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Studies on the respiratory protein hemerythrin Bruce, Robert Emerson 1978

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STUDIES ON THE RESPIRATORY PROTEIN ' HEMERYTHRIN by ROBERT EMERSON BRUCE B.Sc, University of B r i t i s h Columbia, 1970 M.Sc, University of B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY xn THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard The University of B r i t i s h Columbia A p r i l , 1978 © Robert Emerson Bruce, 1978 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes i s fo r f i nanc ia l gain sha l l not be allowed without my writ ten permission. Department of j The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date \ X \ C \ , V \ ^ S <0 J 6 - i i -ABSTRACT Hemerythrin from the marine worm Phascolosoma lurco has been i s o l a t e d and characterized. The protein appears to have the uncommon tr i m e r i c structure reported for Phascolosoma agas s i z i i hemerythrin, but i s more homogeneous on disc gel electrophoresis, and can be c r y s t a l l i z e d from ethanol. P. lurco hemerythrin has no free cysteine residue and ha l f the number of tryptophans of octameric hemerythrins. I t appears to have a s i m i l a r secondary structure to Phascolopsis g o u l d i i hemerythrin, with a high degree ('-75%) of alpha h e l i x . Proteins which appear to be hemerythrins have been i s o l a t e d from the sipunculid Phascolosoma noduliferum and the brachiopod Glott i d e a pyramidata, although they have not been as well characterized as the hemerythrin from P. lurco. Comparative studies using UV-visible and CD spectroscopies of oxy- and metazidohemerythrins from several species of sipuncu-l i d s have shown that the proteins from a l l species examined have very s i m i l a r a c t i v e s i t e s , despite differences i n primary and quaternary structures. New methemerythrin d e r i v a t i v e s with selenocyanate and dicyanamide, and the adduct formed between deoxyhemerythrin and n i t r i c oxide, have a l l been characterized by UV-visible and c i r c u l a r dichroism spectroscopies. Tricyanomethide has been shown to bind " n o n - s p e c i f i c a l l y " to P. g o u l d i i hemerythrin and t h i s has been r e l a t e d to the a c c e s s i b i l i t y of the active s i t e of the p rotein. - i i i -Fluorescence and nuclear magnetic resonance spectroscopies have been shown to be a p p l i c a b l e to comparative studies of heme-r y t h r i n , with nmr of paramagnetically s h i f t e d resonances perhaps providing the best means of i n v e s t i g a t i n g the p h y s i o l o g i c a l l y important colourless deoxy protein. Metal atom replacement studies and protein redox poten-t i a l determinations were not able to be c a r r i e d out s u c c e s s f u l l y , and possible reasons for t h i s as well as suggestions for future research are discussed. - i v -TABLE OF CONTENTS Page Abstract i i Table of Contents i v L i s t of Tables v i L i s t of Figures 1 v i i Acknowledgment x Chapter One - INTRODUCTION 1 Chapter Two - ISOLATION AND PURIFICATION 22 I s o l a t i o n 26 Results 34 Subunit Size and Quaternary Structure 47 Subunit Size 48 Quaternary Structure 51 Summary 57 Chapter Three - SPECTROSCOPIC STUDIES 59 UV-Visible Spectroscopy 59 C i r c u l a r Dichroism Spectroscopy 73 Nuclear Magnetic Resonance (NMR) Spectroscopy 84 Fluorescence Spectroscopy 99 Chapter Four - CHEMICAL STUDIES N-Bromosuccinimide 104 105 Page DTNB 110 Electrochemistry 113 Results and Discussion 116 Metal Atom Replacement 119 Results 121 N i t r i c Oxide Binding 125 Results 127 Chapter Five - CONCLUSIONS AND FUTURE CONSIDERATIONS 131 References 143 - v i -LIST OF TABLES Table Page I C h a r a c t e r i s t i c s of Some Dioxygen-Carrying Proteins 4 II Procedure for Disc Gel Electrophoresis 31 III Procedure for Sodium Dodecyl Sulphate Gel Electrophoresis 32 IV Molecular Sizes of Various Hemerythrins 52 V O p t i c a l Absorption Parameters of Oxyhemerythrin and Metazidohemerythrin from Five Species of Sipunculids.... 63 VI O p t i c a l Absorption Spectral Parameters for New P. l u r c o Methemerythrin Derivatives 65 VII P r i n c i p a l C i r c u l a r Dichroic Bands i n Oxyhemerythrin and Metazidohemerythrin from Five Species of Marine Sipunculids 78 VIII C i r c u l a r Dichroism Spectroscopic Parameters f o r New P. lurco Methemerythrin Derivatives 81 IX Modification of Tryptophans i n Hemerythrin with N-Bromosuccinimide 107 X Tryptophan Content and Sequence Location i n Hemerythrins from Six Species of Sipunculids 108 XI Iron Analyses of Apohemerythrins 124 - v i i -LIST OF FIGURES Figure Page 1 Iron Protoporphyrin-IX 5 2 Primary Structure of P. gouldii Hemerythrin 9 3 Forms of Hemerythrin (1971) 12 4 Arrangement of Subunits in T. dyscritum Hemerythrin... 18 5 Polypeptide Chain Folding i n Hemerythrin Subunit 19 6 Proposed Active Site Structures 21 7 Disc Gel Electrophoresis Profiles for P. lurco and P. agassizii Hemerythrins 41 8 Elution Profile of P. lurco Hemerythrin on a Sepharose 6B Column 44 9 Calibration Curve and Molecular Weight Determination for P. lurco Hemerythrin Monomers by Means of SDS Gel Electrophoresis 49 10 Calibration Curve and Molecular Weight Determination 'for P. lurco Multimeric Hemerythrin on a Sephadex G-100 Column > 54 11 Elution Profiles of Several Hemerythrins on a Small Sephadex G-75 Column 56 12 UV Absorption Spectra Around 280 nm for Hemerythrins from Four Species of Sipunculids 61 13 Absorption Spectra of Two New P. lurco Methemerythrin Derivatives 64 14 Job Plot of Binding of Selenocyanate to P. lurco Me taquo hemerythrin 66 15 Non-Specific Anion Binding by Tricyanomethide to P. gouldii Methemerythrin 68 16 Effect of pH on Near UV Spectrum of P. lurco Methydroxohemerythrin 70 17 Partitioning of Binding of Species to Hemerythrin into Active Site and Non-Active Site Binding 71 - v i i i -Figure Page 18 C i r c u l a r Dichroic Spectra i n the Amide Bond Region of P. g o u l d i i and P. lurco Oxyhemerythrin 75 19 Near UV-Visible C i r c u l a r Dichroic Spectra of Oxyheme-r y t h r i n from Three Species of Sipunculids 77 20 C i r c u l a r Dichroic Spectra of Metazido Protein from P. noduliferum and G. pyramidata 80 21 C i r c u l a r Dichroic Spectra of Two New P. lurco Methemerythrin Derivatives 81 22 UV-Visible (Upper Trace) and C i r c u l a r Dichroic (Lower Trace) Spectra of P. l u r c o Oxy- ( ) and Metseleno-cyanato- ( ) Hemerythrins 83 23 100 MHz Proton NMR Spectrum of P. g o u l d i i Metazido-hemer y thr i n 89 24 Aromatic Proton NMR Spectral Region for P. g o u l d i i Metazidohemerythrin at 100 MHz 90 25 Aromatic Proton NMR Spectral Region of P. g o u l d i i Metazidohemerythrin at 270 MHz 91 26 Downfield NMR Spectrum of H i s t i d i n e and H i s t i d i n e Modified with Diethylpyrocarbonate 93 27 U p f i e l d NMR Spectra of P. g o u l d i i Oxy- and Deoxy-hemerythrin 95 28 Downfield NMR Spectra of P. g o u l d i i Oxy- and Deoxy-hemerythrin 96 29 U p f i e l d NMR Spectra of P. lurco Oxy- and Deoxy-hemerythrin 97 30 Downfield NMR Spectra of P. lurco Oxy- and Deoxy-hemer y thr i n 98 31 Fluorescence Emission Spectra for Hemerythrins from Five Species of Sipunculids 101 32 Absorbance Change at 412 nm for the Reaction of DTNB with Three D i f f e r e n t Hemerythrins 112 33 Apparatus for Protein Redox T i t r a t i o n s 115 - i x -Figure Page 34 Oxidative T i t r a t i o n of PY g o u l d i i Deoxyhemerythrin with K-Fe (CN) _ 118 3 6 35 Denaturation Difference Spectrum of P. g o u l d i i Hemerythrin by 6 M GuHCl i n the Aromatxc Ammo Acids* Absorption Region.... 123 36 Absorption and C i r c u l a r Dichroic Spectra Showing P.  g o u l d i i Hemerythrin Reaction with N i t r i c Oxide 128 37 77°K ESR Signal from Reaction of 3.3 mM P. g o u l d i i Deoxyhemerythrin i n 0.1 M TRIS Acetate Buffer, pH 7.2, with NO 129 - x -ACKNOWLEDGMENT I would l i k e to express my sincere appreciation to Dr. A. W. Addison f o r introducing me to Bio-inorganic Chemistry. His encouragement and enthusiasm made working with him a pleasure, and h i s ideas and advice made i t a r e a l learning experience. Many other people contributed t h e i r time and expertise, and I would l i k e to acknowledge e s p e c i a l l y Drs. D. G. Clark, A. G. Marshall, P. J . Morrod, and D. C. Roe, and Mr. G. Luoma, J. Smith, and G. Webb f o r many valuable discussions and w i l l i n g p r a c t i c a l assistance. F i n a l l y , I am very g r a t e f u l to my wife, Joy, for her support and patience during the l a s t f i v e years, and f o r her many hours of e f f o r t i n producing t h i s t h e s i s . - 1 -CHAPTER ONE INTRODUCTION The f a s t e s t growing f i e l d of Inorganic Chemistry i n the l a s t three years has been the area of Bio-Inorganic Chemistry, as evidenced by the plethora of l i t e r a t u r e and new books on the (1 2 3 4) subject. ' ' ' Within t h i s f i e l d , one of the most i n t e r e s t -ing and most well studied areas i s that of dioxygen transport and storage by a small number of metalloproteins. Dioxygen i s required by most organisms for the metabolic processes which sustain l i f e . In order to carry out the b i o l o -g i c a l oxidations which provide the energy required by the c e l l , a number of enzymes have evolved which use dioxygen e i t h e r for incorporation i n t o a substrate, as i n tryptophan oxygenase, equation (1), - 2 -or as an oxidant, as i n cytochrome oxidase, equation (2). 4 cyt c ( F e l l ) + 0 2 + 4 H + — 4 c y t c (FeIH) + 2 H 2 0 <2> These reactions r e s u l t i n the dioxygen molecule being i r r e v e r -s i b l y changed. Small organisms are able to obtain the dioxygen they require d i r e c t l y from the a i r by d i f f u s i o n . Larger more complex creatures require a means of getting dioxygen from the atmosphere to the s i t e s of oxidation. They also require a means of st o r i n g dioxygen. To perform these functions, a l i -mited number of metalloproteins has evolved. P r i m i t i v e animals which are r e l a t i v e l y small and have slow metabolic rates have dioxygen c a r r i e r s enclosed i n c e l l s within the coelomic f l u i d . Hemerythrin i s t h i s type of molecule. The evolution of a c i r -culatory system led to dioxygen c a r r i e r s which were dissolved d i r e c t l y i n the plasma, such as erythrocruorin and hemocyanin. F i n a l l y , the c a r r i e r molecules were enclosed within c e l l s i n - 3 -the c i r c u l a t o r y system. This provided an increased concentra-t i o n of c a r r i e r s i n the c i r c u l a t o r y f l u i d , and hence a greater amount of dioxygen for the organisms. Hemoglobin t y p i f i e s t h i s c l a s s of c a r r i e r molecule. A number of c h a r a c t e r i s t i c s of several dioxygen-carrying proteins are l i s t e d i n Table I. Three d i f f e r e n t metal atoms are used for the function of rever-s i b l e oxygenation, equation (3). p r o t e i n + 0 2 p r o t e i n — 0 2 c o m p l e x ( In the proteins, the metal to dioxygen mole r a t i o can be ei t h e r 1:1 or 2:1 with i r o n , while copper forms a 2:1 complex. The metals a l l undergo formal redox reactions, and the dramatic colour changes which occur on reaction with dioxygen resulted i n these proteins being among the e a r l i e s t to be recog-• (5,6) nized. ' The most common oxygen-carrying -structure by f a r i s the protoporphyrin-IX macrocycle with an ir o n atom co-ordinated to the four pyrrole nitrogens as represented i n F i g . 1. The porphyrin u n i t with various side chain substituents, when com-bined with d i f f e r e n t polypeptide chains, i s found i n the hemo-proteins, and within t h i s group, hemoglobin, myoglobin, chlo-rocruorin, and erythrocruorin are r e v e r s i b l e dioxygen c a r r i e r s . In these proteins a nitrogen atom of a h i s t i d i n e amino acid Protein Source Molecular Weight # of 0 2binding subunits/molecule Metal atoms/ bound C>2 Myoglobin Mammalian muscle 17,500 1 1 Fe Hemoglobin Mammalian blood 65,000 4 1 Fe Hemocyanin Molluscs 4-8 X 10 6 80-160 2 Cu Arthropods 10 6 13 2 Cu Hemerythrin P. g o u l d i i marxne worms 108,000 8 2 Fe Figure 1 Iron Protoporphyrin-IX - 6 -side chain i s co-ordinated d i r e c t l y to the i r o n atom of the heme on one side of the porphyrin and t h i s leaves a s i x t h co-ordination s i t e vacant at the i r o n which i s where dioxygen or other ligands bind. The l i t e r a t u r e on hemoglobin and myoglo-bin i s extensive, and several review a r t i c l e s and books are he nat (10,11) (7 8 9) av a i l a b l e on the subject. ' ' Investigations on the natu-r a l proteins such as the c l a s s i c a l studies by Perutz, (12) and work on model systems such as that done by Wang and (13) Collman, have le d to a d e t a i l e d p i c t u r e of many aspects of re v e r s i b l e oxygenation of heme proteins. Much l e s s i s known about the other two. most common dioxygen c a r r i e r s ; hemocyanin and hemerythrin. The "heme" p r e f i x r e f e r s to both molecules being found i n blood: neither contains a porphyrin macrocycle. Several reviews of hemocyanin research are a v a i l a -b l e . d 4 ' ! 5 ) The protein i s very large, having a molecular weight of several m i l l i o n , and two d i f f e r e n t types of hemo-cyanin are recognized. The hemocyanin from molluscs has a fu n c t i o n a l l y a c t i v e subunit of about 50,000, while that from arthropods has a molecular weight of around 70,000. In both types of hemocyanin the two copper atoms per subunit are co-ordinated d i r e c t l y to amino acid side chains of the protein and one dioxygen molecule i s bound per copper dimer. The number and nature of the copper ligands and t h e i r geometry about the metal atoms i s not known, although evidence e x i s t s for h i s t i d i n e being co-ordinated to the copper.^ 1 6^ The - 7 -mode of binding of dioxygen i s also undetermined, however, resonance Raman spectroscopy experiments indicate that dioxy-(17) gen i s bound as peroxide with the copper atoms going from Cu(I) i n deoxy- to Cu(II) i n oxyhemocyanin, consistent with (18) spectroscopic evidence. Hemocyanin ex h i b i t s c o - o p e r a t i v i t y of oxygenation and also has a Bohr e f f e c t , two properties (19) which are associated with hemoglobin oxygenation. Much work i s being c a r r i e d out on the quaternary structure of hemo-(20 21) cyanins, ' and the subunit heterogeneity which has been (22) found w i l l add to the d i f f i c u l t i e s of working with such large complex macromolecules. The t h i r d c l a s s of dioxygen c a r r i e r s , hemerythrin, can be considered to be intermediate between the hemoproteins and hemocyanin. Hemerythrin i s comparable i n siz e to hemoglobin and i s an iron-containing metalloprotein. Like hemocyanin, the metal to dioxygen r a t i o i s 2:1 and the i r o n atoms are co-ordinated d i r e c t l y to amino acid side chains. I t seems appro-p r i a t e to begin the discussion of hemerythrin, the subject of t h i s t h e s i s , by reviewing what had been discovered about the protein when t h i s study was i n i t i a t e d i n September, 1973. This w i l l be followed by a consideration of the developments which have occurred i n the l a s t four years, which have par-t i c u l a r relevance to the thes i s work. Hopefully, t h i s w i l l provide a context i n which to r e l a t e the research described i n the the s i s to the current understanding of hemerythrin. The development of knowledge about hemerythrin between - 8 -1955 and 1971 i s perhaps best i l l u s t r a t e d by comparisons of (23 24) review a r t i c l e s written i n these years by I. M. Kl o t z . ' The e a r l i e r a r t i c l e describes the comparative oxygenation of hemoglobin/ hemocyanin and hemerythrin. Hemerythrin was believed to change from a colourless deoxy form with two Fe(II) atoms (which were co-ordinated by sulphur ligands to the protein) and one Fe(III) atom, to a violet-brown oxy form with three Fe(III) atoms and one peroxide molecule (which was bound between two of the i r o n atoms). Enough had been discov-ered by 1971 to make a f i f t y page chapter on hemerythrin with nearly a l l the studies having been c a r r i e d out on hemerythrin from Phascolopsis g o u l d i i sipunculids. Measurements by a (25) number of d i f f e r e n t techniques had established that P. g o u l d i i hemerythrin was a multimeric protein with a molecular weight of around 108,000, composed of eight equivalent sub-units of molecular weight 13,500. The i r o n to dioxygen r a t i o had been established to be 2:1, with one dioxygen molecule binding to each subunit of the pr o t e i n . I t was known that (26) the subunits could be d i s s o c i a t e d by d i l u t i o n or by che-mical modification of the s i n g l e cysteine residue per sub-(27) u n i t . The primary structure of the P. g o u l d i i protein had (28) been determined, and i s shown i n F i g . 2. Sedimentation 36 —7 experiments had res u l t e d i n a value of 3.4 X 10 M being obtained for the a s s o c i a t i o n constant of eight monomers, i n the oxidized form co-ordinated with azide, into one octamer at 5°C.< 2 6> - 9 -a) I. M. Klotz, G. L. Klippenstein and W. A. Hendrickson, Science, 192, 335 (1976). - 10 -A number of spectroscopic techniques had been used to study hemerythrin. The c i r c u l a r d i c h r o i c spectra of d i f f e r e n t l y l i g a t e d forms of the protein, for both octamer and monomer, had shown that the secondary structure remained the same for these d i f f e r e n t forms, and consisted of approximately 75% alpha (29 30) h e l i x , ' which was s i m i l a r i n value to the alpha h e l i x con-(31) tent of the heme dioxygen c a r r i e r s . Absorption spectra measurements showed that the cysteine residue was not an i r o n ligand, due to the lack of UV-visible spectroscopic changes on (27) a l k y l a t i o n of the sulphydryl group. Furthermore, compari-sons of the protein absorption spectra with those of model com-pounds suggested that the ac t i v e s i t e of oxy- and methemery-t h r i n consisted of a pa i r of Fe(III) atoms connected by an oxo bridge and e i t h e r a bridging dioxygen i n oxyhemerythrin, or water or an anionic ligand, depending on the type of methemery-(32) t h r i n . Deoxyhemerythrin had no absorption or c i r c u l a r d i -chroic bands i n the v i s i b l e region, which i s t y p i c a l of two (32) non-interacting high spin Fe(II) atoms. (33) Magnetic measurements ' and Mpssbauer spectrosco-py (34,35) bQth been performed on d i f f e r e n t d e r i v a t i v e s of hemerythrin and had further elucidated the i r o n states and i r o n environments of the d i f f e r e n t forms of the protein. Although the bulk diamagnetism of the protein made i t d i f f i c u l t to mea-sure absolute magnetic s u s c e p t i b i l i t i e s of the i r o n , changes i n magnetic s u s c e p t i b i l i t y , obtained on changing forms of the protein, showed that oxyhemerythrin and methemerythrin had the - 11 -same s u s c e p t i b i l i t y , and that the s u s c e p t i b i l i t y increased on (34) forming deoxyhemerythrin. That the low magnetic moment of the oxy and met forms was due to antiferromagnetic coupling between the Fe(III) atoms rather than being caused by spin p a i r -ing on each atom, was deduced from the isomer s h i f t and quadru-pole s p l i t t i n g parameters for these products which are t y p i c a l of (35) high spin Fe(III) atoms. Deoxyhemerythrin has values for (35) these parameters representative of high spin Fe(II) atoms. The Mossbauer spectra also showed that, while there i s only one kind of i r o n environment i n deoxy- and methemerythrin, as e v i -denced by the presence of only one p a i r of l i n e s i n the Mbss-/ bauer spectra of these species, the i r o n atoms i n oxyhemerythrin are inequivalent since the oxyhemerythrin spectrum consists of two p a i r s of l i n e s . Combining these r e s u l t s , the p i c t u r e of the active s i t e of the d i f f e r e n t forms of hemerythrin i n 1971 was as depicted i n F i g . 