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UBC Theses and Dissertations

Haptoglobins and hemoglobin-haptoglobin complexes Malchy, Barry L. 1970

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HAPTOGLOBINS AND HEMOGLOBIN-HAPTOGLOBIN COMPLEXES by BARRY L. MALCHY B. S c , U n i v e r s i t y of Manitoba, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag ree tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y pu rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d tha t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t hou t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada 7 i Abstract Haptoglobins are serum glycoproteins which form complexes with hemoglobin. Three phenotypes of haptoglobin e x i s t i n serum (Hp 1-1, Hp 2-1, Hp 2-2). The l a t t e r two types e x i s t as a se r i e s of polymers while the.former type e x i s t s as a homogeneous pr o t e i n . A l l three haptoglobin types consist of 3 (heavy), chains and a ( l i g h t ) chains which are attached by disulphides. The haptoglobin types d i f f e r i n t h e i r a chains; Hp 1-1 contains only a"*" chains, while Hp 2-2 contains 2 1 2 only a chains and Hp 2-1 contains a and a chains. The hemoglobin-haptoglobin 1-1 complex consists of one molecule of hemoglobin attached to one molecule of haptoglobin. The thesis has been divided i n t o three parts. The f i r s t part (Section III) i s concerned with the r e a c t i o n of haptoglobin with an octameric (double) hemoglobin obtained from an inbred s t r a i n of mice. In t h i s hemoglobin each of the hemoglobin dimers i s joined together by a disulphide bond. The fac t that haptoglobin binds ag dimers i n d i c a t e s that i t i s a b i v a l e n t molecule l i k e the antibody molecule, immuno-g l o b u l i n G (IgG). This bivalence and re s u l t a n t resemblance to IgG i s examined by studying the r e a c t i o n of haptoglobin with t h i s mouse hemo-globin i n which the aB dimer i s held together by a disulphide bond. The r e s u l t s of both p r e c i p i t a t i o n studies and acrylamide gel e l e c t r o -phoresis confirm the postulated bivalence of haptoglobin and i t s resemblance to an antibody. o The second part (Section IV) of the thesis i s concerned with confirming the r e s u l t s obtained i n studying the disulphides of hapto-gl o b i n which were obtained by the c y s t e i c a c i d diagonal technique. These r e s u l t s predicted a model i n which the two halves of the hapto-globin molecule were held together by a disulphide bond at p o s i t i o n 21a. Also the r e s u l t s predicted an i n t r a c h a i n loop disulphide between h a l f - c y s t i n e s at po s i t i o n s 35 and 69 i n the haptoglobin a chain and an i n t e r c h a i n disulphide between a h a l f - c y s t i n e at p o s i t i o n 73a and the B chain. This s t r u c t u r e has been confirmed by studies on a cyanogen bromide fragment i s o l a t e d from haptoglobin which contains the i n t a c t a chain. Also the structure has been confirmed by studies on a hapto-gl o b i n d e r i v a t i v e i n which the molecule has been s p l i t i n h a l f by the breaking of a disulphide bond. The t h i r d part (Section V) of t h i s thesis i s an i n v e s t i g a t i o n i n t o the nature of the 393 sulphydryl of hemoglobin when hemoglobin i s bound by haptoglobin. The r e s u l t s demonstrate that there i s a d e f i n i t e change i n the environment of t h i s sulphydryl upon formation 14 of the hemoglobin-haptoglobin complex. Studies with C-iodoaceta-mide demonstrate however that 393 can s t i l l react i n the HbHp complex. i i i TABLE OF CONTENTS SECTION PAGE I INTRODUCTION 1 Haptoglobin Structure 1 Hemoglobin Structure 9 Hemoglobin-Haptoglobin Structure 14 Outline of the Present Study 19 II MATERIALS AND METHODS 23 Starch Urea Gel Electrophoresis 23 Polyacrylamide Disc Gel Electrophoresis 24 High Voltage Electrophoresis 26 Acid Hydrolysis and Amino Acid Analysis 27 Amino-Terminal Amino Acid and Carboxy-Terminal Amino Acid A n a l y s i s 29 Enzymatic Digestions of Fragment PC I I I 30 Preparation of Hemoglobins 31 Reactions with Hemoglobin and the Haptoglobin Heuoglobin Complex 32 D i t h i o d i p y r i d i n e s 32 ^C-iodoacetamide 32 Reactions with Haptoglobins 33 Cyanogen Bromide Cleavage of Haptoglobins 33 Reaction of Haptoglobin with a Mixture of Sodium Sul-phite and p-chloromercurisulphonate (pCMS) . . . . 34 i v SECTION PAGE Preparation of Haptoglobin 34 II I HAPTOGLOBIN DOUBLE HEMOGLOBIN (Hb.Hb) Reaction . . . . . . . 36 Introduction . 36 P r e c i p i t a t i o n Studies 38 Acrylamide Gel Elec t r o p h o r e s i s 43 IV THE DISULPHIDES OF HAPTOGLOBINS - Studies on Cyanogen Bromide Reactions with Haptoglobin and on Half-Haptoglobin . . . 58 Introduction 58 Nature of the Reaction as Examined by Disc Gels . . . . . 63 Starch-Urea Gel Analyses of the Reaction Products . . . . 67 Studies of the Reaction of CNBr with the Half-Haptoglobin Molecule 76 P u r i f i c a t i o n and Properties of Cyanogen Bromide Fragments 80 Further Studies on Fragment PC I I I 88 Diagonal Analyses on Fragment PC I I I 99 Further Studies on Half-Haptoglobin 106 Discussion and Conclusions 116 V THE EFFECT OF HAPTOGLOBIN ON THE REACTIVITY OF THE B 93 SULPHYDRYL OF HEMOGLOBIN 121 Pyridine Disulphide and Iodoacetamide Reactions 121 Comparison of Residues Reacting i n Hb.Hp Complex with Those i n Hb 127 V TABLES TABLE PAGE I Amino Acid Analysis of $ Chain Fragment Isolated from Fragment PC I I I A f t e r Reduction and A l k y l a t i o n 93 II Amino Acid Analysis of the Amino Acids Released by Carboxypeptidase A Digestion of Fragment PC I I I A f t e r the Fragment had been Oxidized using Performic Acid . . . . . 98 I I I Amino Acid Analyses and Amino-Terminal Analyses of Peptides B a l , Ba2, and Ba3 . . . . 103 IV Comparison of the Amino Acid Composition of the a-a1 D i s u l -phide Peptide Obtained a f t e r Pepsin Digestion of Hp 1-1 35 with the S-Peptide Obtained a f t e r Pepsin Digestion of 3 5S-Hp/2 I l l vi LIST OF FIGURES FIGURE PAGE 1 2 1 Corrected Sequences of the a and a chains of Haptoglobins 6 2 Structures of the Haptoglobin Disulphide Peptides . . . . . 8 3 Interatomic Contacts Between Residues i n Unlike Hemoglobin Chains , . 12 4 Sedimentation C o e f f i c i e n t s of Mixtures of Hp and Haemo-glob i n Chains as a Function of the Molar Ratio Haem/Hp. . 20 5 P r e c i p i t a t i o n Curves with the Concentration of Hb.Hb Maintained Constant 4 0 6 A Scheme f o r the Cleavage of Hb and Hb.Hb into Halves . . . 42 7 A Photograph (by Reflected Light) of the T u r b i d i t y Observed Shortly a f t e r Mixing Hb.Hb and Hp 4 4 8a Disc Gel Electrophoresis of Mixtures of Hp and Hb.Hb . . . . 46 8b Enlarged Photograph of the F i r s t 6 Gels i n Figure 8A . . . . 48 9 Disc Gel Electrophoresis of Solutions Prepared by Mixing a 12.5 mg/ml Solution of Hb.Hb with a 14 mg/ml Solution of Hp 1-1 51 10 Disc Gels (5%) Showing the E f f e c t of Mercaptoethanol on Hp-(aB-Ba) Complexes 54 11 A Scheme to I l l u s t r a t e the Possib l e Complexes of Haptoglobin with Double Hemoglobin 55 12 The Reaction of Cyanogen Bromide with a Methionyl Peptide . 62 v i i FIGURE PAGE 13 Acrylamide Disc Gel Electrophoresis (7.5%) of the Reaction Products of Cyanogen Bromide with Haptoglobins 64 14 Acrylamide Disc Gel A n a l y s i s of the Reaction of CNBr with Hp 2-1 as a Function of Time 68 15 Analysis of the CNBr Hp Reactions Using Starch Gel E l e c t r o -phoresis i n 8M Urea-Formate Buffer pH 4.0 . 70 16 Two Dimensional Urea-Formate Electrophoresis of CNBr Hp 1-1 . 73 17 Two Dimensional Starch Gel A n a l y s i s of CNBr Hp 2-2 74 18a Demonstration of the Formation of Hp/2 by Starch-Urea Gel Electrophoresis 77 18b Comparison of the Reaction Products of Hp 1-1 and Hp /2 with CNBr by Starch Gel Electrophoresis i n Formate-Urea . . . . 77 19 Phosphocellulose Chromatography of the Fragments Obtained a f t e r the Reaction of Haptoglobin with Cyanogen Bromide . . 81 20 Starch Urea Gel Electrophoresis i n Formate and Aluminum Lactate Buffers of CNBr Peptides a f t e r P u r i f i c a t i o n on Phosphocellulose 84 21 Electrophoresis i n Q.1M. Sodium Phosphate pH 7.0, 0.1% SDS . . 89 22 Separation of Fragment I I I Polypeptides a f t e r Reduction and A l k y l a t i o n 90 23 Electrophoresis i n O.lM Sodium Phosphate pH 7.0, 0.1% SDS . . 92 24 The Sequence of Fragment E 95 25 Analysis by Thin Layer Chromatography of the Dansyl-Amino Acids Obtained from the Amino-Terminal Residues of Frag-ment PC I I I 97 v i i i FIGURE PAGE 26 pH 6.5 Diagonal Map of 1.5 mg of a P e p t i c - T r y p t i c Digest of Fragment PC I I I 101 27 pH 4.0 Diagonal Map of 2.0 mg of a P e p t i c - T r y p t i c Digest of Fragment PC I I I 105 28 Structure of aB Disulphide Peptides Obtained from Peptic T r y p t i c Digests of Fragment PC I I I 107 29 Autoradiogram of the A c i d i c Peptides a f t e r High Voltage 35 Electrophoresis at pH 6.5 of a Peptic Digest of S-half-haptoglobin 108 30 Diagonal Map of Peptic Digests of Haptoglobin and H a l f -Haptoglobin 112 31 Scheme to Explain the Comparitive Diagonals of Haptoglobin and Half-haptoglobin 115 32 Structure of Haptoglobin 1-1 Showing the Disulphide Bonds . 