.4,a,  the  temperature i s l a r g e r than that which would eguaticn  mean  experimentally  the p r e d i c t i o n s of the k i n e t i c theory line)  stress-optical  proportional  that of  anisotropy  decrease  be  with  predicted  by  7.12.  There are two p o s s i b l e sources f o r t h i s d e v i a t i o n . since  dashed  elastin  temperature expected  decreases  (Gosline  1978),  to i n c r e a s e  t h i s increase refractive optical  should  coeffecient.  polarizability  of  the  mean  with  refractive  increasing index  is  with temperature. However, the extent of  i s small,  index  i t ' s volume  First,  and  any  increase  Second, the  in  random  case  the  i t is  increasing  value  of the s t r e s s -  possible  link  the  i°<^-°<2)  that  the  changes  with  temperature, which could account f o r the l a r g e decrease i n C . Figure  7.4,b  polarizability unchanged  with  polarizability 30%  (open of  circles), the  random  temperature. of  shows link It  the  value  for  the  assuming t h a t n remains is  evident  that  the  the random l i n k decreases by approximately  over the temperature range  t h i s change i s discussed  below.  studied.  The  implication  of  189  Figure.7_4: Temperature dependence of photoelasticity. (A) Graph of the s t r e s s - o p t i c a l coeffecient, C , versus 1/T. ( f i l l e d c i r c l e s ) : experimental points. The l i n e a r r e g r e s s i o n has a c o r r e l a t i o n coeffecient of r = 0 . 9 2 . (dashed l i n e ) : expected r e l a t i o n s h i p f o r the temperature dependence of C* calculated from eguation 7 . 1 2 . (E) (open c i r c l e s ) : dependence of the random l i n k a n i s o t r o p y on temperature. (filled circles): the same data p l o t t e d as an Arrhenius r e l a t i o n s h i p a c c o r d i n g to eguation 7 . 2 3 . . The activation energy calculated from the slope o f the l i n e a r r e g r e s s i o n ( r = C 9 4 ) has a value of 1 . 6 kcal/mole. .  190  Figure.7.U.  2 3.0  12  3.4  3.5  10>K  6  —  1  —  3.0  1  1  i  3.4 3.6 3.8  12  ia /°K 3  191  As  pointed  out  c h a i n s , the presence implies  that  e x h i b i t the  a  before, of  when d e a l i n g with r e a l  energy  number  of  barriers  chemical  p r o p e r t i e s of a random  to  bond  bends  link.  are  This  polymer rotation  r e q u i r e d to  also  implies  that as these enerqy b a r r i e r s are overcome with an i n c r e a s e i n temperature, should and be  the number of bonds needed to give a random  decrease.  Hence the l e n g t h of  consequently temperature  dependence elastin, link  dependent..  Studies  on  decreases  with  restrictions  variation  link  of  the  c o n s i s t e n t with an Arrhenius  cn  the  has  anisotropy type  molecules  been  shown  with  (Saunders in  in  the  that  the  temperature  relationship  (°<r*_.)= Aexp(E^/RT) is  an  activation  value of E  value  of  the  is form  of  the  7.23  energy . Hence i t i s p o s s i b l e t c  f r o a a p l o t of activation  l n H , - ^  energy  magnitude of the energy b a r r i e r s of the  A  random  T r e l o a r 1972) :  (Morgan and  polymer  of the  than  t h i s decrease i s thought to r e f l e c t a r e d u c t i o n  above.. I t  o b t a i n the  temperature  i n c r e a s i n g temperature  network, as d i s c u s s e d  The  the  o p t i c a l p r o p e r t i e s cf polymers other  conformational  E^  link  the polymer c h a i n dimensions are expected t o  the  usually  Where  'functional*  have shown that the o p t i c a l a n i s o t r o p y  1957), and the  of  this  link  should  1/T.  versus reflect  restrictions  the  on  the  f o r the l i n k a n i s o t r o p y  as a  chains. plot  function circles).  of the e l a s t i n  of temperature From  is  data shown  in  figure  the slope of the graph one  7.4,b  (filled  o b t a i n s a value  of  192  1.6 kcal/mole comparable resonance  for to  the  the  activation  value  data f o r e l a s t i n  magnitude  energy.  obtained peptides  from  of t h i s a c t i v a t i o n energy  the  activation  energies  value  nuclear  (Urry e t . a l .  is  magnetic  1978d).  The  i s too s m a l l to r e p r e s e n t  the m e l t i n g or the formation of s t a b l e since  This  secondary  for  these  structures,  systems would  2 0 kcal/mole  expected  to have values i n the range  of  (Fraser  and  more p l a u s i b l e t h a t t h i s  McCrae  value, of 1.6 energy  1973).  It  is  15  be  kcal/mole, i s r e p r e s e n t a t i v e  barriers  about  cf*—CO  the  and  to  of  the  C^—N  torsional  bonds of the  p o l y p e p t i d e backbone. T h i s e x p l a n a t i o n i s c o n s i s t e n t with t h e o r e t i c a l a n a l y s i s of random p o l y p e p t i d e s presented by and F l o r y  (1965  analyzed the  non-Gaussian graphically  curves  a c c o r d i n g t o eguations 7.13 is  Figure  e v a l u a t i o n f o r the non-Gaussian properties  of  the  number  7.5,a  and  polymer networks. In each  7.16  reduced  t o 7.19.  of the  to the unswollen  These l i n e s  elastin  p o l y n o m i a l . The  data  to  mechanical  form  of  t o 7.15  random  to for  links  7.5,b, presents t h i s  mechanical  r e p r e s e n t the experimental data obtained fibres,  network were  by comparing the experimental r e s u l t s  generated  cross-links.  network  p r o p e r t i e s of the e l a s t i n  v a r i o u s values of s, which between  Brant  a and b ) .  _(e]_ Non-Gaussian p r o p e r t i e s of the e l a s t i n The  the  and  photoelastic  case the s o l i d for  single  lines  elastin  a c c o r d i n g to eguations  were obtained by a l e a s t sguares f i t regressions and  upto  a  tenth  degree  p h o t o e l a s t i c data were found t c  193  Figure.7.5 : Non-Gaussian p r o p e r t i e s of the e l a s t i n network. (A) Analysis of the non-Gaussian mechanical p r o p e r t i e s . The t h e o r e t i c a l curves (open circles) were generated by e v a l u a t i n g eguation 7.13 f o r v a r i o u s values of s. The f i l l e d circles represent the e l a s t i n data f i t t o a seventh degree polynomial: y= -0.15 X 109 +0.62 X 10*x -0.11 X 1 0 i o 2 +0.11 X 1 0 i o 3 -0.65 X 10«x* +0.22 X 10^x5 0.41 X 108x6 +0.32 X 10 x*. The s o l i d v e r t i c a l bar r e p r e s e n t s t standard d e v i a t i o n . (B) A n a l y s i s of the non-Gaussian photoelastic properties. The t h e o r e t i c a l curves (open c i r c l e s ) were generated by e v a l u a t i n g eguaticns 7. .14 and 7.15 f o r v a r i o u s values of s, using N=1.0 X 1 0 c h a i n s / m and H « - * _ ) =2.84 X 1 0 - 3 o 3 . _he filled circles represent the e l a s t i n data f i t t o a f o u r t h degree polynomial: y= 0.33 X 10~ +0.82 X 10-«x +0.36 X 10-15x2 -0.2 X 1 0 - 2 i x +0.26 X 10-z«x*. The s o l i d v e r t i c a l bar r e p r e s e n t s + standard d e v i a t i o n . X  x  7  28  m  3  3  3  194 •  Figure.7,5.  6r  5  10 Af/NkT  195  have  the  best  respectively evaluation  f i t to seventh and  (see in  legend  for  f o u r t h degree  figure  7.5).  polynomials  The  graphical  both cases y i e l d s a value of approximately  10,  f o r the number of random l i n k s , s, between c r o s s - l i n k s . As i n d i c a t e d above, the value cf the e l a s t i c is  consistent  6,000 to 7,100 elastin  modulus,  with a molecular weight between c r o s s - l i n k s of g/mole. Since the average  i s about 84.5  g/mole  (Sandberg  residue  weight  s i n c e the a n a l y s i s of 10  dividing by  random  the  links  non-Gaussian were  present  Furthermore,  behaviour between  indicated  cross-links,  the number of amino a c i d r e s i d u e s between c r o s s - l i n k s  10 g i v e s a value of 7.1  to 8.4  amino a c i d s per random l i n k .  That i s , i t takes about 7 to 8 amine a c i d r e s i d u e s t o the p r o p e r t i e s of the s t a t i s t i c a l , form  the  validity  basis  of  the  freely rotating  kinetic  theory  l i n k s which  of these numbers obtained from the p r o p e r t i e s of  Ji This  chapter  the  kinetic  elastomers. elasticity  conformation,  the  Conclusions  has presented c o n v i n c i n g evidence t h a t the  theory  Furthermore, are  The  chapter.  p r o p e r t i e s of the e l a s t i n network are best d e s c r i b e d i n  chains  exhibit  relationships.  e l a s t i n network w i l l be evaluated i n the next  of  for  1976), t h i s amounts to  71 to 84 amino a c i d r e s i d u e s between c r o s s - l i n k s .  that  G,  based  i t is  on  relatioships  derived  since  theories  an  these  assumption  reasonable  which make up the e l a s t i n  to  state  terms  for  entropy  of  rubber  of a random network that  the  f i b r e s are themselves  protein devoid  196 of any s t a b l e secondary agreement  This  with the r e s u l t s of b i r e f r i n g e n c e  1980)  and  1980,  Aaron  nuclear  evaluation  of  and  the  1980)  of  the  activation  C*—N  determinants of p r o t e i n random-coil previously  (Schimmel and F l o r y  (Fleming e t ^ a l ^  for  normal  the  are  dimensions,  the as  1968, M i l l e r and Goebel  c  The  network  conditions,  angles  in  Gosline  network.  energy  torsional  is  (Aaron and  elastin  i n d i c a t e s t h a t , under the  conclusion  magnetic resonance s t u d i e s  et^al..  •restrictions' —CO  structures.  the major  proposed 1968).  197  _____________  A __________  TEST FCR ELASTIN  ____________  A_ I n t r o d u c t i o n Having of  the  kinetic  obtained some 'concrete* numbers from the a n a l y s i s  physical theory  properties of  rubber  of  elastin,  elasticity,  according  t o the  i t i s tempting  to  e x t r a p o l a t e from the c h a r a c t e r i s t i c s of the e l a s t i n p r o t e i n t o other p r o t e i n s i n the random c o i l conformation. In order to do so  one  must f i r s t  d e f i n e the ' c h a r a c t e r i s t i c v a r i a b l e ' which  i s t o be p r e d i c t e d using the (and manipulations stumble  experimentally  obtained  of these values) , and i d e a l l y  one must then  a c r o s s some l i t e r a t u r e t h a t has 'observed  the same v a r i a b l e , p r e f e r a b l y technique  of  experimental  attained  analysis.  through The  comparison should allow one to e v a l u a t e the random conformation  values  values' f o r a  results  different of  assumption  this of  a  for elastin.  B_ C h a r a c t e r i z a t i o n of Random P r o t e i n s  _a_ The measurable  dimension  I f a person i s qiven an o b j e c t and asked physical  shape,  he w i l l u s u a l l y respond  to d e s c r i b e i t ' s  with t h e a p p r o p r i a t e  a d j e c t i v e such as round, square, oblcng, weird! to be s p e c i f i c , t h i s person might  then  their  terms  like  l e n g t h , width,  probably  will  not  dimensions  radius, axial progress  with  in  ratio, that  the  usual  but  he  'weird'  object.  proceed  e t c . I f asked to  Random c o i l e d  describe  make  any  polymers  198  would be c l a s s i f i e d the  'weird'  category..  dimension t h a t can polymer. . T h i s  In  r.m.s.  practice  be u t i l i z e d  dimension,  root-mean-sguare this  by t h i s f r u s t r a t e d person as belonging  to  describe  as you  represent  a  there random  is  < r > / . . But 2  in  1  what  2  terms  of  a  a  coiled  know, i s r e f e r r e d to as  (r.m.s.) d i s t a n c e , value  however,  to  the does  measurable  dimension? Consider time  t=0.  i n any  an i d e a l gas molecule l o c a t e d at the  origin  Suppose that the movement of the molecule can  direction  at  occur  (randomly) i n d i s c r e t e steps of l e n g t h , 1. I f  the molecule moves a steps per second, then a f t e r b seconds i t w i l l have moved s number of s t e p s . Where of  s=ab.  