3. Chemical modification of proteins with group-specific reagents i s one method of d i s t i n g u i s h i n g accessible from inac-c e s s i b l e f u n c t i o n a l groups and thus i n d i c a t i n g possible amino aci d residues involved i n co-ordination of, for example, metal ions to proteins. Several studies using t h i s approach had been c a r r i e d out on P. g o u l d i i hemerythrin. Reaction of the protein (36) with trinitrobenzenesulphonic a c i d showed that the l y s i n e residues could a l l be modified without a f f e c t i n g the protein's UV-visible spectrum, implying that the l y s i n e s are not i r o n co-ordinating ligands. In addition, only three h i s t i d i n e - 12 -Figure 3 Forms of Hemerythrin (1971) Fed) Fe(II) deoxy-A Fe'(I) F e d ) •L' m e t -- 13 -residues reacted completely with diazonium-lH-tetrazole, while four h i s t i d i n e s reacted p a r t i a l l y , suggesting that four h i s t i d i n e s might be i r o n ligands. Tetranitromethane t r e a t -ment of hemerythrin res u l t e d i n loss of i r o n and loss of c i r -cular d i c h r o i c s p e c t r a l features, i n d i c a t i n g that tyrosine was (37) l i k e l y to be an i r o n ligand. In summary, i t was believed that h i s t i d i n e and tyrosine l i k e l y provided ligands to the ir o n atoms, but the number of residues, t h e i r o r i e n t a t i o n a-round the i r o n , and the possible p a r t i c i p a t i o n of other groups, such as carboxylates i n i r o n binding, was undetermined. Results from a number of d i f f e r e n t experiments suggested that the si n g l e cysteine residue, the active s i t e , and the subunit contact areas were a l l i n close proximity. A l k y l a t i o n of the cysteine r e s u l t e d i n d i s s o c i a t i o n of the protein, sug-gesting that the sulphydryl was near the subunit i n t e r f a c e . Furthermore, the d i s s o c i a t i o n rate was dependent upon anion (38) co-ordination at the active s i t e . N-ethylmaleimide did not d i s s o c i a t e metaquohemerythrin i n the time i n which i t completely d i s s o c i a t e d metazidohemerythrin, implying an e f f e c t of the ac t i v e s i t e on the sulphur residue. In addition, cer-t a i n anions (A), such as NO^ and ClO^, affected the r e a c t i v i t y at both the cysteine and the active s i t e . While these "non-s p e c i f i c binding" anions do not bind to the i r o n atoms, as judged by t h e i r f a i l u r e to change UV-visible and CD s p e c t r a l c h a r a c t e r i s t i c s of the protein, they do cause the equilibrium between co-ordinated water and hydroxide ( i . e . between met-- 14 -aquohemerythrin and methydroxohemerythrin) to s h i f t towards (39) the metaquo form. Symbolically, i f L represents a ligand co-ordinated to the i r o n atoms and A represents a "non-specific binding" anion, then for the r e a c t i o n : R HQ X ( H S - H r L ) 8 ^ 8 R H g - S - H r L ( 4 ) the rate of the reaction and the value of the equilibrium con-stant depend on both L and A. Non-specific anion binding by ClO^ prevented d i s s o c i a t i o n of P. g o u l d i i hemerythrin octamers by salyrganic acid, and reduced the r e a c t i v i t y of the sulphy-d r y l towards mercurials, i n contrast to the increased sulphy-d r y l r e a c t i v i t y produced by iron-binding anions. The e f f e c t s produced by the non-specific anion binding were explained as being proximal e l e c t r o s t a t i c e f f e c t s . ^ 4 0 ^ Thus C104 binding at the secondary s i t e made binding of water more favourable than hydroxide and reduced the rate of binding of other anions at the active s i t e . Near the Clb^ the pKa of the s u l -phydryl would be r a i s e d , decreasing the concentration and hence the r e a c t i v i t y of the S~ species. O v e r a l l , the active s i t e , the cysteine residue, and the ClO^ binding s i t e seemed to be positioned near to each other i n the t e r t i a r y structure of the subunit. The b i o l o g i c a l l y important reaction of hemerythrin, r e v e r s i b l e oxygenation of the p r o t e i n , has been q u a n t i t a t i v e l y - 15 -(41) studied for over f i f t y years. Most of the measurements on several d i f f e r e n t species have been c a r r i e d out by Man-(42 43 44) w e l l . In almost a l l cases the H i l l p l o t parameter, n, has values of 1.0 or only a l i t t l e greater, implying an i n t e r a c t i o n of at most only a few hundred c a l o r i e s between active s i t e s on oxygenation. Another way of expressing t h i s i s that hemerythrin oxygenation i s a non-co-operative reaction i n contrast to the highly p o s i t i v e c o - o p e r a t i v i t y of oxygena-t i o n of the hemoglobins and hemocyanins. Most hemerythrins do not have a Bohr e f f e c t on oxygenation, the oxygenation curve i s independent of pH, within the s t a b i l i t y range of the protein. Only hemerythrin from the brachiopod Lingula has an oxygenation reaction which e x h i b i t s c o - o p e r a t i v i t y and also a (44) Bohr e f f e c t , making i t a unique hemerythrin. The thermo-dynamics of hemerythrin oxygenation have also been i n v e s t i g a -ted for a few species. Measurements of the oxygenation reac-t i o n as a function of temperature lead to a calcu l a t e d heat of oxygenation of Phascolosoma a g a s s i z i i hemerythrin of -17 Kcal mole \ compared to the value of -13.5 Kcal mole ^ (45) reported for Sipunculus nudus pro t e i n . These values are greater than the corresponding values for oxygenation of P. . g o u l d i i p r o t e i n of -9.2 Kcal mole"^.^ 4 6^ An entropy of oxy-genation of -46 c a l per degree was determined for P. a g a s s i z i i (42) hemerythrin. Not unexpectedly, perchlorate binding near the active s i t e has been shown to a l t e r the shape of the H i l l (47) p l o t for oxygen binding to P. g o u l d i i hemerythrin. - 16 -By 1972, hemerythrins from the sipunculid f a m i l i e s Sipunculus, Z o s t e r i c o l a , and Phasedlopsis, and the f o s s i l bra-chiopod Lingula, had been i s o l a t e d and characterized as octa-meric proteins of molecular weight around 108,000, and i t was believed that t h i s was.likely to be t y p i c a l of a l l hemerythrins. Thus i t appeared that comparative studies on hemerythrins from d i f f e r e n t animals might prove to be of l i m i t e d value, i n con-t r a s t to hemoglobin research, where invest i g a t i o n s on d i f f e r e n t hemoglobins have shown how comparative studies of proteins from d i f f e r e n t sources can lead to a much increased understanding, at the molecular l e v e l , of b i o l o g i c a l molecules and t h e i r reac-t i o n s . The e l u c i d a t i o n of the r o l e of the h i s t i d i n e at p o s i -t i o n E7 i n the/B chains, for example, has been a s s i s t e d by com-parisons of normal human hemoglobin with hemoglobins Saskatoon and Zurich, where t h i s residue i s replaced by tyrosine and ar-ginine r e s p e c t i v e l y . P r o t e i n s from a number of d i f f e r e n t sources are required i n order to carry out comparative studies, so i s o l a t i o n of hemerythrin from new sources would at l e a s t increase the number of proteins being compared, and, at best, might be hoped to reveal new and d i f f e r e n t c h a r a c t e r i s t i c s of the r e s p i r a t o r y molecule. Of hemerythrins which had been i s o l a t e d , several fea-tures were undetermined when t h i s work was begun. The i r o n ligands and the geometry of the a c t i v e s i t e were not known, and the oxidation state of co-ordinated dioxygen had not been d i r e c t l y examined, but was i n f e r r e d from the oxidation state - 17 -of the i r o n . The redox p o t e n t i a l of the i r o n atoms was un-known and only one k i n e t i c study had been published on hemery-t h r i n , an i n v e s t i g a t i o n of the k i n e t i c s of deoxygenation of (45) Sipunculus nudus oxyhemerythrin. While the hemerythrin research, which has been report-ed during the course of the thesis research, w i l l be discussed i n Chapter Five, i t i s u s e f u l to mention, at t h i s point, the i n i t i a l X-ray c r y s t a l l o g r a p h i c r e s u l t s which were obtained on hemerythrin, as they provide a p i c t u r e of the macromolecule which i s h e l p f u l for considering other aspects of the chemistry of the protein. In 1974, the f i r s t reports of c r y s t a l l o g r a p h i c studies on octameric hemerythrin from P. g o u l d i i , and monomeric (49 50) hemerythrin from Themiste z o s t e r i c o l a appeared. ' These i n i t i a l i n v e s t i g a t i o n s indicated that the octameric protein had close to D 4 symmetry, and that the subunits i n the octamer were arranged i n a configuration intermediate between a cube and an antiprism. I t has also proven possible to carry out c r y s t a l l o -graphic studies on a t h i r d hemerythrin, octameric protein from Themiste dyscritum. The structures of these proteins are be-o ing solved at higher r e s o l u t i o n and 5 A structures have been (51 52 53) reported for a l l three proteins, ' ' with the active s i t e o (54) to 2.8 A being reported for T. dyscritum hemerythrin. The c r y s t a l structure of the monomer showed that myohemerythrin consists of four roughly p a r a l l e l segments of alpha h e l i x con-s i s t i n g of ten to twenty amino acids each, with the two i r o n atoms located between and co-ordinated to the four h e l i c a l seg-- 18 -ments. The carboxy terminus closes o f f one end of the c y l i n d e r formed by the alpha h e l i c a l rods, and the f i r s t eighteen r e s i -dues of the amino terminus form an extended polypeptide chain. A representation of the subunit i n myohemerythrin i s shown i n F i g . 4. Despite differences i n primary structures, the same basic f o l d i n g of the subunit polypeptide chain i s observed i n the c r y s t a l structures of the octameric proteins. The eight subunits are then arranged as shown i n F i g . 5, where each sub-unit i s represented as a c y l i n d e r of length 4 nm and of d i -ameter 2 nm. Figure 5 Arrangement of Subunits i n T. dyscritum Hemerythrin 3 a) R. E. Stenkamp, L. C. Sieker, L. H. Jensen and J. S. Loehr, J . Mol. B i o l . , 100, 23 (1976). Eight subunits are arranged into what i s c a l l e d a square dough-nut to make up the multimer, with each octameric uni t being approximately 7 X 7 X 5 nm with a square hole i n the middle Hendrickson, G. L. Klippenstein and K. B. Ward, Proc. Acad. S c i . U.S.A., 72, 2160 (1975). - 20 -2 nm on each side. The N-terminal chain of every subunit ex-tends out from the molecule into s o l u t i o n , and the f i r s t two alpha h e l i c a l rods from the N-terminus i n each subunit are on the i n s i d e of the multimer at the subunit i n t e r f a c e region, o as expected from sequence studies. At 5 A r e s o l u t i o n , the i r o n ligands could not be unambiguously assigned, and two d i f -ferent arrangements of the active s i t e were proposed which are depicted i n F i g . 6 . ( 5 2 / 5 4 ) Given a number of possible areas f o r study of hemery-t h r i n , the reasons we are interested i n t h i s protein are best summarized by the t i t l e of the most recent review a r t i c l e on hemerythrin i n Science of l a s t year: "Hemerythrin: A l t e r n a t i v e ( 5 5 ) Oxygen C a r r i e r " . *1 a b c iq a) W. A. Hendrickson, G. L. Klippenstein and K. B. Ward, Proc. Natl . Acad. S c i . U.S.A., 72, 2160 (1975). b) R. E. Stenkamp, L. C. Sieker and L. H. Jensen, Proc. Natl. Acad. S c i . U.S.A. , 73_, 349 (1976) . c) L. C. Sieker, personal communication. - 22 -CHAPTER TWO ISOLATION AND PURIFICATION The hemerythrin used i n t h i s work, came from a number of d i f f e r e n t sources, and so rather than describe them when r e f e r -r i n g to each hemerythrin, i t i s convenient to c o l l e c t the i n -formation about the d i f f e r e n t animals, how and where they were obtained, and how they w i l l be designated throughout the t h e s i s , at t h i s point. Although hemerythrin can be found i n four marine (55) invertebrate phyla, nearly a l l the research was done with protein obtained from marine worms of the phylum Sipuncula. The standard reference on t h i s phylum used i n c l a s s i f y i n g the a n i -mals i s the book by Stephen and Edmonds.^56^ Phascolopsis (syn. Golfingla) g o u l d i i , (P. goul d i i ) P. g o u l d i i sipunculids are found along the east coast - 23 -of North America, p a r t i c u l a r l y i n Massachusetts. They are about 10 cm i n length, uniform i n shape, a greyish colour, and a good source of the protein which i s by f a r the most well-studied and characterized hemerythrin. Animals were obtained l i v e from the Marine B i o l o g i c a l Laboratory at Woods Hole, Massachusetts. C r y s t a l l i n e protein was also provided by Dr. I. M. Klotz at Northwestern Un i v e r s i t y . Themiste (syn. Dendrostomum) z o s t e r i c o l a , (T. zostericola) T. z o s t e r i c o l a worms are found along the southwest coast of North America. The animals are 10-15 cm i n length, uniform i n shape, and brownish i n colour. Worms were ob-tained from P a c i f i c Bio-Marine at Venice, C a l i f o r n i a . Themiste dyscritum, (T. dyscritum) T. dyscritum marine worms l i v e on the c e n t r a l west coast of North America. P u r i f i e d p r o t e i n from these animals was supplied by Dr. J. S. Loehr at Portland State U n i v e r s i t y . Themiste alutacea, (T. alutacea) T. alutacea sipunculids are found i n the Caribbean. They are small, being about one cm i n length, greyish i n colour, and l i v e i n rocks, i n contrast to the more common - 24 -i n t e r - t i d a l sand dwellers. Animals were obtained from Gulf Specimen Company, Panacea, F l o r i d a . Phascolosoma a g a s s i z i i , (P. a g a s s i z i i ) P. a g a s s i z i i specimens are found along the west coast of North America and are smaller than P. g o u l d i i sipunculids, being t y p i c a l l y a few cm i n length. They are i r r e g u l a r i n shape, being bulged at the pos t e r i o r end, and are speckled brown i n colour, hence the name "peanut worm". Animals were supplied from the west coast of Vancouver Island by Dr. T. Carefoot, U.B.C., and by Peninsula Marine B i o l o g i c a l s , Sand C i t y , C a l i f o r n i a . Phascolosoma lurco, (P. lurco) P. lurco sipunculids are the la r g e s t worms from which hemerythrin was obtained, being 4 0 cm or longer. The animals are speckled brown i n colour, but i n contrast to P. a g a s s i z i i worms, are regular i n shape. They are found i n the southwest P a c i f i c , where they l i v e i n mangrove swamps, and were supplied by Dr. D. Outram, Unive r s i t y of the South P a c i f i c , Suva, F i j i . Phascolosoma noduliferum, (P. noduliferum) Animals of the species P. noduliferum are found i n - 25 -New South Wales, A u s t r a l i a . They are small worms, being a few cm i n length and bulged at the p o s t e r i o r end. Specimens were obtained by Dr. A. W. Addison from Long Reef, New South Wales, A u s t r a l i a . Two members of the phylum Brachiopoda were studied i n view of the f a c t that hemerythrin has been i s o l a t e d from the brachiopod Lingula. Glottidea pyramidata, (G. pyramidata) G. pyramidata i s a l i n g u l i d brachiopod c l o s e l y r e l a t e d to the Indo-Pacific f o s s i l brachiopod Lingula. The animals l i v e i n a s h e l l about one cm i n length, shaped l i k e a lamp-shade, and have a foot which r a i s e s and lowers them within t h e i r mud burrows. G. pyramidata brachiopods are found o f f of the coast of F l o r i d a and were supplied by Gulf Specimen Com-pany, Panacea, F l o r i d a . T e r e b r a t a l i a transversa, (T. transversa) T. transversa i s an a r t i c u l a t e brachiopod found along the P a c i f i c Northwest, e s p e c i a l l y i n the San Juan and Gulf Island area. The animals l i v e i n s h e l l s , somewhat l i k e clam s h e l l s , a few cm i n diameter, and are orange-pink i n colour. Animals were supplied by Dr. W. Austin. - 26 -I s o l a t i o n I s o l a t i o n and p u r i f i c a t i o n of hemerythrins has been performed i n a number of ways, ranging from i n v e s t i g a t i n g (57) (58} packed blood c e l l s to c r y s t a l l i z a t i o n of the pro t e i n . The degree of p u r i f i c a t i o n has been dependent on the type of study being performed and on the source of the protein, for both factors determine the p u r i f i c a t i o n needed and pos s i b l e . Although hemerythrin can be found i n the tentacles and (59) muscles of some sipunculids, a l l the studies reported here were on the more abundant coelomic hemerythrin which i s found i n red blood c e l l s i n the hollow body ca v i t y , or coelom, of the worms. To obtain the protein, an animal was held v e r t i -c a l l y over a funnel l i n e d with cheesecloth and an i n c i s i o n was made, using s c i s s o r s , into the po s t e r i o r end. The coelomic f l u i d , i n i t i a l l y under pressure, was c o l l e c t e d , and f i l t e r e d through the cheesecloth into a vessel packed i n i c e . The i n c i s i o n was then extended the length of the worm, taking care not to cut int o the i n t e r n a l organs. The inside of the animal was rinsed with a wash so l u t i o n c o n s i s t i n g of 0.2 M Na2SO^ plus 10 mM Na2EDTA adjusted to pH 7.3 with NaOH, and the washings were c o l l e c t e d with the coelomic f l u i d . The car-casses were c o l l e c t e d i n the cheesecloth and rinsed once more before being discarded. A l l further processing was c a r r i e d out at 4°C. The washings and coelomic f l u i d were centrifuged i n a bench centrifuge, g i v i n g a c l e a r supernatant, beneath a - 27 -layer of l i p i d material, both of which were removed. The packed erythrocytes were gently s t i r r e d with more wash so l u -t i o n and then re-centrifuged, and t h i s washing of the c e l l s was repeated two or three times. F i n a l l y a l l of the c e l l s were combined, and to them was added a volume of water equal to three to four times the volume of the c e l l s . This mixture was s t i r r e d overnight to lyse the c e l l s . The suspension was then centrifuged at 18,000 rpm for one hour, the red heme-rythrin-containing supernatant was f i l t e r e d through glass wool to remove any low density material f l o a t i n g on top of the sol u t i o n , and the membrane fragments and p r e c i p i t a t e d material were discarded. The water used i n t h i s work was p u r i f i e d by s t e r i l e f i l t r a t i o n followed by exchange r e s i n treatment to remove water-soluble organic compounds and inorganic ions. Since hemerythrin i s a g l o b u l i n protein, some i o n i c strength i s necessary to keep i t i n s o l u t i o n . Furthermore, hemerythrin has a l i m i t e d pH range of s t a b i l i t y from about 6.5 to 9.5, thus the protein was stored and studied i n aqueous buffer solutions. In most of t h i s work the buffer used was. made from 0.1 M TRIS plus 0.05 M sodium acetate adjusted to pH with a c e t i c a c i d . This buffer s o l u t i o n w i l l be r e f e r r e d to as pH 7.0 TRIS or pH 8.2 TRIS, for example. Af t e r hemerythrin-containing supernatant has been ob-tained from the lysed c e l l s , d ifferences i n species become important i n determining what needs to and can be done for p u r i f i c a t i o n . Protein from P. g o u l d i i animals, for example, - 28 -i s usually f a i r l y pure at t h i s stage, and can r e a d i l y be c r y s -(58) t a l l i z e d from 20% ethanol and stored i n t h i s form. P. l u r c o hemerythrin can also be c r y s t a l l i z e d , however, protein from most of the other species cannot. Both T. Z o s t e r i c o l a and P. lurco proteins often had a green low molecular weight «10,000) impurity which could be removed by extensive d i a l y s i s , or by u l t r a f i l t r a t i o n . Ammonium sulphate p r e c i p i t a t i o n has also been reported for protein p u r i f i c a t i o n , and was used suc-c e s s f u l l y . I t was f e l t that avoiding changes of state was desirable to minimize possible s t r u c t u r a l changes, and so the method of choice for p u r i f i c a t i o n was gel permeation chromato-graphy. Gel f i l t r a t i o n p u r i f i e s components on the basis of t h e i r s i z e and shape. The gels used for gel chromatography are manufactured by Pharmacia under the trade names Sephadex, which consists of beads of cross-linked dextran, an anhydro-glucose polymer, and Sepharose, which consists of beads of agarose, a l i n e a r polysaccharide. Both gels are manufactured i n a seri e s of molecular weight ranges of f r a c t i o n a t i o n and the appropriate gels were chosen by reference to the Phar-, (60,61) macia manuals. ' Since P. g o u l d i i hemerythrin has a molecular weight (25) of 108,000, a Sepharose 6B column was used for the p u r i f i c a -t i o n . The hydrated gel was poured into a piece of s i l y l a t e d glass tubing to make a column 55-70 X 5 cm. The void volume for large molecular weight (>10^) molecules was determined using - 29 -Blue Dextran, a coloured dextran of molecular weight approxi-mately 