117 33 The Reaction of 4-PDS with Hemoglobin, the Hemoglobin-Haptoglobin (Hb-Hp) Complex, and Free Haptoglobin . . . . 123 34 The Reaction of 4-PDS and 2-PDS with Methemoglobin i n the Presence of Increasing Amounts of Haptoglobin 124 14 35 The Reaction of C-iodoacetamide with Hemoglobin and Hemo-globin Haptoglobin Mixtures 125 36 High Voltage Electrophoresis at pH 6.5 a f t e r 16 Hour Acid 14 14 Hydrolysis of C-carboxymethyl-Hb and C-carboxymethyl-Hb-Hp 126 i x FIGURE PAGE 37 High Voltage Electrophoresis at pH 3.6 A f t e r 20 Minute 14 14 Acid Hydrolysis of C-carboxymethyl-Hb, C-carboxymethyl Hb-Hp and ^C-carboxymethyl Hp 128 Acknowledgments F i r s t I would l i k e to thank Dr. Dixon for h i s excel l e n t guid-ance, support and supervision during the course of t h i s work. His perceptive ideas and invaluable assistance have provided a major con-t r i b u t i o n to the t h e s i s . I would also l i k e to thank Choy Hew, Otto Rorstad, Gwen Chan, John Black, and Dorothy Kaufmann who have worked on the haptoglobin problem during my stay i n Dr. Dixon's laboratory. Their informative discussions and r e s u l t s have also provided impetus for many experi-ments . I s i n c e r e l y thank Joe Durgo f or t e c h n i c a l and general assistance which was provided with the utmost generosity. In a d d i t i o n to the above I owe thanks to many other people i n Dr. Dixon's laboratory who I have met over the past four years. I would l i k e to acknowledge Dave Gibson, Jim Ingles, Francisco Eng, John T r e v i t h i c k , V i c t o r Ling, David Pulleyblank, Bengt J e r g i l , Andy Louie, Peter Candido, Stuart Gilmore, Ken Stevenson, George Bailey, Michael Sung, Don Wigle, Sa-nga Pootrakul, K e i j i Marushige and Marilyn Saunders, a l l of whom I have had the p r i v i l e g e or working with. I am also indebted to many people who have not worked i n t h i s labor-atory but who have been of assistance to the work described. Of these people I would l i k e to acknowledge here, Ian Gillam, Ian Smith and Don Rainnie. F i n a l l y I would l i k e to acknowledge both the National Research Council and the Medical Research Council without whose f i n a n c i a l assistance t h i s t h e s i s would not have been p o s s i b l e . I INTRODUCTION Haptoglobin Structure Haptoglobin was discovered i n 1938 (1) when i t was found that serum had the property of increasing the peroxidase a c t i v i t y of hemo-globin. U t i l i z i n g the measurement of the oxidation of potassium iodide by e t h y l hydroperoxide, Polonowski and Jayle found that, while hemoglobin has a low peroxidase a c t i v i t y with a pH optimum of 5.6, the ad d i t i o n of serum s h i f t e d the optimum to 4.2 with a considerable increase i n peroxidase a c t i v i t y . The existence of haptoglobin was confirmed s e v e r a l years l a t e r by paper electrophoresis (2) which demonstrated that a hemoglobin-haptoglobin (HbHp) complex could be separated from hemoglobin. The early work on haptoglobin also demon-strated that t h i s p r o t e i n existed i n more than one form (3). Haptoglobin 1 was found to p r e c i p i t a t e between 54 to 64 per cent ammonium sulphate and to have a molecular weight around 100,000 while haptoglobin 2 p r e c i p i t a t e d between 40 and 51 per cent ammonium sulphate and had a molecular weight greater than 200,000 (4). Haptoglobin 1 passed the physico-chemical tests of homogeneity— s o l u b i l i t y curve, e l e c t r o p h o r e t i c migration, and u l t r a - c e n t r i f u g a t i o n while haptoglobin 2 was found to be heterogeneous (5,6). A great breakthrough i n haptoglobin research came with the development of starch gel electrophoresis (7). Smithies and Walker 2 found that serum could be c l a s s i f i e d i n t o three types (8). Type 1 produced and g l o b u l i n bands of about equal i n t e n s i t y . Type 2A produced s e v e r a l more bands which migrated on the starch gels between and 3 g l o b u l i n bands while the band decreased i n i n t e n s i t y . In type 2B the pattern was s i m i l a r to the 2A pattern except that the bands between the and 3 globulins moved more slowly. Using p a r t i a l -l y hemolyzed serum i t was observed that these proteins were pink before s t a i n i n g and were i n f a c t the hemoglobin-binding haptoblobins (9). The existence of these patterns i n sera was explained by the r e s u l t s of family studies (10). I t was postulated that there was a Hp"^  gene 2 and a Hp gene which were autosomal and exhibited incomplete dominance. Thus the phenotypes observed would be Hp 1-1, Hp 2-1 and Hp 2-2. These would correspond to the previously observed serum patterns type 1, type 2A and type 2B r e s p e c t i v e l y . P u r i f i c a t i o n of the haptoglobin components of serum confirmed these observations (9). Haptoglobin 1-1 ran on starch as a s i n g l e hemoglobin binding component while hapto-globins 2-1 and 2-2 ran as a s e r i e s of polymers with the 2-2 polymers running more slowly than the 2-1 polymers. At t h i s point the meaning of the multiple haptoglobin forms was a mystery. Although the r e s u l t s could be completely explained by 1 2 2 po s t u l a t i n g a Hp and an Hp gene, the presence of the Hp gene i n some unexplained way led to the formation of a s e r i e s of polymers upon gel e l e c t r o p h o r e s i s . Some authors (9,11) suggested that the polymer observation was an aggregation a r t i f a c t but i t was generally accepted to be a r e a l phenomenon. Another break-through came when 3 h a p t o g l o b i n was reduced w i t h mercaptoethanol i n 8 M urea and, a f t e r r e d u c t i o n , a l k y l a t e d w i t h iodoacetamide (12). The polypeptides produced were analysed by s t a r c h g e l e l e c t r o p h o r e s i s i n 8 M urea u s i n g sodium formate b u f f e r at pH 4.0. A l l three c l a s s e s of h a p t o g l o b i n produced a slow running very dark band which was c a l l e d the 3 chain of h a p t o g l o b i n and a f a s t e r running l i g h t e r band c a l l e d the a chain. I t was observed that the a chain band produced by h a p t o g l o b i n 1-1 ran more q u i c k l y than the corresponding a chain band i n h a p t o g l o b i n 2-2 w h i l e h a p t o g l o b i n 2-1 produced both bands. Thus the polymorphism of h a p t o g l o b i n could now be seen to be a s s o c i a t e d w i t h v a r i a t i o n s i n a chains and the occurrence of m u l t i p l e bands appeared to c o r r e l a t e w i t h the presence of the more 2 s l o w l y running band (a chain) observed upon g e l e l e c t r o p h o r e s i s . A l s o i t was found that the h a p t o g l o b i n 1-1 1s could be subdivided i n t o two IF IS 1 c a t e g o r i e s (a and a ) on the b a s i s of the m o b i l i t y of t h e i r a chains. Amino a c i d analyses of the p u r i f i e d a chains demonstrated the IF IS replacement of a l y s i n e i n Hp a by an a c i d i c amino a c i d i n a . The 1 2 amino a c i d analyses of the a and a chains were a l s o very s i m i l a r but they could not be e x p l a i n e d on the b a s i s of a simple p o i n t mutation. Instead f i n g e r p r i n t analyses i n d i c a t e d the presence of a new peptide 2 i n the a chain (13). This peptide had p r o p e r t i e s c o n s i s t e n t w i t h i t s r e s u l t i n g from the j o i n i n g of the c a r b o x y l t e r m i n a l of the a"*" chain to the amino t e r m i n a l of another a"*" chain. Amino a c i d analyses showed that t h i s j u n c t i o n peptide i s s l i g h t l y s m a l l e r than one which would occur by j o i n i n g the c a r b o x y l t e r m i n a l peptide to the amino t e r m i n a l 2 peptide. Since the a chain was a l s o shown to have about twice the molecular weight of the the evidence s t r o n g l y i n d i c a t e d that the polymeric haptoglobins had r e s u l t e d from a g e n e t i c event i n which a crossing-over had occurred at the DNA l e v e l w i t h a r e s u l t i n g p a r t i a l gene d u p l i c a t i o n . This e x c i t i n g r e s u l t represented, f o r the f i r s t time, the d e t e c t i o n at a molecular l e v e l of a p a r t i a l gene d u p l i c a t i o n and a l s o served to i n d i c a t e how the h a p t o g l o b i n polymers could be formed. The e x i s t e n c e of gene d u p l i c a t i o n s had been p o s t u l a t e d before and was based upon c y t o l o g i c a l observations on the bar locus i n D r o s o p h i l a (14). Since the time of the h a p t o g l o b i n d i s c o v e r y the presence of a p a r t i a l gene d u p l i c a t i o n has been detected i n many other p r o t e i n s and i n 5s RNA (15,16). 