He-evaluation  the molecules p o s i t i o n a f t e r s steps w i l l show that i t has  been  displaced  characterized new <r>  away  from  the  origin  8.1a).. In  distance  one  has  sucessive  p o s i t i o n s c f the gas  made  of  a  tc  simply  number  this  analogy  to  molecules by an  of chemical  * ideal'  which  now  The  the d i s t a n c e between the two  two link,  bonds, of l e n g t h , 1.  i t ' s c h a r a c t e r i z a t i o n by the measurable dimension  molecule  real  r e p l a c e the spaces between  allows  represents  this  the end-to-end d i s t a n c e ,  transforming  polymers  up  a  by the l i n e a r v e c t o r between the o r i g i n and  p o s i t i o n . T h i s vector r e p r e s e n t s (figure  by  This <r>,  ends of  the  forms  the  ( f i g u r e 8.1b). c l u e , as to the conceptual  analysis  of  'random  walk*  the  adjective  definition  of  dictionary  describes  the  framework t h a t  systems,  can  be  'random'.. The  word random i n two  found  i n the  Miriam-Webster ways. One  i s to  199  £igi?rei8_.Jj_ The random walk. (A) A 10 step random walk f o r a gas molecule starting at the o r i g i n and moving i n d i s t i n c t steps of l e n g t h 1 . <r> i s the end-to-end d i s t a n c e . (B) Replacing the d i s t a n c e between two successive p o s i t i o n s by an ' i d e a l ' l i n k of length 1, allows the extension of the Gaussian d e r i v a t i o n s f o r random walks to r e a l polymers. .  200-  <  20 1  think it  of random as being haphazard. The other i s to  think  of  i n terms cf chance. I t i s t h i s second d e f i n i t i o n of random  that  describes  occurrence' ,  i t i n terms of a which  allows  yields  the end-to-end  of the  2  1  2  cf  derivations  their  molecules  can  as  1,  Gaussian  characterizes  the  number  non-ideality  f o r random c o i l s assumes an i d e a l  volume,  essentially  occupy  of  1953):  (Flory  does not occur i n r e a l polymers,  finite  volume e f f e c t ' . This  an  8. 1  2  Accounting f o r the  system. T h i s obviously because  through  2  of the l i n k ,  <r >=sl  above  problem  < r > / , i n terms of  l i n k s , s, and the length  The  observing  a f a i r l y simple r e s u l t which  distance,  Jb__  p r o b a b i l i t y of  a p r a c t i c a l method f o r d e s c r i b i n g  the random walk. E v a l u a t i o n statistics  1  w i l l display states  that  which  an 'excluded no  two  real  the same volume element. The r e s u l t of  t h i s e f f e c t i s t c expand  the domain of the random  coil  by  a  f a c t o r oL , which accounts f o r the n c n - i d e a l i t y of the system ( i.e.  polymer-polymer  interactions,  and  polymer-solvent  interactions) : <r > V 2  where the  r  0  and  2 =  <K <r2^V  8.2  2  r are the unperturbed and a c t u a l dimensions of  molecule r e s p e c t i v e l y . In the case  where  the  balances  non-ideality out  interacticns,  the <=<  of  egual  a  theta  solvent,  the polymer-solvent i n t e r a c t i o n s  non-ideality is  of  of  the  polymer-polymer  t c u n i t y , and the above e g u a t i o n  202  reduces  t o eguation 8.1.  C_ ______  ______  From  Viscosity  (a) V i s c o s i t y of random c o i l s There are present i n the papers  that  polymers  deal  (Flory  with  the  literature  a  vast  number  of  v i s c c s u t y of random hydrocarbon  1949, Fox ______  1951,  Kurata  ______  1960,  Kurata and Stockmayer 1963, Stockmayer and Fixman 1963). These studies  have  developed  obtained experimental  theoretical  relationships  and have  parameters f o r the p r e d i c t i o n of random-  c o i l dimensions.  Only r e c e n t l y have there  made  these v i s c o s i t y r e l a t i o n s h i p s t o the study of  to  extend  random p r o t e i n polymers Tanford  ______  (Brant  1966,1967,  and  Reisner  r e s u l t s i n d i c a t e the a p p l i c a b i l i t y  been  Flory and  any  1965, a  attempts  and  b,  Eowe 1969), and t h e  of t h i s type of approach t o  proteins Tanford e t . a l . viscosity  (1966)  have  published  studies  on the  of p r o t e i n s i n 6M GuHCl, where these polymers behave  as random c o i l s . Applying the a p p r o p r i a t e r e a l t i o n s h i p s , authors  have  evaluated the r.m.s.  p r o t e i n s . This i s f o r t u n a t e s i n c e ,  these  d i s t a n c e s f o r a number of as  mentioned  before, i t  w i l l allow a comparison f o r the p r e d i c t e d values obtained from the  properties  of  the e l a s t i n p r o t e i n . However, i n order t o  ensure t h a t t h i s comparison variables, published  a  number  values. .  of  takes  place  corrections  between  must  be  eguivalent made  t o the  20 3  (b) C o r r e c t i o n of visccsit_y The  eguation  dependence  of  that  the  takes the form  intrinsic  (Yang  constant  for  molecular a  a  conformation, theta  convenient  to  polymers  viscosity,  value  1961).  deal  proteins  and  0.67  obtained f o r X  is  an  system,  empirical M  In  with  them  the  have  f o r random polymers  greater the  case in  actual  X  than of  terms  1  of  assigned a  r e s p e c t i v e l y . The  elastin I  domain and,  c o e f f e c i e n t , <Xs , comparison  to  and  a  is  since  weights. values  hydrochloride  evaluated the  it  value of  hence, i t i s necessary be  in  weight,  average r e s i d u e  i n d i c a t e s t h a t guanidine  before making the  in  the number of  good s o l v e n t f o r p r o t e i n s . T h i s should r e s u l t i n the  expansion  the  for rod-like  proteins,  mclecular  different  f o r K and  of the randcm-coil  is  (or the number of r e s i d u e s  Doing t h i s , Tanford e t . a l . . (1966, 1967) 0.684  K  polymer-sclvent  a  r e s i d u e s as opposed to different  for linear  a value of 0.5  and  (Tanford  viscosity  i s the exponent t h a t d e s c r i b e s the polymer  having  solvent,  proteins  given  X  weight  8.3  weight of the polymer  p r o t e i n ) , and  molecular  =KM*  D  i s the i n t r i n s i c  Q  the  1961) : £n]  Where [ n ]  describes  values  of 0.67  is  a  expansion t h a t the  corrected for  predictions  from  the  network. have  chosen  data by an a l t e r n a t e random-coils  to do t h i s by manipulating  the  analysis.  viscosity  can be d e s c r i b e d by  The  intrinsic  viscosity of  (Kurata and Stockmayer 1963):  20 4  [n]=KMV <* 2  In  this  f o r m u l a t i o n -»  8.4  3  i s taken to be egual t o 0.5,  d e v i a t i o n s due to s o l v e n t e f f e c t s  is  expansion  forms  coeffecient  e v a l u a t i o n of c<  °<- .  This  accounted the  for  basis  any  in  the  for  the  according t o : o< = [ n ] / [ n ] _  8.5  3  Hence i f one  can evaluate  theoretical  value  for  [  n  K,  it  is  possible  to  ]_  by  assuming <x =1.  obtain  One  u t i l i z e the a c t u a l value measured f o r the i n t r i n s i c £n],  and  the c a l c u l a t e d  can  a  then  viscosity,  value f o r [ n ] , and equation 8.5,  to get a  value f o r <K . The method  e v a l u a t i o n of K f o r the Tanford data of Stockmayer and Fixman  1.3 cc/gm f o r t h i s parameter the  (1963),  was  done by  which gave a value of  (see appendix 3). The r e s u l t s  subseguent c a l c u l a t i o n s are presented i n t a b l e 8.1,  with the c o r r e c t e d values f o r the end-tc-end by Tanford e t _ a l _ the  high  are  consistent  (1966, 1967). I t should  values  Furthermore,  the  c o n s i s t e n t with the treatment  6molar  molecular results  f o r random polymers  GuHCl weight  (Flory  _a_ 8.1  along  mentioned  that  c o e f f e c i e n t , <X , a  good  solvent.  dependence of o< i s a l s o  expected  0". P r e d i c t i o n s From The  Eguation  be  being  of  d i s t a n c e obtained  obtained f o r the expansion with  the  from  the  theoretical  1953).  E l a s t i n Network  C a l c u l a t i o n of s  s t a t e s t h a t i f the number of random l i n k s ,  205  Table. 8..1.: P r e d i c t ions f o r random-coil p r o t e i n s .  PREP1CTI0HS FOR THE PIKENS10HS OF RAWDOH-COIL PR0TE1WS. nusber o f  expansion coeffecient:  residues  cr  corrected < r V ' V  preaicwa-r r">- ~A" b c  <r*> b  1 / 2  *rV/ obs. c 2  Insulin  26  44  0.97  45  38  41  .84  .92  rlbonuclease  124  101  1.03  98  83  90  .85  .92  hemoglobin  144  112  1.07  106  89  97  .84  .92  myoglobin  153  120  1.09  1.10  22  100  .84  .91  J-Iactoglobulln  162  126  1.11  113  95  103  .84  .91  chymotrypslnogen  242  148  1.10  134  116  126  .87  .94  aldolase  365  189  1.12  168  142  154  .85  .92  serum albumin  627  258  1.17  220  1S6  202  .85  .92  thyreoglobulin  1500  401  1.18  341  288  313  .85  .92  myosin  1790  443  1.19  372  314  342  .84  .92  "data from Tanford e t . a l 1966, 1967. ''for 7aa/random l i n k . f o r 8aa/randoni l i n k .  c  206  and  the l e n g t h of the l i n k i s known, then, i t i s  predict  a  value  for  the  possible  to  end-to-end d i s t a n c e , < r > / .  The  2  a n a l y s i s of the p r o p e r t i e s of the that  between 7 and  elastin  network  1  2  indicated  8 amino a c i d r e s i d u e s are r e g u i r e d to g i v e  a • f u n c t i o n a l ' random l i n k . Hence, i t i s p o s s i b l e to o b t a i n value of s f o r any residues  in  per l i n k the  a  given p r o t e i n by d i v i d i n g the the number of  the p r o t e i n by the number of amino a c i d r e s i d u e s  ( i_e_  length  of  7 to 8). In t r y i n g to evaluate the  1,  link,  I  have  a  value  for  chosen a q u a l i t a t i v e  approach which i s based on the t h e o r e t i c a l d e r i v a t i o n s f o r the dimensions cf random p r o t e i n s .  _b_ F l o r y and evaluation  resulted  of in  random random  energy  (Schimmel and Goebel  h i s co-workers have d e a l t with the  of  energetics  C a l c u l a t i o n of 1  Fiery  1968).  proteins protein  and  have  evaluated  dimensions,  which  minima f o r bond angles at 1968,  Although  Miller the  ______  actual  random polymers w i l l be d i s t r i b u t e d  bend  the  distribution.. I  (^=270, (1^ = 120) link  using  Schellman  range  the (1 963).  acids  therefore  used  to c a l c u l a t e the displacement relationships  provided  Doing t h i s I obtained  23.4A<> f o r the l e n g t h , 1, amino  have  (J^ = 120  Miller  and  angles present  the value at the e n e r g e t i c minimum should r e f l e c t for  the  usually  ^=270,  1967,  over a  theoretical  by  of an  values, 'average'  this  value  along the random Schellman  and  values of 19.8A° and  cf a random l i n k made up of 7 and  r e s p e c t i v e l y , which allowed  in  8  me t c p r e d i c t values  20 7  of r.m.s.  d i s t a n c e s f o r v a r i o u s p r o t e i n s using  and  a value  to  the  c f 1 f or c{ • This should  observed values  eguation  permit a v a l i d  (Tanford e t . a l .  1966,  1967)  8.1  comparison since  they  have been c o r r e c t e d f c r the expansion c o e f f e c i e n t as d e s c r i b e d above.  |. D i s c u s s i o n and The 8.3,  r e s u l t s , which are summarized i n t a b l e 8.1  indicate  properties within  Conclusions  that  predictions  obtained  from  the  o f the e l a s t i n network are i n e x c e l l e n t agreement,  limits  calculation,  of  the  with  statistical (Miller  the  and f i g u r e  uncertainties  Goebel  observations  in  both the t h e o r e t i c a l e x p e c t a t i o n s  mechanical  and  inherent  treatment  1968)  of  peptide  ( f i g u r e 8.3,a) and  ( f i g u r e 8.3,b) on  random  their from  the  conformation  the  coiled  experimental  proteins.  The  f a c t t h a t the p r e d i c t i o n s from the e l a s t i n data are lower than the  expected  values can  be reasonably  g l y c i n e content  of t h i s p r o t e i n . G l y c i n e  third  amine a c i d content  and  of  one  the  cn  the p r o t e i n c h a i n  T h i s would lower the  makes  of e l a s t i n  would expect t h i s to r e s u l t  restrictions  a t t r i b u t e d to the  in  a  up  high  almost  (Sandberg  reduction  a  1976), of  the  m o b i l i t i e s i n the network.  