2 X 10 . The small molecule e l u t i o n volume was deter-mined using K^Fe(CN)g. Standards and samples were layered onto the top of the column i n a 10% sucrose s o l u t i o n , and eluted i n t o a Gilson Micro Fractionator c o l l e c t i n g 6-7 ml f r a c t i o n s . Protein was monitored by absorbance at 280 nm and hemerythrin was monitored by absorbance at 500 nm, where the oxygenated form of the protein absorbs strongly. The centre part of the hemerythrin peak was pooled and the leading and t r a i l i n g edges were c o l l e c t e d together. I f necessary, protein was concentrated on an Amicon u l t r a f i l t r a t i o n apparatus of the appropriate volume using a PM 10 membrane which r e t a i n s spe-(62) c i e s of molecular weight >10,000. Since ammonium sulphate p r e c i p i t a t i o n has been report-(59) ed for i s o l a t i n g hemerythrin, t h i s method of p u r i f i c a t i o n was sometimes used i n the case of T. z o s t e r i c o l a p r o t e i n . The protein i n pH 8.2 TRIS was dialyzed against 60% saturated ammonium sulphate, the sol u t i o n was centrifuged and the p e l l e t , containing the hemerythrin, was redissolved i n buffer and re-p r e c i p i t a t e d . One of the most important and often most d i f f i c u l t steps i n protein research i s i s o l a t i n g and determining that one has obtained a pure preparation of a protein. In the case of hemerythrin, obtaining protein turns out to be tedious, but not notably d i f f i c u l t . To demonstrate that pure hemerythrin had been obtained, several techniques were employed. - 30 -One s e n s i t i v e procedure f o r determining protein p u r i t y i s electrophoresis on a medium which r e s u l t s i n protein migra-t i o n across an applied e l e c t r i c f i e l d . Since the r e l a t i v e m o b i l i t y of a molecule i n an e l e c t r i c f i e l d depends on both i t s s i z e and charge, electrophoresis can be quite s e n s i t i v e to small changes i n proteins such as amino ac i d s u b s t i t u t i o n s . Thus, as a preparative technique, i t complements g e l chromato-graphy i n protein p u r i f i c a t i o n and as an a n a l y t i c a l technique i t provides a good c r i t e r i o n of p u r i t y since more than one component w i l l usually give more than one band on the g e l . Two types of electrophoresis were performed, d i s c gel e l e c t r o -phoresis on native p r o t e i n , and sodium dodecyl sulphate g e l electrophoresis on denatured protein. The d i s c gels were run according to the method of Dietz (63) and Lubrano. The procedure for making, running and s t a i n -ing the gels i s ou t l i n e d i n Table I I . Gels were made i n s i l y -l a ted pyrex tubes, 4 mm insi d e diameter, pre-rinsed with "Pho-to-Flo 200" s o l u t i o n , and polymerized within an hour. The gels were made to be 9 cm i n length, as t h i s s i z e f i t s the c e l l used f o r scanning the gels. The gels were stained and destained as described i n Table I I , and scanned for p r o t e i n -bound dye at 550 nm on a G i l f o r d 240 spectrophotometer with scanning attachment. Sodium dodecyl sulphate gels were made, according to the procedure of Fairbanks et a l . ( s e e Table III) i n the same tubes and of the same length as d i s c gels, and were - 31 -Table II Procedure for Disc Gel Electrophoresis STOCK SOLUTIONS A: 48 ml IN HCl 36.3 gm TRIS 0.23 ml N, N, N'/N 1-tetramethylenediamine H2O to a f i n a l volume of 100 ml B: 30 gm acrylamide 0.8 gm N,N'-methylenebisacrylamide H2O to a f i n a l volume of 100 ml C: 140 mg ammonium persulphate H 20 to a f i n a l volume of 100 ml ELECTROPHORESIS BUFFER 6.0 gm TRIS 28.8 gm glycine H 20 to a f i n a l volume of 1 1 (use 0.002% Bromophenol Blue i n the upper r e s e r v o i r as a tracking dye.) TO MAKE 5.5% GELS 2 ml s o l u t i o n A 4 ml s o l u t i o n B 8 ml s o l u t i o n C 7.8 ml H 20 (Mix together and pour in t o gel tubes, then l e t polymerize.) TO RUN GELS Layer on <100 /*gm protein i n 30% sucrose. Run at 2.5 mA/tube, u n t i l Bromophenol Blue reaches bottom of gels. TO FIX, STAIN AND DESTAIN GELS 12.5% t r i c h l o r o a c e t i c acid s o l u t i o n with a g i t a t i o n for t h i r t y minutes 12.5% t r i c h l o r o a c e t i c a c i d s o l u t i o n with 0.05% Coomassie Blue for one hour 10% t r i c h l o r o a c e t i c a c i d s o l u t i o n for two to three days to develop i n t e n s i t y of gels a) A. A. Dietz and T. Lubrano, Anal. Biochem., 20, 246 (1967). - 32 -Table III Procedure for Sodium Dodecyl Sulphate Gel Electro-p h o r e s i s 3 STOCK SOLUTIONS A: Concentrated AcBis 40.0 gm acrylamide 1.5 gm N/N 1-methylenebisacrylamide H 20 to a f i n a l volume of 100 ml B: 10X buffer 0.4 M TRIS 0.2 M sodium acetate 0.02 M EDTA pH 7.4 with a c e t i c a c i d C: 20% SDS (weight/weight) ELECTROPHORESIS BUFFER 100 ml 10X buffer 50 ml 20% SDS H2O to a f i n a l volume of 1 1 DENATURING SOLUTION 2 gm SDS 10 gm sucrose 75 mg EDTA 2 mg pyronin Y 0.24 gm TRIS H 20 to a f i n a l volume of 100 ml (pH 8.0 with HC1) OVERLAY SOLUTION 0.1% SDS 0.15% ammonium persulphate 0.05% TEMED TO MAKE 4 GELS 1.4 ml 1.0 ml 5.6 ml 0.5 ml A B H,0 or degassed 1.0 ml ammonium persulphate (15 mg/ml) 0.5 ml TEMED (0.5%) (Cover gels with overlay s o l u t i o n and polymerize overnight.) Table III (continued) TO MAKE PROTEIN SOLUTION Mix equal volumes of protein and denaturing solution (80 mM in dithioerythritol) and heat for 20 minutes (37°C sample, 60°C standards). TO RUN GELS Prerun gels for 1 hour at 5 mA/gel. Layer < 100 JJLL cooled protein solution onto gels. Electrophorese at 5 mA/gel. TO FIX, STAIN, AND DESTAIN GELS 25% isopropanol, 10% acetic acid, 0.025% Coomassie Blue (overnight). 10% isopropanol, 10% acetic acid, 0.0025% Coomassie Blue (~12 hours). 10% acetic acid, 0.0013% Coomassie Blue (-12 hours). 10% acetic acid (overnight). a) P. J. Morrod, Ph.D. thesis, University of B r i t i s h Columbia (1975). - 34 -scanned i n the same manner. The other c r i t e r i o n of protein p u r i t y used was spectro-scopic. The molar a b s o r p t i v i t i e s , e s p e c i a l l y at 280 nm, are c h a r a c t e r i s t i c of the proteins, and as w i l l be discussed i n Chapter Three, the shapes of the spectra i n t h i s region are i n d i c a t i v e of protein p u r i t y . Iron analyses, to obtain the 280 nm molar a b s o r p t i v i t i e s per i r o n dimer, were performed ac-cording to R i l l and Klotz modified by s u b s t i t u t i n g 1 M NaHSO^ for hydroxylamine to improve r e p r o d u c i b i l i t y . Protein s i z e was determined from gel chromatography by determining the K of the protein on a given column. V -V a v e K = T T e T T° where V = e l u t i o n volume of the protein, V = ave V -V e c o t o void volume of the column measured using Blue Dextran, and V"t = the gel bed volume. Results P. g o u l d i i Since hemerythrin from P. g o u l d i i sipunculids has been very well characterized, t h i s protein was used as a reference hemerythrin, both f o r checking experimental r e s u l t s , and for comparative studies. Three shipments, a t o t a l of 260 worms, were processed as described up to the stage of c e n t r i f u g a t i o n of the lysed red blood c e l l s . At t h i s point the protein was dialyzed against f i r s t 10% and then 20% ethanol i n e i t h e r pH - 35 -8.1 TRIS or pH 7.0 TRIS to obtain needle-like c r y s t a l s of oxy-hemerythrin. This was stored i n a sealed septum b o t t l e under N 2 u n t i l needed. To use the protein, an a l i q u o t was dialyzed into pH 7.1 TRIS and any white p r e c i p i t a t e was centrifuged down and discarded. The r e s u l t i n g s o l u t i o n was r o u t i n e l y d i a -lyzed against the desired buffer and ligand, or passed down a Sephadex G-25 column e q u i l i b r a t e d i n the buffer and ligand, and used. This procedure has been found to be suit a b l e for obtaining pure protein, and as an added check, the spectro-scopic parameters of d i f f e r e n t forms of the protein agreed with published values (see Chapter Three). F i n a l l y , i r o n analyses yielded a molar a b s o r p t i v i t y per Fe dimer or subunit at 280 nm of 35,000 (± 3% 1 mole~ 1cm~ 1). T. z o s t e r i c o l a The f i r s t species investigated was T. z o s t e r i c o l a . During t h i s work, 400 worms were processed i n four d i f f e r e n t batches. I n i t i a l l y , the procedure used s u c c e s s f u l l y for P. g o u l d i i hemerythrin was t r i e d . A f t e r c e n t r i f u g a t i o n , the protein s o l u t i o n was f i l t e r e d under N 2 pressure through a 0.45 micron f i l t e r to remove microorganisms. On d i a l y s i s against 10% ethanol, protein denaturation and p r e c i p i t a t i o n resulted. At t h i s point the Sepharose 6B column was used, and the protein eluted o f f the column with K =0.42, cor-c ave responding to a molecular weight of ju s t over 100,000, i n - 36 -agreement with published v a l u e s . v u u ' Two l o t s of T. z o s t e r i c o l a protein had a small molecular weight contaminant which was removed by the Sepharose column, however, even the column-puri-f i e d p r o t e i n could not be c r y s t a l l i z e d from ethanol. On s t o r -ing some of the protein, without' chromatographing i t , i n a serum b o t t l e with e i t h e r a few drops of toluene or octanol, extensive p r o t e i n p r e c i p i t a t i o n ocurred, and t u r b i d i t y i n d i c a t i v e of b a c t e r i a l contamination was noticed. In the course of studying t h i s protein, a temporal, seemingly i r r e v e r s i b l e change occurred which res u l t e d i n the chromatographic procedure being checked i n the following way. One batch of f r e s h l y i s o l a t e d p r o t e i n was divided, with h a l f the protein s o l u t i o n being p u r i f i e d on the Sepharose 6B column, and ha l f being p u r i f i e d by 60% ammonium sulphate p r e c i p i t a t i o n . Aliquots from each p u r i f i c a t i o n were then run on di s c gels. Both preparations gave i d e n t i c a l r e s u l t s , only one band being observed on s t a i n -ing the gels, which migrated the same distance i n both gels. Iron analyses of T. z o s t e r i c o l a hemerythrin resulted i n a 280 nm molar a b s o r p t i v i t y of 32,300 (± 3% 1 mole ^cm per dimeric i r o n u n i t . T. dyscritum T. dyscritum hemerythrin was supplied by Dr. J . S. Loehr as a s o l u t i o n of oxidized met protein with azide anion c o - o r d i -nated. I t was dialyzed into the appropriate buffer and used as - 37 -received with the concentration being determined from the molar a b s o r p t i v i t y at 280 nm of 33,200 (± 3% 1 mole" 1cm~ 1).^ 6 7^ T. alutacea One shipment of 44 animals was processed. The animals were very small (a few mm) and the usual i s o l a t i o n procedure yielded only a small volume of s o l u t i o n which had no v i s i b l e colour and i n s u f f i c i e n t protein absorbance to be worth further work. In addition, shipment delays res u l t e d i n some of the animals being dead on a r r i v a l , and i t i s important to ensure that the animals are received a l i v e , since protein degradation ensues following death. P. a g a s s i z i i Five d i f f e r e n t shipments of P. a g a s s i z i i sipunculids were processed, representing 860 worms. As t h i s was an unknown source at the time, the y i e l d per worm was determined for the various shipments. I t turned out to be quite v a r i a b l e , ranging from 0.5 to 10.0 mg of hemerythrin per worm. This seems to be a function of two f a c t o r s . On one hand, larger animals seem to r e s u l t i n a y i e l d p r o p o r t i o n a l l y greater than the increased s i z e (this i s i l l u s t r a t e d by the hemerythrin y i e l d from P. lurco s i p u n c u l i d s ) . In addition, some of the P. a g a s s i z i i specimens from C a l i f o r n i a contained a l o t of orange coloured p a r t i c l e s - 38 -i n the coelomic f l u i d and l e s s hemerythrin. I t was subsequently determined that these p a r t i c l e s were c i r c u l a r eggs of about 110-140 microns i n diameter. A recent review on the reproduction (68) of sipunculids, reports that P. a g a s s i z i i worms from C a l i -f o r n i a breed i n the spring, while specimens from Washington breed i n the summer months, hence these are periods i n which to avoid c o l l e c t i n g animals i n order to maximize protein y i e l d . A f t e r the red blood c e l l s had been lysed and centrifuged, an a l i q u o t of supernatant containing hemerythrin was dialyzed against 10% ethanol. As with T. z o s t e r i c o l a hemerythrin, hemerythrin from P. a g a s s i z i i could not be c r y s t a l l i z e d i n t h i s way; the protein p r e c i p i t a t e d out, e s p e c i a l l y on being dialyzed against 15% or 20% ethanol. Thus p u r i f i c a t i o n was performed on the Sepharose 6B column. Although the protein eluted as one band on the column, i t eluted i n a much larger volume than the other hemerythrins discussed so f a r , suggesting i t to be s i g n i f i c a n t l y smaller. In f a c t , a K of 0.60 was found, implying a protein s i z e of about 40,000. This was determined at the same time as a report appeared i n the l i t e r a t u r e des-(69) c r i b i n g the quaternary structure of P. a g a s s i z i i hemerythrin. Although protein p u r i f i e d on the column i s stable i n so l u t i o n i n the cold, i t tends to slowly oxidize to the met form. To prevent t h i s , some of the hemerythrin was stored sealed under N 2 with an excess of formamidinesulphinic acid to reduce the protein to the Fe(II) deoxy form. The colourless s o l u t i o n thus obtained i s very stable, and on exposure to a i r , spectro-- 39 -s c o p i c a l l y native oxyhemerythrin has been obtained even a f t e r up to two years 1 storage. This then seems to be a very good method of keeping the protein. Disc gel electrophoresis of p u r i f i e d P. a g a s s i z i i heme-r y t h r i n gave a multi-banded pattern shown i n F i g . 7. However, sodium dodecyl sulphate g e l electrophoresis of the unfolded subunits r e s u l t e d i n only one band being observed, implying that only one s i z e polypeptide chain was present. The m u l t i -banded d i s c g e l pattern i s due not to the presence of impurities, but i s caused by protein heterogeneity. Indeed, i t has been (69) shown that P. a g a s s i z i i hemerythrin has at l e a s t four e l e c t r o p h o r e t i c a l l y d i s t i n c t types of subunits, caused by amino acid s u b s t i t u t i o n s i n the subunit polypeptide chain. The presence of only one band on sodium dodecyl sulphate gels of P. a g a s s i z i i hemerythrin i s further evidence of pure protein being present. Iron analyses re s u l t e d i n a value at 280 nm of 28,700 (± 3% 1 mole~^cm~^) per i r o n dimer, somewhat lower than the values for the previous hemerythrins. P. lurco Two batches of P. lurco sipunculids were obtained, and protein i s o l a t e d from them. In t o t a l , f o r t y - f i v e worms were used and the y i e l d from these large animals was by far the highest obtained. The smaller worms gave about 75 mg per worm while the shipment of large animals, which was obtained i n June, - 4 0 -Figure 7 Disc Gel Electrophoresis P r o f i l e s for P. lurco and P. a g a s s i z i i Hemerythrins Disc gel electrophoresis absorption p r o f i l e at 550 nm f o r P. lurco (upper trace) and P. a g a s s i z i i (low-er trace! t r i m e r i c hemerythrins. Gels were run at 5°C and stained for protein with Coomassie blue. The arrows in d i c a t e the tracking dye. - 41 -Figure 7 Disc Gel Electrophoresis P r o f i l e s for P. lurco and P. a g a s s i z i i Hemerythrins D I S T A N C E A L O N G G E L - 42 -y i e l d e d an enormous 600 mg per worm. The protein from one animal was c o l l e c t e d and processed separately. A f t e r c e n t r i -fuging the lysed erythrocytes, a portion of the supernatant was dialyzed against 10% ethanol. In contrast to P. a g a s s i z i i p rotein, the P. lurco hemerythrin c r y s t a l l i z e s from ethanol as long needles. In order to e s t a b l i s h further the protein crys-t a l l i z a t i o n , the p r o t e i n was c r y s t a l l i z e d by vapour phase d i f f u s i o n ^ 7 ^ at room temperature. Using 5 //l drops of 1.0 mM metazidohemerythrin i n pH 8.1 TRIS, allowed to e q u i l i b r a t e with 10 pX of organic solvent such as ethanol or 2-methyl-2,4-pentane-d i o l , the organic molecules d i f f u s e into the protein drop and over the course of a few days t h i n needles of protein c r y s t a l s could be prepared. The other method involved e q u i l i b r a t i n g the protein with a 60% ammonium sulphate s o l u t i o n , i n which case solvent water d i f f u s e s from the protein drop to the s a l t droplet. This procedure also resulted i n t h i n needles of hemerythrin being obtained. C r y s t a l l i z a t i o n within a few days causes twinning and clumping of the needles, and should be done more slowly to obtain large s i n g l e c r y s t a l s , or performed using seed c r y s t a l s . One cautionary note i s needed. A f t e r storage under 20% ethanol for several months, the protein was found to have i r r e v e r s i b l y denatured. The cause of t h i s i s undetermined at t h i s time, however, ethanol i s known to disrupt (71) hydrophobic i n t e r a c t i o n s between molecules which could re-s u l t i n protein denaturation. In view of t h i s development, subsequent protein was treated i n several d i f f e r e n t ways. Part - 43 -of the protein was chromatographed on the Sepharose 6B column, a t y p i c a l e l u t i o n i s shown i n F i g . 8. The protein eluted i n the same volume as hemerythrin from P. a g a s s i z i i , suggesting that i t , too, i s smaller than octameric P. g o u l d i i hemerythrin. Another portion of the protein was c r y s t a l l i z e d from 10% ethanol and stored under N 2» A t h i r d portion was made into the metazido form and p r e c i p i t a t e d from 30-50% (NH 4) 2S0 4 i n pH 8.1 TRIS and stored. Since the y i e l d of protein was so high, storage of the protein by l y o p h i l i z i n g i t from NH 4C0 3 buffer was t r i e d . This resulted i n i r r e v e r s i b l e changes i n the protein, the red oxyhemerythrin s o l u t i o n being converted to a brown-green powder which, on being redissolved i n buffer, only p a r t i a l l y regenerated oxyhemerythrin on chemical reduction of the i r o n and exposure to a i r . Another storage method was more successful. An a l i q u o t of the centrifuged supernatant was quickly frozen i n l i q u i d nitrogen, i n a p l a s t i c s c i n t i l -l a t i o n v i a l , and stored i n a freezer. On allowing t h i s to thaw at 4°C, the s o l u t i o n obtained had the same spectrum as before thawing. Several aliquots of the protein were stored at -10°C a f t e r l i q u i d nitrogen freezing. F i n a l l y , as was the case with P. a g a s s i z i i hemerythrin, some of the P. lurco protein, a f t e r passage through the Sepha-rose column and concentration on an Amicon PM 10 membrane, was stored under nitrogen as the deoxy form with excess formami-dinesulphinic a c i d . Disc g e l electrophoresis of P. lurco hemerythrin, which - 45 -had been through the Sepharose column, gave the p r o f i l e shown i n F i g . 