2 F i n g e r p r i n t analyses of the a chain a l s o i n d i c a t e d that the IS IF chain r e s u l t e d from the f u s i o n of the genes f o r the a and the a 2 (F S) chain. Thus once the d u p l i c a t i o n had occurred to form the Hp ' gene then a much more l i k e l y g e netic event than t h i s i n i t i a l occurrence 2FF 2SS would be the formation of Hp and Hp genes by c r o s s i n g over. S i m i l a r l y the formation of a t r i p l e c hain gene by m i s p a i r i n g should occur. Nance and Smithies have obtained evidence f o r the former p r e d i c t i o n (18) w h i l e the d i s c o v e r y of h a p t o g l o b i n Johnson appears to provide evidence f o r the l a t t e r (19). S e v e r a l other h a p t o g l o b i n phenotypes have been i d e n t i f i e d . These i n c l u d e Hp C a r l b e r g and Hp 2-1M i n which i t appears i n the former case that there i s an underproduction of a"*" chains and so 2-2 polymers appear along w i t h the 2-1 polymers w h i l e i n the l a t t e r case 2 there i s an underproduction of a chains so that the f a s t e r running polymers of s m a l l e r molecular weight are present i n greater p r o p o r t i o n (20). Other abnormal hap t o g l o b i n phenotypes are b e l i e v e d to be P H L caused by the Hp , Hp , and Hp genes but the gene a b n o r m a l i t i e s have not been c h a r a c t e r i z e d (21). With the Hp gene a d i f f e r e n t m o b i l i t y has been observed f o r the a chain i n a c i d i c urea s t a r c h g e l s (22). Both Hp Marburg and Hp Bellevue (23,24) are b e l i e v e d to r e s u l t from mutations i n the 8 chain of haptoglobin. Haptoglobin 2-1 Johnson, which migrates as a s e r i e s of polymers moving, more s l o w l y than hapto-g l o b i n 2-2 polymers, when examined i n the urea gels produces a normal 1 2 a chain and a new band which migrates more s l o w l y than the a band (25). 1 2 The complete sequence of both the a and a haptoglobin chains has now been e s t a b l i s h e d (26). The a"*" chain contains 84 amino acid s 2 and the a chain has 143 (Figure 1). Both chains have an amino t e r m i n a l v a l i n e and a c a r b o x y l t e r m i n a l glutamine and c o n t a i n no methionine or phenylalanine. There are four h a l f - c y s t i n e s i n the a^ " and seven i n the 2 a chain. The sequence of the h a p t o g l o b i n a chains has been compared w i t h known sequences of some of the l i g h t chains of a n t i b o d i e s (Bence-Jones p r o t e i n s ) using a computer program developed by F i t c h (27). The r e s u l t s i n d i c a t e d a homology between the r e g i o n around h a l f - c y s t i n e 86 i n the Bence-Jones p r o t e i n s and h a l f - c y s t i n e 35 of the h a p t o g l o b i n a chain. This r e s u l t i n d i c a t e d that there was p o s s i b l y a common e v o l u t i o n a r y o r i g i n f o r haptoglobins and a n t i b o d i e s . Haptoglobin a 1 84 residues o^F^Lys at p o s i t i o n 54 a 1S=Glu at p o s i t i o n 54 1 10 20 -Val-Asn-Asp-Ser-Gly-Asn-Asp-Val-Thr-Asp-Ile-Ala-Asp-Asp-Gly-Gln-Pro-Pro-Pro-Lys-30 40 -Cys-Ile-Ala-His-Gly-Tyr-Val-Glu-His-Ser-Val-Arg-Tyr-Gln-Cys-Lys-Asn-Tyr-Tyr-Lys-50 60 -Leu-Arg-Thr-Gln-Gly-Asp-Gly-Val-Tyr-Thr-Leu-Asn-Asn-Glu-Lys-Gln-Trp-Ile-Asn-Lys-70 80 -Ala-Val-Gly-Asp-Lys-Leu-Pro-Glu-Cys-Glu-Ala-Val-Cys-Gly-Lys-Pro-Lys-Asn-Pro-Ala-84 -Asn-Pro-Val-Gln-COOH Haptoglobin a 2 143 residues 1 10 20 -Val-Asn-Asp-Ser-Gly-Asn-Asp-Val-Thr-Asp-Ile-Ala-Asp-Asp-Gly-Gln-Pro-Pro-Pro-Lys-30 40 -Cys-Ile-Ala-His-Gly-Tyr-Val-Glu-His-Ser-Val-Arg-Tyr-Gln-Cys-Lys-Asn-Tyr-Tyr-Lys-50 -Lys- 60 -Leu-Arg-Thr-Gln-Gly-Asp-Gly-Val-Tyr-Thr-Leu-Asn-Asn-Glu-Lys-Gln-Trp-Ile-Asn-Lys-70 80 -Ala-Val-Gly-Asp-Lys-Leu-Pro-Glu-Cys-Glu-Ala-Asp-Asp-Gly-Gln-Pro-Pro-Pro-Lys-Cys-90 100 -Ile-Ala-His-Gly-Tyr-Val-Glu-His-Ser-Val-Arg-Tyr-Gln-Cys-Lys-Asn-Tyr-Tyr-Lys-Leu-110 -Lys- 120 -Arg-Thr-Gln-Gly-Asp-Gly-Val-Tyr-Thr-Leu-Asn-Asn-Glu-Lys-Gln-Trp-Ile-Asn-Lys-Ala-130 140 -Val-Gly-Asp-Lys-Leu-Pro-Glu-Cys-Glu-Ala-Val-Cys-Gly-Lys-Pro-Lys-Asn-Pro-Ala-Asn-143 -Pro-Val-Gln-COOH FIGURE 1 CORRECTED SEQUENCES OF THE a 1 AND a 2 CHAINS OF HAPTOGLOBINS 7 The h e a v i e r 3 chains of h a p t o g l o b i n have a molecular weight of 40,000 to 42,000 (28,29,30) and c o n t a i n a l l of the carbohydrate attached to the molecule. Haptoglobin contains 14 to 16 per cent carbohydrate which has the composition, 4.6 per cent s i a l i c a c i d , 4.2 per cent glucosamine, 0.2 per cent fucose, and 5.6 per cent hexose (31). The 3 chain has an amino t e r m i n a l i s o l e u c i n e (32,33) and t h i s c h ain appears to have a very s i m i l a r s t r u c t u r e i n a l l three major c l a s s e s of haptoglobins (34). The d i s u l p h i d e bonds of h a p t o g l o b i n (35) have been i n v e s t i g a t e d u s i n g the c y s t e i c a c i d d i a g o n a l technique (36). When these s t u d i e s were performed a f t e r h a p t o g l o b i n was d i g e s t e d w i t h pepsin only one d i s u l p h i d e peptide could be i s o l a t e d i n good y i e l d . This peptide corresponded to a r e g i o n of the a chain of h a p t o g l o b i n which contained the h a l f - c y s t i n e at p o s i t i o n 21 (Figure 2). A peptide a l s o running o f f the d i a g o n a l i n a corresponding p o s i t i o n was not observed and so i t appeared that t h i s peptide was l i n k e d to i t s e l f and thus a symmetrical l i n k a g e occurred between the a chains of h a p t o g l o b i n . By performing c y s t e i c a c i d diagonals on h a p t o g l o b i n peptides a f t e r t h e r m o l y s i n d i g e s t s of h a p t o g l o b i n 1-1 i t was p o s s i b l e to account f o r a l l of the d i s u l p h i d e s i n haptoglobin (Figure 2). Peptide T h l represents a sequence of the a chain from p o s i t i o n 11 to 21 and again confirms that the h a l f - c y s t i n y l group at p o s i t i o n 21 i s j o i n e d i n a symmetrical i n t e r c h a i n l i n k a g e i n the h a p t o g l o b i n molecule. Peptide Th2 A corresponds to residues 61 to 71 i n the a chain w h i l e Th2 B corresponds to residues 31 to 38 i n the a chain. Thus t h i s d i s u l p h i d e PEPSIN DISULPHIDE H e A l a Asp Asp Gly Glu Pro Pro Pro Lys Cys H e A l a His Gly PI j H e A l a Asp Asp Gly Glu Pro Pro Pro Lys Cys H e A l a His Gly THERMOLYSIN DISULPHIDES H e A l a Asp Asp Gly Glu Pro Pro Pro Lys Cys TH1 | H e A l a Asp Asp Gly Glu Pro Pro Pro Lys Cys A l a V a l Gly Asp Lys Leu Pro Glu Cys Glu A l a Th2a TH2 | V a l Arg Tyr Gin Cys Lys Asn Tyr Th2b TH3 V a l Cys Gly Lys Pro (Pro Lys Asp) T h 3 a Th3b r H e Cys Pro Leu Ser ( Asp Lys ) T H 4 Tyr Gin Glu Asp Thr Cys T Phe Asp Lys Cys(Ser A l a ) V a l A l a Asp Gin Asp Glu Cys TH5 r Phe Cys F i g u r e 2 STRUCTURES OF THE HAPTOGLOBIN DISULPHIDE PEPTIDES 9 peptide demonstrates that the h a l f - c y s t i n y l group at p o s i t i o n 35 i n the a chain i s j o i n e d to a h a l f - c y s t i n y l group at p o s i t i o n 69 i n the a chain. Peptide Th3 A i s i d e n t i c a l to an a chain peptide corresponding to residues 72 and 78 i f an a d d i t i o n a l h a l f - c y s t i n y l group i s placed at p o s i t i o n 73 i n the a chain sequence. R e i n v e s t i g a t i o n of the a chain sequence has shown that i n f a c t a h a l f c y s t i n y l group i s present at t h i s p o s i t i o n . Thus the 3 chain i s j o i n e d to the a chain by a h a l f -c y s t i n y l group at p o s i t i o n 73 i n the a chain. Peptides Th3 B, Th4, and Th5 cannot be assigned to the a chain and so must be present i n the 3 c h a i n of haptoglobin. The d i s u l p h i d e s of h a p t o g l o b i n w i l l be discussed f u r t h e r i n S e c t i o n IV of the t h e s i s . Hemoglobin S t r u c t u r e Hemoglobin without doubt has been the most a s s i d u o u s l y s t u d i e d p r o t e i n . This i s p r i m a r i l y because i t could be e a s i l y obtained i n l a r g e q u a n t i t i e s i n a high s t a t e of p u r i t y (3). Hemoglobin c o n s i s t s of an apoprotein p a r t , g l o b i n , and an oxygen c a r r y i n g chromophore, heme. I t c o n s i s t s of 4 polypeptide c h a i n s , two a chains and two 3 chains each w i t h a molecular weight of about 16,500 r e s u l t i n g i n a hemoglobin molecular weight of 65,000 (38). I t was demonstrated by sequencing the hemoglobin chains (39,40) that the two types of chains had homologous s t r u c t u r e s (41). In comparing v a r i o u s g l o b i n chains i t has been shown that the chains have r e l a t e d sequences i n a l l regions and that 64 residues are i d e n t i c a l i n the a and 3 hemoglobin chains. However these chains 10 only share 21 i d e n t i c a l residues w i t h myoglobin. The three dimensional s t r u c t u r e of hemoglobin i s now known at a r e s o l u t i o n of 2.8A° (42). The four chains are arranged t e t r a h e d -r a l l y around an a x i s of two-fold symmetry and the conformation of the chains c l o s e l y resembles the conformation of myoglobin (43). In general terms, the nonpolar residues r e s i d e i n the i n t