number of amino a c i d r e s i d u e s r e g u i r e d to  give a random l i n k , as compared to Host p r o t e i n s which c o n t a i n l e s s e r amounts of g l y c i n e . I t i s more  than  8  amino a c i d r e s i d u e s  reasonable  to  expect  that  would be r e g u i r e d to g i v e a  random l i n k f c r p r o t e i n s which do not e x h i b i t such extremes i n amino a c i d composition  as e l a s t i n . T h i s would have the  effect  208  Figure.8.2: P r e d i c t i o n s f o r the dimensions of randcm-coil protein, Boot-mean-sguare d i s t a n c e f o r various p r o t e i n s as a f u n c t i o n of the number o f amine a c i d r e s i d u e s . (a) T h e o r e t i c a l p r e d i c t i o n s from M i l l e r and Gcebel 1968. (b) Experimentally obtained values from Tanford e t _ a l . 1966 and 1.967, c o r r e c t e d f o r n o n - i d e a l i t y (see Table 8.1). (c) P r e d i c t i o n s from the e l a s t i n system a c c o r d i n g t o eguation 8.1: ( s o l i d line) for 7aa/random l i n k , (broken l i n e ) f o r 8aa/random l i n k .  209  210  of  increasing  random l i n k ,  the  value  of  1,  which i s the length of the  r e s u l t i n g i n even c l o s e r  p r e d i c t e d and observed  values.  agreement  between  the  21 1  Chapter. IX. . CONCLUSIONS^ Unlike  most  cf the  number of p r o t e i n s that being  random. The  this  thesis,  The  biological  belong to t h i s group of  and  movements  of  muscles.  because of t h i s there r e s t o r e the  high  muscles as i n s k e l e t a l  Muscles  proteins  probably  passive  evolved  metabolic needs, and  turnover.  On  the  due  to t h e i r low  are  describes  in  their  the only  metabolic  f u n c t i o n . But  simply  rubber-like  does not t e l l  proposed  years, in  mechanism but  stating  several  an only  attempt one  provided  characterized  that  proteins  by  needs,  metabolic c o s t t o  the  p l a c e . Hence,  passive to the  as s t a t e d i n the  last  passive  by metabolic  savings  The  performance  other hand, elastomers being  this thesis,  Over the  be  in their  are g e n e r a l l y  turnover  the  opposing  i n response to t h i s  seems p l a u s i b l e that the advantages of realized  only  e l a s t i c elements.  organism i s t h a t of s y n t h e s i z i n g i t i n the f i r s t it  can  movements, h y d r o s t a t i c systems  mechanical components, are not r e s t r i c t e d and  as shown i n  i s a need f o r an  need. Furthermore, muscles are r e s t r i c t e d by t h e i r  by  muscle to i t ' s f u n c t i o n a l s t a t e  as i n many marine organisms, and elastin  roles  biomolecules.  each c o n t r a c t i o n c y c l e . T h i s antagonism can  by other  are a  f u n c t i o n performed by these e l a s t i c p r o t e i n s i s  mechanism t h a t can after  their  elastomers R e s i l i n , Abductin and  to antagonize the contract,  fulfill  Elastin,  biological  bio-polymers known t o us, there  elasticity animal,.This  i n t r o d u c t i o n to  like  elastin  are  us about the f u n c t i o n i n g mechanism. theories to  theory.  of  elasticity  characterize The  Kinetic  the Theory  have the of  been  elastic Rubber  Elasticity,  has  been  able t o account f o r and,  p r e d i c t , the macroscopic Like  most  mathematical  p r o p e r t i e s of e l a s t o m e r i c m a t e r i a l s .  theoretical  derivations  convenient and c r u c i a l  i n most cases  frameworks  the  that  are  the  most  important  which  i s t h a t of a random conformation. I t i s t h i s  which  has  chemists.  The  on  k i n e t i c theory makes some very  assumptions,  prevented  based  i t ' s general  c u r r e n t thought  acceptance  of  assumption  by  i n biochemistry and  protein biopolymer  r e s e a r c h i s summarized by the s t a t e m e n t — - " i f i t has a primary seguence i t must have a statement account  f o r the e x c e p t i o n a l c l a s s of  basis  that  the  of  this  approach  unacceptable  since,  'phantom' chains and conformation  Although  statement adopted  by  protein  conformations  in ARE  the  kinetic  theory  'ideal' l i n k s . This b i t t e r p i l l  terms  of  real  EQUALLY ACCESSIBLE  polymers (WHICH  IS  of  of random  to  chains  whom a l l  THE  SAME  AS  STABLE conformation). This allows  networks that are devoid of s t a b l e secondary  had  is  i n t h e i r minds i t invokes the concept  THAI THERE IS NO ONE  polymer  structures.  using the k i n e t i c theory r e l a t i o n s h i p s to e x p l a i n  macroscopic  On  a number cf people have s t a t e d  the mathematics of random walks to be a p p l i e d to r e a l  In  this  elastomers.  might be e a s i e r to swallow i f the phantom  restated  SAYING  structure".  might be v a l i d f o r p r o t e i n s i n g e n e r a l , i t does not  the  are  tertiary  p r o p e r t i e s of s i n g l e e l a s t i n  fibres,  I  have  the also  t o assume a random network conformation f o r e l a s t i n . . T h i s  assumption  was  t e s t e d using d i f f e r e n t  methods of  (see summary f i g u r e , 9 . 1 ) . V i s c o s i t y experiments  investigation were used  tc  213  f i g a r e . 9 . 1:  Summarj f i g u r e  for  the  thesis.  214  Figure.9.1  SUMMARY.  SOLUBLE  PEPTIDE  STUDIES: a. viscosity. b. n . m . r .  BIREFRINGENCE  SCANNING ELECTRON  STUDY OF  MICROSCOPY.  SINGLE  ELASTIN  FIBRES.  / RANDOM-COIL  /  /  CONFORMATION  FOR THE E L A S T I N NETWORK.  K I N E T I C THEORY  RELATIONSHIPS:  a.  mechanical  b.  photoelastic  properties.  c.  non-Gaussian  properties.  PREDICTIONS  properties.  FOR  RANDOM-COIL  PROTEINS.  /  /  215  evaluate  the  shape cf the s o l u b l e e l a s t i n . These s t u d i e s d i d  not support the presence of any microscopy was results  rod-like structures. Polarized  used to t e s t f o r  indicated  a  random  molecular  organization.  conformation.  resonance s t u d i e s were used to i n v e s t i g a t e the elastin  network". The  The  Nuclear magnetic m o b i l i t y of  a n a l y s i s showed that the e l a s t i n  the  network  i s c h a r a c t e r i z e d by r a p i d motions. A s s i m i l a t i n g these r e s u l t s , one  should  be  able  to  state  guite  confidently  that  the  assumption of a random network f o r e l a s t i n i s  justified.  fact  f o r random-coil  that  proteins In  elastin  can  serve  as  a  o f f e r s f u r t h e r support i n favour  conclusion  elasticity  it  provides  seems a  that  valid  the  proteins  conformations.  which,  i n general  of t h i s  assumption.  k i n e t i c theory  theoretical  a n a l y s i s of the e n t r o p i c e l a s t o m e r i c like  model  The  of rubber  framework  p r o p e r t i e s of the  for  the  rubber-  are c h a r a c t e r i z e d by random  216  APPENDIX.I: T h e r m o g l a s t i c i t y  ___  Ther a d y n a m i c R e l a t i o n s h i p s  Since an e l a s t i c system, i n involved it  i t ' s functional  state,  i n the storage and d i s s a p a t i o n of mechanical  i s p o s s i b l e t o evaluate t h e  basis  cf  this  is  energy,  mechanism  by  using thermodynamic r e l a t i o n s h i p s . According  to the f i r s t law of thermodynamics, energy, E,  can be converted from one form t o another, but  i t cannot  c r e a t e d or d e s t r o y e d . Hence when c o n s i d e r i n g an e l a s t i c  be  sample  and i t ' s s u r r o u n d i n g s , f o r a given deformation, dE i s egual t o zero.  I f , however,  one  considers  only  the  sample, f o r a  r e v e r s i b l e process one can w r i t e : A. 1 . 1  dE=dg +dw where g r e p r e s e n t s the heat evolved represents positive and  the  work.  Both  dg  when heat flows from  work  i s done  b_  by  and  the  sample  and  w  dw are considered to be  the sample to  the  process,  on  the the  surroundings surroundings,  respectively. Eor  a  thermodynamics the heat  reversible defines  process,  second  law  of  the change i n entropy, S, i n terms of  and the a b s o l u t e temperature, dS = dg/T  S o l v i n g f c r dg, and  the  substituting  T, a c c o r d i n g t o :  A. 1 . 2 into  eguation  A.1.1,  one  gets: dE=TdS+dw., Using  A. 1 . 3  these equations i t i s p o s s i b l e d e f i n e a s t a t e  function.  217  r e f e r r e d  to  as  the  H e l m h c l t z  f r e e  energy,  F=E-TS At  c o n s t a n t  takes  t e m p e r a t u r e ,  the  and  S u b s t i t u t i n g  A . 1.  A.1.3  e g u a t i o n  i n t o  dF=dw U t i l i z i n g  t h i s  e g u a t i o n  e g u a t i o n s  A.1.2  5  t h e  above  A . 1.  6  and  A.1.6  g i v e s :  one  can  w r i t e  e g u a t i o n  as: dw = d E - d g  T h i s  e g u a t i o n  r e v e r s i b l e  forms  the  systems,  b a s i s  and  of  i t t o  of  p r o c e s s .  t h e  g i v e n  m e c h a n i c a l t h e  response,  In  i t a l l o w s  c o n t r i b u t i o n t c  energy,  f e ,  and  the  the  the  i s  book-keeping,  a  The  The  p o s s i b l e ,  one  f s ,  t o  v a r i o u s  determine  o f  o f of  components a  m a t e r i a l s  the  magnitudes f ,  from  the  i n  two  components: A . 1.  experiment  means  r e t r a c t i v e f o r c e ,  Thermodynamic  t h e r m o d y n a m i c  the  a n a l y s i s  by  p a r t i c u l a r case  f=f e+fs  JB1  t h e r m o d y n a m i c  e v a l u a t e  t o t a l  entropy,  1.7  A.  t h e r m o d y n a m i c  of  volume,  form: dF=dE-TdS  A.1.5  as:  1.4  A.  p r e s s u r e ,  F,  8  Experiment  can  be  c a r r i e d  out  ways: J.. a  The  sample  can  f i x e d e x t e n s i o n ,  f o r c e ,  f ,  e v a l u a t e d  as  a  be  immersed  one  f u n c t i o n  a c c o r d i n g  to:  can of  i n  f o l l o w  a  d i l u e n t , and  the  temperature.  change  i n  The  d a t a  k e e p i n g the can  i t  a t  r e t r a c t i v e then  be  218  f=fe + T (df/dT) ........ A. 1.9 2±  The  sample,  temperature,  in  a  diluent,  is  extended  at a constant  and the heat r e l e a s e d , along with the work ( i.e_.  the area under the f o r c e e x t e n s i o n graph) i s monitored. energy  component  is  then  evaluated  according  The  to eguation  A. 1.7. For the sake of convenience, above  will  temperature  be  referred  to  the  two  t h e r m o e l a s t i c experiments,  respectively.  With r e f e r e n c e to the e l a s t i c i t y  system  responsible  Elastomers  of  materials,  that i n v o l v e s the deformation of a r i g i d l a t t i c e  the energy  internal  the  (as i n  where  most  i s stored i n the a l t e r a t i o n of bond l e n g t h s and  o r b i t a l s , the r e s t o r i n g f o r c e a r i s e s  energy  if  f o r the r e t r a c t i v e f o r c e c o n s i s t s of a  the case of metals, c o l l a g e n , k e r a t i n , and s i l k ) , of  outlined  as constant s t r a i n and constant  (C) Thermoelasticity. of K i n e t i c  mechanism  methods  energy  component  as  from  a  change  in  the  i n d i c a t e d by the magnitude of  component, f e , obtained from a thermodynamic  experiment  (fe/f=1). In the case of n a t u r a l shown  that  the  rubbers,  fs/f  ratio  cf  approximately  one,  theory of rubber e l a s t i c i t y ,  kinetically  has  These m a t e r i a l s  been  agitated  conformation  exhibit  with the f e term being  s m a l l . M a t e r i a l s of t h i s type are thought t o kinetic  it  s t r a i n energy i s s t o r e d as a decrease i n the  c o n f o r m a t i o n a l entropy of the system. a  however,  conform  to  the  which r e g u i r e s a random, at  the  molecular  level  (Treloar  1 S75) .  