7 for e i t h e r pooled or s i n g l e animal protein. The protein consists of e s s e n t i a l l y one band with a minor compo-nent, a much more homogeneous hemerythrin than that from P.  a g a s s i z i i worms. Iron analysis resulted i n a molar a b s o r p t i v i t y at 280 nm of 27,500 (± 3% 1 mole~^cm~^") per dimeric i r o n , s i m i l a r to the value obtained for P. a g a s s i z i i hemerythrin. P. hoduliferum Forty-four animals were c o l l e c t e d , cut open, and the coelomic f l u i d and washings pooled as previously described. The erythrocytes were rinsed and centrifuged and the packed c e l l s were transported i n i c e from A u s t r a l i a to Vancouver. The c e l l s were lysed by adding water, and a f t e r c e n t r i f u g a t i o n the s o l u t i o n was dialyzed against pH 8.2 TRIS plus 10 mM NaN^. Although no pink colour i n d i c a t i v e of oxyhemerythrin could be seen i n the l y s a t e , the d i a l y s a t e had a.pale orange colour c h a r a c t e r i s t i c of metazidohemerythrin; however, there were only three ml of s o l u t i o n , and i n s u f f i c i e n t protein to deter-mine molar a b s o r p t i v i t i e s . G. pyramidata Since the brachiopods were very small and opening one - 46 -animal gave only a few drops of colo u r l e s s s o l u t i o n , a l l the whole animals were homogenized i n a Waring blender with excess buffer. This mixture was then poured through cheesecloth to remove pieces of s h e l l and other p a r t i c u l a t e material and the sol u t i o n was centrifuged. The supernatant was concentrated and run down the Sepharose column. Two peaks eluted with K a v e = 0.54 and 0.74, corresponding to molecular weights of >50,000 and about 13,000. D i a l y s i s of the bands against 5 mM Na2EDTA and 5 mM NaN^ i n pH 8.1 TRIS caused the smaller weight band to become pale orange, suggesting that some metazidoheme-r y t h r i n was present. The larger weight band had no absorbing peaks between 300 and 400 nm and so was discarded. When material, which had not been through the Sepharose column, was run on sodium dodecyl sulphate gels, two bands ap-peared of about the same molecular weight as the column bands. Since there was not enough of the protein for i r o n analyses, (66) a sodium dodecyl sulphate gel was stained for i r o n . The 13,000 molecular weight band only, gave a p o s i t i v e r e s u l t , i n -di c a t i n g the presence of iro n i n t h i s component. T. transversa F i f t e e n brachiopods were pried open and the insides scraped out and pooled together with the r i n s e s o l u t i o n . This volume of material and three times the volume of water were combined. This mixture was homogenized i n a blender, f i l t e r e d - 47 -through cheesecloth, and centrifuged. The r e s u l t i n g pink so-l u t i o n eluted i n the void volume of a Sephadex G-75 column, implying a molecular weight of >7G,000 for a globular p r o t e i n . The s o l u t i o n had no absorption peaks between 300 and 400 nm, gave eight bands on d i s c g e l electrophoresis, and i r o n analyses revealed no i r o n i n s o l u t i o n . Thus i t seems that there was e i t h e r no, or only a trace of, hemerythrin, and no further studies of t h i s material were performed. Subunit Size and Quaternary Structure A number of techniques have been applied for determining (72 73 74) the s i z e of b i o l o g i c a l macromolecules. ' ' Considerations of cost, convenience, and usefulness resulted i n the use of two procedures: sodium dodecyl sulphate gel electrophoresis for de-termination of subunit s i z e , and Sephadex gel permeation chro-matography for determining native protein s i z e , and thus quater-nary structure. As i n other aspects of t h i s work, P. g o u l d i i hemerythrin was used, both as a standard and for species compa-ris o n s . The sodium dodecyl sulphate gels were run as described e a r l i e r (see page 32). To use them for molecular weight deter-mination, standard proteins of known molecular weight were run i n duplicate, i n p a r a l l e l with hemerythrin runs. A c a l i b r a t i o n curve was constructed by p l o t t i n g the logarithm of molecular weight against m o b i l i t y on the g e l . The protein m o b i l i t y was determined, and by i n t e r p o l a t i o n , the molecular weight of the - 48 -subunits was c a l c u l a t e d . Native protein s i z e was determined on a 73 X 2 cm Sephadex G-100 column c a l i b r a t e d with standard globular proteins. A c a l i -b ration curve was made by p l o t t i n g the logarithm of molecular weight versus ^- a v e defined as before. The hemerythrin e l u t i o n volume was determined and the molecular weight obtained from the c a l i b r a t i o n curve. Protein e l u t i o n was monitored by measuring absorbance at 280 nm on a Cary 14 spectrophotometer, and the column was eluted with 2 ml f r a c t i o n s being c o l l e c t e d i n the Gi l s o n Micro Fractionator. Columns were run i n a cold box at 4°C. When not i n use, columns were stored i n buffer plus 10 mM azide anion to prevent b a c t e r i a l contamination. Void volume and evenness of column packing were determined with Blue Dextran. The columns were loaded e i t h e r : by layering on protein i n 10% sucrose, or by running the column j u s t dry and running the pro-t e i n into the column, and then they were eluted with buffer under gr a v i t y . Care was taken to keep the column pressure within the l i m i t s s p e c i f i e d by Pharmacia, and any noticeable change i n flow rate r e s u l t e d i n the column being repacked. Subunit Size A t y p i c a l c a l i b r a t i o n curve, and subunit molecular weight determination for P. lurco hemerythrin are shown i n F i g . 9. The curve tends to become non-linear at low molecular weight, introducing some uncertainty into the measurement; a - 49 -Figure 9 C a l i b r a t i o n Curve and Molecular Weight Determination fo r P. lurco Hemerythrin Monomers by Means of SDS Gel Electrophoresis M o b i l i t y i s determined as the distance of protein migration along the g e l , divided by the distance of tracking dye migration. Standards are bovine serum albumin (•) , ovalbumin (•), papain (A), myoglobin (O) , and RNase-A (A)• P. lurco hemery-t h r i n i s indicated by (•). The points represent the mean of duplicate determinations, and the l i n e i s a least-squares f i t to the points. - 51 -10% error l i m i t would represent two standard deviations. Re-s u l t s for the other proteins have been c o l l e c t e d i n Table IV. As can be seen from the table, a l l species of Sipunculids and Brachiopods containing hemerythrin have protein subunits of molecular weight s i m i l a r to that of P. 'gouldii hemerythrin subunits. I t i s c l e a r that hemerythrin from any source i s made up of sing l e polypeptide chains of around 115 amino acids. Quaternary Structure A t y p i c a l standardization curve and molecular weight determination f o r P. lurco hemerythrin are shown i n F i g . 10, and the r e s u l t s for other species are presented i n Table IV. It i s immediately obvious that hemerythrin from Phascolosoma sipunculids i s d i f f e r e n t from the " t y p i c a l " P. g o u l d i i protein, indeed, the Phascolosoma hemerythrins are unusual i n having a tr i m e r i c quaternary s t r u c t u r e . ^ 6 9 ^ The e l u t i o n p o s i t i o n and agreement between the two Phascolosoma proteins implies that they are both globular structures and of s i m i l a r i f not the same s p a t i a l arrangement of subunits. Given the known arrange-ment of subunits of octameric hemerythrin (see page 18), i t seems u n l i k e l y that the subunits i n the trimer would be arranged i n a l i n e a r manner. The sequence of P. a g a s s i z i i hemerythrin (75) i s p a r t i a l l y known, and the f i n a l seventeen residues show very high homology with the octameric protein, suggesting that t h i s region of the subunit i s not involved i n subunit i n t e r -- 52 -Table IV Molecular Sizes of Various Hemerythrins Species Mol. Wt. Subunit Mol. Wt. # Subunits/ multimer Ref. Phascolopsis g o u l d i i 108,000 13,500 8 a Themiste dyscritum 103,000 12,600 8 b Themiste z o s t e r i c o l a (from tentacles) (from muscles) 100,000 100,000 15,000 13,000 13,000 15,000 8 8 1 c c c Sipunculus nudus 100,000 12,800 8 d Phascolosoma a g a s s i z i i 40,600 12,800 3 e Phascolosoma lurco 40,000 14,000 3 f Phascolosoma noduliferum 40,000 - - -Lingula unguis 110,000 13,500 8 g Lingula r e e v i -110,000 - - h Gl o t t i d e a pyramidata - -14,000 - -Priapulus caudatus - - 1 i Table IV (continued) a) I. M. Klotz and S. Keresztes-Nagy, Biochemistry, 2_, 445 (1963) . b) K. Weber and M. Osborn, J . B i o l . Chem., 244, 4406 (1969). c) G. L. Klippenstein, D. A. Van Riper and E. A. Oosterom, J. B i o l . Chem., 247, 5959 (1972). d) G. Bates, M. Brunori, G. Amiconi, E. Antonini and J . Wyman, Biochemistry, 7, 3016 (1968). e) F. A. Liberatore, M. F. Truby and G. L. Klippenstein, Arch. Biochem. Biophys., 160, 223 (1974). f) A. W. Addison and R. E. Bruce, Arch. Biochem. Biophys., 183, 328 (1977). g) J . G. Joshi and B. S u l l i v a n , Comp. Biochem. Physiol., 44B, 857 (1973). h) I. M. Klotz i n "Subunits i n B i o l o g i c a l Systems" Part A, p.100 Ed. S. H. Timasheff and G. D. Fasman, Marcel Dekker New York, 1971. i) G. L. Klippenstein, p r i v a t e communication. - 54 -Figure 10 C a l i b r a t i o n Curve and Molecular Weight Determination for P. lurco Multimeric Hemerythrin on a Sephadex G-100 Column 3 a) Standards are P. g o u l d i i hemerythrin octamer (•), bovine serum albumin (A) , ovalbumin (B), P. a g a s s i z i i hemerythrin trimer (•) , and myoglobin (A), P. lurco hemerythrin i s indicated by the empty c i r c l e (O)• The l i n e i s a l e a s t -squares f i t to the points. - 55 -actions. Furthermore, the three chain-bending p r o l i n e residues i n the f i r s t ten amino acids from the N-terminal are conserved (75) xn the P. agassizxx protexn. Since t h i s region i s not i n -volved i n subunit i n t e r a c t i o n s i n the octamer, the subunits of the trimer may well be arranged head to t a i l i n a manner s i m i l a r to one layer of the octameric structure. Experimental determi-nation of the t r i m e r i c structure w i l l require X-ray d i f f r a c t i o n studies and for t h i s , p r o t e i n that i s c r y s t a l l i z a b l e (such as the P. lurco hemerythrin) w i l l be required. In addition, the heterogeneity evidenced by d i s c g e l electrophoresis of P. a g a s s i z i i hemerythrin has hindered the complete sequencing of (75) thxs protein. Here again, hemerythrin from P. lurco should be more amenable to determination of the primary structure of the t r i m e r i c subunits. Since there was not enough protein from P. noduliferum animals to determine the quaternary structure on the c a l i b r a t e d Sephadex column, a small (38 X 0.5 cm) Sephadex G-75 column was prepared, and the P. noduliferum protein e l u t i o n volume o f f of t h i s column was compared with the e l u t i o n volumes of other characterized hemerythrins. F i g . 11 shows the e l u t i o n p r o f i l e s of P. g o u l d i i octameric and monomeric hemerythrins, and P. lurco t r i m e r i c hemerythrin, on the small column, and also the e l u t i o n p r o f i l e of the P. noduliferum p r o t e i n . The P. noduliferum protein elutes i n a volume between that of octameric and monomeric hemerythrins, implying that i t has a quaternary structure between these two types. In f a c t , - 56 -Figure 11 E l u t i o n P r o f i l e s of Several Hemerythrins on a Small Sephadex G-75 Column P. g o u l d i i hemerythrin hemerythrin I O O O O P. nodu 1 iferum hemerythrin 8 12 Fraction number - 57 -the P. noduliferum protein elutes i n a s i m i l a r volume to that of t r i m e r i c P. lurco hemerythrin. While one cannot r u l e out the protein being a tetramer, the absence of any published r e -port of t h i s quaternary arrangement for hemerythrin, the t r i -meric nature of both Phascolosoma lurco and Phascolosoma  a g a s s i z i i hemerythrins, and the f a c t that P. noduliferum i s also a Phascolosoma sipunculid, make i t l i k e l y that the P.  noduliferum p r o t e i n i s also a t r i m e r i c hemerythrin. Summary In t h i s chapter a number of d i f f e r e n t procedures for i s o l a t i n g and stor i n g hemerythrin have been described. Column chromatography on 6B Sepharose i s a simple, routine procedure for obtaining p u r i f i e d p r o t e i n and at the same time giv i n g an i n d i c a t i o n of molecular weight of the protein. C r y s t a l l i z a t i o n from 10% ethanol of hemerythrin from P. g o u l d i i i s known to provide a su i t a b l e way of s t o r i n g t h i s p rotein. Another pre-ferred method i s to store the chromatographed protein i n solu-t i o n with excess reducing agent such as formamidinesulphinic acid as the deoxy form under nitrogen. Freeze drying i s unsuit-able for stor i n g hemerythrin as changes occur which are i r r e v e r -s i b l e as judged by UV-visible absorption spectroscopy. However, the protein can be stored frozen a f t e r quick freezing i n l i q u i d nitrogen. Disc g e l electrophoresis experiments have shown that - 58 -pooled hemerythrin from P. lurco animals i s i d e n t i c a l to that from a s i n g l e specimen, and that the p r o t e i n i s composed pre-dominantly of one elec t r o p h o r e t i c component i n contrast to the four components found for P. a g a s s i z i i hemerythrin. I t has been determined that hemerythrins from the family Phascolosoma, while having subunits s i m i l a r i n si z e to those of other hemerythrins, with molecular weights around 13,500, have a d i f f e r e n t native arrangement, composed of three subunits per molecule i n contrast to the eight subunits per multimer arrangement found i n Phascolopsis, Themiste, and Sipunculus. A l l the proteins contain i r o n ; the molar a b s o r p t i v i t y at 280 nm i s lower for the Phascolosoma hemerythrins than f o r the others, being around 28,000 compared to 33,000-35,000 (± 3% 1 mole cm ). No hemerythrin was i s o l a t e d from the a r t i c u l a t e brachio-pod T e r e b r e t a l i a transversa. However, for the lampshell brach-iopod G. pyramidata, s o l u t i o n with a protein of molecular weight s i m i l a r to that of other hemerythrin subunits was ob-tained. In addition, t h i s p r o t e i n contains i r o n and, as w i l l be discussed i n Chapter Three, spectroscopic evidence i s strong-l y suggestive of the presence of hemerythrin i n t h i s animal. - 59 -CHAPTER THREE SPECTROSCOPIC STUDIES A number of d i f f e r e n t types of spectroscopy have been employed i n order to obtain structure and function informa-t i o n about hemerythrin. In t h i s study, u l t r a - v i o l e t (UV)-v i s i b l e and c i r c u l a r dichroism (CD) techniques were used to compare hemerythrin from new animals with the protein from more well characterized species. Nuclear magnetic resonance (nmr), and fluorescence spectroscopies were used as new tools i n studying hemerythrin. UV-Visible Spectroscopy UV-visible spectroscopy was performed using e i t h e r Cary 14 or Cary 15 spectrophotometers and Northumberland TSL matched c e l l s of 0.5 or 1.0 cm path length. Unless stated, - 60 -spectra were recorded at ambient temperatures. In a l l cases, baselines were run with buffer and anion i n both c e l l s , f o l -lowed by protein s o l u t i o n versus buffer scans. Oxy protein was obtained by reducing the oxidized he-merythrin using excess d i t h i o n i t e and then removing excess d i t h i o n i t e e i t h e r by d i a l y s i s , or by passage down a short G-25 Sephadex column. The met anion d e r i v a t i v e s were pre-pared by d i a l y z i n g a known concentration of approximately -4 -1 10 M protein against 10 M anion i n buffer. NaN^ and NaN(CN)2> from MCB and A l d r i c h r e s p e c t i v e l y , were used as received. KSeCN, obtained from ICN/K&K, was r e c r y s t a l l i z e d from ethanol and dried i n vacuo over P4°^g before use. Po-tassium tricyanomethide was a g i f t from Dr. M. Wicholas. The absorption spectra around 280 nm of four hemery-th r i n s are shown i n F i g . 12. Several features can be noted. In common with many proteins, hemerythrin has an absorption maximum near 280 nm with a shoulder at 290 nm. This absorption i s p r i m a r i l y caused by 7T-7C* t r a n s i t i o n s of the aromatic amino acids (76) tyrosine and tryptophan, but i n the case of metalloproteins, there may also be contributions due to metal-ligand charge trans-fer bands. The absorption maxima for the Phascolosoma proteins occur at a s l i g h t l y shorter wavelength than for the octameric proteins, and as reported i n Chapter Two, the molar a b s o r p t i v i -t i e s are also lower. Both observations are consistent with Phascolosoma hemerythrin containing l e s s tryptophan than heme-r y t h r i n from the other species, and chemical modification - 61 -Figure 12 UV Absorption Spectra Around 280 nm for Hemerythrins from Four Species of Sipunculids T. z o s t e r i c o l a hemerythrin P. g o u l d i i hemerythrin P. lurco hemerythrin P. a g a s s i z i i hemerythrin - 62 -r e s u l t s discussed i n Chapter Four support t h i s i n t e r p r e t a t i o n . F i n a l l y , a l l the spectra have minima at about 250 nm and the absorbance at 240 nm i s s i m i l a r i n i n t e n s i t y to that at 280 nm. Both these features can be used as a rough guide to protein p u r i t y , e s p e c i a l l y during i s o l a t i o n , when many of the b i o l o g i c a l contaminants contribute to the absorption i n t h i s region, a l t e r -ing these features. The s p e c t r a l features between 300 and 600 nm have been used to characterize a number of d i f f e r e n t d e r i v a t i v e s of P. (32) g o u l d i i hemerythrin. The most usefu l forms for comparative purposes are the b i o l o g i c a l l y relevant oxyhemerythrin, and the oxidized form with the l a r g e s t measured ass o c i a t i o n constant, that with azide anion co-ordinated. Since deoxy protein has (32) no absorption bands above 300 nm, i t s spectrum i s uninforma-t i v e . The UV-visible s p e c t r a l features of the oxy and metazido forms fo r a number of d i f f e r e n t species are c o l l e c t e d i n Table V. Despite v a r i a t i o n s i n primary and quaternary structures, as a r e s u l t of v a r i a t i o n s i n species, the positions and strengths of the d i f f e r e n t bands are very s i m i l a r ;for a l l the hemerythrins, and t h i s betokens a s i m i l a r a c t i v e s i t e and i r o n atom co-ordi -nation to the protein, since the bands involve the i r o n atoms, having been assigned as e i t h e r raetal-ligand charge t r a n s f e r b a n d s , o r simultaneous p a i r e l e c t r o n i c e x c i t a t i o n s . This uniformity of a c t i v e s i t e s i s , i n general, further support-ed by CD spectroscopy. Several new oxidized forms of hemerythrin were prepared, - 63 -Table V Optical Absorption Parameters of Oxyhemerythrin and Metazidohemerythrin from Five Species of S i p u n c u l i d s a Species Oxyhemerythrin P. g o u l d i i 280 (35,400) 326 (6900) 360sh (5400) 500 (2300) p. g o u l d i i ^ - - 330 (6800) 360sh (5440) 500 (2200) T. dyscritum 280 (33,000) 329 (6700) 360sh (5100) 500 (2350) T. z o s t e r i c o l a 0 280 (32,800) - - - - 500 (2050) P. a g a s s i z i i 280 (29,200) 328 (6100) 360sh (4850) 500 (2000) P. lurco 280 (27,500) 330 (6370) 360sh (5150) 500 (2150) Species Metazidohemerythrin P. g o u l d i i 327 (7200) 370sh (4800) 446 (3800) P. g o u l d i i ^ 326 (6750) 380sh (4300) 446 (3700) T. dyscritum 325 (7750) 375sh (4750) 446 (3600) T. z o s t e r i c o l a 326 (7400) 380sh (4500) 446 (3500) P. a g a s s i z i i 327 (6700) 375sh (4450) 446 (3500) P. lurco 326 (7360) - - 480 (3640) d a) ^ m a x i n nm ± 2 nm f£ i n M (iron dimers) cm ± 3%J. b) K. Garbett, D. W. Darnall, I. M. Klotz and R. J. P. Williams, Arch. Biochem. Biophys., 135, 419 (1969). c) , T. z o s t e r i c o l a oxyhemerythrin near UV data were not quan t i f i e d . d) P. lurco metazidohemerythrin has no well-defined maximum. - 64 -and the absorption spectra of two of these forms f o r P. lurco pro-t e i n are shown i n F i g . 13 and s p e c t r a l parameters are l i s t e d i n Table VI. The selenocyanate d e r i v a t i v e i s notable f o r the charge t r a n s f e r band at 508 nm which i s at longer wavelengths than such bands f o r other met d e r i v a t i v e s , and causes t h i s form to be very s i m i l a r ( v i s u a l l y ) to oxyhemerythrin. Figure 13 Absorption Spectra of Two New P. lurco Methemerythrin D e r i v a t i v e s a 6000 E u \ A000\ O E 2000 0 350 400 4§0~ X(nm) 500 a) Absorption spectra of metselenocyanato- ( ) , and metdicy-anamido- ( ) d e r i v a t i v e s of P. lurco hemerythrin, i n 0.1 M TRIS acetate b u f f e r , pH 8.2. - 65 -Table VI Op t i c a l Absorption Spectral Parameters for New P. lurco Methemerythrin D e r i v a t i v e s 3 UV-Visible Spectra Ligand X € X e X € SeCN~ b 508 3850 363(Sh) 5650 331 6440 N(CN)~ b 480 880 380 6040 328 6220 a) \ ,„ i n nm ± 2 nm. 'vmax b) Oxidized p r o t e i n was dialyzed against TRIS acetate, pH 8.2, 0.1 M i n the appropriate ligand and the di a l y s a t e used as reference f o r the spectra. In order to determine the stoichiometry of binding of selenocyanate to hemerythrin, a binding experiment was conducted according to Job's method of continuous v a r i a t i o n . Varying amounts of equal concentrations of 1 mM metaquohemerythrin and selenocyanate, both i n pH 7.0 TRIS, were mixed together so that the t o t a l amount of protein plus anion remained constant. The amount of complex formed was determined from the increase i n absorbance at 508 nm, and a p l o t of the amount of complex formed versus varying mole f r a c t i o n of P. g o u l d i i hemerythrin i s shown i n F i g . 