e r i o r of the 0 molecule forming Van der Waals contacts and the p o l a r residues are at the surface. G l y c i n e residues and a l a n i n e residues a l s o appear to r e s i d e at the surface of the molecule. Each chain c o n s i s t s of e i g h t h e l i c a l regions (A-H) w i t h seven corners and some n o n h e l i c a l areas. When the three dimensional s t r u c t u r e of hemoglobin was d e t e r -mined (43) i t was found that the s t r u c t u r e of each of the hemoglobin chains was remarkably l i k e the three dimensional s t r u c t u r e of myoglobin. In f a c t more recent r e s u l t s i n d i c a t e that the three dimensional s t r u c t u r e of myoglobin provides a model around which a l l hemoglobin and myoglobin conformations f i t (44). However i n comparing the primary s t r u c t u r e s of a l l of the known v e r t e b r a t e g l o b i n chains only nine residues remain i d e n t i c a l . A l l of the three dimensional s t r u c t u r e s appear to be s i m i l a r because of the maintenance of a p a t t e r n of nonpolar and p o l a r residues (44) of which the nonpolar residues appear to be the most i n v a r i a n t . The evidence i s based on the r e s u l t s of x-ray s t u d i e s and an a n a l y s i s of the sequences of many hemoglobin chains of the v e r t e b r a t e s . C h a r a c t e r i s t i c amino a c i d s are u s u a l l y found between the h e l i c a l regions i n the hemoglobin chains. For example, i n the B chain each of the f i v e p r o l i n e s occurs i n p o s i t i o n two of a h e l i x . Model b u i l d i n g demonstrates t h a t , because of i t s imino group, i n any a h e l i x c o n t a i n i n g N residues p r o l i n e can only e x i s t at p o s i t i o n s 1, 2, 3 or N+l (44). P o s i t i o n 1 i s o f t e n occupied by a s e r i n e , threonine, a s p a r t i c a c i d or asparagine. The hemes are l o c a t e d i n hydrophobic pockets and are attached by coordinate covalent bonds to h i s t i d i n e s at p o s i t i o n F8.. There are s i x t y i n t e r a c t i o n s between g l o b i n chain atoms and heme atoms which are w i t h i n 4A° and a l l but three are non-p o l a r . The s i m i l a r i t i e s i n the s t r u c t u r e s of g l o b i n chains i n the regions corresponding to these i n t e r a c t i o n s i s s t r i k i n g . Each a chain of hemoglobin i s i n contact w i t h two 3 chains and w i t h the other a chain. The converse i s true f o r each 3 chain. The contact a j 31 i s more extensive than the contact a^32- I t c o n s i s t s of 110 atomic i n t e r a c t i o n s which are mainly nonpolar. The contact a^32 has only 80 i n t e r a c t i o n s (Figure 3). Again they are mainly nonpolar but there i s one c l e a r hydrogen bond between a s p a r t i c 94 and aspara-gine 102. Upon deoxygenation of hemoglobin there i s a l a r g e movement i n the a^32 area w i t h a displacement by as much as 5.7A° f o r some con-t a c t atoms w h i l e movement i n the r e g i o n of the a j $ i contact i s s l i g h t w i t h the contact becoming more ext e n s i v e . The ai$2 contact i s such th a t i t allows the two subunits to s l i d e past each other w i t h a r e s u l t a n t e f f e c t on the environment of the hemes. The e l e c t r o n d e n s i t y maps do not show d e f i n i t e contacts between l i k e chains a l -though they probably e x i s t . The contact a^B^ =1 In 131 H9 ala 128 H6 1-gln 127 H5 3-glu 125 H3 pro 124 H2 thr 123 HI phe 122 GH5 3 ^ g l y 119 GH2 arg 116 G18 ala 115 G17 leu 112 G14 asn 108 G10 2 1—met 55 D6 —1—pro 51 D2 j -—• tyr 35 CI val 35 B16 •2 -1 \ ^ v a l 33 B15 ^ a r g 30 B12 Thirty-four residues, including about 110 atoms in contact. Figure 3 Interatomic contacts between Plain connecting lines indicate van der Waals contacts; broken ones indicate that the contact includes a hydrogen bond. The numbers on the lines wive the number of atoms contributed to the contact by the two residues on each side. The contact n,p, comprises thirty-four residues. Twenty-one. of these are common to all the normal mammalian haemoglobin sequences analysed so far. The contact « A comprises nineteen residues. All but one of these are common. The one replacement [C!i(M)/J (tin—»arg], reported for llama,, would not afl'ect the stereochemistry of the contact. sidues i n unl i k e hemoglobin chains 13 Hemoglobin has three s u l p h y d r y l groups per dimer which are lo c a t e d at p o s i t i o n s 393, 3112 and al04. Of these only one, 393, w i l l r e a c t w i t h iodoacetamide under normal c o n d i t i o n s (45,46). In deoxyhemoglobin t h i s c y s t e i n e becomes u n r e a c t i v e (47). This e f f e c t upon the r e a c t i v i t y of 393 a l s o appears r e l a t e d to the d i s s o c i a t i o n of hemoglobin from tetramers to dimers which occurs to a s m a l l extent under p h y s i o l o g i c a l c o n d i t i o n s (48) and can be increased by i n c r e a s i n g the s a l t c o n c e n t r a t i o n or by extremes of pH (48). D i s s o c i a t i o n occurs to a much s m a l l e r extent i n deoxyhemoglobin than i n oxyhemoglobin (49). X-ray c r y s t a l l o g r a p h y has revealed that the 393 SH i n t e r a c t s w i t h h i s t i d y l 397 and that t h i s h i s t i d y l r e s i d u e i s i n v o l v e d i n the contact which i s broken upon d i s s o c i a t i o n from tetramers to dimers (50,42). The decreased r e a c t i v i t y of 393 has now been exp l a i n e d as r e s u l t i n g from r e s t r i c t e d access to the exposed SH. This r e s t r i c t i o n i s caused by the i n t e r a c t i o n of h i s t i d y l 3146 w i t h the 3 c a r b o x y l of a s p a r t y l 394 (51). Over 100 v a r i e n t s of the normal hemoglobin s t r u c t u r e are now known (52). Most of these have been detected by abnormal e l e c t r o -p h o r e t i c m o b i l i t y and the modified s t r u c t u r e can be expl a i n e d by s i n g l e base changes i n the gen e t i c code. In a European p o p u l a t i o n , a study showed the frequency of mutant hemoglobins to occur w i t h a 1 frequency of about one i n two hundred (52). Se v e r a l abnormal hemo-gl o b i n s have been observed which a f f e c t the subunit contacts i n hemoglobin (53). Those a f f e c t i n g the ai&2 c o n t a c t , which i s broken (ar£~^gln) when hemoglobin d i s s o c i a t e s i n s o l u t i o n , are Hb J Capetown a92 Hb Chesapeake a 9 2 ( a r g ^ l e u ) , Hb Yakima 3 9 9 ( a s P ^ h l s ) , Hb Kempsey Q Q O (asp->asn) u u (asn-*thr) 899 r , Hb Kansas 8102 Hemoglobin-Haptoglobin S t r u c t u r e The r e a c t i o n of haptoglobin w i t h hemoglobin can be detected by s t a r c h , paper, acrylamide or c e l l u l o s e acetate e l e c t r o p h o r e s i s , by u l t r a c e n t r i f u g a t i o n or by Sephadex chromatography (54). A l l of these techniques produce a s e p a r a t i o n of the HbHp complex from hemoglobin or h a p t o g l o b i n and the complex can be detected by i t s absorbance at 407 or 540 nm or by i t s peroxidose a c t i v i t y . The complex i s extremely s t a b l e and does not d i s s o c i a t e at a l l under normal c o n d i t i o n s as there appears to be no exchange between i s o t o p i c a l l y l a b e l l e d hemoglobin and the complex (56). A l s o the complex w i l l form under c o n d i t i o n s of pH from 4 to 9 and i n 2M sodium c h l o r i d e (54). Human haptoglobin w i l l r e a c t w i t h hemoglobins from a s e r i e s of r e l a t e d animals and w i l l b i n d g l o b i n but w i l l not bi n d myoglobin (54). I t can be observed upon s t a r c h g e l e l e c t r o p h o r e s i s that a l l of the hapto g l o b i n polymers bind hemoglobin (9). There was a re p o r t that the l a r g e polymers bound l e s s hemoglobin per gram (56) but t h i s has been disproven and i t now appears that a l l polymers bind 0.7 grams of hemoglobin per gram of hapt o g l o b i n (54). In 1964, Nagel and Ranney reacted a v a r i e t y of hemoglobins w i t h h a p t o g l o b i n and t e s t e d the r e a c t i o n by s t a r c h g e l e l e c t r o p h o r e s i s (57). They found that hemoglobins A^, F, I , and Lepore bound haptoglobin w h i l e H and Bart's f a i l e d to show b i n d i n g . These l a s t two hemoglobins are tetramers of the 3 chain and the a chain of hemoglobin r e s -p e c t i v e l y . Since oxyhemoglobin H resembled deoxyhemoglobin A i n c r y s t a l s t r u c t u r e (58) they went on to t e s t t h i s p r o t e i n f o r r e a c t i o n w i t h h a p t o g l o b i n (59). I f hemoglobin were completely deoxygenated w i t h a s m a l l amount of d i t h i o n i t e , no b i n d i n g to hap t o g l o b i n was observed. This l a c k of b i n d i n g of deoxyhemoglobin was confirmed w i t h h a p t o g l o b i n 2-2 by a n a l y s i s i n the u l t r a c e n t r i f u g e (60). Deoxygenation of the oxygenated HbHp complex d i d not reverse the com-b i n a t i o n and r e l e a s e deoxyhemoglobin and