220  ____________  ___________  OF  A_ Recently there has properties  and  the  tropoelastin.  SOLUBLE ELASTIN BY  PHOTOLYSIS.  Introduction  been a l o t cf i n t e r e s t i n the  physical  primary s t r u c t u r e of the p r e c u r s o r  protein,  This  research  has  created  a  need  for  convenient method to i s o l a t e the t r o p o e l a s t i n molecule upto  now,  has  been i s o l a t e d from l a t h y r i t i c  of a b i o l o g i c a l source c a r r i e s with i t the high  expense  and  lew  which i n turn y i e l d e d point  guite  to get  Hence  procedure to i s o l a t e the  of  Foster  et.al.  55gms of wet  tissue  35mg of t r o p o e l a s t i n ,  clearly.  use  problems  p r o d u c t i v i t y . A paper by  (1975), where they used 2000 c h i c k s  which,  animals. The  usual  a  illustrates  i t seemed v a l u a b l e  precursor p r o t e i n ,  this  to develop a  from  the  freely  a v a i l a b l e , i n s o l u b l e e l a s t i n by chemical means. This  chapter  absorption induce  first  l y s i s and  the  of  a  method  the  that  elastin  utilizes  the  cross-links  to  subseguent r e l e a s e  of the  soluble  Since t h i s procedure i s a r e v e r s a l of the b i o l o g i c a l  pathway that the  with  characteristics  their  peptides.  deals  r e s u l t s i n formation of the place  (by  i n s o l u b l e network), i t  the  linking  should  hopefully  peptide t h a t r e p r e s e n t s the p r e c u r s c r  B_  Methodology  ja)  of  Rationale  insoluble e l a s t i n tropoelastin into yield  protein.  a  in an  monomeric  221  The  rationale  for  the  use c f p h o t o l y s i s to c l e a v e the  c r o s s - l i n k s of e l a s t i n i s based and  Houdard  r i n g s by  (1968)  who  on a paper  studied  by  Joussot-Dubien  the cleavage of p y r i d i n i u m  u l t r a - v i o l e t r a d i a t i o n . The relevance to the  cross-links  lies  in  the  fact  that the  (iso)desmosines  t e t r a - s u b s t i t u t e d p y r i d i n i u m r i n g s , as demonstrated by et_.a 1... (1963). et.ah  This  ( 1976, 19 77)  purpose  of  to attempt  solubilizing  but  they  the use of  photolysis  an  the  additional  (iso)desmosine  in  Dubien  1968  links  with  cleavage  figure and  A.2.1  Baurain  in  this  the were  crossfrom  chapter  step which r e s u l t s i n the r e l e a s e of  hypothetical reaction  shown  for  c c u l d not i s o l a t e any s o l u b l e peptides  s o l u b l e p e p t i d e s from the photolysed The  Thomas  f i b r o u s e l a s t i n . These authors  t h e i r p r e p a r a t i o n s . The methology presented utilizes  are  aspect of e l a s t i n chemistry led Baurain  able t o demonstrate a cleavage of links,  elastin  elastin.  pathway  (as based  for  this  light  is  on the the work of Joussot-  1976). The i r r a d i a t i o n of  ultra-violet  method  (275-285nm)  the  cross-  results  i n the  of the s i n g l e bond between the n i t r o g e n  and  carbon  6  ( f i g u r e A. 2.1). This product i s unstable at low ph and/or high temperature  and  can  be broken down ( f i g u r e A.2.1).  s t e p i n v o l v e s the cleavage of the  unsaturated  carbon  The  next  bonds,  using standard o x i d a t i v e t e c h n i g u e s , t o give s o l u b l e p e p t i d e s . This  pathway  is  and iso-desmosine The  e s s e n t i a l l y the same f o r both the desmosine as o u t l i n e d i n f i g u r e A.2.1.  obvious guestion to ask at t h i s time  i s : why  o x i d i z e the unsaturated c r o s s - l i n k s without having  to  not  just  bother  222  Figure.A.2. 1: P h o t o l y s i s of ________ (I) o x i d a t i o n pathway f o r e l a s t i n . (II) p h o t o l y s i s of e l a s t i n f o l l o w e d by o x i d a t i o n . (A: pathway for desmosines, E: pathway f o r isodesmosines.)  223.  II  224  with  the  the  photolysis?  pathway  desmosine  The reason f o r t h i s i s demonstrated by  outlined  A.2.1.  i n figure  by dihydroborate f o l l o w e d by potassium  o x i d a t i o n r e s u l t s i n the s e p a r a t i o n of peptides  Seduction  A. 2 . 1 ) .  (figure  the two  of the  permanganate cross-linked  The same procedure, i f u t i l i z e d on  the iso-desmosines, does not g i v e s a t i s f a c t o r y r e s u l t s , the  movement  carbon  6  of  the c r o s s - l i n k i n g  prevents the r e l e a s e  of  p o s i t i o n from carbon* t o  the  two  t r o p o e l a s t i n molecule i s l i n k e d at about length be  (Gray et_al_.  an  1973),  isc-desmosine,  since  peptides.  As the  ten p o i n t s along i t ' s  i f any of these c r o s s - l i n k s were t o  the  peptides would be r e t a i n e d i n the  i n s o l u b l e network. I t i s t h i s problem  which i s cicumvented  by  the p h o t o l y s i s , a l l o w i n g the combination of the two procedures to g i v e s o l u b l e p e p t i d e s .  Jb_ Elastin  from  ligament nuchae was p u r i f i e d  with 0 . 1 N NaOH at 1 0 0 ° C resulting  Procedure  material  f o r 45  was d r i e d and ground  washed with copious g u a n t a t i e s 50Omg  of HC1.  minutes  of  by e x t r a c t i o n  (Lansing  The  1952).  t o a f i n e powder and  boiling  distilled  water,.  of t h i s powdered e l a s t i n was hydrated o v e r n i g h t i n 200ml  distilled  water  This solution  ultraviolet  lamp  A Corex f i l t e r  that  was  had been adjusted to ph4.4 with 2N  photolysed  using  an  immersion  type  (Hanovia 450W, medium pressure, u.v. Model).  was used to remove wavelenghts  below  270nm  to  prevent the p h o t o l y s i s of peptide bends and, a t the same time, provide  adeguate  irradiation  f o r the cleavage of the c r o s s -  225  l i n k s which  absorb a t 275-285nm  The samples which was  the then  solution  (Thcmas et. al._. 1963) .  were photolysed f o r 6 hours, at  solid  e l a s t i n was c o l l e c t e d  stirred  into  a  (Lemieux  1 955)  by c e n t r i f u g a t i o n . I t  periodate-permanganate  which  contained  mostly  f o r 24  distilled  water  hours,  followed  by  aspirated.  insoluble  d i s c a r d e d . The supernatant was d i a l y z e d water  oxidation  a t 48<>C f o r 24 hours. The mixture was  c e n t r i f u g e d and the supernatant was c a r e f u l l y precipitate,  the end of  against  dialysis  e l a s t i n was running  against  (1L X 24 hrs.) and l y o p h i l i z e d . The  peptides had a c l e a n white appearance.  The  tap 4L o f  resulting  A c o n t r o l c o n s i s t i n g of  the o x i d a t i o n step without p h o t o l y s i s was a l s o  conducted.  C_. R e s u l t s and D i s c u s s i o n  Ja)_ Y i e l d The  amount  lyophilizaticn Although  this  the  is a  yield  as  last  weight.  compared t o the  1974), i t i s probably  f o r t h i s procedure. Examination of the  peptides  such  as  tropoelastin  undergo an i n v e r s e temperature  which upon s t a n d i n g f o r 12 or  irreversible  the  (in r e t r o s p e c t ) r e v e a l s a very c r u c i a l blunder.  alpha-elastin,  at 3 7 0 C ,  after  40mg/gm of o r i g i n a l dry  (Sykes and P a r t r i d g e  S p e c i f i c a l l y , soluble elastin and  recovered  substantial  optimum y i e l d  o x i d a t i v e step  peptide  step was about  b i o l o g i c a l sources not  of  precipitate.  As  more  i s clear  transition  hours from  s e c t i o n , the o x i d a t i v e step was c a r r i e d out a t  forms  the 45°C  an  methods f o r 24  226  hours,  and  it  is  p o s s i b l e that a major part of the  e l a s t i n s prepared i n t h i s manner lost  during  the s e p e r a t i o n  of the  supernatant, which contained proteins.  Oxidation  of  precipitated  the  the  and  were  i n s o l u b l e e l a s t i n from remainder  photclysed  temperature f o r longer p e r i o d s should The  out  soluble  of  the  elastin  the  soluble  at a lower  r e s u l t i n better y i e l d s .  o x i d a t i o n of unphctolysed e l a s t i n gave no  detectable  results.  Jb_  C h a r a c t e r i z a t i o n of the s o l u b l e  Molecular  weight  performed on a 15% presented there  in  determination  pclyacrylimide-SDS  weight. This could  released  mean  peptide  or  procedures  aspect  has  that (b)  (a) that  was  results  s u r p r i s e , and  are  delight,  the  procedure  there not  yields  are other  a  peptides  be detected  by  the  used i n t h i s i n i t i a l c h a r a c t e r i z a t i o n . T h i s  not been i n v e s t i g a t e d  Although the  The  peptides  p r o t e i n band at 66,000 molecular  i n s m a l l g u a n i t i e s t h a t could  crude  the  gel.  f i g u r e A.2.2. Much to my  appeared to be only one  homogeneous  of  peptides  felly.  amino a c i d analyses  has  not been done, i t i s  p o s s i b l e to make some p r e d i c t i o n s based on the work of  Davril  e t . a l . , (1 577).  should  have a higher due  to the  I t i s expected t h a t the  soluble protein  l y s i n e content as compared t c i n s o l u b l e e l a s t i n ,  l i b e r a t i o n of a f r e e l y s i n e a f t e r the  p h o t o l y s i s of  the c r o s s - l i n k s . .In a d d i t i o n to t h i s , one  would expect to have  a lower content of t y r o s i n e due  destruction  wavelength  used  to t h e i r  at  the  f o r the p h o t o l y s i s , which i s i n the range of  227  the  absorption  acids  are not The  been  peak f c r t h i s expected  branching  studied  as y e t , but  t u r n s out  incomplete  photolysis  f o r the  attained an  by  other  on  First,  72,000 peptide  modification  the  the  of  66,000  the  the  peptide  might  i t  this  due  be  more  to not  easily  out  to  interesting  the  peptide  be to  66,000  Of  implies  insoluble  elastin  (Foster e t . a l ,  the r e s u l t a n t 1969)  and  i s about for  the  post-translational  protein  before  it's  This i s supported  1975). has  by  necessary  to obtain a  product  has  protein.  obtained  a  state.  procedure  represents  cf tropoelastin  tropoelastin  72,000  In the  in  weight  absence  a molecular  been shown t o be  of  weight  missing  a  peptide.  c o n c l u s i o n , the  possibility  If  method i s  turns  be  insoluble  weight  value  isolation  (Sandberg  N-terminal  that  high  they/it  protein  argues f o r the  for  isolation  the  of  study,  molecule  inhibitors  In  this  which r e p o r t t h a t enzyme i n h i b i t o r s a r e  tropoelastin  of  i t  not  seme mention h e r e .  that can  i f this  molecular  into  biological  these  of p e p t i d e s ,  to the  this  incorporation studies  amino  have  polymer, e i t h e r  possible that  component  compared in  deserves  polymer  i t c o u l d be  since as  of t h e  the i m p l i c a t i o n s .  the p r e c u r s o r Second,  rest  peptide  o x i d a t i o n , then  methods. But  linear  cf this  a branched  or  The  altered.  this  t o be  production  unbranched  speculate  t o be  characteristics  the p e p t i d e  useful  amino a c i d .  yield might  cf soluble peptides turn  out  t o be  f u r t h e r d e v e l o p m e n t of  of e l a s t i n  fraqments,  t o be  the  this  and  the  precursor technique  followed  up  with  228  P i ^ f l E i - A ° 2.2: Molecular ______ cf __________ peptides. SDS-polyacrylimide gel electrophoresis results for the p h o t o l y s i s products: (o) molecular weight markers: Albumin (66,000), Ovalbumin (45,000), Trypsincgen (26,000), Betal a c t o g l o b u l i n (18,000), Lysozyme (14,000). (•) p h o t o l y s i s p e p t i d e .  230  f a r t h e r c h a r a c t e r i z a t i o n of the s o l u b l e  products.  