14. I t can be shown that the.position of the maximum at a mole f r a c t i o n value of 0.50 indicates a protein to anion r a t i o of one to one, while a maximum at 0.33 mole f r a c t i o n p r o t e i n implies a protein to anion r a t i o of one to two. The experimental maximum at a protein mole f r a c t i o n of 0.54 - 66 -Figure 14 Job P l o t of Binding of Selenocyanate to P. lurco Metaquohemerythrin a 2 . 1 0 0 Mo le F r a c t i o n of H e m e r y t h r i n a) Hemerythrin and SeCN~ i n i t i a l l y 1.12 mM i n pH 7.1 TRIS acetate buffer. - 67 -indicates that one selenocyanate anion co-ordinates to each subunit of the protein,, i n agreement with the c i r c u l a r d i c h r o i c spectrum i n t e r p r e t a t i o n (see page 84). The s l i g h t l y high value of the p o s i t i o n of the maximum could be due to a small amount of non-native protein, or to incomplete complex formation due to a low binding constant. When metselenocyanatohemerythrin was dialyzed overnight against pH 8.2 TRIS, the selenocyanate co-ordinated to the i r o n was replaced by hydroxide and methy-droxohemerythrin was obtained. Addition of tricyanomethide does not cause the UV-v i s i b l e s p e c t r a l c h a r a c t e r i s t i c s of metaquohemerythrin to change, suggesting that i t does not bind to the i r o n atoms i n the protein. However, when tricyanomethide was added to methydroxohemerythrin from P. g o u l d i i , the spectrum was a l -tered towards that of the metaquo form, as shown i n F i g . 15. Addition of perchlorate to P. g o u l d i i methydroxohemerythrin caused the spectrum to s h i f t i n the same way towards the metaquo form, an e f f e c t associated with "non-specific" anion (39) binding near the active s i t e i n P. g o u l d i i hemerythrin. Hence, i t appears as i f tricyanomethide behaves l i k e a "non-s p e c i f i c " binding anion with P. g o u l d i i hemerythrin. When tricyanomethide was added to P. lurco methydroxohemerythrin, no s p e c t r a l change was observed, nor was a s h i f t from the methydroxohemerythrin spectrum to that of metaquohemerythrin produced by adding perchlorate to P. lurco methydroxohemery-t h r i n . The UV-visible s p e c t r a l d i f f e r e n c e between samples of - 68 -Figure 15 Non-Specific Anion Binding by Tricyanomethide to P g o u l d i i Methemeryr.hrin a \ \ \ 300 320 340 360 380 400 X ( n m ) a) P. g o u l d i i protein, 0.2 mM, i n 0.1 M TRIS acetate pH 8.2 and with 0.1 M KC(CN), ( ). - 69 -P. lurco hemerythrin i n pH 8.1 TRIS and i n pH 6.6 TRIS, as shown i n F i g . 16, i s very small, and t h i s implies that the co-ordinated water at the active s i t e has a d i f f e r e n t pKa i n P. lurco heme-r y t h r i n than i n P. g o u l d i i p r o t e i n . Thus with P. lurco heme-r y t h r i n one cannot detect "non-specific" anion binding i n t h i s way, and so one cannot determine from these experiments whether or not tricyanomethide binds " n o n - s p e c i f i c a l l y " to the hemery-t h r i n . More important than the f a c t that new methemerythrin d e r i v a t i v e s have been made, i s the observation that a number of d e r i v a t i v e s can be f i t t e d into a pattern represented i n F i g . 17. A l l of the active s i t e binding species are elongated molecules, much larger i n one d i r e c t i o n i n space ( i . e . tending towards being one dimensional), while the "non-specific" binding molecules are b u l k i e r and have much more two or three dimensional character (see F i g . 17). In other words, i t i s easy to imagine that a s p a t i a l l y r e s t r i c t e d access to the ac t i v e s i t e of heme-r y t h r i n could allow long, t h i n molecules to reach the binuclear i r o n s i t e while preventing larger molecules from getting to the i r o n atoms. This p a r t i t i o n i n g of binding must r e f l e c t geometric constraints imposed by the protein on the a c c e s s i b i l i t y of the active s i t e to small molecules. A cautionary note must be added which applies i n general to the i n t e r p r e t a t i o n of mo d i f i -cation and binding studies on proteins. The s i z e and complexity of these molecules means that care must be exercised i n a t t r i -buting causal r e l a t i o n s h i p s to any one f a c t o r . In the above - 70 -a) Spectrum of P. lurco hemerythrin ~0.2 mM i n 0.1 M TRIS acetate buffer, pH 8.1 ( ) and pH 6.9 C ) i n a 0.5 cm c e l l . - 71 -Figure 17 P a r t i t i o n i n g of Binding of Species to Hemerythrin into Active S i t e and Non-Active S i t e Binding - 72 -Figure 17 (continued) a) J. H. Enemark and R. H. Holm, Inorg. Chem., 3_, 1516 (1964) . b) P. Andersen, J. Klewe and E. Thorn, Acta Chem. Scand., 21, 1530 (1967). - 73 -example, a recent report that formate forms a met d e r i v a t i v e v ' ' ' i l l u s t r a t e s the f a c t that geometry cannot be taken as the only c r i t e r i o n for determining active s i t e versus "non-specific" binding of small molecules to hemerythrin. C i r c u l a r Dichroism Spectroscopy CD spectroscopy i s used i n several d i f f e r e n t ways i n protein studies. The CD spectra, i n the f a r u l t r a - v i o l e t be-tween 190 and 220 nm, are r e l a t e d to K—TC* and n-K* t r a n s i t i o n s i n the amide bonds of the polypeptide chain, and are used to estimate r e l a t i v e amounts of d i f f e r e n t types of secondary (78 79) structure i n proteins, based on model compound studies. ' S p e c i f i c forms of a protein are often characterized by the i n t e n s i t i e s and p o s i t i o n s of the s p e c t r a l features i n the near u l t r a - v i o l e t and v i s i b l e CD spectra of the macromolecule. One can then compare the spectra of d i f f e r e n t forms of a protein or the spectra of proteins from d i f f e r e n t sources. From the s i m i l a r i t i e s or d ifferences of the spectra, comparative i n f o r -mation can be obtained about the d i f f e r e n t samples being inves-t i g a t e d . A l l of these approaches have been used i n t h i s study of hemerythrin. CD spectra were recorded on JASCO J-20 s p e c t r o p o l a r i -meters operated i n the CD mode. The f a r u l t r a - v i o l e t spectra were obtained at the U n i v e r s i t y of Calgary with Dr. R. S. Roche. A l l other spectra were recorded by courtesy of Dr. L. D. Hayward. - 74 -Instrument standardization was performed using the 290 nm band of d-camphor-10-sulphonate i n water. In a l l cases, baselines were run using buffer or buffer plus anion. The CD spectra of the polypeptide chains of P. g o u l d i i and P. lurco hemerythrins are shown i n F i g . 18. Although se-v e r a l d i f f e r e n t methods of determining secondary structure from sp e c t r a l parameters i n t h i s region have been used, the proce-dures are empirical and only p a r t i a l l y r e l i a b l e . Compari-son of the alpha h e l i x content from the c r y s t a l structure of P. g o u l d i i hemerythrin with the alpha h e l i x content calcu l a t e d from the CD spectrum shows the spectroscopic determination of secondary structure f o r t h i s p r o t e i n to be quite good. The s p e c t r o s c o p i c a l l y predicted value of 75% for the amount of alpha (29) h e l i c a l secondary structure i s the same as the value deter-(52) mined from X-ray studies. The s i m i l a r i t y i n subunit mole-cular weights of P. lurco and P. g o u l d i i hemerythrins means that the s i m i l a r i t y i n the spectra i n F i g . 18 implies a s i m i l a r secondary structure i n the subunits of the two. proteins, and thus a high degree (about 75%) of a l p h a 1 h e l i x i n the t r i m e r i c hemerythrin. The subunits i n the trimer are a n t i c i p a t e d to c o n s i s t of four p a r a l l e l alpha h e l i c a l segments arranged i n what Argos et a l . have re f e r r e d to as a "super secondary" structure,^ with the i r o n dimer located between the h e l i c e s holding them together. As with the UV-visible spectra, the CD spectra of oxy- and metazidohemerythrin are the most relevant for compa-- 75 -Figure 18 C i r c u l a r Dichroic Spectra i n the Amide Bond Region of P. g o u l d i i and P. lurco Oxyhemerythrin 200 2T6 22*6 25b" 2~3b~ Anm a) Spectra were obtained using oxyhemerythrin from P. g o u l d i i ( ) and P. lurco ( ) sipunculids of around 10~ 5 M i n subunits i n 0.1 M TRIS acetate buffer, i n a 0.98 mm c e l l . - 76 -r a t i v e purposes. The CD spectra of oxyhemerythrins from several species of sipunculids are shown i n F i g . 19, and the s p e c t r a l parameters of both forms are c o l l e c t e d i n Table VII for f i v e hemerythrins. As i s the case for the UV-visible spectra, a s t r i k i n g feature of the data i s the high degree of s i m i l a r i t y between hemerythrins from d i f f e r e n t sipunculids. CD bands are (82 83) quite s e n s i t i v e to ligand geometry, ' and thus the agree-ment i n p o s i t i o n and i n t e n s i t y of the bands among the proteins from d i f f e r e n t species i s strong evidence for a common act i v e s i t e c onfiguration. In f a c t , the only diff e r e n c e i n band p o s i -tions i n the CD spectra i s a s h i f t , of around 10 nm, to longer wavelength, of the shorter wavelength band i n oxyhemerythrin from P. a g a s s i z i i animals compared with the band p o s i t i o n i n the other oxyhemerythrins. Since the short wavelength peak i n the P. lurco oxyhemerythrin spectrum occurs i n the same p o s i t i o n as i n the spectra of octameric hemerythrins, the s h i f t i n the P.  a g a s s i z i i hemerythrin spectrum cannot be associated with the t r i m e r i c quaternary structure, and i t s cause i s not yet deter-mined . The brachiopod G. pyramidata and the sipunculid P. no- duliferum were two species investigated from which very l i t t l e p r o t e i n was obtained. Not enough material was i s o l a t a b l e to characterize a hemerythrin e i t h e r by a quantitative i r o n analysis or by UV-visible spectroscopy. In order to convert any hemery-t h r i n present in t o the most stable metazido form, and because the CD spectrum of metazidohemerythrin i s quite c h a r a c t e r i s t i c , Figure 19 Near UV-Visible C i r c u l a r Dichroic Spectra aof Oxyheme-r y t h r i n from Three Species of Sipunculids a) Spectra for P.. g o u l d i i ( ) i n pH 8.2 TRIS buffer and for P. agassizii~T ) and T. z o s t e r i c o l a (....), both i n pH 7.1 TRIS acetate buffer. Table VII P r i n c i p a l C i r c u l a r Dichroic Bands i n Oxyhemerythrin and Metazidohemerythrin from Fi v e Species of Marine Sipunculids 3-Oxyhemerythrin Metazidohemerythrin Species* 3 A(nm) A £ A(nm) P. g o u l d i i 520 -2.46 500 -4.09 Q P. g o u l d i i 520 -2.48 500 -4.30 T. dyscritum 520 -2.29 500 -3.98 T. z o s t e r i c o l a 520 -2.65 500 -3.55 P. a g a s s i z i i 520 -2.03 500 -2.68 P. lurco 520 -2.02 500 -3.50 P. g o u l d i i 336 -3.78 370 -9.02 P. g o u l d i i 336 -3.72 37 0 -8.92 T. dyscritum 336 -4.01 370 -9.41 T. z o s t e r i c o l a 336 -4.12 370 -8.42 P. a g a s s i z i i 346 -4.06 370 -6.22 P. lurco 338 -4.92 368 -8.75 a) Avalues ± 1 nm; A € (per dimeric i r o n unit) + 1%. b) Both oxy- and metazidohemerythrin from P. g o u l d i i and P. lurco were at pH 8.2, while those from T. dyscritum were at pH 7.5. T. z o s t e r i c o l a and P. a g a s s i z i i samples had oxyhemerythrin at pH 8.2 and metazidohemerythrin at pH 7.1. c) K. Garbett, D. W. Darnall, I. M. Klotz and R. J. P. Williams, Arch. Biochem. Biophys., 135, 419 (1969). - 79 -sodium azide was added to the supernatant a f t e r l y s i n g of the coelomic c e l l s and c e n t r i f u g a t i o n . In both cases a pale orange colour was observed, t y p i c a l of metazidohemerythrin. The CD spectra of these solutions a f t e r d i a l y s i s against buffer plus azide are shown i n F i g . 20.- A € values were determined for the P. noduliferum protein by assuming t h i s hemerythrin to have the same molar a b s o r p t i v i t y at 280 nm as P. lurco hemerythrin and using t h i s to determine P. noduliferum protein concentration. The A 6 values obtained are s i m i l a r to those of hemerythrins from other species (see Table V I I ) . The s i m i l a r i t y i n i n t e n s i t y and p o s i t i o n of the peaks i n the spectra i n F i g . 20 to the mag-nitude and peak positions of the other metazidohemerythrin spectra i s strongly suggestive that a hemerythrin protein has been obtained from G. pyramidata brachiopods and P. noduliferum sipunculids. Gel chromatography of P. noduliferum protein i s also i n d i c a t i v e of t h i s p r o t e i n being a t y p i c a l Phascolosoma t r i m e r i c hemerythrin (see Chapter Two). Several attempts to obtain more specimens of G. pyramidata were unsuccessful, so the r e s p i r a t o r y protein from t h i s source i s not yet characterized. The CD spectra of the two met d e r i v a t i v e s , dicyanami-dohemerythrin and selenocyanatohemerythrin from P. lurco worms, are shown i n F i g . 21, and the s p e c t r a l parameters l i s t e d i n Table VIII. The CD spectrum of the selenocyanate d e r i v a t i v e i s much more d i s t i n c t from oxyhemerythrin than the UV-visible spectrum, as shown i n F i g . 22, and confirms the presence of a new d e r i v a t i v e . The CD spectrum i s of the type suggested to be 350 400 450 500 X ( n m ) CD Spectra of protein from P. noduliferum ( ) and G. pyramidata ( ) i n pH 8.2 TRIS acetate buffer with >10 mM NaN^ added. AS was calcul a t e d for P. noduliferum using € 2 8 0 = 27/500 to determine the protein concentration. - 81 -Figure 21 C i r c u l a r Dichxoic Spectra gf Two New P. lurco Methemerythrin Derivatives  + 2.0-- 4 . 0 -350 4^0 4*50 50~0 X(nm) a) CD spectra of metselenocyanato- ( ) , and metdicyanamido-( ) d e r i v a t i v e s of P. lurco hemerythrins i n 0.1 M TRIS acetate buffer, pH 8.2. Table VIII Circular- Dichroism Spectroscopic Parameters for New P. lurco Methemerythrin Derivatives Ligand X A e X A e X A e X A e SeCN~ b 510 -1.97 415 -1.67 350 +0.17 305 -1.50 N(CN)~ b 485 -0.63 392 -3.63 345 +0.15 300 -2.20 a) A€ i n M (iron dimers) -1 cm ± 3%. b) Oxidized protein was dialyzed against TRIS acetate, pH 8.2, 0.1 M i n the appropriate ligand and the d i a l y s a t e used as reference for the spectra. - 82 -Figure 22 UV-Visible (Upper Trace) and C i r c u l a r Dichroic (Lower Trace) Spectra of P. lurco Oxy- ( ) and Metseleno-cyanato- ( ) Hemerythrins -4 Oxyhemerythrin, ~10 M i n 0.1 TRIS acetate, pH 8.2. Metselenocyanatohemerythrin, -IO""4 M i n 0.1 M TRIS acetate, pH 8.2, with -0.1 M KSeCN added. - 83 -Figure 22 UV-Visible (Upper Trace) and C i r c u l a r Dichroic (Lower Trace) Spectra of P. l u r c o Oxy- ( ) and Metseleno-cyanato- ( ) Hemerythrins 400. 450 X ( n m ) - 84 -representative of a one to one adduct between protein and ( 3 2 ) anion, i n agreement with the Job p l o t r e s u l t s f o r the s e l e -nocyanate d e r i v a t i v e . Nuclear Magnetic Resonance (NMR) Spectroscopy The use of nmr i n studying b i o l o g i c a l macromolecules i s (84 85 86) described i n several recent books. ' ' I n i t i a l nmr studies of protons i n proteins were followed by inves t i g a t i o n s by Mildvan, (87) Cohn, and others, on paramagnetic systems. Nmr research on (88) paramagnetic species has been extended by La Mar, and other signals besides proton resonances have also become useful for b i o l o g i c a l studies. Protons are so numerous i n proteins that most proton resonances are overlapped i n a broad diamagnetic envelope between 0 and 10 parts per m i l l i o n (ppm) downfield from tetramethylsilane (TMS). However, i n some cases, the c l a s s i c a l example being ribonuclease, i n d i v i d u a l resonances have been assigned, and h i s t i d i n e proton resonances have been most r e a d i l y (89) i d e n t i f i e d . Paramagnetic metal ions can cause large s h i f t s i n resonances of protons near to the metal ion u p f i e l d or down-f i e l d from the normal resonance positions and t h i s has enabled, for example, the proximal h i s t i d i n e resonance near the i r o n i n myoglobin to be i d e n t i f i e d . The proton spectra of P. g o u l d i i octameric and P. a g a s s i z i i t r i m e r i c hemerythrins were compared to t r y to r e l a t e s p e c t r a l d ifferences to known differences i n protein composition. Spectra of P. g o u l d i i hemerythrin were run - 85 -at varying pH values to t r y to resolve h i s t i d i n e resonances; and spectra obtained by courtesy of Dr. G. La Mar were used to t r y to locate paramagnetically s h i f t e d s i g n a l s . Most nmr spectra were run with assistance from Drs. A. G. Marshall and D. C. Roe, on a Varian XL-100 spectrometer using a proton probe. One spectrum was obtained by courtesy of Dr. B. D. Sykes, Uni v e r s i t y of Alberta, on a Bruker 270 MHz instrument. A l l the spectra were run using samples i n 5 mm sample tubes at temperatures of between 5° and 15°C with spectra being obtained i n the Fourier Transform mode of operation. Pro-t e i n samples were prepared i n the following manner. One half ml of known concentration of hemerythrin of about 1.0 mM was dialyzed into Sorenson's r^ M phosphate buffer of the desired (91) pH. A f t e r two changes of d i a l y s a t e , the protein was dialyzed against two to four changes of twenty-five ml aliquots of the same pH phosphate buffer made up i n D2O. A l l dialyses were per-formed at 4°C. The convention followed for assigning peak p o s i -tions w i l l be that for which chemical s h i f t s are given i n terms of 5 i n PP m u p f i e l d (-) or downfield (+) from TMS where X = 10 6 X — ^ ^- (5) Vo and l / r e f i s the frequency of TMS, 1/ i s the frequency of the reso-nance observed, and V i s the spectrometer operating frequency. In p r a c t i c e , sodium 2,2-dimethyl-2-silapentane-5-sulphonate (DSS) - 86 -was added to some of the aqueous pr o t e i n samples and peak p o s i -tions w i l l be quoted r e l a t i v e to t h i s standard or r e l a t i v e to the HDO peak. In addition to using P. g o u l d i i octameric hemerythrin and P. a g a s s i z i i and P. lurco t r i m e r i c hemerythrins, some P.  g o u l d i i metazidohemerythrin was modified overnight with 5-fold excess N-ethylmaleimide to convert the protein into monomeric hemerythrin with a modified cysteine residue. Since the p o s i t i o n of resonance of an i n d i v i d u a l proton depends upon i t s microscopic environment, the seven h i s t i d i n e C 2 protons i n native hemerythrin should, i n theory, give seven resonances corresponding to the i n d i v i d u a l environments of each residue i n the native conformation of the pro t e i n . Several fac-tors may a f f e c t the a b i l i t y to observe these s i g n a l s . H i s t i d i n e (92 C 2 and proton signals s h i f t downfield as the pH i s lowered, and thus t i t r a t i o n curves of h i s t i d i n e C 2 protons i n proteins (89) have been used to make peak assignments. As hemerythrin i s only stable above pH 6.0, the protein h i s t i d i n e resonances may be overlapped by signals from other aromatic amino acids, and i t i s u n l i k e l y that complete t i t r a t i o n curves w i l l be able to be obtained for the protein h i s t i d i n e s i g n a l s . H i s t i d i n e s co-ordinated to or near to the i r o n atoms w i l l , have t h e i r resonances paramagnetically s h i f t e d , which may enable i d e n t i f i c a t i o n of these signals i f they can be located. Octameric hemerythrin i s large enough that the rate of r o t a t i o n of the protein may broaden the resonances, making them d i f f i c u l t or impossible to detect. - 87 -A t y p i c a l proton nmr spectrum of P. g o u l d i i metazido-hemerythrin i s shown i n F i g . 23. I t shows the c h a r a c t e r i s t i c protein proton envelope with a broad peak around +7.0 ppm downfield due to aromatic protons, the solvent peak at +5.0 ppm, and resonances due to the a l i p h a t i c protons further u p f i e l d . At pH 8.2 (meter reading) no s i g n a l corresponding to an i n d i v i d u a l h i s t i d i n e resonance i s apparent i n the 7 to 8 ppm downfield region where such resonances are usually found. Spectra such as that i n F i g . 23 were also obtained for oxyhemerythrin, t r i m e r i c P. agas- s i z i i hemerythrin, and cysteine-modified monomeric P. g o u l d i i metazidohemerythrin. In no case was an i s o l a t e d h i s t i d i n e proton sig n a l observed. F i g . 