haptoglobin. Carboxypep-t i d a s e A (CpA) t r e a t e d hemoglobin reacted w i t h h a p t o g l o b i n whether in' the deoxy or oxy form (60). In CpA-treated Hb the C-terminal h i s t i d i n e i s removed and conformational changes accompanying deoxygenation cannot occur (51). Two p h y s i o l o g i c a l l y s i g n i f i c a n t p r o p e r t i e s of hemoglobin are i t s reduced oxygen-carrying a b i l i t y under a c i d i c c o n d i t i o n s , "Bohr e f f e c t " , and the s i g m o i d a l nature of i t s oxygen b i n d i n g as a f u n c t i o n of oxygen t e n s i o n , u s u a l l y a s c r i b e d to "heme-heme i n t e r a c t i o n " (61). The hemoglobin-haptoglobin complex has r a d i c a l l y a l t e r e d oxygen-b i n d i n g p r o p e r t i e s when compared w i t h f r e e hemoglobin (62) i n c l u d i n g a 3 0 - f o l d i n c r e a s e i n oxygen a f f i n i t y and a nonsigmoidal oxygen b i n d i n g curve. Further s t u d i e s showed the absence of a Bohr e f f e c t , no change i n the carbon monoxide combination r a t e w i t h pH, and a decrease i n the molar e x t i n c t i o n c o e f f i c i e n t (e) at 430 nm (63). These f i n d i n g s a l l suggest that i n the complex the hemoglobin can no longer undergo conformational changes. I n t e r e s t i n g l y , i t was a l s o found that HbHp 2-2 combines w i t h carbon monoxide f a s t e r than HbHp 1-1. Recently hemoglobins which show an impaired b i n d i n g to hapto-g l o b i n have been reported (64). The f i r s t of these was a hemoglobin obtained a f t e r r e a c t i o n w i t h b i s (N-maleimidomethyl) ether (65). This b i f u n c t i o n a l maleimide reagent f i r s t adds to the 393 SH and then r e a c t s w i t h h i s t i d y l 397 (66). When prepared from human hemo-g l o b i n t h i s d e r i v a t i v e shows reduced d i s s o c i a t i o n i n t o dimers i n a s i m i l a r manner to deoxyhemoglobin. However i t has been shown that horse b i s (N-maleimidomethyl) ether-Hb i s c r y s t a l l o g r a p h i c a l l y s i m i l a r to oxyhemoglobin and has s i m i l a r d i s s o c i a t i o n p r o p e r t i e s whether i n the oxy or deoxy form (67). In a d d i t i o n to the standard methods f o r stu d y i n g the hemoglobin-h a p t o g l o b i n r e a c t i o n , Bunn (64) has devised an i n t e r e s t i n g new method. In t h i s method h a p t o g l o b i n i s added to a s o l u t i o n c o n t a i n i n g equal amounts of r a d i o a c t i v e and n o n r a d i o a c t i v e hemoglobin and then the complex i s separated from excess hemoglobin by g e l f i l t r a t i o n and the r e l a t i v e s p e c i f i c a c t i v i t i e s i n the two p r o t e i n s determined. Since the hemoglobin to be t e s t e d f o r b i n d i n g i s n o n r a d i o a c t i v e , i f the s p e c i f i c a c t i v i t y i n the complex peak i s grea t e r than i n the hemo-g l o b i n peak, then the l a b e l l e d hemoglobin bound more r a p i d l y to hapto-g l o b i n . Bunn (64) has t e s t e d a s e r i e s of hemoglobins, a l l m odified a t 393 by treatment w i t h e i t h e r iodoacetamide, p-hydroxymercuribenzoate o I t s s t r u c t u r e i s 17 (pHMB), c y s t i n e , cystamine, or N-ethylmaleimide. In a l l cases he found that t h e i r combination w i t h h a p t o g l o b i n was e s s e n t i a l l y unchanged except f o r a s l i g h t l y f a s t e r r a t e of r e a c t i o n w i t h pHMB-hemoglobin. However both l e s s complete and l e s s r a p i d b i n d i n g f o r b i s (N-maleimidom-e t h y l ) ether-hemoglobin (BME-Hb) to h a p t o g l o b i n were detected by g e l f i l t r a t i o n , peroxidase assays, and by the r a d i o a c t i v e hemoglobin method. When the BME-HbHp was rechromatographed on Sephadex G-100 no d i s s o c i a t i o n of the complex was observed. Bunn suggests that t h i s BME-hemoglobin shows decreased b i n d i n g because i t i s l e s s d i s s o c i a t e d and h a p t o g l o b i n only r e a c t s w i t h the d i s s o c i a t e d a3 dimer of hemo-g l o b i n . Since i t has not been demonstrated that human BME-hemoglobin has the same conformation as human oxyhemoglobin t h i s c o n c l u s i o n cannot be drawn d e f i n i t e l y . However i t should be p o s s i b l e to t e s t t h i s i n t e r p r e t a t i o n by u s i n g human BME-hemoglobin or deoxyhemoglobin under c o n d i t i o n s of pH and i o n i c s t r e n g t h i n which they are as d i s s o c i a t e d as oxyhemoglobin. Since the hemoglobin-haptoglobin b i n d i n g i s so very strong i t seems l i k e l y that even a s m a l l degree of d i s s o c i a t i o n of hemoglobin would lead to a complete r e a c t i o n and so BME-hemoglobin and deoxyhemoglobin must b i n d l e s s t i g h t l y to h a p t o g l o b i n because of an a l t e r e d conformation. Thus i f hemoglobin d i s s o c i a t e s l e s s r e a d i l y , t h i s could e x p l a i n an e f f e c t on the r a t e of r e a c t i o n w i t h haptoglobin but not on the e q u i l i b r i u m . In a f u r t h e r study, Bunn has provided more evidence f o r the r e l a t i o n s h i p between hemoglobin d i s s o c i a t i o n and b i n d i n g to hapto-g l o b i n (68). He found that Hb Kansas, which i s more h i g h l y d i s s o c i a t e d 18 than hemoglobin A, bound haptoglobin more r a p i d l y and that f o r Hb Chesapeake, which i s l e s s h i g h l y d i s s o c i a t e d , the reverse i s t r u e . Since the hemoglobin-haptoglobin complex has a molecular weight of 170,000 (4) i t was f e l t t hat 1 molecule of hapt o g l o b i n combines w i t h 1 molecule of hemoglobin. The f i r s t i n d i c a t i o n that h a p t o g l o b i n might b i n d "half-hemoglobin", the a3 dimer, came from L a u r e l l ' s l a b o r a t o r y when he observed t h a t , i f excess h a p t o g l o b i n was mixed w i t h hemoglobin, a new species was observed a f t e r s t a r c h g e l e l e c t r o -p h oresis at pH 7 (69). The new peroxidase p o s i t i v e band migrated between the HbHp complex and f r e e h a p t o g l o b i n and L a u r e l l suggested that i t was a complex of one hapt o g l o b i n and one a3 hemoglobin dimer. S e v e r a l years l a t e r , Hamaguchi (70) p u r i f i e d t h i s HbHp intermediate and showed that i t s molecular weight of 134,000 and heme content were c o n s i s t e n t w i t h L a u r e l l ' s p o s t u l a t e . The f a c t that h a p t o g l o b i n can re a c t w i t h f i r s t one a3 hemo-g l o b i n dimer and then a second i n d i c a t e s that h a p t o g l o b i n i s b i v a l e n t and that the f u l l complex c o n s i s t s of ha p t o g l o b i n plus a p a i r of hemoglobin dimers. Further c o n f i r m a t i o n of t h i s model comes from the work of Nagel and Gibson (71). They were able to measure the r a t e of r e a c t i o n of hemoglobin w i t h h a p t o g l o b i n by measuring quench-ing of the tryptophane fluorescence of haptoglobin by the heme groups. T h e i r r e s u l t s showed that the r a t e of r e a c t i o n d i d not inc r e a s e l i n e a r l y w i t h hemoglobin c o n c e n t r a t i o n and thus a d i s s o c i a -t i o n appeared to precede r e a c t i o n . They a l s o t e s t e d the r e a c t i o n of haptoglobin w i t h the i s o l a t e d a and 3 hemoglobin chains and observed a r e a c t i o n w i t h i s o l a t e d a but not w i t h 3 chains. When haptoglobin was incubated w i t h a chains and then 3 chains added, an i n i t i a l r a p i d r e a c t i o n was observed, but when the i n c u b a t i o n was w i t h 3 chains and then a chains were added, the r a t e was s i m i l a r to that w i t h a chains alone. This i n d i c a t e d that the a chains form a complex w i t h h a p t o g l o b i n which can then r e a c t r a p i d l y w i t h 3 chains. More r e c e n t l y another d e t a i l e d study of the r e a c t i o n of hemo-g l o b i n chains w i t h h a p t o g l o b i n has a l s o provided i n t e r e s t i n g r e s u l t s (72). Both a and 3 hemoglobin chains were found to b i n d to hapto-g l o b i n but to a much s m a l l e r extent than hemoglobin i t s e l f . The a chains had a higher a f f i n i t y than the 3 chains and four a chains could be bound per hapt o g l o b i n (Figure 4). The r e a c t i o n w i t h the chains appears to be r e v e r s i b l e and they can be d i s p l a c e d by adding hemoglobin. O u t l i n e of the Present Study The present study represents attempts to f u r t h e r understand both the nature of hemoglobin-haptoglobin b i n d i n g and some aspects of hapto g l o b i n s t r u c t u r e . This study has been d i v i d e d i n t o three p a r t s , one p e r t a i n i n g to s t u d i e s on hapt o g l o b i n b i v a l e n c e , one to s t u d i e s on the d i s u l p h i d e s of ha p t o g l o b i n , and the l a s t to s t u d i e s on the s u l p h y d r y l s of the hemoglobin-haptoglobin complex. The f i r s t p a r t of the t h e s i s ( S e c t i o