231  '  AEjoendix^IIIj. PEECICTIONS FOR  7W The Relevant The  molecular  weight  TEOPGELASTIN VISCOSITY  Equation  dependence  for  the  intrinsic  v i s c o s i t y , £ n ] , of unlranched, random pointers, can in  terms  of  an  r e s i d u e s , M, as  empirical  constant,  (Stockmayer and Kurata [n]=KMV  K,  and  be  stated  the number of  1963):  A.3.1  2  o  This eguation a p p l i e s t o i d e a l polyners and polymers i n t h e t a s o l v e n t systems. A more u s e f u l provided  by  Stockmayer  derivation  and  Fixman  c h a r a c t e r i z a t i o n of the v i s c o s i t y i n any  of  this  (1963) solvent,  eguation  allows and  the takes  the form: [n ]= KM V where  is  (c.g.s.)  and  2  + 0. 51<[BM  A. 3.2  a u n i v e r s a l c o n s t a n t with a value of 2.1  X  10 3 2  E i s defined according t o : B=v2 (1-2XJ/VN  A.3. 3  where v, i s the p a r t i a l s p e c i f i c volume of the polymer,  V  is  the molar volume of the s o l v e n t , N i s Avcgadro's number, and X i s the F l o r y i n t e r a c t i o n parameter  (Flory  1953).  .implication to Tropoelastin According M/, 1  to  equation  A.3.2, a p l o t of [ n ] / M / 1  should give a s t r a i g h t l i n e  2  with an i n t e r c e p t  2  versus  equal  to  K. T h i s has been done f o r the data obtained f o r p r o t e i n s ( i n a random was  coil  conformation)  by Tanford e t , a l . .. (1966, 1967)  c a l c u l a t e d to have a value of  1.3  cc/gm  (figure  and  A.3.1).  232  F__!LT2________ V i s c o s i t y of _ _ _ _ c j _ _ c c i l p r o t e i n s . P l o t of data frcm Tanford e t . a l . 1 9 6 6 , a c c o r d i n g eguation A. 3. 2. .  233  Fig ere. A.  3.  1.  234.  T a b l e . A . 3 , 1:  Peediction  for  tropoelastin  PREDICTIONS FOR TROPOELASTIN VISC0.TTY. T°C  V n  *  B (10" ) 26  I n ] cc/g  0  .C99  -2. 66  35 .48  10  .786  -3. 83  34..42  20  .875  -5. 02  33. 33  30  .943  -5. 93  32. 50  40  1.01  -6. 80  31. 71  i 1977.  viscosity.  235  What  makes t h i s manipulation  obtain X values  f o r the  useful i s that i t i s possible to  elastin  protein  from  studies  of  network s w e l l i n g (Gosline 1978). E l a s t i n decreases i t ' s volume with  an  increase  in  temperature  and a c c o r d i n g to eguation  A.3.2 and A.3.3,'this should l e a d t c a decrease subsequent  decrease  eguations allow  the  decrease  in  intrinsic  viscosity  temperature, 850 equal  interesting  the  prediction  [ n ] . The  results  intrinsic  to  and  magnitude  a  These  of  the  cf  the c a l c u l a t i o n s f o r the  of  this  of t r o p o e l a s t i n i n water, as a f u n c t i o n o f  1976) , X values taken frcm G o s l i n e  .725  B  viscosity.  are presented i n t a b l e A.3.1, using  (Sandberg to  in  in  (Partridge compare  et.al.  the  1955).  actual  egual  to  (1977) and v  It  should  be  experimental r e s u l t s t o  these t h e o r e t i c a l p r e d i c t i o n s i n order  to  c o i l conformation  molecule.  f o r the t r o p o e l a s t i n  M  support  a  random  236  ____________ In  chapter  _V_L___I__ 8  I  OF JROTEIN CONFORMATION  have  presented  seme  values  f o r the  parameters t h a t c h a r a c t e r i z e random p r o t e i n s . Eeing encouraged by t h e i r  apparent  g e n e r a l i t y , I have  ventured  to  e m p i r i c a l method f o r the e v a l u a t i o n of p r o t e i n Using and eguation end  i n chapter 8)  8.1 i t should be p o s s i b l e t o p r e d i c t the  distance  an  conformation.  values of s and 1 (as presented  the  devise  end-to-  f o r any given p r o t e i n made up o f a given number  of. r e s i d u e s . From t h i s  value one can c a l c u l a t e the  radius  of  g y r a t i o n , Eg ( f i g u r e A.4.1), f o r t h e random molecule a c c o r d i n g to  (Tanford  1961): Rg2 = <r>2/6  If  A.4.1  the d e n s i t y of the p r o t e i n i s known, one can c a l c u l a t e the  volume, v, of the s i n g l e v= (molecular  molecule:  weight)/(density)  6.02 X 1 0  A. 4. 2  2 3  (although t h i s a n a l y s i s assumes that t h e r e i s no h y d r a t i o n the  molecule,  the hydrodynamic volume could be used i n p l a c e  of the volume given by eguation Using  this  dimensions  and  value  of  A. 4.2) .  volume  one  can  given by (Tanford  2  can  the  of  gyration  is  1961): Eg =l2/12  length  calculate  the r a d i u s o f g y r a t i o n f o r a number of shapes  as f o l l o w s . For rods of l e n g t h 1 , the r a d i u s  The  of  be  A. 4. 3  c a l c u l a t e d from the volume f o r d i f f e r e n t  diameter reds according t o : l=v/( TTr ) 2  For  spheres  A. 4. 4  of r a d i u s , r , the r a d i u s of g y r a t i o n i s given  237  Figure.A.4. 1: Radius of g y r a t i o n f o r v a r i o u s shapes. Radius of g y r a t i o n f o r r o d s , random-coils, and spheres, from Tanford 1961.  238-  239  F i g u r e . A . 4 . 2 : E v a l a a t i o n of p r o t e i n conf ormati_gn_. P l o t s of r a d i u s of g y r a t i o n Eg, versus number r e s i d u e s f o r d i f f e r e n t shapes: (A) lew molecular weight range. (B) high molecular weight range.  240  24 1  by  (Tanford  196 1) : Eg = ( 3 / 5 ) r 2  and s i m i l a r l y following  A. 4.5  2  r could be c a l c u l a t e d from the  and  A. 4. 6  3  abcve  eguaticns  can  be  used  to  generate g r a p h i c a l  r e l a t i o n s h i p s f o r the d i f f e r e n t shapes. T h i s has been done figure  the  eguation: r = (v) (3/4 T )  These  volume  in  A.4.2a f o r the dependence of the r a d i u s of g y r a t i o n on  the number c f r e s i d u e s . Given  a  composition,  protein a  of  known  comparison  molecular  of  to  conformational  figure  the  provide weight  values  obtained  from  (Timasheff  and  Townend  the standard curves should allow a r a p i d e v a l u a t i o n  of the p r o t e i n s A,4.2a,  molecular  and  Rg  experiments such as l i g h t s c a t t e r r i n g 1970)  weight  state.  uncertainties  at  weight are high, but t h i s type reasonable  answers  ( f i g u r e A. 4. 2b).  for  As the cf  proteins  is  evident  lower  limit  analysis of high  from of  should  molecular  242  A££endix___V_: DETEBMINATION OF SOLVENT EFFECTS ON RANDOM COILS A s i m i l a r approach as the one presented above, used  could  t o e v a l u a t e the e f f e c t s of s o l v e n t s cn p r o t e i n s t h a t are  .known to be i n the random c o i l conformation. Bg  as  and  A.4.1, and generate  2  standard curves f o r  << Bg 2  values obtained from ether evaluation  of c< ,  A.5.1.  Stockmayer  sources  f o r any  (1949),  will  which  would  allow  allow  a  f o r the arguments  provided this  Zimm  method  pointed  length of the l i n k , an  of  a  presented  random l i n k  assumption  A.4.2a  and  A.5.1,  obtained  (8.4aa/link) and t h e This i s  f c r p r o t e i n s t h a t show  extremes i n t h e i r amino a c i d composition. Figures  t o be  i n t h i s , and the  1 , apply t c p r o t e i n s i n g e n e r a l .  unreasonable  and  out that the  p r e v i o u s , appendix assumes that the absolute numbers the composition  graphical  polymers.  As a f i n a l comment, i t should be  probably  values  given p r o t e i n . I t might a l s o be  extended t o the a n a l y s i s o f branched  basis  different  Again, a comparison of Bg  f e a s i b l e t c i n c o r p o r a t e the r e l a t i o n s h i p s  of  predict  A. 5.1  2  o  has been done i n f i g u r e  entire  can  f a c t o r , <K : Bg =  This  One  a f u n c t i o n o f the number of r e s i d u e s from eguation 8.1  of the expansion  for  be  Also the comparison  indicates  that  i t would be  i m p o s s i b l e t c d i s t i n g u i s h between reds and random c o i l s a t low molecular  weights,  where the i n t e r f e r e n c e  effects,  from  the  s o l v e n t i n t e r a c t i o n s with the random c o i l s , would tend t o mask the  shape  reduced  anisotropy  of  the reds.  a t the higher molecular  weights.  This problem should be  243  F i g u r e . A . 5 . 1: Evaluation of s o l v e n t effects on ______ ______ P l o t s of r a d i u s of g y r a t i o n Eg, versus number of residues f o r random-coils f o r d i f f e r e n t values of the expansion c o e f f e c i e n t .  244  F i g u r e . JW5. 1..  500 ^residues  1000  245  ____________ In  a  recent  presented a  wide  AMINO ACID COMPOSITION AND publication  Sage  ELASTIN EVOLUTION  and  Gray  data f o r the amino a c i d composition variety  substantial  cf  sources.  In  their  the bony f i s h e s , and I have attempted  of  the e l a s t i n  evolutionary  from  the  succession  numerical a n a l y s i s i t s e l f  different  have  of e l a s t i n  data  their  amount of i n f o r m a t i o n f o r the e l a s t i n  of  is  a  composition  to use the  f o r the e l a s t i n ox" these f i s h e s .  The  carried  out  to  'likeness' an  was  sources  from  develop  (Matsumura e t . § 1 .  expression  (1979)  by  the  following  1 579) :  h  | AA,j -AA |  DIJK = 5>  where AAjj acid for  i ,  A. 6.1  JL<  i s the number of r e s i d u e s per thousand in  a protein s p e c i f i e d  by  j , and AA  to  evaluate  the  relatedness  Holonan  1971,  1973). .Equation A.6.1 Fortran to  Metzger  workers  between p r o t e i n s and,  then, to  ______  species  1968,  H a r r i s and  cn  computer  Teller  me  difference (table  e v o l u t i o n a r y scheme was was  index  A.6.1)..  a  (Fondy  program, f o r 13 groups of f i s h e s , and t h i s allowed a  evaluated  source  a  composition  attempt  Similar  with  compile  was  by k.  by a number of  e x t r a p o l a t e to the r e l a t e d n e s s of the and  i s the number  1K  the same amino a c i d i n the p r o t e i n s p e c i f i e d  e x p r e s s i o n s t o t h i s have been u t i l i z e d  of the amine  matrix Based  for on  their  elastin  this  result  an  derived as shown i n f i g u r e  A.6,1.  No  made t o analyze the r e s u l t s beyond t h i s p o i n t .  246  Table.B,6. 1: D i f f e r e n c e index  O  ~H  i - l  —  <3  ft  £  o;  —  —  —  -i  fy, _,  <n  t-n  m  ' 3  —H  N c_  »n csi  o; CO  composition  r-»  u*> rtca •—• r-.— CO  for e l a s t i n  ^  o  r»  -H  — • " - «  in  .-4  *r  5  «  —  8  *r  ^  *r  f—•  i- p. oi cn « L Ql c  «  s s _ to  I  m  V c c TD-- r  •-•_*»- _i ro  LO C>  r*.  Q. * — 3 (U O  O "3  o>  o  --«  CL  «—  cn o O U U U C ' f •Q <Q C ( J  «— <— -r~ ^> __ f —  Q.  n3  T  247  F i g are. A. 6. 1_: E l a s t i n e v o l u t i o n . E v o l u t i o n a r y seguence f o r e l a s t i n based on t a b l e A.6. 1.  of  higher  fishes  248  Figure* A.  Elastin  13  9  \  6.1.  Evolution  10  12 3  249  I.ITEBATOBE CITED Aaron, B.E., and G o s l i n e , J.M. 1980. O p t i c a l p r o p e r t i e s of s i n g l e e l a s t i n f i b r e s i n d i c a t e a random conformation. Nature i n press. Aaron, B.B., and G c s l i n e , J.M. 1980. E l a s t i n as a random network elastomer: a mechanical and o p t i c a l a n a l y s i s of s i n g l e e l a s t i n f i b r e s . Submitted t o B i o p g l Y ? r s , m  Aaron, B.B., Burns, P.D., 1980. .In p r e p a r a t i o n .  M a r s h a l l , A.G.,  and  Gosline,  J.M.  Alexander, R. McN. 1966. B u b b e r - l i k e p r o p e r t i e s of the inner hinge-ligament of P e c t i n i d a e . J . Exp. Biol.. 44: pp. 119-130. Anderson, J.C. 1976. G l y c o p r o t e i n s of the connective matrix. Int.. Bey, .Conn. . Tiss,.. Res. 7: pp. .251-322. . Anderson, S.O. pp. 633-657.  tisuue  1971. R e s i l i n . Comprehensive Biochemistry.  26C:  Astbury, W.T. 194C. The molecular s t r u c t u r e of the f i b r e s of the c o l l a g e n group. J... I nt... S ocj. Leather Trades' Chemists. 24: pp. 69-92. Ayer, J. P. , 1964. . E l a s t i c t i s s u e . . Int.. Be v. Conn. . T i s s . Beg. 2: pp. 33-100. B a i r a t i , A., and Gotte, 1 . 1977. C a r a t t e r i d e l l a b i r e f r a n g e n z a d e l l e f i g r e e l a s t i c h e . XXXIV Conqresso Nazionale d e l l a S o c i e t a Italiana d i Anatomia. B i a s s u n t i d e l l e relazicni e delle ccsisicazionii Trieste, Italy. Banga, I . , and Balo, J . 