24 shows the s p e c t r a l region where resonances from protons on aromatic residues occur for a spectrum of P. g o u l d i i metazidohemerythrin at pH 6.0 (meter reading). At +8.4 ppm there i s a resonance which i s l i k e l y due to the C 2 proton of a h i s t i d i n e residue. This assignment i s consistent with the peak being seen only at low pH values. If i t i s due to a h i s t i d i n e C 2 proton, i t would l i k e l y be s h i f t e d u p f i e l d and be unresolved from the other aromatic resonances at higher pH. In an attempt to further resolve t h i s s i g n a l , a spectrum was obtained at 270 MHz, however, as shown i n F i g . 25, l i t t l e improvement i s seen. The most promising approach to r e s o l v i n g h i s t i d i n e proton signals i n hemerythrin may be to compare the aromatic s p e c t r a l regions of t r i m e r i c and octameric proteins by nmr diff e r e n c e spectroscopy, e s p e c i a l l y as the h i s t i d i n e content i s d i f f e r e n t i n , for example, - 88 -Figure 23 100 MHz Proton NMR Spectrum of P. g o u l d i i Metazido-hemerythrin 3.5 mM P. g o u l d i i hemerythrin i n fjM phosphate buffer pH = 8.0 (meter reading). Spectrum was recorded with a recycle time of 1 sec. and represents an accumula-t i o n of 10^ t r a n s i e n t s . - 89 -Figure 24 Aromatic Proton NMR Spectral R|gion f o r P. g o u l d i i Metazidohemerythrin at 100 MHz a) Spectrum of ~3.5 mM protein i n ^M phosphate buffer i n D 20, pH = 6.0 (meter reading). The spectrum represents 1000 t r a n -sients with a recycle time of 1 sec. - 91 -a) Spectrum of ~3.5 mM protein i n £M phosphate buffer i n D 20, pH = 6.0 (meter reading). - 92 -P. g o u l d i i and P. a g a s s i z i i hemerythrins. K 1 Another approach to i n v e s t i g a t i n g h i s t i d i n e resonances involves modifying the h i s t i d i n e residues with d i e t h y l p y r o c a r -bonate (DEP), (equation 6) and comparing nmr spectra before and a f t e r m o d i f i c a t i o n . Spectra of L - h i s t i d i n e hydrochloride and L - h i s t i d i n e hydrochloride plus DEP were recorded, and the r e s u l t s are shown i n F i g . 26. The two CH r i n g protons i n the h i s t i d i n e , are decreased i n i n t e n s i t y by d i f f e r e n t amounts, and two or three new resonances of unequal i n t e n s i t y are present i n the ethoxyformylated product. DEP has r e c e n t l y been reported to r e a c t i n both 1:1 and 2:1 r a t i o s with (93) h i s t i d i n e , and can react at e i t h e r or U^, thus i t i s not as straightforward a modifying reagent as was i n i t i a l l y b e l i e v e d . Since s p l i t t i n g of the small peak due to h i s t i d i n e i n the p r o t e i n spectrum would make t h i s s i g n a l even more d i f f i c u l t to observe, nmr studies of DEP modified p r o t e i n were not pursued. In f a c t , DEP has r e c e n t l y been used i n conjunction with nmr to look not at - 93 -Figure 26 Downfield NMR Spectrum of H i s t i d i n | and H i s t i d i n e Modified with Diethylpyrocarbonate a) 10 mM h i s t i d i n e * H C l (upper trace) and a f t e r adding ~50 mM DEP (lower trace) i n pH 6.0 (meter reading) phosphate buffer i n D 20. Spectra were recorded at 100 MHz with a recycle time of 6 sec. and represent 65 scans. - 94 -aromatic protons, but at NH resonances."'*' Spectra showing paramagnetically s h i f t e d resonances i n P.  g o u l d i i hemerythrin are shown i n F i g s . 27 and 28. Two signals can be seen, at +12.5 ppm and -25 ppm from the solvent peak. These s h i f t e d resonances are probably due to protons near to the i r o n atoms as a change i n i r o n state from oxy- to deoxyhemerythrin a l t e r s the i n t e n s i t y of the s i g n a l s . When the same experiment i s performed with P. lurco hemerythrin, the oxyhemerythrin nmr spec-trum i s s i m i l a r to that for P. g o u l d i i hemerythrin, however, the spectrum of deoxyhemerythrin shows not the two signals seen i n the octameric deoxyhemerythrin spectrum, but instead, four down-f i e l d resonances are observed at +31.7, +43.2, +57.2, and +59.8 ppm from HDO (see F i g s . 29 and 30). Investigations of these paramagnetically s h i f t e d reso-nances may be the most informative use of nmr i n e l u c i d a t i n g structure-function information about hemerythrin because the resonances observed provide a d i r e c t probe of the active s i t e and can be examined as a function of species of pro t e i n , form of the protein, and quaternary structure of the hemerythrin. Further studies are needed to determine whether the differences i n the deoxy spectra are r e a l or are due to s i g n a l broadening i n the octamer, or to a d i f f e r e n t amount of solvent protons i n the two samples. Nevertheless, t h i s observation of resonances i n deoxyhemerythrin provides a method of i n v e s t i g a t i n g t h i s p h y s i o l o g i c a l l y important form of the protein which has no v i s i b l e absorption or c i r c u l a r dichroism and so i s not amenable - 95 -Figure 27 U p f i e l d NMR Spectra of P. g o u l d i i Oxy- and Deoxy-hemerythrin 3 a) Spectra obtained by Dr. G. N. La Mar on 3.3 mM hemerythrin i n £M phosphate buffer, pH ~ 7.7 (meter reading). Peak p o s i -tions from HDO.' - 96 -a) Spectra obtained by Dr. G. N. La Mar on 3.3 mM hemerythrin i n [5M phosphate buffer, pH ~ 7.7 (meter reading). Peak posi t i o n s from HDO. - 97 -a) Spectra obtained by Dr. G. N. La Mar on 1.6 mM hemerythrin i n rgM phosphate buffer, pH ~ 7.7 (meter reading). Peak positions from HDO. - 98 -a) Spectra obtained by Dr. G. N. La Mar on 1.6 mM hemerythrin i n TJM phosphate buffer, pH ~ 7.7 (meter reading). Peak positions from HDO. - 99 -to the studies with these techniques that have been c a r r i e d out on oxy and met forms. Fluorescence Spectroscopy Fluorescence spectroscopy as a t o o l for studying b i o l o -(95) g i c a l molecules, i s described i n a recent review. In general, two approaches are used. E i t h e r e x t r i n s i c fluorophores are i n -troduced into the macromolecule and t h e i r fluorescence i s inves-ti g a t e d , or the i n t r i n s i c fluorescence of, for example, the aro-matic amino acids i s studied. The l a t t e r approach has been used with hemerythrin, the fluorescence of tryptophan being examined. Tryptophan fluorescence has been most studied since phenylalanine has a very low quantum y i e l d , and tyrosine fluorescence i s often quenched. Spectra were run on an Aminco Bowman spectrofluorimeter using hemerythrins from f i v e d i f f e r e n t sipunculid species. In addition, spectra were obtained by courtesy of Dr. R. S. Roche, at the University of Calgary, on P. g o u l d i i and P. lurco heme-r y t h r i n s . Protein solutions were t y p i c a l l y about 3 JLM, which corresponds to an absorbance at 280 nm of around 0.05, i n the h a l f cm c e l l used, depending on the species, i n order to prevent the s o l u t i o n absorbance from acting as an o p t i c a l f i l t e r for the fluorescence. Studies were performed at room temperature using metazidohemerythrin i n order to prevent b a c t e r i a l contamination. Spectra of water, buffer, and buffer plus azide were run i n order - 100 -to provide a baseline. The 0.1 M TRIS acetate buffer and 10 mM NaN^ solutions did not fluoresce under the conditions used f o r examining the hemerythrins, and a small fluorescence i n the water was subtracted manually from the protein fluorescence spectra. Q u a l i t a t i v e emission spectra of f i v e hemerythrins are presented i n F i g . 31 and show that the fluorescence maximum varies from about 334 to 347 nm and also that the octameric pro-teins have a more intense emission than the t r i m e r i c proteins. The decreased emission fluorescence i n t e n s i t y for the Phascolo- soma proteins compared to the octameric hemerythrins i s consis-tent with a decreased tryptophan content (see Chapters Three and Four), i n the t r i m e r i c proteins. However, i t may also be par-t i a l l y due to d i f f e r i n g environments of tryptophans i n the d i f f e r e n t proteins since factors such as solvent p o l a r i t y around the residue, or distance or o r i e n t a t i o n of the tryptophans with respect to the i r o n atoms, could a f f e c t the fluorescence inten-s i t y . The wavelength of maximum fluorescence i n the 335 to 345 nm region of the emission spectra of P. g o u l d i i and P. lurco heme-ryt h r i n s s h i f t s with changing e x c i t a t i o n wavelength, and the peak maxima and i n t e n s i t i e s of the e x c i t a t i o n spectra of the two heme-ry t h r i n s change i n the 282 to 291 nm region but not between 260 and 280 nm with varying emission wavelength. Variations i n positions and shape of e x c i t a t i o n and emission fluorescence spectra with varying emission and e x c i t a t i o n wavelength respect-- 101 -Figure 31 Fluorescence Emission Spectra for Hemerythrins from Five Species of Sipunculids 310 326 33<j 340 350 3^0 376 3c?6 3910 X ( n m ) a) Spectra were obtained on 3.0 jM metazidohemerythrin i n pH 8.2 TRIS acetate buffer. A e x c i t a t i o n was 280 nm. - 102 -i v e l y , imply that the fluorescence i s heterogeneous, i . e . that there are d i s s i m i l a r fluorophores i n the sample. The v a r i a t i o n s i n fluorescence occur i n the s p e c t r a l regions corresponding to tryptophan fluorescence and so suggest that some tryptophans d i f f e r i n t h e i r fluorescence i n these hemerythrins. The f a c t that the heterogeneity i s more pronounced for P. lurco hemery-t h r i n than for P. g o u l d i i protein, could be i n d i c a t i v e of one anomalous tryptophan which could represent h a l f the trimer fluorescence, but only one quarter the octamer fluorescence, thus having a more pronounced influence on the fluorescence from P. lurco hemerythrin. Another observation about hemerythrin fluorescence i s that i t i s highly quenched. In f a c t , r e l a t i v e to a quantum y i e l d of 0.02 for tryptophan, the quantum y i e l d for P. g o u l d i i -3 hemerythrin i s about 5 X 10 . This observation i s consistent with examination of stereo s l i d e s of a subunit of P. g o u l d i i protein, which shows at l e a s t one tryptophan l y i n g near the two i r o n atoms i n the active s i t e , which could quench the f l u o r e s -cence. Furthermore, the quantum y i e l d of P. g o u l d i i monomeric hemerythrin prepared by N-ethylmaleimide modification of the octa-mer i s very s i m i l a r to that of the native octamer. This i s also i n agreement with models of the octamer i n which there are three tryptophan residues l y i n g on the outside of the molecule, not i n -volved i n subunit i n t e r a c t i o n s . These residues would have a s i m i -l a r environment i n the monomer or multimer, and so t h e i r . f l u o r e s -cence would not be a l t e r e d by changing the quaternary structure. - 103 -The low quantum y i e l d means that an o r i g i n a l objective of using fluorescence to measure low concentrations of pr o t e i n and a s s o c i -ation or d i s s o c i a t i o n e q u i l i b r i a i s not p r a c t i c a l . The v a r i a t i o n of fluorescence with oxidation state of the i r o n , and with anion co-ordination should both be i n v e s t i -gated i n order to determine whether changes occur which might be r e l a t e d , e s p e c i a l l y f o r changes i n i r o n oxidation state, to changes i n protein t e r t i a r y structure. - 104 -CHAPTER FOUR CHEMICAL STUDIES As a means for e l u c i d a t i n g information about structure and function, chemical modification of proteins constitutes one of the most widely used methods, and often provides the i n t e r -d i s c i p l i n a r y connection between chemistry and biology. A number of references are a v a i l a b l e on chemical modifications of pro-(96 97) teins ' which i l l u s t r a t e the breadth of studies undertaken. There are two aspects to t h i s type of i n v e s t i g a t i o n . One approach i s the development of new methods of modifying macro-molecules, e i t h e r by making new reagents, or by employing d i f -ferent conditions from established modification procedures, and determining the effectiveness of reagent or conditions for the protein modification. This type of study has elements of inge-nious science and c r e a t i v i t y . The other approach i s to use established techniques on new proteins, and i n f a c t , t h i s - 1 0 5 -approach, while being more straightforward, has, i n many cases, l e d to new appreciations about the s u i t a b i l i t y or otherwise of a p r o t e i n m o d i f i c a t i o n procedure. This l a t t e r method has been used i n t h i s work i n the context of comparisons of hemerythrins from d i f f e r e n t species. N-Bromosuccinimide N-bromosuccinimide i s a u s e f u l reagent f o r modifying tryptophan residues. The m o d i f i c a t i o n involves tryptophanyl peptide bond cleavage and can be represented as The reagent reacts with free sulphydryl groups and also with t y r o s i n e and h i s t i d i n e side chains, but at a much slower rate (98) than the r e a c t i o n with tryptophan. The u t i l i t y of the reagent i s due to the f a c t that the m o d i f i c a t i o n of tryptophan - 106 -i s accompanied by a decrease i n absorbance around 280 nm, which can be used to quantify the number of tryptophans modified. I t (99) has been shown that the number of oxidized tryptophans, n, can be obtained from the equation 1.31 X AA0_Q  n = 278 ( 8 ) 5500 c where A A 2 y g i s the decrease i n absorbance at 278 nm, c i s the molar concentration of protein, 5500 i s the molar e x t i n c t i o n c o e f f i c i e n t for tryptophan at 278 nm, and 1.31 i s an empirical factor based on model studies. The modification of tryptophans i n hemerythrin was per-formed by taking protein solutions of known concentration of around 1.6 X 10 ^ M i n pH 7.1 TRIS i n the sample c e l l and buffer i n the reference c e l l , adding 25 JULL aliquots of f r e s h l y prepared 0.01 M N-bromosuccinimide i n water, and measuring the absorption spectrum between 240 and 300 nm. Further aliquots of N-bromosuc-cinimide were added to both c e l l s , and the spectra were recorded u n t i l no further changes occurred. The number of tryptophans mo-d i f i e d was c a l c u l a t e d by using equation (8), and by knowing the number of subunits i n the sample, the number of tryptophans per subunit was determined. York and Fan have reported that N-bromosuccinimide modi-f i e s the four tryptophans i n P. g o u l d i i hemerythrin without affect-ing the near UV spectrum of the p r o t e i n . W h e n the procedure described above was used with P. g o u l d i i hemerythrin, four tryp-- 107 -tophans were modified i n the protein. When the same method was applied to P. lurco hemerythrin, the absorbance change correspond-ed to the modification of only two tryptophans (see Table IX). Table IX Mod i f i c a t i o n of Tryptophans i n Hemerythrin with N-Bromosuccinimide a Species [Hr] subunits A A b s 2 7 8 .#. Trp/subunit P. g o u l d i i 1.59 X 10" 5 0.255 3.8 P. lurco 1.57 X 10~ 5 0.130 2.0 a) Modification was c a r r i e d out and q u a n t i f i e d as described i n the text. The amino ac i d sequence around the tryptophans i s shown i n Table X f o r P. g o u l d i i , and T. z o s t e r i c o l a octameric hemerythrins, P. a g a s s i z i i t r i m e r i c hemerythrin, and T. z o s t e r i c o l a monomeric hemerythrin. C l e a r l y , the t r i m e r i c Phascolosoma hemerythrins have a lower tryptophan content than the octameric proteins. Furthermore, i t would appear from Table X that there are two i n v a r i a n t tryptophans i n hemerythrin subunits, those at p o s i -tions 10 and 97. In examining stereo s l i d e s of molecular models of the subunit of P. g o u l d i i hemerythrin, the tryptophan at p o s i t i o n 97 l i e s along the outer side of the subunit and blocks o f f the a c t i v e s i t e making i t hydrophobic and r e s t r i c t i n g access to i t . In terms of the known hydrophobic and s t e r i c a l l y hindered active s i t e s of hemoglobin and myoglobin, i t seems reasonable - 108 -Table X Tryptophan Content and Sequence Location i n Hemerythrins from Six Species of Sipunculids rH rd 4-> o En • P •r | C 3 Xi 3 W \ 04 P4 GO CM vo CM CM n CM CN CM 00 00 03 vo C O CM cn cu • H o CD Cu • H • H 'd rH o CM I 3 cu rH* CM En S H <u to cd rH < CM OH E H cn > i cn rH* ~or OH E H C cn CM cn • < CM OH EH > o •rH n OJ g rd -p o o rd •r l r l X ! -P >i r l CU g cu Xi (d rH o o • r | M CU •+J cn o N 3 (U a CM CH E H CU CO rd rH 4! CM 05 EH cn > i r H CO >i r H CM OH1 E H G cn < CM cn < CM Oi E H O •r l r l CU g rd - P O 0 Xi c • H M JG - P >i r l <U fi CU cd rH o o •r l U CU -P cn o N cn 3 CU rH CM OH E H 3 rH o cn >1 u T T >1 E H cn < cu co ~3~ 0) rH >1 rH C J CM CO rtj CM OS E H rd > o • H r l <u g o C 0 g o c • H r l r l CU g CU Xi -P • H M O cn >i CM cn -< OH « E H r l CU CO o • r l r l CU - P O o T 3 • r l M . C -P >i M CU g xi • r l • r l N • r l cn cn cd cn rd CM I CN 3 cu r H OH OH E H • CM cn < rd rH < "TT >1 EH rd rH CM cn rt! CM OH EH > O •r l u (U g •rl U • P CN (U c •rl r l O o 4-> O •rl >l u r l U 3 cu (U rH g 6 •r l cu • r l X ! CU +> •rl r l -P >1 u cu e cu xi - 109 -Table X (continued) a) G. L. Klippenstein, Biochemistry, 11, 372 (1972). b) R. E. F e r r e l l and G. B. Kitto, Biochemistry, 10, 2923 (1971). c) G. L. Klippenstein, J. C. Cote and S. E. Ludlam, Biochemistry, 15, 1128 (1976). d) J. S. Loehr, personal communication. e) G. L. Klippenstein, personal communication. - 110 -that t h i s tryptophan i s r e l a t e d to the function of hemerythrin. The tryptophan at p o s i t i o n 10 i s i n the N-terminal chain and may extend outward from the subunit. A report on the structure of P. g o u l d i i hemerythrin does not suggest any strong i n t e r a c t i o n between t h i s residue and other amino acids i n the octamer. In addition, since protein absorbance at 280 nm i s due p r i m a r i l y to tryptophan, the lower 280 nm e x t i n c t i o n c o e f f i c i e n t s found for the Phascolosoma proteins (see Chapter Two) are consistent with decreased tryptophan content. In summary, the N-bromosuccinimide reaction y i e l d s h a l f the number of tryptophans for P. lurco hemerythrin as for P.  g o u l d i i hemerythrin, consistent with the 280 nm molar absorp-t i v i t y of the proteins. In conjunction with sequencing work i t i s suggested that residues 10 and 97 are in v a r i a n t tryptophans i n P. lurco protein and most probably a l l other hemerythrins, and that hemerythrin from Phascolosoma sipunculids l i k e l y contains only two tryptophans. DTNB Ellman's reagent, 5,5' d i t h i o b i s 2-nitrobenzoic acid, (DTNB) i s a widely used compound for determination of sulphydryl residues i n p r o t e i n s . T h e reaction involved i s represented i n equation (9), and involves a disulphide exchange. The u t i l i t y of the reagent comes from the f a c t that the thionitrobenzoate anion formed i s strongly coloured, hence the number of modified - I l l -sulphydryls can be determined spectrophotometrically. The mo d i f i c a t i o n has two other features which make i t a t t r a c t i v e for p r o t e i n s t u d i e s . F i r s t , the r e a c t i o n i s quite s p e c i f i c ; DTNB does not react with any other side chains to give the coloured anion. Second, the r e a c t i o n i s r e a d i l y r e v e r s i b l e and the o r i g i n a l sulphydryl can be regenerated with d i t h i o l s such as d i t h i o t h r e i t o l . The r e a c t i o n was c a r r i e d out according to a modified Ellman procedure. To a s o l u t i o n of about 10 ^ M p r o t e i n i n -2 pH 8.1 TRIS i n a one cm c e l l was added 0.2 ml of 10 M DTNB. The same amount of DTNB was also added to the reference c e l l and the absorbance at 412 nm was monitored as a funct i o n of time. The number of modified sulphydryls was determined from the molar e x t i n c t i o n c o e f f i c i e n t f o r the thionitrobenzoate anion of 13,600 M^cm" 1 at 412 nm. ( 1 0 1 ) The absorbance change at 120 minutes i n F i g . 32 cor r e s -ponds to the m o d i f i c a t i o n of 0.95 of a sulphydryl group. Thus - 112 -Figure 32 Absorbance Change at 412 nm for the Reaction of DTNB with Three D i f f e r e n t Hemerythrins 0.5 Time (minutes) a) Direct spectrophotometric trace obtained on reaction of 3.7 X 10 M hemerythrin with excess DTNB i n pH 8.1 TRIS acetate buffer. - 113 -P. g o u l d i i hemerythrin reacts with DTNB to give one sulphydryl group modified per subunit of protein. This i s i n agreement with the known amino ac i d content (see F i g . 