n I I I ) i s concerned w i t h the r e a c t i o n of hapt o g l o b i n w i t h an octameric (double) hemoglobin obtained from an inbred s t r a i n of mice. In t h i s hemoglobin each of 20 T 1 r r 4| i i i ' I 0 2 4 6 8 Molar ratio haem/Hp Fio.'t. Sedimentation coefficients of mixtures of Hp and haemoglobin chains as a function of tho molar ratio haem/Hp. Hp concentration 2 to 4X 10"8 M. aPMB (Qj 0 X y o r carboxy; (©) deoxy. a 8 H (•) oxy or carboxy; (a) deoxy. / J P M B (A) ° x y or carboxy; (A) deoxy. (3SB (V) oxy or carboxy; (J) deoxy, the hemoglobin dimers i s j o i n e d together by a d i s u l p h i d e bond. The f a c t that haptoglobin binds a8 dimers i n d i c a t e s that i t i s a b i v a l e n t molecule l i k e the antibody molecule, immunoglobulin G (IgG). This b i v a l e n c e and r e s u l t a n t resemblance to IgG i s examined by studying the r e a c t i o n of haptoglobin w i t h t h i s mouse hemoglobin i n which the aB dimer i s h e l d together by a d i s u l p h i d e bond. The r e s u l t s of both p r e c i p i t a t i o n s t u d i e s and acrylamide g e l e l e c t r o p h o r e s i s confirm the po s t u l a t e d b i v a l e n c e of haptoglobin and i t s resemblance to an antibody. The second p a r t ( S e c t i o n IV) of the t h e s i s i s concerned w i t h confirming the r e s u l t s obtained i n studying the d i s u l p h i d e s of hapto-g l o b i n which were obtained by the c y s t e i c a c i d d i a g o n a l technique. These r e s u l t s p r e d i c t e d a model i n which the two halves of the hapto-g l o b i n molecule were he l d together by a d i s u l p h i d e bond at p o s i t i o n 21a. A l s o the r e s u l t s p r e d i c t e d an i n t r a c h a i n loop d i s u l p h i d e between h a l f - c y s t i n e s at p o s i t i o n s 35 and 69 i n the hapt o g l o b i n a chain and an i n t e r c h a i n d i s u l p h i d e between a h a l f - c y s t i n e at p o s i t i o n 73a and the B chain. This s t r u c t u r e has been confirmed by s t u d i e s on a cyanogen bromide fragment i s o l a t e d from haptoglobin which contains the i n t a c t a chain. A l s o the s t r u c t u r e has been confirmed by s t u d i e s on a hapto-g l o b i n d e r i v a t i v e i n which the molecule has been s p l i t i n h a l f by the breaking of a d i s u l p h i d e bond. The t h i r d p a r t ( S e c t i o n V) of t h i s t h e s i s i s an i n v e s t i g a t i o n i n t o the nature of the B93 s u l p h y d r y l of hemoglobin when hemoglobin i s bound by haptoglobin. The r e s u l t s demonstrate that there i s a d e f i n i t e change i n the environment of t h i s s u l p h y d r y l upon formation 14 of the hemoglobin-haptoglobin complex. Studies w i t h C-iodoaceta-mide demonstrate however that 393 can s t i l l r e a c t i n the HbHp complex. II MATERIALS AND METHODS Starch-Urea Gel E l e c t r o p h o r e s i s The v e r t i c a l method of Smithies was employed (73). E i t h e r 80 to 83 g of s t a r c h (Connaught Me d i c a l Research L a b o r a t o r i e s , Toronto, Canada) or 67 to 70 g (Otto H i l l e r , E l e c t r o s t a r c h Company, Madison, Wisconsin, l o t 682) were weighed i n t o a one l i t r e beaker. Two hundred and f o r t y grams of urea were added and the two powders thoroughly mixed. Three hundred m i l l i l i t e r s of 0.083M sodium formate b u f f e r pH 3.0 (prepared from 50 ml of s o l u t i o n c o n t a i n i n g 0.5M formic a c i d and 0.1M sodium hydroxide) or 300 ml of aluminum l a c t a t e b u f f e r pH 3.7 (stock s o l u t i o n of Sung and Smithies (74) d i l u t e d four f o l d ) were added to the mixture of s t a r c h and urea. Subsequent steps i n the pre-p a r a t i o n of the gels c o n t a i n i n g urea followed the p r e v i o u s l y described method (75) except that degassing i s omitted (74). Bridge s o l u t i o n s f o r the formate gels were the same as those p r e v i o u s l y described (75) and f o r the aluminum l a c t a t e g e l s they were the four f o l d d i l u t e d stock aluminum l a c t a t e pH 3.7. The gels were s l i c e d and s t a i n e d by the method of Smithies, C o n n e l l and Dixon (75). Both the Amido Black s t a i n and the Wool Fast Blue S t a i n were used. The Wool Fast Blue s t a i n was not as s e n s i t i v e as the Amido Black s t a i n but the g e l could be destained more q u i c k l y . Thus, except when a pre-l i m i n a r y a n a l y s i s was r e q u i r e d , the Amido Black s t a i n was used i n preference to the Wool Fast Blue s t a i n . Two dimensional s t a r c h urea g e l e l e c t r o p h o r e s i s was performed according to the method of Smithies, C o n n e l l and Dixon (76). However a 2-bladed c u t t i n g t o o l was used i n which the blades were 0.7 cm apart i n s t e a d of 0.4 cm. In the second dimension, g e l markers of r e -duced and a l k y l a t e d haptoglobins were i n s e r t e d at both ends of the g e l at the s t a r t i n g l i n e by means of a s m a l l p i e c e of Whatmann 3MM paper (73). Polyacrylamide D i s c Gel E l e c t r o p h o r e s i s Polyacrylamide g e l s were prepared f o l l o w i n g c o n d i t i o n s des-c r i b e d by Davis (77). Although the c o n c e n t r a t i o n of acrylamide was a l t e r e d to s u i t p a r t i c u l a r experiments, the p r e p a r a t i o n of a f i v e per cent polyacrylamide g e l serves as an example of the experimental technique. Glass tubes approximately 10 cm i n len g t h and 5 mm diameter were used as moulds f o r the d i s c g e l s . The bottom of the tubes was sealed w i t h a rubber plug and the tubes were clamped i n a rack f o r f i l l i n g . Then 1.3 g acrylamide and 63 mg N, N^-methylenebisacrylamide were added to 25 ml of 0.1M sodium phosphate b u f f e r . Immediately before c a s t i n g of the gels 10 u l of tetramethylethylenediamine (TEMED) and 15 mg of ammonium persulphate were added to the acrylamide s o l u t i o n . The g e l l i n g s o l u t i o n was added to the glass tubes w i t h a Pasteur p i p -e t t e , t a k i n g care that i n each tube the l e v e l s of the g e l s o l u t i o n s were equal. Water was c a r e f u l l y l a y e r e d on top of the acrylamide 25 s o l u t i o n surface w i t h a m i c r o p i p e t t e i n order that the polyacrylamide g e l would have a f l a t meniscus. The g e l l i n g time v a r i e d c o n s i d e r a b l y w i t h the composition of the b u f f e r but by v a r y i n g the ammonium s u l -phate c o n c e n t r a t i o n the g e l l i n g time can be adjusted to about one-half hour. The polymerized g e l was washed on the surface w i t h d i s t i l l e d water to remove any unge l l e d m a t e r i a l . E l e c t r o p h o r e s i s was performed i n e i t h e r of two sets of apparatus which both were s i m i l a r to that described by Davis (77) and which could h o l d e i t h e r 8 or 20 g e l s . The gels i n the gl a s s tubes made e l e c t r i c a l contact w i t h the upper and lower c o n t a i n e r s which h e l d the e l e c t r o p h o r e s i s b u f f e r . Rubber plugs sealed the openings through which the gels penetrated the bottom of the upper c o n t a i n e r . U s u a l l y sucrose was d i s s o l v e d i n the e l e c t r o p h o r e s i s sample to i n c r e a s e i t s d e n s i t y . The sur f a c e of the g e l was f i r s t covered w i t h the e l e c t r o -p h o r e s i s b u f f e r and then 5 to 20 u l of sample were a p p l i e d onto the g e l s u r f a c e by a m i c r o p i p e t t e . The compartments of the apparatus were f i l l e d w i t h sodium phosphate b u f f e r u n t i l the platinum e l e c t r o d e s were covered. E l e c t r i c a l contact was made to a Heathkit IP-32 power source and e l e c t r o p h o r e s i s was c a r r i e d out at a maximum of 200 to 300 v o l t s . A f i n e w i r e o r , when a v a i l a b l e , the inner s h a f t of a 22-gauge hypo-dermic needle was used to remove the g e l from the gl a s s tube. Gels were s t a i n e d by keeping them f o r approximately 45 minutes i n a 0.1 per cent Amido Black (w/v) i n 10 per cent a c e t i c a c i d (v/v) s o l u t i o n . A f t e r the s t a i n i n g p e r i o d the dye s o l u t i o n was removed and the ge l s were destained i n 10 per cent a c e t i c a c i d . 