1960. The e l a s t i c i t y i n c r e a s i n g property of elastomuco p r o t e i n a s e . Biochim. Biophys. A c t a 40: pp. 367-368. r  Bashaw, J . , and Smith, K.J. 1968. . T h e r m o e l a s t i c i t y of networks i n s w e l l i n g e g u i l i b r i u m . J . Polymer S c i 6: pp. 1041-1050.. A  Baurain, R. , l a R o c h e l l e , J.F., and Lamy, F. 1976.. P h o t o l y s i s of desmosine and isodesmosine by u.v. L i g h t . , E u r , J. Biochem. 67: pp. 155-164. Bear, R.S., Schmitt, F. C. , • and Young, J . A. . 1937. The ultrastructure of nerve axoplasm. Eroc.. Roy.. Soc.. Lon.. Ser^JU 123: pp..505- 515 Bear, R.S. 1942. The long X-ray d i f f r a c t i o n c o l l a g e n . J . . Am... Chgm._ .Soc. 64: pg. 727. . Bear,  R.S.  spacings  1944. .X-ray d i f f r a c t i o n s t u d i e s on p r o t e i n  of  fibres.  250  I: the l a r g e f i b r e - a x i s p e r i o d of c o l l a g e n . . J ^ .Am .Chem___. Soc. 66: pp 1297-1305. :  Bedford, G.E., and K a t r i t z k y , A.R. 1963. Proton magnetic resonance s p e c t r a of degradation products from e l a s t i n . Nature 200: pg. . 652. Bennett, H.S.. 1950.. The m i c r o s c o p i c a l investigation of biological m a t e r i a l s with polarized light, i n "McLunq s handbook of m i c r o s c o p i c a l technique". T h i r d e d i t i o n . Ruth M.J. Ed. Paul B. Hoeber, Inc. New York. pp. 591-677. 1  Boucek, R.J., Noble, N.C., and Woessner, J.F. 1959. P r o p e r t i e s of f i b r o b l a s t s , i n "Connective tissue, thrombosis, and a t h e r o s c l e r o s i s " . Page, I.H. Ed. Academic Press. New York. Pp. 193-21 1. . ;  Bowen, I . J . 1953. P h y s i c a l s t u d i e s on a s o l u b l e p r o t e i n obtained by the degradation o f e l a s t i n with urea. Biochem. J.. 55: pp. 766-768. Bradbury, E.M., and R a t t l e , H.W.E., 1972..Simple computeraided approach f o r the analyses of the n u c l e a r magnetic resonance s p e c t r a c f h i s t o n e s . Eur.. J_. Biochem. 27: pp, 270281. Brant, D.A., and F l o r y , P.J.. 1965a. The c o n f i g u r a t i o n of random p o l y p e p t i d e c h a i n s . . I : experimental r e s u l t s . . J . . Am.. Chem. Soc.. 87: pp. . 2788-279 1. _ Brant, D.A., and F l o r y , P.J. 1965b. The c o n f i g u r a t i o n of random p o l y p e p t i d e c h a i n s . I I : t h e o r y . J_. Am.. Chem.. Soc._. 87: pp. .2791-2800. Bugenberg de Jong, H.G. . 1949. C r y s t a l l i z a t i o n - c o a c e r v a t i o n flocculaticn, i n " C o l l o i d s c i e n c e " volume 2*. Kruyt, H.R. Ed. E l s e v i e r P u b l i s h i n g Co. Amsterdam. Pp. 232-258. Carnes,W.H., Hart, M. L.,, and Hodgkin, N.M. 1977. . Conformation of a o r t i c e l a s t i n r e v e a l e d by scanning e l e c t r o n microscopy o f d i s s e c t e d s u r f a c e s . Advances i n Exj__. Med.. And B i o l . . 79: pp. 61-70. Carton, R.W. , Dainauskas, J . , and C l a r k , J.W. 1962. The i s o l a t i o n and p h y s i c a l p r o p e r t i e s df s i n g l e elastic fibres. Z§ii§£ation P r c c . 19: pg. 383. Carton, R.W., Dainauskas, J . , and C l a r k , J.W. 1962. E l a s t i c p r o p e r t i e s c f s i n g l e e l a s t i c f i b r e s . J . -Appl, Physiology. 17: pp. 547-551. Ceccorulli, G., Scandola, M., C a l o r i m e t r i c i n v e s t i g a t i o n of some M o f l . Y S § § t 16: pp. 150 5- 1512. c  r  and P e z z i n , G.. 1977. elastin-solvent systems.  251  Charm, S.E., and Kurland, G.S. 1974. "Elood flow and M i c r o c i r c u l a t i o n " . . John-Wiley and Sons L t d . New York. 312 pp. Chrambach, A., and Ecdbard, D. 1971. P o l y a c r y l i m i d e g e l e l e c t r o p h o r e s i s . S c i e n c e ^ 172: pp 440-451. Clark, G., Coalson, E.E., and Nordguist, E.E. 1973. Methods f o r c o n n e c t i v e t i s s u e , i n _________ procedures". C l a r k , G. Ed. Williams and W i l k i n s Co. . E a l t i m o r e . Pp. 215-217.. C l e a r y , E.G., and C l i f f , W.J. 1978. The s u b s t r u c t u r e e l a s t i n . . Exp_ And Molec. Path. 28: pp. 227-246.  of  Cohn, E.S., and E d s a l l , J . I . 1943. Density and apparent s p e c i f i c volume cf p r o t e i n s , i n " P r o t e i n s , amino acids_ and £_£ti<l____ Cohn, E.J. , and E d s a l l , J.T. Eds. Hafner P u b l i s h i n g Co. New York. .Pp. . 370-396. . Cotta-Pereira, G. E c d r i g o , F.G., and D a v i d - F e r r e i r a , J.F. . 1977. The e l a s t i n system f i b r e s . Advances i n Exp_ Med_ And _____ 79: pp. 19-30. Cox, S.C., and L i t t l e , K. 1962. An e l e c t r o n microscope study of e l a s t i c t i s s u e . Prop. Boy. Soc. Ser.B. . 155: pp. 232-242. Cox, B., S t a r c h e r , B., and alpha-elastin results i n _____ 317: pp. 209-213.  Urry, D. 1973, f i b r e formation.  Coacervation o f Biochim..Biophys.  Cox, B., S t a r c h e r , B. , and Urry, B. 1974.. Coacervation of tropoelastin results i n f i b r e f o r m a t i o n . J _ B _ o l _ Che__-249: 997-998. Cozzone, P., T o n i o l o , C , and J a r d e t z k y , 0. 1980. Nmr study o f the main components o f clupienes and their possible i n t e r a c t i o n with n u c l e i c a c i d s . Febs. ________.110: pp..21-24. Davril, M., Han, K., Guay, M., and Lamy, F. . 1979. P h o t o l y s i s of c r o s s l i n k e d peptides from e l a s t i n of porcine aorta. Febs. L e t t e r s . 98: pp. 128-134. Dayhoff, M. 1976. A t l a s o f p r o t e i n sequence and s t r u c t u r e , _ol_ 5_ supp. 2_ N a t i o n a l Biomedical Bes. Foundation. Washington D.C. Pg.267. Dempsey, E.W. submicroscopic 520.  1952. . The chemical c h a r a c t e r i z a t i o n and the s t r u c t u r e of e l a s t i c t i s s u e . S c i e n c e , 116: pg. .  Diehl, J.M., and I t e r s o n , G. 1935. Die doppelbrechung von c h i t i n s e h n e n . __________ 73: pp..142-146. D o r r i n g t o n , K., Grut, W., and McCrum,  N.G.  1975.  Mechanical  252  s t a t e of e l a s t i n . Nature. 255:  pp.  Dorrington, K.L., and McCrum, N.G. Biopolymers 16: pp. . 120 1-1222.  476-478. 1977.  E l a s t i n as a rubber.  Dwek, B.A. 1973. HNuclear magnetic resonance i n biochemistry.^ Oxford U n i v e r s i t y Press. Oxford._629 pp..  1  Egmond, M.S., Rees, D., Welsh, J. , and W i l l i a m s , B.J.P. 1979. ifi-nmr s t u d i e s on g l y c o p h o r i n and i t ' s c a r b o h y d r a t e - c o n t a i n i n g t r y p t i c p e p t i d e s . Eur. J._. Biochem._ . 97: pp. .73-83. Ellis, G.E., and Packer, K.J. 1976. Nuclear s p i n - r e l a x a t i o n s t u d i e s of hydrated e l a s t i n . Biopolymers^ 15: pp. 813-832.. F i e l d , J.M., Eodger, G.W., Hunter, J . C , Seraf i n i - F r a c a s s i n i , A., and Spina, M. . 1 9 7 8 . . I s o l a t i o n of e l a s t i n from the bovine a u r i c u l a r c a r t i l a g e . . Arch. Biochem. Biophys^ 191: pp. 705-713.. F i n l a y , J.B., and Stevens, F.S. 1973. . The f i b r o u s component of the bovine lig.ament.um nuchae observed i n the scanning e l e c t r o n microscope. J_. Microscopy^. 99: pp. 57-63. Fisher, J. 1979. Ultrastructure Histochemica. 65: p p . 8 7-98.  of  elastic  fibres.  Fleming, W.W., Sullivan, C.E., and Torchia, C h a r a c t e r i z a t i o n of the molecular motions in aortic elastin by C- H double magnetic Biopolymers. 19: pp. 59 7-617. l 3  l 3  l  F l o r y , P.J. 1949. The c o n f i g u r a t i o n of r e a l polymer Chenu PhySi 17: pp. 303-310. F l o r y , P.J. 1953. " P r i n c i p l e s of polymer U n i v e r s i t y P r e s s . London. 672 pp.  Acta..  D. A. 1980. C-labelled resonance. chains,. J»  chemistry."  Cornell  Fondy, T.P., and Holonan, P.D. 1 9 7 1 . . S t r u c t u r a l s i m i l a r i t i e s w i t h i n groups of p y r i d i n e n u c l e o t i d e - l i n k e d dehydrogenases. J ^ Theor. . B i d . 31: pp. . 229-244. . F o s t e r , J.A., Bruenger, E. , Gray, W.B. , and 1973.. I s o l a t i o n and amino a c i d seguences p e p t i d e s . C . Biol^.Chem^.2 48: pp. .2876-2879.  Sandberg, L.B. . of t r o p o e l a s t i n  F o s t e r , J.A., S h a p i r o , E., Voynon, E., Crcmbie, G., F a r i s , B., and F r a n z b l a u , C. 1975.. I s o l a t i o n of s o l u b l e elastin from lathyritic chicks. Comparison to t r o p o e l a s t i n from copper d e f f e c i e n t p i g s . B i o c h e m i s t r y . 14: pp. 5343-5347. F o s t e r , J.A., Mecham, E.P., and F r a n z b l a u , C. 1976. A high molecular weight s p e c i e s of s o l u b l e e l a s t i n . Biochem. Biophys. Re s C c m m u n 7 2 ; pp. . 1399- 1406. .  253  Foster, J.A., Mecham, E., Imberman,M., F a r i s , B., and F r a n z b l a u , C. 1970. A high molecular weight s p e c i e s of s o l u b l e e l a s t i n . Advances i n Exp. .Med. And E i o l ^ 79: pp. .351-369. Fox, T.G., Fcx, J . C , and F l o r y , P.O. 1951. The e f f e c t of r a t e of shear on the viscosity of dilute solutions of p o l y i s o b u t y l e n e . J.. Am. Chem. Soc. 73: pp. 1901-1904.. Franzblau, 659-712.  C. .1971. E l a s t i n . .Comprehensive Biochemistry^.26C:  Eraser, E.B.D., and McCrae, T.P. 1973.. "Conformation f i b r o u s p r o t e i n s . " Academic Press Inc. New York..628 pp..  in  Frey-Wyssling, A. 1953. "Submicroscopic protoplasm." E l s e v i e r , Amsterdam. 237 pp.  of  morphology  G o s l i n e , J.M., Weis-Fcgh, T., and Yew, F.F. 1975. E e v e r s i b l e structural changes i n a hydrophobic protein, e l a s t i n , as i n d i c a t e d by f l o u r e s c e n c e probe a n a l y s i s . . Eiopolymers. .14: pp. 1811-1826. . G o s l i n e , J.M. 1977. The r o l e of hydrophobic interactions i n the s w e l l i n g of e l a s t i n . . Advances i n Exp__. Med. And Biol.. 79: pp. 415-421. G o s l i n e , J.M. . 1978.. The temperature-dependent e l a s t i n . Biopolymers^ 17: pp..697-707.  swelling  of  Gosline, J.M., and French, C.J.. 1979.. Dynamic mechanical p r o p e r t i e s of e l a s t i n . Biopolymers. 18: pp. 2091-2103. Gotte, L., M e n e g h e l l i , V., and C a s t e l l a n i , A. 1965. E l e c t r o n microscope observations and chemical analyses of human e l a s t i n , i n " S t r u c t u r e and f u n c t i o n of connective and s k e l e t a l tissue].'. Jackson, S.F., Harkness, £.D., P a r t r i d g e , S. M. , and T r i s t a m , G.B. Eds. Butterworths. London. Pp. 93-100. Gotte,. L. , P e z z i n , G., and S t e l l a , G.D..1966. Mechanical p r o p e r t i e s of e l a s t i n , dry and swollen i n d i f f e r e n t solvents, in *J_ Bipchimie et p h y s i o l c q i e du t i s s u con-jonctif." Comte, P. Ed. Ormeco. Lyon. Pp. 145-154. G o t t e , L., G i r o , u l t r a s t r u c t u r a l  E e s . .46: p p .  M.G., V o l p i n , D. , of o r g a n i z a t i o n  and Home, E.W. 1974.. The elastin. J Ultrastructure x  23-33..  Gotte, L. , V o l p i n , D., Home, B.W., and Mammi, M. 1976. Electron microscopy and o p t i c a l diffraction of e l a s t i n . Micron. 7: pp. 95-102. Grant. M.E., Steven, F.S., Jackson, D.S., and Sandberg, L.B.. 1971. Carbohydrate.content of i n s o l u b l e e l a s t i n s prepared from adult bovine and c a l f ligament urn nuchae and t r o p o e l a s t i n  254  i s o l a t e d from c o p p e r - d e f f e c i e n t 121: pp. 1S7-203.  porcine  aorta.  Biochem.  J_  Gray, W.H., Sandberg, L.B., and F e s t e r , J.A..1973. Molecular model f o r e l a s t i n s t r u c t u r e and f u n c t i o n . Nature^ 246: pp. 461-466. Gross. J . 1949. The s t r u c t u r e of e l a s t i c t i s s u e as s t u d i e d with the e l e c t r o n microscope..J_ Ex__ Med_ 89: pp. 699-708. Grut,W., and McCrum, N.G. Nature. ,251: pg. 165.  1974. L i g u i d drop model of  elastin.  Guth, £., James, H.M. , and Mark, H. .1946. The k i n e t i c theory of rubber e l a s t i c i t y , i n "Advances i n c c l l o i d science^. I I _ I n t e r s c i e n c e . New York. . Pp. . 253-281. _ Hall, D.A., and Czerkawski, J.W. 1961. The r e a c t i o n betweeen e l a s t a s e and e l s a t i c t i s s u e , 4. S o l u b l e elastins..Biochem. J_ 80: pp. 121-127. HA11, D.A. . 