2). When P. lurco oxyhemerythrin was allowed to react with DTNB, no change i n absorbance occurred, implying that the protein contains no free cysteine residues. Protein from P. a g a s s i z i i gave no reaction with DTNB, and since i t i s k n o w n t h a t P. a g a s s i z i i hemerythrin contains no cysteine i t appears l i k e l y that the Phascolosoma proteins do not have t h i s amino a c i d . In view of the spectroscopic s i m i l a r i t y of the a c t i v e s i t e s of a l l hemerythrins (see Chapter Three), t h i s excludes cysteine from being an i r o n co-ordinating ligand. While the single cysteine i n octameric hemerythrin i s in v a r i a n t at p o s i t i o n 50 and con-t r i b u t e s to the in t e r a c t i o n s s t a b i l i z i n g the octameric quater-nary structure, i t s absence i n the t r i m e r i c proteins shows that i t i s not required for the formation of quaternary s t r u c -ture. Electrochemistry The a p p l i c a t i o n of the techniques 'of electrochemistry to the study of b i o l o g i c a l molecules i s a l o g i c a l extension of the r e a l i z a t i o n that one of the fundamental functions c a r r i e d out by metalloenzymes i n l i v i n g systems i s electron t r a n s f e r . Oxidative phosphorylation, which involves the pro-duction of adenosine triphosphate coupled to the reduction - 114 -of dioxygen to water, by the passage of electrons through the e l e c t r o n transport system, i s perhaps the best known case, but there are many examples of proteins being involved i n redox r e a c t i o n s . ^ 0 2 ^ often these are metalloproteins i n which the metal atom undergoes sequential oxidation and reduction. Measurements of t h i s process can lead to the determination of the number of electrons being transferred, and the redox p o t e n t i a l of the metal, and t h i s can be r e l a t e d to the free energy change associated with the redox re a c t i o n . The i r o n atoms i n hemerythrin may be considered to undergo a p a r t i a l redox change during the p h y s i o l o g i c a l reaction, compared to the complete electron t r a n s f e r of, for example, the oxygen-ases. The t r a n s i t i o n from deoxyhemerythrin to methemerythrin involves the protein i r o n going from an Fe(II) to an Fe(III) state. The numerical value of the redox p o t e n t i a l of a metal ion i n a protein can be r e l a t e d to the r o l e of the metal. At a more basic l e v e l , the redox p o t e n t i a l and i t s r e l a t i o n s h i p to the environment of the metal, both the types of ligands and t h e i r d i s t r i b u t i o n around the metal, and other possible e f f e c t s of the protein on the value of the p o t e n t i a l , constitute one of the most ac t i v e areas of bio-inorganic chemical re-s e a r c h . ' 1 0 3 ' 1 0 4 ' Measurements of the redox p o t e n t i a l of a metal ion i n a protein are usually performed i n one of two d i f f e r e n t ways. The d i r e c t method involves measuring the s o l u t i o n redox po-t e n t i a l , v i a an electrode immersed i n a protein s o l u t i o n , as - 115 -a function of the r e l a t i v e amounts of the oxidized and r e -duced forms present i n . s o l u t i o n . The i n d i r e c t method consists of measuring the r e l a t i v e amounts of oxidized and reduced forms of protein, and a redox i n d i c a t o r , spectrophotometrie-a l l y . The s o l u t i o n p o t e n t i a l i s then determined from the r e l a t i v e amounts of the forms of the i n d i c a t o r using the Nernst equation. This l a t t e r method requires a lack of spec-trophotometric interference among the d i f f e r e n t components, a redox i n d i c a t o r with a redox p o t e n t i a l near that of the protein metal (which p o t e n t i a l i s unknown), and the occurrence of thermodynamic r e v e r s i b i l i t y amongst a l l the species i n s o l u t i o n . Thus the d i r e c t method was employed i n studies on hemerythrin. Experiments were performed using the apparatus i l l u s -t rated i n F i g . 33. Figure 33 Apparatus for Protein Redox T i t r a t i o n s Pt flag electrode. N 2 r e fe rence electrode —Teflon cap 100ml beaker water jacket magnet ic s t i r re r - 116 -At l e a s t twenty ml of protein s o l u t i o n were placed i n the c e l l and brought to thermal equilibrium. Depending on whether an oxidative or reductive t i t r a t i o n was being performed, known amounts of K,Fe(CN)., or Na-S^O. (standardized by t i t r a t i o n with 3 6 2 2 4 _ 3 Fe(CN), ) were added to the protein s o l u t i o n , and the s o l u t i o n b p o t e n t i a l was measured using a platinum mesh or f l a g electrode and a standard calomel electrode as reference. P o t e n t i a l s were e i t h e r read o f f of the d i g i t a l s i g n a l of a Beckman pHasar-1 pH meter, or were recorded on a Leeds and Northrup X-Y recorder. P o s i t i v e nitrogen pressure was maintained over the s o l u t i o n , and the i n i t i a l s olutions were deoxygenated by s t i r r i n g f o r extended periods under a stream of nitrogen. The apparatus and procedure were tested by carrying out an oxidative t i t r a t i o n of Fe(II) trans 1,2-diaminocyclohexane-N,N,N',N'-tetraacetate (CDTA) with KFe 3(CN)g. Electrodes were cleaned before and a f t e r use by alternate r i n s i n g i n hydrazine and aqua r e g i a followed by a water r i n s e . Results and Discussion The oxidative t i t r a t i o n of Fe(II) CDTA resulted i n a p l o t of s o l u t i o n p o t e n t i a l vs. logarithm of the r a t i o of o x i -dized to reduced form which was l i n e a r with a slope of 58 ± 2 mV, which indicates that the apparatus could be used to measure a r e v e r s i b l e one-electron transfer between the i r o n atoms. The best oxidative t i t r a t i o n of octameric P. g o u l d i i - 117 -hemerythrin i s shown i n F i g . 34. Unfortunately, t h i s p l o t was non-reproducible, and could not be duplicated by reduc-t i v e t i t r a t i o n , a common t e s t of thermodynamic r e v e r s i b i l i t y . In f a c t , a number of t i t r a t i o n s , both oxidative and reductive, on hemerythrin from P. g o u l d i i , T. z o s t e r i c o l a , and P. agas- s i z i i , at temperatures between 10°C and 25°C, resulted re-peatedly i n measurements of s o l u t i o n p o t e n t i a l s which changed i n the expected d i r e c t i o n on addition of oxidant or reductant, but which were non-Nernstian and, i n most cases, unstable with respect to time. The i n t e r p r e t a t i o n of these r e s u l t s i s that the electrode was determining a meaningless p o t e n t i a l i n the sense that the p o t e n t i a l d i d not correspond to the protein i r o n . Since the stoichiometric oxidative t i t r a t i o n of hemerythrin i s slow,^05) solutions were l e f t for more than twelve hours, but a stable p o t e n t i a l was s t i l l not obtained. Since oxidation i s promoted by the presence of co-ordinating anions, oxidative t i t r a t i o n s were performed with excess azide ion i n siolution, but t h i s d i d not r e s u l t i n stable reproducible p o t e n t i a l s being obtained. The f a c t that none of these (octameric, t r i -meric, or octameric di s s o c i a t e d into monomeric) hemerythrins gave meaningful s o l u t i o n p o t e n t i a l s , implies that the f a i l u r e of the electrode to sense the protein Fe(II/III) couple i s due to the secondary or t e r t i a r y structure rather than the quaternary structure of the protein. Presumably, the amino aci d residues s h i e l d the i r o n atoms inside a somewhat hydro-phobic environment i n s o l u t i o n . This type of probl€;m has been - 118 -Figure 34 Oxidative T i t r a t i o n of P. 'gouldii Deoxyhemerythrin with K 3Fe(CN) 6 -140 -120 -100 -80 -60 E(mV)vs.SCE - 1 1 9 -encountered i n redox t i t r a t i o n s of other proteins, for example hemoproteins, ( 1 0 6> and one possible s o l u t i o n which has been employed i s to use small amounts of a molecule, which acts as a mediator for the protein metal s i t e and the electrode. Seve-r a l organic dyes have been used as mediators i n redox studies of h e m o g l o b i n , a n d methylene blue was t r i e d i n the heme-r y t h r i n s o l u t i o n s . The addition of mediator did n o t . a l t e r the s o l u t i o n p o t e n t i a l , nor d i d i t speed up any approach to a stable p o t e n t i a l on subsequent addition of reactant. I t has been suggested that the organic dye mediators, which are con-jugated aromatic systems, act through overlap between t h e i r 7T systems and the n cloud of the porphyrin r i n g , and i f so, t h i s explanation would r a t i o n a l i z e t h e i r f a i l u r e to mediate hemerythrin t i t r a t i o n s . In summary, we have not been able to measure a redox p o t e n t i a l for the change i n i r o n oxidation state from Fe(II) to Fe(III) for hemerythrin i n s o l u t i o n , by using metal e l e c -trodes, despite using protein from d i f f e r e n t animals, using mediators, and performing d i f f e r e n t types of t i t r a t i o n s . Further comments w i l l be deferred u n t i l Chapter Five. Metal Atom Replacement When successful, one of the most informative experiments to perform on metalloproteins i s to remove the metal atoms making apoprotein, and then to r e c o n s t i t u t e native holoprotein - 120 -with the metal reincorporated. This type of experiment: a) demonstrates c o n c l u s i v e l y the e s s e n t i a l nature of the metal atoms; b) often enables the r o l e , s t r u c t u r a l , c a t a l y t i c or both, of the metal atoms to be deduced; c) may enable the metal ion ligands of the protein to be determined; and d) by inv e s t i g a t i o n s of the e f f e c t s of metal s u b s t i t u t i o n on enzyme a c t i v i t y , may enable mechanistic i n t e r p r e t a t i o n s of the cata-l y t i c r o l e of the metal to be made. In view of the usefulness of such an experiment, a number of d i f f e r e n t attempts at pre-paring apo- and reconstituted hemerythrin were made. The i r o n atoms i n hemerythrin are bound to the protein such that d i a l y s i s for many days against chelating ligands such as b i p y r i d y l or 1,10-phenanthroline does not r e s u l t i n loss of i r o n from the pr o t e i n . Thus, i n order to make apo-hemerythrin, the protein must f i r s t be unfolded, di s r u p t i n g the secondary, t e r t i a r y , and quaternary structures. This was r o u t i n e l y accomplished by d i a l y s i s of the hemerythrin i n buffer with e i t h e r 6 M guanidine hydrochloride, or 8 M urea, both of which are well known protein unfolding agents. S o l i d 1,10-phenanthroline or b i p y r i d y l was then added to the unfolded protein in s i d e the d i a l y s i s tubing and the solu t i o n was dialyzed against several changes of denaturant i n buffer. This was repeated u n t i l addition of complexing agent produced no more coloured i r o n complex in s i d e the d i a l y s i s tubing. I n i t i a l l y , t h i s procedure was performed i n the stock pH 7.1 TRIS buffer, however, following suggested procedures f o r - 121 -making other apoproteins, V A J - U ; experiments were performed i n which the pH was adjusted to pH-3.7 before or during removal of the i r o n . At f i r s t , the i r o n - f r e e p r o t e i n was then dialyzed against buffer without denaturing agent. In l a t e r experiments, i r o n cations (both Fe(II) and Fe(III) s a l t s were used) were added to the d i a l y s a t e at e i t h e r neutral or a c i d i c pH, a f t e r which several changes of neutral buffer and denaturant were used with EDTA to remove n o n - s p e c i f i c a l l y bound i r o n . F i n a l l y , the denaturant was removed by d i a l y s i s against the stock pH 7.1 TRIS bu f f e r . A l l experiments were performed at e i t h e r 4°C or 0°C. To check that the 6 M guanidine hydrochloride would denature hemerythrin, a seri e s of solutions of hemerythrin i n d i f f e r e n t concentrations of guanidine were prepared and the 330 nm peak of the metchlorohemerythrin spectrum was measured for each s o l u t i o n . The peak at 330 nm was used rather than the more common procedure of monitoring the e f f e c t of dena-turant at 280 nm, because, while the l a t t e r absorbance i s caused by hydrophobic aromatic amino acids, the 330 nm peak i s r e l a t e d to the active s i t e of the protein, the relevant area to monitor for attempting i r o n atom removal. Results A f t e r twenty hours, the spectra of solutions of met-chlorohemerythrin i n varying guanidine concentrations from - 122 -0 M up to 2 M were superimposable. At 4 M guanidine, the 330 nm peak had disappeared and the spectrum was the same as that ob-tained i n 6 M guanidine. Thus i t appears that at low guanidine concentrations « 2 M) the ac t i v e s i t e i s not disrupted, but that above 4 M guanidine the protein i s unfolded. The diff e r e n c e spectrum i n the aromatic amino acids' absorption region for P.  g o u l d i i hemerythrin i n 6 M guanidine hydrochloride vs. protein i n 0.1 M TRIS acetate buffer i s shown i n F i g . 35. This spectrum i s s i m i l a r to that obtained for other unfolded proteins, with the three minima being a t t r i b u t e d to exposure to solvent of tryptophan and tyrosine residues i n the unfolded random c o i l form of the polypeptide chain. The i r o n could be removed from unfolded hemerythrin, as indicated by the formation of the coloured b i p y r i d y l or 1,10-phenanthroline i r o n complex, on adding the chelating agent to random c o i l chains of the p r o t e i n i n 6 M guanidine hydro-ch l o r i d e . D i a l y s i s against buffer and s o l u b i l i z i n g agent resulted i n removal of the i r o n complex from the protein . so l u t i o n . In addition, i r o n analyses performed on the protein s o l u t i o n r e s u l t e d i n l e s s than 0.1% of the o r i g i n a l i r o n con-tent s t i l l being present (see Table XI). Thus, making unfolded apohemerythrin i n denaturant i s straightforward. Under a l l conditions t r i e d , removal of the denaturant resulted i n copious p r e c i p i t a t i o n of the apoprotein, and i r r e v e r s i b l e denaturation. The apohemerythrin could only be maintained i n s o l u t i o n when s o l u b i l i z e d with urea or guanidine hydrochloride. This r e s u l t - 123 -Figure 35 Denaturation Difference Spectrum of P. g o u l d i i Hemerythrin by 6 M GuHCl &in the Aromatic Ammo Acids* Absorption Region ' 27X) 2815 2^ 0 3b~6 X ( n m ) —5 a) P. g o u l d i i methydroxohemerythrin, -10 M, i n pH 8.2 TRIS ace-tate buffer with denaturant i n the sample c e l l (1.0 cm) vs. an equal concentration of protein i n the same buffer without dena-turant i n the reference c e l l (1.0 cm). - 124 -Table XI Iron Analyses of Apohemerythrins Species I n i t i a l (Fe) F i n a l (Fe) % of I n i t i a l Fe P. g o u l d i i 1.0 X 10~ 3 M 5 X I O - 7 M .05% T. z o s t e r i c o l a 5.9 X 10~ 4 M 5 X 10~ 7 M .08% P. a g a s s i z i i 7.0 X I O - 4 M 4 X 10~ 7 M .06% i n combination with knowledge of the k i n e t i c s of r e f o l d i n g , VJ-J-*t,/ implies a s t r u c t u r a l r o l e for the i r o n atoms i n hemerythrin i n addition to the characterized functional purpose of the metal. Amino aci d co-ordination to the i r o n r e s u l t s i n the protein being held folded i n a correct conformation for s o l u t i o n s t a b i l i -ty, i . e . having, for example, hydroph i l i c groups ex t e r n a l l y and hydrophobic ones i n t e r n a l l y positioned. The p o s s i b i l i t y of p r e c i p i t a t i o n being due to formation of i n t e r - c h a i n disulphide bridges was excluded i n two ways. In one case, a reductant, d i t h i o t h r e i t o l , was added before removing the denaturant to reduce any disulphide bonds. In the other case, P. a g a s s i z i i hemerythrin was used, which has no cysteine residue. In both cases, the r e s u l t of removing the denaturant was the same as before, extensive p r e c i p i t a t i o n . I t thus appears that making apohemerythrin r e s u l t s i n the sub-units e x i s t i n g as random c o i l polypeptide chains, which do not remain i n s o l u t i o n i n the absence of a s o l u b i l i z i n g agent. Addition of metal ion, as e i t h e r Fe(II) or F e ( I I I ) , i n e i t h e r a c i d i c or neutral s o l u t i o n , followed by d i a l y s i s i n - 125 -neutral buffer and removal of the denaturant, resulted, i n a l l cases, i n protein p r e c i p i t a t i o n on removal of the denaturant. This would seem to suggest that, during protein synthesis on the ribosome, i n i t i a l f o l d i n g of the polypeptide chain creates a p a r t i a l l y formed metal bonding s i t e , and metal binding causes the molecule to f o l d into the native three-dimensional conformation. In contrast, the unfolded random c o i l form of the protein represents a Gibbs free energy minimum, and spon-taneous r e f o l d i n g does not occur even with i r o n atoms present. N i t r i c Oxide Binding In view of the f a c t that a l l the r e s p i r a t o r y proteins bind the small gaseous diatomic dioxygen r e v e r s i b l y , i t i s reasonable to inquire into t h e i r r e a c t i v i t y with other gaseous diatomics. In f a c t , i t i s well known that carbon monoxide binds about two hundred times more strongly to hemoglobin than does dioxygen, and i t has been shown that n i t r i c oxide also binds to the ir o n i n the heme re s p i r a t o r y pro-t e i n s . ^ ^ 4 ^ CO binding to the heme re s p i r a t o r y proteins has been studied both d i r e c t l y , and by measuring i t s e f f e c t on the binding of 02« NO co-ordination has been investigated by monitoring the v i s i b l e absorption spectrum and esr spectrum of, for example, nitrosylhemoglobin. ^-^^ I t appears that NO reacts with e i t h e r reduced or oxidized hemoglobin to form an Fe(II) d e r i v a t i v e and causes weakening or cleavage of the iron -126 -proximal-histidine co-ordination, so that both f i v e and six co-ordinate i r o n can be obtained, judging by the esr s p e c t r a . ^ x x 6 ^ In addition, NO binds more t i g h t l y to Fe(II) hemoglobin than -12 (117) any other ligand CK^^gg =10 ) . Hemocyanin has been shown to i n t e r a c t with NO, again using esr spectroscopy, but i n t h i s case, the NO acts d i f f e r e n t l y on the protein metal. Upon addition of NO to hemocyanin, i t appears that e i t h e r one or both Cu(I) ions per subunit can be oxidized to Cu(II), and that the f i n a l product does not have NO bound to the copper.^ x x*^ This i n t e r e s t i n g behaviour prompted i n v e s t i g a t i o n of a possible n i t r i c oxide reaction with hemerythrin. Deoxyhemerythrin was prepared by blowing N 2 saturated with buffer over a s t i r r e d s o l u t i o n of oxy protein at 4°C u n t i l the co-l o u r l e s s deoxy form was obtained, t y p i c a l l y for 8 to 12 hours. This s o l u t i o n was transferred anaerobically, using a nitrogen-flushed syringe, to a tonometer which had also been thoroughly flushed with N 2. The UV-visible and CD spectra of t h i s s t a r t i n g s o l u t i o n were recorded, a f t e r which NO was passed over the solu-t i o n for two to f i v e minutes, and the spectra of the s o l u t i o n were remeasured. Esr spectra were obtained by syringing an a l i q u o t of s o l u t i o n anaerobically into an esr c e l l , and freezing the sample to 77°K. F i n a l l y , the excess NO was displaced from the tonometer using N 2, then a i r or 0 2 was allowed into the tonometer, and spec-t r a were once more recorded. Mass spectrometrically pure n i t r i c oxide was obtained by courtesy of Dr. P. Legzdins and B. Koltham-mer by passing NO from a c y l i n d e r (Matheson 99.0% pure) through a - 127 -dry ice-acetone trap containing s i l i c a g e l to remove any moisture and higher nitrogen oxides. Results When NO was blown over a sol u t i o n of deoxyhemerythrin, the co l o u r l e s s s o l u t i o n became green within a minute of exposure to the gas, as has been reported for h e m o c y a n i n . O n recording the UV-visible absorption and CD spectra of the sol u -t i o n before and a f t e r NO addition, s p e c t r a l features appeared as shown i n F i g . 36 for P. g o u l d i i hemerythrin. The peaks i n the spectrum before NO treatment are due to small amounts of oxy-hemerythrin present, however, the hemerythrin-NO spectrum did not change on being l e f t i n an NO atmosphere for up to f o r t y - f i v e minutes. Thus i t seems probable that spectrum B i n F i g . 36 i s that of the protein f u l l y reacted with n i t r i c oxide. I n i t i a l l y , when esr spectra were recorded for the protein-NO so l u t i o n , the rather d i s t i n c t i v e spectrum shown i n F i g . 