2 6 Three d i f f e r e n t acrylamide g e l b u f f e r s ; 0.05M g l y c i n e , 0.01M T r i s , pH 8.5; O.IM sodium phosphate, pH 7.0; and 0.11M T r i s , 0.062M b o r i c a c i d , 2.5mM disodium [ e t h y l e n e d i n i t r i l o ] t e t r a a c e t a t e (EDTA), and a v a r i e t y of g e l lengths were employed i n the experiments described i n t h i s t h e s i s . However, where app r o p r i a t e the p a r t i c u l a r m o d i f i -c a t i o n s are discussed along w i t h the experiment. A more d e t a i l e d d e s c r i p t i o n of the technique of polyacrylamide g e l e l e c t r o p h o r e s i s can be found i n the a r t i c l e by Davis (77). E i g h t molar urea-polyacrylamide gels were prepared i n a s i m i l a r manner to that described above. However, because urea increases the volume of aqueous s o l u t i o n s the g e l s o l u t i o n was made up to volume a f t e r the urea was d i s s o l v e d . A l s o , s i n c e urea decreased the polymer-i z a t i o n time f o r the g e l s , the c o n c e n t r a t i o n of ammonium persulphate and TEMED used was one-half that used f o r making gels that contained no urea. High Voltage E l e c t r o p h o r e s i s High v o l t a g e e l e c t r o p h o r e s i s was performed i n a v e r t i c a l apparatus using four b u f f e r s ; p y r i d i n i u m acetate pH 6.5 (100 ml p y r i d i n e — 4 ml a c e t i c a c i d — 9 0 0 ml wa t e r ) , p y r i d i n i u m acetate pH 3.6 (10 ml p y r i d i n e — 1 0 0 ml a c e t i c a c i d — 8 9 0 ml wa t e r ) , f o r m i c - a c e t i c a c i d s o l u t i o n pH 1.9 (20 ml formic a c i d — 8 0 ml a c e t i c a c i d — 9 9 0 ml w a t e r ) , and p y r i d i n i u m formate pH 4.0 (48 ml p y r i d i n e — 3 5 ml formic a c i d — 3920 ml water) on Whatmann 3MM paper. The apparatus i s described by Ryle (78) and the technique i s discussed i n d e t a i l by J . Legget 27 B a i l y (79). A s e r i e s of coloured markers, xylene cyanol FF (XCFF), methyl green (MG), orange G (OG), and c r y s t a l v i o l e t (CV) were used i n order to help monitor the progress of the e l e c t r o p h o r e s i s . The electrophoretograms were s t a i n e d f o r peptides or amino ac i d s w i t h a 0.5 per cent n i n h y d r i n i n acetone s o l u t i o n . For a per-manent s t a i n a cadmium acetate n i n h y d r i n d i p p i n g reagent was used (80). Amino-terminal p r o l i n e was detected by d i p p i n g the electrophoretogram i n an i s a t i n s o l u t i o n (0.2 g i s a t i n , 100 ml acetone, and 4 ml a c e t i c a c i d ) . H i s t i d i n e was detected by spraying w i t h Pauly reagent (79). I t was p o s s i b l e to s t a i n f i r s t w i t h i s a t i n , then w i t h n i n h y d r i n and then Pauly reagent. However, i n order to remove most of the i s a t i n from the 3MM paper before n i n h y d r i n s t a i n i n g the 3MM paper was dipped twice i n ethanol. A c i d H y d r o l y s i s (82) and Amino A c i d A n a l y s i s (83) For a p r o t e i n h y d r o l y s i s 100 to 200 u l of 6N HCl (1:1 d i l u t i o n of reagent concentrated HCl) was added to 0.5 to 2 mg of l y o p h i l i z e d p r o t e i n i n a 10 by 70 mm Pyrex t e s t tube. A f t e r h e a t i n g i n an oxygen flame a s e c t i o n of the tube 2 to 3 cm from the top was p u l l e d to a bore of about 2 mm. The sample was then cooled i n an a l c o h o l dry i c e bath and the tube was connected by means of an adaptor to an o i l pump. The sample was evacuated and allowed to warm to room temperature. When bubbles of a i r ceased to form i n the sample the Pyrex tube was sealed under vacuum w i t h the oxygen flame. The sample was hydrolyzed f o r 15 to 20 hours i n a oven at 110° C. A f t e r h y d r o l y s i s the evacuated tube was cooled, opened, and then d r i e d i n a vacuum d e s i c -c a t o r over sodium hydroxide. P a r t i a l a c i d hydrolyses were performed i n 100 y l of 6N HC1 (1:1 d i l u t i o n of reagent concentrated HC1) at 110° C. The hydrolyses were performed i n Pyrex tubes (10 by 70 mm). The samples were heated to 100° C f o r 1 minute i n a b o i l i n g water bath and then hydrolyzed f o r 19 minutes i n a 110° C oven. Then the samples were cooled, d i l u t e d f i v e - f o l d w i t h water, and l y o p h i l i z e d . Peptide h y d r o l y s i s was performed a f t e r e l u t i o n of the peptide w i t h water (0.3 to 0.4 ml) from a paper electrophoretogram. The peptide was e l u t e d i n t o a t e s t tube (84) and then d r i e d at 50° C i n a Buchler Rotary Evapomix. One hundred m i c r o l i t e r s of 6N HC1 was added to the d r i e d peptide and h y d r o l y s i s was performed i n the same manner as used f o r p r o t e i n s . Amino a c i d analyses were performed on a Beckmann 120 C amino a c i d a n a l y z e r according to the method of Spackman, S t e i n and Moore (83). The d r i e d h y d r o l y s a t e s were d i s s o l v e d i n 0.2 ml to 0.4 ml of 0.2N sodium c i t r a t e b u f f e r , pH 2.2. For p r o t e i n h y d r o l y s a t e s the p r e c i p i -t a t e r e s u l t i n g from the degradation of tryptophane by HC1 was removed by c e n t r i f u g a t i o n or f i l t r a t i o n through a M i l l i p o r e f i l t e r . Then a 50 to 75 per cent a l i q u o t of the sample was used f o r a n a l y s i s . Amino a c i d a n a l y s i s of peptides was performed using a s i n g l e column procedure developed by Devenyi (85). Amino a c i d analyses of homoserine peptides were performed f o l l o w i n g the method of Tang and H a r t l e y (87). Dry h y d r o l y s a t e s were d i s s o l v e d i n 100 y l of 2N NH.0H and incubated at 37 C f o r one hour to convert homoserine l a c t o n e to homoserine. The samples were then d r i e d on a Buchler Rotary Evapomix and analyzed as described above. Amino-Terminal Amino A c i d and Carboxy-Terminal Amino A c i d Analyses Amino-terminal a n a l y s i s were performed f o l l o w i n g the dansyl c h l o r i d e method of Gray (88). A f t e r r e a c t i o n of the polypeptides w i t h dansyl c h l o r i d e (l-Dimethylaminonaphthalene-5-sulphonyl c h l o r i d e ) the sample was d r i e d on a r o t a r y evaporator and hydrolyzed i n 6N HC1 as described i n the procedure f o r amino a c i d a n a l y s i s . In order to i d e n t i f y d a n s y l - p r o l i n e the sample was only allowed to hydrolyze f o r 6 hours i n s t e a d of the normal 15 to 20 hour h y d r o l y s i s time. Dansyl-amino acids were i d e n t i f i e d by the t h i n l a y e r chromatographic method of Black and Dixon (89). Carboxy-terminal a n a l y s i s was determined a f t e r d i g e s t i o n w i t h carboxypeptidase A. The enzyme was prepared by the method of P o t t s , Berger, Cooke and Anfinsen (90). Four m i l l i g r a m s of fragment PC I I I were d i s s o l v e d i n 200 y l of performic a c i d at 0° C and o x i d i z e d f o r 90 minutes at 0° C (91). Then 1.0 ml of water at 2° C was added and the r e s u l t i n g s o l u t i o n was then f r o z e n and l y o p h i l i z e d . P e r f o r m i c - a c i d . o x i d a t i o n converted the fragment i n t o a denatured form which was s u i t a b l e f o r d i g e s t i o n w i t h carboxypeptidase. Carboxypeptidase d i g e s -t i o n of non-oxidized fragment r e s u l t e d i n only a very low y i e l d of amino a c i d s . Two m i l l i g r a m s of p e r f o r m i c - o x i d i z e d PC I I I were d i s s o l v e d 30 i n 100 y l 2N NH^ f o r 1 hour at 37° C to convert any homoserine lactone to homoserine and subsequently the s o l u t i o n was d r i e d . The fragment was then r e d i s s o l v e d i n 0.5 ml of 0.2N ammonium bicarbonate to which 10 y l of carboxypeptidase A s o l u t i o n (44 yg) were added. D i g e s t i o n was allowed to proceed f o r 6 hours at 37° C. A f t e r d i g e s t i o n the amino a c i d s which were r e l e a s e d were absorbed on Dowex 50 and subse-quently e l u t e d w i t h 5N NH^ (92). The amino a c i d c o n t a i n i n g s o l u t i o n was then d r i e d and analyzed on the amino a c i d analyzer as described p r e v i o u s l y . Enzymatic D i g e s t i o n s of Fragment PC I I I One per cent s o l u t i o n s of fragment PC I I I and pepsin (3 x c r y s t a l -l i z e d , N u t r i t i o n a l Biochemicals Corporation) were prepared by d i s s o l v i n g the p r o t e i n s i n an appropriate volume of 5 per cent (v/v) formic a c i d . To a given volume of PC I I I s o l u t i o n was added a 1/10 volume of pepsin s o l u t i o n . D i g e s t i o n was allowed to proceed f o r 16 to 18 hours at 37° C. A f t e r t h i s time the sample was d r i e d on a r o t a r y evaporator and then r e d i s s o l v e d i n pH 6.5 b u f f e r (100 ml p y r i d i n e — 4 ml a c e t i c a c i d — 900 ml water) equal i n volume to the volume of 5 per cent (v/v) formic a c i d used i n i n i t i a l l y d i s s o l v i n g the fragment. To t h i s s o l u t i o n was added a 1/20 volume of 1 per cent (w/v) porcine t r y p s i n (Novo I n d u s t r i ) s o l u t i o n (weight r a t i o enzyme to fragment = 1 to 20). Then d i g e s t i o n was allowed to proceed f o r from 5 to 7 hours at 37° C. A f t e r the See S e c t i o n IV f o r a d e s c r i p t i o n of PC I I I . 