1976. . The p r e p a r a t i o n of s o l u b l e e l a s t i n s , i n _The methodology of connective t i s s u e r e s e a r c h . " Hall, D.A.. Ed. Joyson-Bruvvers Ltd..Oxford..Pp. 105-108. Harkness, M.L.R., Harkness, R.D., and McDonald, D.A. 1957..The collagen and e l a s t i n content of the a r t e r i a l w a l l i n the dog. Proc. Roy. Soc. Ser.B. 146: pp. .54 1-551. H A r r i s , G.E., and T e l l e r , D.S..1973..Estimation of the primary seguence homology from amino acid composition of evolutionarily r e l a t e d p r o t e i n s . J _ Theor_ B i c l . 38: pp. 347362. Hart, M.L., Beydler, S.A., and Carnes, W.H.. 1978.. The fibrillar structure of a o r t i c e l a s t i n . Scanning electron microscopy_ V o l _ I I _ S.E.M. .Inc. AMF O'Hare, I l l o n o i s . Pp. 2125. . . Hass, G.M. 365. .  1939. E l a s t i c t i s s u e . Arch_ Pathology. 27: pp. .334-  Henderson, L.E., O r s o l l a n , S., and Konigsberg, W. 1979. A micromethod f o r the complete removal c f dodecyl sulphate from p r o t e i n s by i o n - p a i r e x t r a c t i o n . Anal_ Biochem. 9 3: pp. 153157. Hoeve, C.A.J., and F l o r y , P.J. 1958. The e l a s t i c e l a s t i n . J_ Am. Chejn. Scc_ 80: pp. 6523-6525. .  p r o p e r t i e s of  Hoeve, C.A., and F i e r y , P.J. 1974. The e l a s t i c p r o p e r t i e s of e l a s t i n . Biopclymers. 13: pp. 677-686. Huggins, M.L.  1942. Ihe v i s c o s i t y of d i l u t e s o l u t i o n s of l o n g -  255  chain molecules. IV: Dependence on c o n c e n t r a t i o n . J _ Am. _____ Soc_ 6 4: pp. 2 716-2718 Jamiescn, A.M., Downs, C.E., and Walton, A.G. 1972. S t u d i e s o f elastin c o a c e r v a t i o n by guasielastic light scattering. ________.Eiophys. . Acta. .27 1: 34-47. Jamieson, A.M., S i m i l - G l o v a s k i , E., Tansey, K., and Walton, A.G. 1S76. Studies of e l a s t i n c o a c e r v a t i o n by g u a s i e l a s t i c l i g h t s c a t t e r i n g . .Faraday Disc. .Chem. Soc_ 61: pp. 194-204. Joussot-Dubien, J . , and Houdard-Pereyre, J . 1969. Photolyse de la pyridine en s o l u t i o n aguese. Bull._Soc..Chim. France, 8: pp. 2619-2623. Kadar, A. 1977. Scanning electron microscopy of p u r i f i e d e l a s t i n with and without ezzymatic d i g e s t i o n . Advances i n Ex__ Med_ And B i o l . 79: pp. 45-53. Kakivaya, S.R., and Hoeve, C.A.J. 1975. The g l a s s point of e l a s t i n . ______ _____ Acad. S c i ^ 72: pp. 3505-3507.. K a r r e r , H.E., and Cox, J . 196 1. An e l e c t r o n microscope study of the a o r t a i n young and ageing mice. J _ i n f r a s t r u c t u r e Res. . 5: pp. .1-13. . K e i t h , D.A., Paz, M.A., and G a l l o p , P.M. 1979. D i f f e r e n c e s i n valyl-proline seguence content i n e l a s t i n from v a r i o u s bovine t i s s u e s . .Biochim. Bicphys. Res. Commun. 87: pp. . 1214-1217. K e l l y , R.E., and R i c e , R.V.. 1967. Abductin: A rubber-like p r o t e i n from the i n t e r n a l t r i a n g u l a r hinge ligament og _______ S c i e _ c e _ 155: pp. 2C8-21C. Khaled, Md. A., Renugopalakrishnan, V., and Urry, D.W. 1976. Pmr and c o n f o r m a t i o n a l energy c a l c u l a t i o n s of repeat p e p t i d e s of t r o p o e l a s t i n : The t e t r a p e p t i d e . J_ Am_ Chem. Soc. 98: 75477553. Kirk, J . E . 1959. Mucopolysaccharides of a r t e r i a l t i s s u e , i n "The a r t e r i a l ______.Lansing, A.S..Ed..Williams and W i l k i n s . B a l t i m o r e . Pp. 161-191. . Kolpak, H. 1935. Rontgenstrukturuntersuchungen uber e l a s t i c h e s gewebe unter besonderer b e r u c k s i c h t i g u n g der dehnung und e n t g u e l l u n g . K o l l o i d Z_ 73: pp. . 129-142. . Kornfeld-Poullain, N., and Robert, L.. 1968. Effet de differents s o l v a n t s organiques s u r l a degradation a l k a l i n e de l ' e l a s t i n e . . B u l l _ .Soc_ _____ B i o l . 50: pp. 759-771. Kurata, M., Stockmayer, W.H., and Roig, A.. 1960. Excluded volume e f f e c t of l i n e a r polymer molecules. ,J..Chem. Ph_s_.33: pp. 151-155.  256  Kurata, M., and Stockmayer, W.H. 1962. Polymer Phys. Japan.,5: pp..23-26..  gept.  On  Prqgr.  In  Lansing, A.L., Eosenthal, T., Alex, M. , and Dempsey, E..1952. The s t r u c t u r e and chemical c h a r a c t e r i z a t i o n of e l a s t i c fibres as r e v e a l e d by e l a s t a s e and by e l e c t r o n microscopy. Anat. Rec. 114: pp. 555-576. Lemieux, R.O., and von R u d l o f f , E. 1955. Periodatepermanganate o x i d a t i o n s . I. O x i d a t i o n s of o l e f i n s . Can.. J_ Chem_ 33: Fp. 17011709. Lent, R.W. , Smith, B. , S a l l e d o , B., F a r r i s , G., and F r a n z b l a u , C. .1969. Studies on the r e d u c t i o n of e l a s t i n . I I . Evidence f o r the presence of a l p h a - a m i n o a d i p i c a c i d -semialdehyde and i t ' s a l d o l condensation product. B i o c h e m i s t r y . . 8 : pp..2837-2845. Lo, S., R u s s e l l , J.C., and T a y l o r , A.W. . 1970. .Determination of glycogen in s m a l l t i s s u e samples. J_ _ p p l _ Physiology. 28: pp. 234-236. Long, M.M.  . 1980.  Personal  communication.  L y e r l a , J.R., and T o r c h i a , D.A..1975. Molecular mobility and structure of e l a s t i n deduced from the s o l v e n t and temperature dependence cf C-magnetic resonance relaxation data. B i o c h e m i s t r y . 14: pp. . 5 175-5183. . 13  Mammi, M., Gotte, L., and P e z z i n , G. 1970..Comparison of s o l u b l e and n a t i v e e l a s t i n conformations by f a r u.v. Circular d i c h r o i s m . Nature_.225: pp. 380-381. Mark, J.E. 1S76. The dependence of the s w e l l i n g of e l a s t i n on e l o n g a t i o n and i t ' s importance i n f l o u r e s c e n c e probe a n a l y s i s . Biopolymers. 15: pp. 1853-1856. Marshall, A.G. 1979.. S p e c t r o s c o p i c dispersion versus absorption. A new method f o r d i s t i n g u i s h i n g a d i s t r i b u t i o n i n peak p o s i t i o n from a d i s t r i b u t i o n in line width. J_ Ph_s_ Chem.. 83: pp. 52 1-524. Maruyama, K., N a t o r i , R., and Nonomura, Y. 1976. p r o t e i n from muscle. Nature. 262: pp. .58-60. .  A new  elastic  Matsumura, T., Hasegawa, M., and S h i g e i , M. 1979. C o l l a g e n b i o c h e m i s t r y and the phylogeny of echinoderms. . Cgmp. Biochem.. ________ E_ 62: pp. 101-105. Metcalfe, J.C. 1970. Nuclear magnetic resonance spectroscopy, i n ^ P h y s i c a l p r i n c i p l e s and techniques cf p r o t e i n chemistry^. _art B_ Sydney, J.L. Ed. Academic P r e s s . New Ycrk. Pp. 275363. .  257  Metzger, H. , Shapiro, M.B,, Mosimann, J.E.,a nd Vinton, J.E. 1968. Assessment of compositional relatedness between p r o t e i n s . .Nature. .219: pp. .1166-1168.. Meyer, K.H., and F e r r i , C. 1936. Die e l a s t i s c h e n e i g e n s c h a f t e n der e l a s t i s c h e n und der kollagenen f a s e r n und i h r e molekulare deutung. _________ Arch. Ges_ P h y s i o l . 238: pp.. 78-90. Meyer, K., Davidson, E., L i n k e r , A., and Hoffman, P..1956..The acid mucopolysaccharide of connective-tissue. Biochim. Biop__s_ Acta. 21: pp. 506-518. M i l l e r , W.G., Brant, D.A., and configurations of polypeptide pp. 67-80.  Flory,P.J. 1967. .Random-coil co-polymers. , J . Mol. . B i o l . 23:  M i l l e r , W.G., and Goebel. 1968. Dimensions of c o i l s . Biochemistry. 7: pp. 3925-3935.  protein  random  Minns, J.R., and Steven, S.F. 1974. Scanning electron microscopy of s t r e t c h e d e l a s t i c f i b r e s . Micron. 5: pp. 127133. Mistrali, L. , V o l p i n , D., G a r i b a l d o , G.B., and C i f f e r r i , A. 1971. Thermodynamics of e l a s t i c i t y i n open systems. . J_. Phys. Chem. 75: pp. . 142-149. . Moczar, M., Mcczar, E., and Robert, L. 1S79. Peptides obtained from elastin by h y d r o l y s i s with agueous e t h a n o l i c potassium hydroxide. __________ Res_ 6: pp. 207-213. Morgan, R.J., and T r e o l a r , L.R.G. 1972. Photoelastic studies of polymers and co-polymers i n the rubbery s t a t e . J_.Pol.y__ S c i . A2. 10: pp. 51-69. Mukherjee, D.P., Kagan, H. M., Robert, E.J., and Franzblau, C. 1976. E f f e c t s of hydrophobic e l a s t i n l i g a n d s on the s t r e s s s t r a i n p r o p e r t i e s of e l a s t i n f i b r e s . _____ T i s s . _es_ 4: pp. 177-179. Narayanan, A.S. , and Page, R.C. . 1976._ Demonstration of a precursor-product r e l a t i o n s h i p between s o l u b l e and crosslinked elastin, and the b i o s y s n t h e s i s of the desmosines i n v i t r o . J . _____ _____.251: pp. . 112 5-11 30. . OPlatka, A., Michaeli, I., and Katchalsky, A.. Thermoelasticity of cpen systems. J_ Polym. . S c i . .46: pp. 371. P a r t r i d g e , S.M., Davis, H.F., and A d a i r , G.S. 1955. proteins derived from partial hydrolysates of Biochenu J . 61: pp. .11-21. Partridge,  S.M.  . 1962.. E l a s t i n ,  in  "Advances  in  1960. 365-  Soluble elastin. protein  258  chemistry...^. 17:  pp.  227-  302..  Partridge, S.M. , Thomas, J . . E l s d e n , D.F. 1965. .The nature of the c r o s s - l i n k a g e s i n e l a s t i n , i n " S t r u c t u r e and function of connective and s k e l e t a l t i s s u e " . Jackscn, S.F., Harkness, R.D., P a r t r i d g e , S.M., and T r i s t r a m , G.R. Eds. Butterworths. London. Pp. . 88-92. . P a r t r i d g e , S.M. 1967a. D i f f u s i o n of s o l u t e s i n e l a s t i n Biochinu BiP£h._y_s.i Acta.. 140: pp. 132-135.  fibres.  Partridge, S.M. 1967b. Gel f i l t r a t i o n using a column packed with e l a s t i n f i b r e s . . Nature, 213: pg. 1123... P a r t r i d g e , S.M. 1968. . E l a s t i n s t r u c t u r e and biosynthesis, in "Symposium on f i b r e us proteins'.'. Crewther, W.G. Ed. Plenum Press. New York. Pp. 246-264. P a r t r i d g e , £.M., and Whiting, A.H. . 1979. Molecular weights and stokes r a d i i of s o l u b l e e l a s t i n s . Bigchim. .Biophys. Acta. .576: pp. .71-80. P a s g u a l i - R o n c h e t t i , I., F o r i n i e r i , C., Baccarini-Contri, M., and V o l p i n , D. .1979. The u l t r a s t r u c t u r e of e l a s t i n revealed by f r e e z e - f r a c t u r e e l e c t r o n microscopy. Micrcn.. 10: pp. 88-99. Pathrapamkel, A.A., Hart, M.L., Winge, A.R., and Carnes, W.H. . 1977. The b i o s y n t h e s i s of e l a s t i n by an a o r t i c medial cell c u l t u r e . Advances i n Exp. . Med. And E i o l . . 79: pp. 397-412, Pfeiffer, H.H. 1943. P o l a r i s a t i o n s mikreskopische messugen an k o l l a g e n f i b r i l l e n i n v i t r o ^ . Arch. . Exper.. Z e l l f p r s c h . Besonders Gewebeluecht, 25: pp..92-104.. Q u i n t a r e l l i , G., B e l l o c i , M., and Z i t o , E. 1973.. S t r u c t u r a l f e a t u r e s cf I n s o l u b l e e l a s t i n . His tcchemie^.. 37: pp. 49-60. . Ramachandran, G,N. , and Santhanaot, e l a s t i n . . Proc. Ind.. Acad., S c i . A.. 45: Ramachandran, G.N. 1963. ^Aspects Academic Press. London. Pg. 39,.  M.S. 1957. S t r u c t u r e pp. 124- 132. of  protein  of  structure,  11  Rapaka, R.S. , and Urry, D.W. . 1978. Coacervation of s e g u e n t i a l p o l y p e p t i d e models of t r o p o e l a s t i n . Int.. J.. Pep. Prot^ Res.. 11 : pp. 97-108. Reisner, A.H., and Rowe, J . 1969. Intrinsic v i s c o s t y of randomly c o i l e d polypeptide of 30,000 daltons and i t ' s e f f e c t on the s o l u t i o n of the Mark-Houwink equation..Nature. 222: pp. 558-559. Robert, B., S z i g e t i , M. , Derouette, J . C , and Robert, L. 1971.. Studies on the nature of the m i c r c f i b r i l l a r component of  259  elastic  f i b r e s . _____ J ^ . Bicchem. 21: pp. .507-516.  Robert, L., and Hornebeck, W. . 1976. Preparation of i n s o l u b l e and s o l u b l e e l a s t i n s , i n '_'The _ _ _ _ _ _ o l c _ _ of c o n n e c t i v e t i s s u e r e s e a r c h " . H a l l , D.A. Ed. Joyscn-Bruvvers l t d . Oxford. Pp. 81103. Robert, L. , 1977. E l a s t i c chemistry. Advances i n Exp. Med. And B i o l . .79: pp. 139-144.. Romhanyi, G. 1958. Submicroscopic s t r u c u t r e of e l a s t i c f i b r e s as observed i n the p o l a r i z a t i o n microscope. Nature. 