37 was obtained. How-ever, the s i g n a l i n t e n s i t y varied with d i f f e r e n t samples, and when the reaction was c a r r i e d out on f r e s h l y i s o l a t e d hemerythrin, no s i g n a l was observed. Thus i t i s presumed that the si g n a l i s an a r t i f a c t not associated with the f u l l y formed native NO-heme-r y t h r i n complex. The subsequent reaction of the so l u t i o n with dioxygen i s most i n t e r e s t i n g i n that on removing excess NO and passing dioxygen over the so l u t i o n , the spectra l a b e l l e d C i n F i g . 36 were obtained which are c l e a r l y due to oxyhemerythrin. - 128 -Figure 36 Absorption and C i r c u l a r Dichroic Spectra Showing P. g o u l d i i Hemerythrin Reaction with N i t r i c Oxide 3 360 336 430 4610 500 540 X ( n m ) a) Absorption (upper traces) and c i r c u l a r d i c h r o i c (lower traces) spectra of: (A) deoxyhemerythrin, (B) a f t e r exposure to NO, and (C) a f t e r passing 0, over the NO-hemerythrin. - 129 -- 130 -Thus, i n contrast to the binuclear copper hemocyanin-NO reaction, NO does not i r r e v e r s i b l y o x i d i z e e i t h e r of the i r o n atoms i n hemerythrin, nor does i t bind p a r t i c u l a r l y t i g h t l y to the Fe atoms as i n the heme r e s p i r a t o r y proteins. U n t i l the s t o i c h i o -metry of binding of NO to the protein i s determined, d e t a i l e d considerations of the binding of NO are premature. I t appears, however, that the n i t r i c oxide forms an i n t e r e s t i n g mimic of the r e v e r s i b l e dioxygen binding. The spectra shown i n F i g . 36 could also be obtained using t r i m e r i c hemerythrin from P. lurco sipunculids, thus here again the quaternary structure of the protein, at l e a s t to a f i r s t approximation, does not a f f e c t the r e a c t i v i t y of the active s i t e . F i n a l l y , i t i s noted that on blowing CO over a s o l u t i o n of deoxyhemerythrin, no colour change occurs, consistent with an e a r l y report that carbon monoxide does not a f f e c t dioxygen binding to hemerythrin.^ x x 9^ - 131 -CHAPTER FIVE CONCLUSIONS AND FUTURE CONSIDERATIONS In order to bring together the work described i n t h i s t h e s i s with our current knowledge of hemerythrin, the hemerythrin research reported i n the l i t e r a t u r e i n the l a s t few years w i l l be summarized. This w i l l be followed by discussion of the contribu-tions which the t h e s i s r e s u l t s have made, combined with sugges-tions for areas of study a r i s i n g out of t h i s work, which seem l i k e l y to be most p r o f i t a b l e for continued i n v e s t i g a t i o n . During the l a s t f i v e years, continuing chemical studies on hemerythrin have been complemented by several developments which are leading towards a molecular understanding of the r e l a -tionship between the structure and the function of the protein. These advances may be grouped for consideration, into three areas; c r y s t a l l o g r a p h i c studies, k i n e t i c studies, and phylogene-t i c comparisons. - 132 -Considering f i r s t the continuing chemical studies of hemerythrin, chemical modification of tyrosines i n P. g o u l d i i hemerythrin with tetranitromethane and N-acetyl imidazole indicated that the two tyrosines at positions 8 and 109 i n the amino acid sequence were p o t e n t i a l ligands to the i r o n atoms.^ i 2^ The other three tyrosines were not co-ordinated to the metal atoms, but t h e i r modification resulted i n d i s s o -c i a t i o n of the octamer, implying that at l e a s t one of the tyrosines was i n or near the subunit i n t e r f a c e s . Modification of a l l eighteen carboxyl groups i n P. g o u l d i i hemerythrin with-out changing the UV-visible absorption spectrum of the met-chloro protein caused Klippenstein to exclude these groups from being i r o n ligands. He also found that modification of at l e a s t one carboxyl group aff e c t e d the monomer-multimer equilibrium. Recent solvent perturbation and chemical modifi-cation studies of P. g o u l d i i hemerythrin showed that three of seven h i s t i d i n e s could be modified without changing the active s i t e absorption spectrum, and that two of four tryptophans were exposed to the solvent with at l e a s t one tryptophan near the active s i t e and subunit i n t e r f a c e . ^ x 2 2 ^ The technique of resonance Raman spectroscopy has been used to help to determine the state of the dioxygen molecule i n oxyhemerythrin. Comparisons of the stretching frequency of dioxygen bound to the protein with that of dioxygen i n well defined oxidation states i n other molecules showed that the molecule i s co-ordinated as peroxide i n oxyhemerythrin both - 133 -(123 57) within the erythrocytes and i n v i t r o . ' Further studies with i s o t o p i c a l l y substituted dioxygen and azide showed that these species are e i t h e r co-ordinated through one atom as a bridge between the i r o n atoms, or through one atom to only one of the ir o n atoms, i . e . the ligands are not co-ordinated by two oxygen or nitrogen atoms to e i t h e r the same or d i f f e r e n t i r o n atoms. ^ 2 4 ^ The most recent X-ray s t r u c t u r a l study reports on the o (54) active s i t e at 2.8 A r e s o l u t i o n for T. dyscritum hemerythrin. At t h i s refinement, the i r o n atoms appear to be co-ordinated octahedrally, forming two antiprisms which share a common face. One i r o n atom i s co-ordinated to nitrogens of three h i s t i d i n e amino acids while the other i r o n i s l i g a t e d to two h i s t i d i n e nitrogens and the oxygen of a tyrosine residue. There appear to be two carboxylate bridges between the i r o n atoms, one each with an as p a r t i c and glutamic residue and t h i s would leave one co-ordination s i t e vacant. Presumably t h i s i s where dioxygen i n oxyhemerythrin and other anions i n methemerythrins bind. Since the amino acid, sequence for T. dyscritum hemerythrin i s not reported, the assignment of i r o n ligands was based on the sequence of the P. g o u l d i i protein, however, the low r e s o l u t i o n electron density maps of the two proteins are s i m i l a r . The spectroscopic comparisons of oxyhemerythrin and metazidohemerythrin from these and other sipunculids, described i n Chapter Three, have contributed to the proposal that the active s i t e s of a l l the proteins are very s i m i l a r and suggest - 134 -that the differences i n a c t i v e s i t e s of hemerythrins from d i f -ferent species are u n l i k e l y to be as great as those proposed from the i n i t i a l X-ray studies. In f a c t , i t appears from spec-troscopic studies that the dioxygen binding s i t e i s very s i m i l a r i n a l l hemerythrins despite differences i n primary, t e r t i a r y , and quaternary structures. K i n e t i c studies of some of the reactions of P. g o u l d i i (125 126) hemerythrin have recently been reported ' using stopped flow spectrophotometry and monitoring the marked colour changes which occur on changing forms of the pr o t e i n . These studies showed that for reaction with e i t h e r dioxygen, or co-ordinating anions, the protein displays no c o - o p e r a t i v i t y . The subunits behave as independent, equivalent e n t i t i e s . The k i n e t i c s of r e a c t i o n of deoxyhemerythrin with dioxygen, and of metaquoheme-r y t h r i n with SCN~ are both e s s e n t i a l l y independent of pH. The oxygenation reaction has an equilibrium constant of 1.5 X 10^ M A with a large formation rate constant of 7 X 10^ M~ Asec~ A. "* a 2 5^ Thermodynamic parameters for oxygenation were also determined and agreed with e a r l i e r r e s u l t s (see page 15). Replacement of one anion by another i n methemerythrin, for a number of d i f f e r e n t anions and d i f f e r e n t conditions of pH, was shown to occur almost always by a d i s s o c i a t i v e mechanism v i a the aquated methemerythrin form.^ x 2^^ The reaction of metaquohemerythrin with d i f f e r e n t anions was suggested to occur v i a an a s s o c i a t i v e mechanism, due to the wide range of pseudo f i r s t - o r d e r rate constants obtained by using a large excess of anion i n the k i n e t i c measurements.^ x 2^^ - 135 -The k i n e t i c s of both oxygenation and anation are influenced by secondary s i t e binding, a fa c t o r not u s u a l l y encountered i n simp-l e r inorganic systems and a complication which must always be considered i n protein studies. The t h i r d recent development i n hemerythrin research i s the i n v e s t i g a t i o n of hemerythrin from d i f f e r e n t sources. In addition to the octameric hemerythrins from the sipunculid fami-l i e s Phascolopsis, Z o s t e r i c o l a , and Sipunculus, and the brachio-(127) pod Lingula, hemerythrin has been i s o l a t e d and characterized from two species of Phascolosoma sipunculids, P. a g a s s i z i i ^ ^ (128) and P. l u r c o . What i s l i k e l y to be hemerythrin has been obtained from the sipunculid P. noduliferum and the brachiopod G. pyramidata, although the protein from each of these sources has not yet been characterized. The Phascolosoma hemerythrins occur with the uncommon t r i m e r i c quaternary structure. Neither of the t r i m e r i c hemerythrins appears to have a cysteine residue, unlike the octameric hemerythrins, and the t r i m e r i c hemerythrins appear to have h a l f the number of tryptophans found i n the octameric proteins, with two tryptophans seeming to be conserved i n the primary structure. Several spectroscopic comparisons of d i f f e r e n t sipunculid hemerythrins show very s i m i l a r properties, and, by imp l i c a t i o n , s i m i l a r a c t i v e s i t e s for both oxyhemerythrin (129) and metazidohemerythrin. Thus, the r e s u l t s obtained have borne out the expectation that i s o l a t i o n and ch a r a c t e r i z a t i o n of hemerythrin from new sources i s a worthwhile p r o j e c t and t h i s aspect of protein study should be continued for hemerythrin, with - 136 -the book by Stephen and Edmonds 1 3 0 1 suggesting that there are many new hemerythrins to be discovered. S p e c i f i c a l l y , charac-t e r i z a t i o n of the protein from other species of Phascolosoma sipunculids should be pursued i n order to examine the proposal that the protein from t h i s genus has a t r i m e r i c quaternary s t r u c -ture, no cysteine residue, and le s s tryptophans than octameric hemerythrin. Experience with P. noduliferum sipunculids, and two species of brachiopods suggests that obtaining s u f f i c i e n t protein to be able to characterize i t may prove rather d i f f i c u l t for small animals, and hence e s p e c i a l l y for non-local creatures, specimens of a few cm i n length are d e s i r a b l e . As part of the comparative theme, i s o l a t i o n of protein from other than the coelomic c e l l s i s one area which should be investigated further. (59) I t has already been shown to be f e a s i b l e i n one case, and the r e l a t i o n s h i p between pr o t e i n from d i f f e r e n t parts of an animal i s an aspect of hemerythrin research which has not been examined. In p a r t i c u l a r , future studies of s t r u c t u r a l and spec-troscopic properties of tentacle hemerythrins should be a worth-while area for i n v e s t i g a t i o n . Two procedures, whose a p p l i c a t i o n to studying hemerythrin has only been i n i t i a t e d i n t h i s work, are the techniques of fluorescence spectroscopy, and nuclear magnetic resonance spec-troscopy. In both cases, experiments described i n Chapter Three have provided some new information on hemerythrin, and shown the usefulness of further studies of t h i s kind. While the i r o n atoms of the protein reduce the natural aromatic amino acid fluorescence, - 137 -i t i s possible to measure the protein fluorescence and, for the tryptophan residues i n P. g o u l d i i hemerythrin, the fluorescence has been r e l a t e d to an external or hydrophilic environment for most of these groups i n s o l u t i o n . Furthermore, the fluorescence of octameric and t r i m e r i c hemerythrins has been shown to be of d i f f e r i n g i n t e n s i t i e s , and chemical modification studies i n d i c a t e that t h i s v a r i a t i o n i s due, at l e a s t i n part, to a d i f f e r e n t tryptophan content i n the d i f f e r e n t proteins. In the future, measurements of the fluorescence of the d i f f e r e n t forms of the protein, i . e . deoxy- vs. oxyhemerythrin, may provide a s e n s i t i v e probe with which to investigate conformational changes i n the protein on oxygenation. Another, l i k e l y more u s e f u l , probe of such changes w i l l be provided by nmr spectroscopy. I t has been demonstrated that appreciable changes i n the proton nmr spectrum occur on oxygenation of hemerythrin, and i n addition, that these changes are d i f f e r e n t for the reaction of protein from d i f f e r e n t sources with dioxygen. Thus the general active s i t e s t r u c t u r a l s i m i l a r i t y evidenced by the UV-visible, CD, and resonance Raman spectroscopic comparisons does not extend to the d i s p o s i t i o n of i n d i v i d u a l hydrogen atoms i n the active s i t e . While the s i z e and the s o l u t i o n s t a b i l i t y of the protein l i m i t s the p o s s i b i l i t y of obtaining useful information by means of conventional proton nmr, the e f f e c t s due to the i r o n atoms on nearby protons, and the changes i n the i r o n atoms on reaction with dioxygen, means that comparative nmr studies are also f e a s i b l e to carry out on hemerythrin, and again, by using d i f f e r e n t forms of the protein - 138 -and protein from d i f f e r e n t species, a p o t e n t i a l wealth of i n f o r -mation becomes a v a i l a b l e about the d e t a i l e d behaviour of the molecule on oxygenation and oxidation. Further a p p l i c a t i o n of both fluorescence and nmr techniques to studying hemerythrin are current areas of i n v e s t i g a t i o n . Chemical studies on hemerythrin, both reactions of pro-t e i n amino acid groups, and reactions of the a c t i v e s i t e , have been c a r r i e d out with some success. In terms of chemical modi-f i c a t i o n of the pro t e i n , standard procedures have been used i n a comparative manner i n order to compare and to contrast heme-r y t h r i n from d i f f e r e n t sources. S p e c i f i c a l l y , i t has been shown that hemerythrin from P. lurco does not contain a cysteine r e s i -due modifiable under conditions for which the cysteine i n P.  g o u l d i i protein can be modified. As P. a g a s s i z i i hemerythrin does not contain cysteine, the absence of t h i s residue may be c h a r a c t e r i s t i c of t r i m e r i c hemerythrins. Another d i f f e r e n c e between the protein from the Phascolosoma sipunculids and other hemerythrins i s q u a n t i t a t i v e l y l e s s reaction with N-bromosucci-nimide i n d i c a t i n g a lower tryptophan content i n these hemery-t h r i n s , i n keeping with other spectroscopic evidence. I t has been suggested that two tryptophans are i n v a r i a n t i n hemerythrin, those at positions 10 and 97 i n the P. g o u l d i i p r o t e i n sequence (see F i g . 2). In the future, one would a n t i c i p a t e amino acid a n a l y s i s , followed by primary structure studies to be c a r r i e d out on a t r i m e r i c hemerythrin. For t h i s work the p r o t e i n from P. lurco, of the t r i m e r i c proteins so f a r characterized, appears, - 139 -by the c r i t e r i o n of di s c g e l electrophoresis, to be more homoge-neous and thus more amenable to such i n v e s t i g a t i o n s . The reac-t i v i t y of octameric and t r i m e r i c hemerythrins with anions such as dicyanamide and selenocyanate, but not with tricyanomethide, while not perhaps being highly s u r p r i s i n g , contributes to the pic t u r e of a uniformity of a c t i v e s i t e s i n hemerythrins from d i f f e r e n t species, and also provides supportive evidence for the postulate of a s t e r i c a l l y hindered access to the a c t i v e s i t e which has been demonstrated for the s o l i d state by X-ray crys-t a l l o g r a p h i c r e s u l t s . F i n a l l y , the establishment of a r e v e r s i b l e r e a c t i v i t y of hemerythrin with another gaseous diatomic molecule, NO, opens up the p o s s i b i l i t y of performing some of the types of experiments c a r r i e d out on hemoglobin or hemocyanin co-ordinated to NO. Having considered some of the p o s i t i v e r e s u l t s which have come from t h i s research, i t i s important to also consider the l e s s successful experiments both with a view to how they may be improved, and as a guide to aspects of research on hemerythrin which may have a low p r o b a b i l i t y of being p r o f i t a b l y pursued. Despite a number of d i f f e r e n t attempts, over a period of many months, i t was not possible to prepare buffer-soluble apoheme-r y t h r i n , or metal-substituted protein, nor was i t possible to determine the redox p o t e n t i a l of the i r o n atoms. I t seems that an e s s e n t i a l s t r u c t u r a l role- i s served by the i r o n atoms i n hemerythrin, such that t h e i r removal i r r e v e r s i b l y denatures the protein. Given the serendipity involved i n metal atom replace-- 140 -merit studies, the purs u i t of t h i s goal for hemerythrin i s l i k e l y to prove f u t i l e , despite the incentive provided by the value of metal atom replacement studies i n terms of improved understanding of the protein. On the other hand, while the i r o n redox proper-t i e s were not measured, useful suggestions can be derived from t h i s work as to how to proceed i n t h i s area. Two approaches seem possible . To perform an electrode redox t i t r a t i o n , i t w i l l be necessary to have a mediator present which can "sense" the i r o n atoms, thus providing a l i n k between the protein metal and the electrode. The ligand binding r e s u l t s from t h i s work and e l s e -where suggest that a comparatively small, elongated molecule i s most l i k e l y to be successful as a mediator. Hence the problem becomes one of f i n d i n g or making a small, preferably anionic, species, soluble i n neutral pH aqueous buffer, which undergoes a redox rea c t i o n with an c of around +100 mV with respect to a standard hydrogen electrode. A l t e r n a t i v e l y , a spectrophoto-metric t i t r a t i o n would be worth t r y i n g using a s o l u t i o n poten-t i a l i n d i c a t o r , again with an £ of around +100 mV, which i s s p e c t r a l l y d i f f e r e n t i n the oxidized and reduced forms but which could i n t e r a c t with, for example, the oxidant rather than the protein, and so sense the s o l u t i o n p o t e n t i a l . Monitoring the changes i n s o l u t i o n p o t e n t i a l as a function of r e l a t i v e amounts of oxidized and reduced hemerythrin, would enable an average £ 0 / for the p r o t e i n to be obtained. Of the many aspects of research i n protein bio-inorganic chamistry which have not been considered i n t h i s t h e s i s , i t i s - 141 -us e f u l , i f admittedly speculative, to t r y to suggest a few areas which might be most i n s t r u c t i v e to pursue i n future studies on hemerythrin. For the newly characterized proteins, a most useful next step w i l l be the determination of the parameters of the oxygenation reaction such as the H i l l c o e f f i c i e n t , the p ^ and i t s temperature dependence. K i n e t i c studies of the t r i m e r i c protein reactions v i s - a - v i s those of P. g o u l d i i hemerythrin w i l l help to answer the question of the r o l e of the quaternary structure i n these proteins. F i n a l l y , i t would seem to be time, both from the acquired knowledge of the hemerythrin active s i t e , and from recent developments i n synthetic inorganic chemistry, to a n t i c i p a t e that within the next few years, models for the act i v e s i t e of hemerythrin w i l l be synthesized and characterized. Already, binuclear metal-atom-containing units with d i f f e r e n t types and geometries of ligands are known,d^O) and as better models for the ac t i v e s i t e of hemerythrin are devised, studies of such e n t i t i e s w i l l contribute to e l u c i d a t i o n of the r o l e of the protein i n t h i s r e s p i r a t o r y c a r r i e r . Hopefully, i t w i l l be apparent that having had well characterized p r o t e i n from one source, P. g o u l d i i sipunculids, has enabled a great deal of comparative protein chemistry and i n t e r -p r e t a t i o n to be c a r r i e d out. 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