182: pp. 929-930. . Rhodin, J . , and Dalhamn, T. 1955. E l e c t r o n microscopy of c o l l a g e n and e l a s t i n i n the lamina p r o p r i a of the t r a c h e a l mucosa of r a t . Exp. . C e l l . Res. 9: pp. 371-375. . Ross, R., and B o r n s t e i n , P.. 1969. The e l a s t i c f i b r e . . I . The s e p e r a t i o n and p a r t i a l c h a r a c t e r i z a t i o n of i t ' s macromolecular components. J_ C e l l B i o l . . 4 0 : pp. 366-379. Ross, R., F i a l k o w , P.J., and Altman, 1.. 1977. morphogenesis of e l a s t i c f i b r e s . . Advances i n Exp_._ed_ _____ .79: pp. 7- 17. .  The And  Rosenbloom, J . , Harsch, M., and C y w i n s k i , A.. 1980. Evidence that tropoelastin i s the primary precursor in elastin b i o s y n t h e s i s . . J . B i o l . .Chem. .255: pp. 100-106. Sage, H., and Gray, W.R. e l a s t i n . I.. Phylcgenetic B_ 64: pp. . 3 13-327.  1979. Studies on the e v o l u t i o n of d i s t r i b u t i o n . Cc_p_ ________ ________  Sandberg, L.B., Weissman, N. , and Smith, D.W. 1969..The purification and p a r t i a l characterization of a soluble elastin-like protein from copper d e f f e c i e n t porcine a o r t a . B i o c h e m i s t r y . 8: pp..2940-2945.. Sandberg, L.B., Weissman, N., and Gray. W.R. . 1971.. S t r u c t u r a l features of t r o p o e l a s t i n r e l a t e d to the s i t e s of c r o s s - l i n k s i n a o r t i c e l a s t i n . . B i o c h e m i s t r y . 10: pp. 52-56. Sandberg, L.B., Gray, W.R., and Bruenger, E. 1972. S t r u c t u r a l studies of a l a n i n e and l y s i n e r i c h r e g i o n s of porcine a o r t i c t r o p o e l a s t i n . _________ Biophys. Acta. . 285: pp. . 453-458. Sandberg, L.B. .1976. E l a s t i n s t r u c t u r e i n h e a l t h and Int_ Rey_ __________ ____ 7: pp. 160-210.  disease.  Sandberg, L.B., Gray, W.R., F o s t e r , J.A., T o r r e s , A. R. , and A l v a r e z , V.L..1977. Advances i n Exp_ _____ And B i o l . 79: pp. 277-284.  260  Saunders, D.W. 1957.. The p h o t o e l a s t i c p r o p e r t i e s of c r o s s l i n k e d amorphous polymers. I I I . I n t e r p r e t a t i o n of r e s u l t s on polythene, polymethylene, natural rubber, and gutta-percha. Trans, .Faraday Soc...53: pp. .860-870. S c h e i n , J . , C a r p o u s i s , A., and Eosenbloom, J . . 1966. Evidence that tropoelastin e x i s t s as a random-coil. Advances i n Ex£. MecL And Eiol.. 79: pp. .727-740. Schellman, J.A., Schellman, C. . 1963.. The conformation of p o l y p e p t i d e c h a i n s i n p r o t e i n s , i n 2_The p r o t e i n s " , Neurath, H. . Ed. Academic Press. .New York. Pp. . 1-37. Schimmel, P.R., and F l o r y , P . J . 1S68. Conformational e n e r g i e s and c o n f i g u r a t i o n a l s t a t i s t i c s of c o - p o l y p e p t i d e s c o n t a i n i n g 1 - p r o l i n e . J_, Mcl.. Bicl_. 34: pp. 105-120. . Schmidt, W.J. 1939.. Einge u n t e r r i c h t s v e r s u c h e zur doppelbrechung der e l a s t i n f a s e r n . K o l l o i d JZ. 89: pp. 233-237. S e r a f i n i - F r a c a s s i n i , A., and T r i s t a m , G.R. 1966. E l e c t r o n microscope study and amino acid analysis on human a o r t i c e l a s t i n . Prgc. Roy... Sec.. Edinburgh, J3..69: pp..334-337. S e r a f i n i - F r a c a s s i n i , A., F i e l d , J . M. , and Spina, M. 1976. . The macromolecular organization of the e l a s t i n f i b r i l . J;. Hoi.. B i o l , . 100: pp. 73-84. S e r a f i n i - F r a c a s s i n i , A., F i e l d , J. M., Smith, J.W., W.G.S. . 1977. . The u l t r a s t r u c t u r e and mechanics ligaments. J± U l t r a s t r u c t r . Res, 58: pp. 244-251.  Stephens, of e l a s t i c  S e r a f i n i - F r a c a s s i n i , A., F i e l d , J.M., and Hinnie, J..1978..The primary f i l a m e n t of bovine e l a s t i n . J_. U l t r a s t r u c t r . Res. 65: pp. . 190- 193. Shad wick, R. .1980. .A p r o t e i n elastomer from Octupus a r t e r i e s . In p r e p a r a t i o n . Simha, R. .1940. The i n f l u e n c e o f Brownian movements v i s c o s i t y of s o l u t i o n s . J ^ Phys.. Chem^. 44: pp. .25-34.  on the  Smith, D.W., Abraham, P.A., and Carnes, W.H. 1975. C r o s s linkage of s a l t - s o l u b l e e l a s t i n i n v i t r o . Biochem. Biophys,, C c y a n . 66: pp. 693-899. Starcher, B.C., Saccomuni, G., and Urry, D.W. 1973,. Coacervation and i o n - b i n d i n g s t u d i e s on a o r t i c elastin. Biochim. Biophys. . Acta. .310; pp. 481-486. Stockmayer, W.H., and Fixman, M.J. 1963. On the e s t i m a t i o n o f unperturbed dimensions from i n t r i n s i c v i s c o s i t i e s . J.. Polyiru . S c i . C 1 : pp. .137-141. ;  261  Stokes, A.E. . 1963. . "The theory of the o p t i c a l p r o p e r t i e s of inhomoqeneous materials.". . E. And E.N. Spon L t d . . London. 17 2 pp. Stothers, Press. New  J.B. 1972. Carbon-13 York. .253 pp. .  nmr  spectroscopy. Academic  Sykes, B.C., and P a r t r i d g e , S.M. . 1S72. I s o l a t i o n of a soluble elastin from lathyritic chicks. Bioche__ J _ 130: pp. 11711172. Tamburro, A.M., Guantieri, V., Daga-Gordini, Abatangelo, G. 1977. Conformational transitions e l a s t i n . BiccJ_im_.. Bioph_s__ Acta_ 492: pp. 370-376. Tanford, C. 1961. "Physical Wiley. New York. 710 pp.  chemistry  of  D., and of a l p h a -  macromolecules".  T a n f o r d , C. , Kawahara, K., and Lapanje, S. . 1966. of random-coil b e h a v i c u r . J . Bigl_.Chem. 241: pp.  Demonstration 1921-1923.  Tanford, C., Kawahara, K., and Lapanje, S. .1967. P r o t e i n s as random coils. I. I n t r i n s i c viscosities and sedimentation coeffecients i n concentrated guanidine h y d r o c h l o r i d e . J_ Am_ Chem_ Soc_ 89: pp. 729-736. Tanford, C. 1973. "The hydrophobic Sons. New York. 200 pp.  effect__  John  Thomas, J . , E l s d e n , D., and Partridge, S.. structure of two aajor degradation products l i n k a g e s i n e l a s t i n . Nature. 200: pp.651-652.  Wiley  1963. from  and  Partial cross-  Timasheff, S.N., Susi, H., Tcwnend, E., Stevens, L., Gorbunoff, M.J., and Kymosinski, T.F. 1967.. A p p l i c a t i o n of C D . . And I.E. Spectroscopy t o the conformation of p r o t e i n s i n s o l u t i o n , i n "Conformation of biopolymers__ Eamachandran, G.N.. Ed. Academic Press. London. Pp. 173-196. T o r c h i a , D.A., and P i e z , K.A..1973..Mobility of e l a s t i n c h a i n s as determined by C-nmr. J_ Mgl_ B i o l _ . 7 6 : pp. 419-423.. 13  T r e l o a r , L.E.G. .1975.. _The ph_sics Clarendon Press. Oxford. .310 pp. .  cf  rubber  elasticity.^  Urry, D.W., and O h n i s h i , T. 1574. Eecurrence of t u r n s i n repeat p e p t i d e s cf e l a s t i n : the hexapeptide ala-pro-gly-valg l y - v a l seguences and d e r i v a t i v e s , i n " P e p t i d e s , p o l y p e p t i d e s ^ and proteins__. Blcut, E.E. , Bovey, F.A., Goodman, M. , and Lotan, N. Eds. John Wiley and Sons. New York. Urry, D.W., Long, M.M. 1976a. Conformations of the r e p e a t p e p t i d e s of e l a s t i n i n s o l u t i o n . C_E_C_ C r i t . . B e y . Bigche__ 4: pp. 1-45.  26 2  Urry, D.W., Okamotc, K. , H a r r i s , R.D., Hendrix, C.F., and Long, M.M. 1976b. S y n t h e t i c c r o s s - l i n k e d polypeptide of tropoelastin: an anisotropic fibrillar elastomer. B i o c h e m i s t r y ^ 15: pp. 4083-40 89. Urry, D.W., Khaled, M. A. , Bapaka, B.S., and Okamoto, K. . 1 97 7a. Nuclear overhauser enhancement evidence for inverse temperature dependence of hydrophobic s i d e c h a i n proximity i n the p o l y t e t r a p e p t i d e c f t r o p o e l a s t i n . Biochenu Biophys.. Res Commun^ 7 9: pp. 700-705. Urry, D.W., and Long, M. M. _ 1977b. .Advances i n Exp. Med. And Biol.. 79: pp. .685-714. . Urry, D.W., Khaled, M.A., Benugopalakrishnan, V., and Bapaka, fi*S. 1978a. Proton magnetic resonance and c o n f o r m a t i o n a l energy c a l c u l a t i o n s of the repeat peptides of t r o p o e l a s t i n . The hexapeptide. , J . . Am. .Chem. , Soc. , 100: pp. 696-705. Urry, D.W. , Long, M.M., and Sugano, H. 1 S78b. . C y c l i c analog of e l a s t i n hexapeptide e x h i b i t s an i n v e r s e temperature t r a n s i t i o n l e a d i n g t c c r y s t a l l i z a t i o n . J._ B i o l . Chenu 253: pp. 6301-6302. Urry, D.W. 1978c. Molecular p e r s p e c t i v e s of vascular wall s t r u c t u r e and d i s e a s e : the e l a s t i c component, i n " P e r s p e c t i v e s i n b i o logy and medicine". .21: pp. .26 5-295. Urry, D.W., Trapane, T.L., and Abu Khaled, M.. 1978d. Temperature dependence c f r o t a t i o n a l c o r r e l a t i o n times f o r an inverse temperature transition. A fundamental c h a r a c t e r i z a t i o n . J . Am. . Chem... Soc., 100: pp. 7744-7746. Volpin, elastin.  D., and C i f f e r r i , A. Nature. 225: pg. 382.  1970. . T h e r m o e l a s t i c i t y  of  V o l p i n , D., P a s g u a l i - B o n c h e t t i , I . , Urry, D.W., and Gotte, 1976a.. Banded f i b r e s i n high temperature coacervates e l a s t i n p e p t i d e s . J_. B i o l . Chem. .251: pp. 6871-6873.  L. of  V o l p i n , D., Urry, D.W., Cox, B . A . , and Gotte, L. 1976b. Optical diffraction cf tropoelastin and a l p h a - e l a s t i n c o a c e r v a t e s . . Bipchim..Biophys..Acta. 439: pp. 253-258. V o l p i n , D., Urry, D.W., P a s g u a l i - B o n c h e t t i , I . , and Gotte, L. 1 9 7 6 c . Studies by e l e c t r o n micrcsccpy on the s t r u c t u r e of c o a c e r v a t e s of s y n t h e t i c p o l y p e p t i d e s of t r o p o e l a s t i n . Micron. 7: pp. 193-198. Walton, A.G., and B l a c k w e l l , J . 1973. "Biopolymers". Press I n c . New York. 604 pp.  Academic  Weber, K., and Osbcrn, M. . 1969. The r e l i a b i l i t y of molecular weight determination by dodecyl sulphate-polyacrylimide g e l  263  electrophoresis. Weis-Fogh, rubber-like  J _ E i o l _ Che__ 244: pp. 4406-4412.  T. 1961a. Thermodynamic p r o p e r t i e s of E e s i l i n , a p r o t e i n . .J..Mol. . B i o l . .3: pp. 520-531 .  Weis-Fogh, T. 1961b. Molecular interpretation e l a s t i c i t y of r e s i l i n . J_ Mol_ B i o l . 3: pp. 648-667.  of  the  Weis-Fogh, T., and Anderson, S.O. 1970. New molecular model f o r the lcng-range e l a s t i c i t y of e l a s t i n . Nature. 277: pp. 718-721. Wilkes, G.L. 1S71. The measurement c f molecular o r i e n t a t i o n i n polymeric s o l i d s . Advances i n Pglym_ . S c i _ 8.: pp. 91-136. Wohlisch, E., Wetnauer, E., Gruning, W.D., and Rohrbach, R. 1943..Thermodynamische analyse der dehnung des elastichen gewebes vom standpunkt der s t a t i s t i s c h - k i n e t i s c h e n t h e o r i e der k a u t s c h u k e - l a s t i z i t a t . K o l l o i d Z_ 104: pp. 14-24. Wolinski, H. , and Glagov. S. 1964. S t r u c t u r a l b a s i s f o r the s t a t i c mechanical p r o p e r t i e s of the a o r t i c media. C i r c _ Res_ 14: pp. 400-413. Wood, G.C. .1958. Biochem J . 69: pp. 538-546. Yang, J.T. 1961..The v i s c o s i t y of macrcmolecules i n r e l a t i o n to molecular conformation. Advances i n p r o t e i n chemistry._ 16: pp. 323-400. Zimm, B.H., and Stockmayer, W.H. molecules c o n t a i n i n g tranches pp. 1301-1314.  1949. The dimensions of chain and r i n g s . J . Phys. ..Chem. .17:  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Country Views Downloads
United States 19 0
Canada 13 4
Poland 5 0
China 4 8
Russia 3 0
Japan 2 0
Ukraine 1 0
France 1 0
Zimbabwe 1 0
City Views Downloads
London 9 0
Unknown 8 8
Ashburn 8 0
Jackson 5 0
Kelowna 3 0
Saint Petersburg 3 0
Tokyo 2 0
Shenzhen 2 8
Seattle 2 0
Beijing 2 0
Montreal 1 0
Cleveland 1 0
Redmond 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094905/manifest

Comment

Related Items