31 d i g e s t i o n p e r i o d was over the sample was again d r i e d and then d i s s o l v e d i n a s m a l l volume s u i t a b l e f o r e l e c t r o p h o r e s i s . P r e p a r a t i o n of Hemoglobins The hemoglobin used i n t h i s t h e s i s (except where i n d i c a t e d ) was prepared by Chan (54) f o l l o w i n g the method of Drabkin (37). Although the hemoglobin had been prepared as carbonmonoxyhemoglobin i t had been sto r e d as a powder at -20° C f o r s e v e r a l years and when r e d i s s o l v e d produced a spectrum i d e n t i c a l w i t h methemoglobin. Double molecules of mouse hemoglobin were prepared f o l l o w i n g the method of Riggs (93). Mouse blood of inbred s t r a i n DBA/2J was obtained from the Roscoe B. Jackson Memorial Laboratory, Bar Harbour, Maine. The c e l l s were washed 3 times i n 0.9 per cent NaCl and then were l y s e d i n an approximately equal volume of d i s t i l l e d water. The l y s a t e was then d i a l y s e d a g a i n s t 0.2M sodium c h l o r i d e f o r 24 hours w i t h four changes of s a l i n e s o l u t i o n . This s o l u t i o n was then s t o r e d f o r 1 to 2 weeks at 4° C. Double hemoglobin (HbHb) was separated from s i n g l e hemoglobin by chromatography on a Sephadex G-100 column (2.2 by 90 cm) usin g a sample volume of 3 ml (88). The column f r a c t i o n s c o n t a i n i n g HbHb were d i a l y z e d , l y o p h i l i z e d , and st o r e d at -20° C. Upon r e d i s s o l v i n g , t h i s m a t e r i a l produced the spectrum of methemo-g l o b i n . In order to prepare l a r g e r amounts of HbHb, 15 ml of d i a l y z e d l y s a t e were f r a c t i o n a t e d on a Sephadex G-100 column (5 by 90 cm). 14 For the study of the r e a c t i o n of C -iodoacetamide w i t h oxy-hemoglobin the oxyhemoglobin s o l u t i o n s were prepared from human red blood c e l l s (37), d i a l y s e d against 0.1M sodium phosphate, 0.2M sodium c h l o r i d e , pH 7.2, stored a t 4° C and used w i t h i n three weeks of pr e p a r a t i o n . Reactions w i t h Hemoglobin and the Hemoglobin-Haptoglobin Complex D i t h i o d i p y r i d i n e s The d i t h i o d i p y r i d i n e s (2-PDS and 4-PDS) were obtained from A l d r i c h Chemical Company, Milwaukee, Wisconsin. The r e a c t i o n between 2-PDS or 4-PDS and hemoglobin (Hb) or the hemoglobin-haptoglobin (Hb-Hp) complex was followed by the absorbance of the s o l u t i o n s at 343 and 324 nm, r e s p e c t i v e l y , w i t h a Model 15 Cary spectrophotometer (94). An a l i q u o t of. stock d i t h i o d i p y r i d i n e s o l u t i o n was mixed w i t h the hemo-g l o b i n s o l u t i o n i n a spectrophotometer cuvette and the change i n A - j ^ or A^24 as a f u n c t i o n of time was determined using the hemoglobin s o l u t i o n as blank. A l l r e a c t a n t s were d i s s o l v e d i n 0.05M sodium phosphate, 0.05 sodium c h l o r i d e , pH 6.0. The f i n a l c o n c e n t r a t i o n of rea c t a n t s were Hb, 7.0 x 10~ 6M ( i n heme); 4-PDS, 3.3 x 10 _ 5M, f o r the r e a c t i o n of Hb and 4-PDS; and Hb 2.4 x 10~ 5M and 2-PDS, 2.5 x -4 10 M w i t h the r e a c t i o n w i t h 2-PDS. 14 C-iodoacetamide 14 C-iodoacetamide (1.53 mCi/mMole) was obtained from Volk Radio-chemical Company and reacted a t room temperature i n 0.05M sodium phos--4 phate, 0.1M sodium c h l o r i d e , pH 7.3, w i t h Hb (5.7 x 10 M i n heme) or -4 Hb-Hp (Hb 5.7 x 10 M i n heme to which an excess of Hp i s added) using a c o n c e n t r a t i o n of 4 x 10 M C-iodoacetamide. C o n t r o l r e a c t i o n s w i t h -4 f r e e h a p t o g l o b i n were performed at 1.2 and 2.2 x 10 M haptoglobin. The r e a c t i o n s were terminated at v a r i o u s times by d i l u t i n g a 15 or 25 y l a l i q u o t i n t o 0.2 ml of 0.07M 3-mercaptoethanol. The excess iodoa-cetamide-mercapthoethanol adduct was removed on a Sephadex G-25 column (0.7 cm by 70 cm) usin g 0.05M T r i s - H C l , pH 8.0, or 0.01M NH^tKX^ as b u f f e r and the amount of r a d i o a c t i v i t y i n the p r o t e i n determined by mixing a 1.2 ml a l i q u o t w i t h Bray's s o l u t i o n (95) and counting i n a U n i l u x 1 l i q u i d s c i n t i l l a t i o n counter. The amount of the iodoacetamide attached to the hemoglobin was determined by the r a t i o of the c.p.m. to the absorbance at 407 nm. Reactions w i t h Haptoglobins Cyanogen Bromide Cleavage of Haptoglobins Cyanogen bromide (CNBr) cleavages were attempted i n three m e d i a — 70 per cent formic a c i d or 0.1 or 0.01N sodium acetate b u f f e r , pH 4.7. For the formic a c i d r e a c t i o n a 3.3 per cent aqueous s o l u t i o n of hapto-g l o b i n (Hp) was prepared and 3 pa r t s of t h i s s o l u t i o n was mixed w i t h 7 p a r t s of a 14 mg/ml cyanogen bromide i n 98 per cent formic a c i d s o l u t i o n . The f i n a l c o n c e n t r a t i o n s of Hp and CNBr were 1 per cent and the molar r a t i o of CNBr to Hp was 1000 to 1. In g e n e r a l , the r e a c t i o n s were allowed to proceed f o r greater than 15 hours and then the s o l u -t i o n s were d i l u t e d w i t h water at l e a s t 4 - f o l d so that they could be e a s i l y f r o z e n and l y o p h i l i z e d . A s i m i l a r p r o t o c o l was f o l l o w e d f o r the acetate r e a c t i o n s . 34 Reaction of Haptoglobin w i t h a Mixture of Sodium S u l p h i t e and p-chloromercurisulphonate (pCMS) The 'half-molecule' of haptoglobin was prepared by the method of Rorstad and Dixon (96) which used sodium s u l p h i t e and p a r a c h l o r o -mercurisulphonate. The r e a c t i o n was allowed to proceed f o r from 30 to 60 minutes and the s o l u t i o n was then d e s a l t e d . In the p r e p a r a t i o n 35 of l a b e l l e d h a l f - h a p t o g l o b i n the s p e c i f i c a c t i v i t y of the S - s u l p h i t e was 11 mCi/mMole. Haptoglobin P r e p a r a t i o n Haptoglobin was prepared f o l l o w i n g the method of Chan (54). The s t a r t i n g m a t e r i a l was a s c i t e s f l u i d , a r i c h source of h a p t o g l o b i n , to which ammonium sulphate was added to give 55 per cent s a t u r a t i o n . The p r e c i p i t a t e was d i s s o l v e d i n 0.01M sodium acetate b u f f e r , pH 4.7, and d i a l y z e d a g a i n s t t h i s b u f f e r to remove the sulphate. Any p r e c i p i -t a t e forming during d i a l y s i s was removed by c e n t r i f u g a t i o n and the supernatant a p p l i e d to a DEAE-cellulose column e q u i l i b r a t ed w i t h 0.01M sodium acetate b u f f e r at pH 4.7. The column was then washed w i t h a l a r g e volume of b u f f e r and then e l u t e d w i t h a grad i e n t of 0.01M NaCl to 0.3M NaCl i n the same acetate b u f f e r . The p r o t e i n peak obtained was then d i a l y z e d against d i s t i l l e d water and l y p o h i l i z e d . The p r o t e i n was then d i s s o l v e d 0.05M ammonium acetate at pH 8.6 and run on a Sephadex G-200 column. One major peak of hapt o g l o b i n was u s u a l l y obtained w i t h a minor f a s t e r - r u n n i n g peak of caeruloplasmin and a minor slower-running peak of albumin and postalbumin. The h a p t o g l o b i n was then c h a r a c t e r i z e d f o r p u r i t y and hemoglobin b i n d i n g by s t a r c h g e l e l e c t r o p h o r e s i s (54) or by polyacrylamide d i s c g e l e l e c t r o p h o r e s i s . I l l HAPTOGLOBIN DOUBLE HEMOGLOBIN (Hb.Hb) REACTION Introduction Human haptoglobin of phenotype 1-1 i s constructed of two d i s -s i m i l a r p a i r s of polypeptide chains held together by disulphide bonds (12). The smaller a-chains have a molecular weight of 8,800 (92) while the l a r g e r , carbohydrate-containing 3-chains, possess a molecular weight of 40,000 to 43,000 (28,29,30) f o r a t o t a l of 98,000 ± 1,000 f o r the i n t a c t haptoglobin 1-1 molecule (28,30). The o v e r a l l s tructure of haptoglobin 1-1 thus bears a strong resemblance to that of immuno-gl o b i n G. Since the complete amino acid sequence of haptoglobin a-chains has been determined and a d e t a i l e d comparison between t h i s sequence and those of a s e r i e s of Bence-Jones proteins indicated homology between portions of the haptoglobin a-chains and Bence-Jones sequences (26), i t i s reasonable to examine whether haptoglobin and the immunoglobins possess any f u n c t i o n a l s i m i l a r i t i e s . The major function of hapto-globin appears r e l a t e d to i t s remarkable property of binding hemoglobin extremely t i g h t l y and s p e c i f i c a l l y g i ving r i s e to a complex of M.W. 163,000 with a stoichiometry of 65,000 gm of hemoglobin to 98,000 gm of haptoglobin 1-1 (4). Since hemoglobin i s normally contained with-i n the red blood c e l l s i t can be considered that when i t i s released by hemolysis into the plasma, the l o c a t i o n of haptoglobin, i t becomes a p r o t e i n f o r e i g n to that p a r t i c u l a r compartment of the body. Thus hapt o g l o b i n i n complexing w i t h i t acts i n a manner analogous to that of an antibody b i n d i n g to a f o r e i g n p r o t e i n . Thus, i n some ways, hap t o g l o b i n can be considered f u n c t i o n a l l y as a c o n s t i t u t i v e hemoglobin antibody although there are a number of d i f f e r e n c e s between hap t o g l o b i n 1-1 and immunoglobulin G (IgG). These i n c l u d e d i f f e r e n t s i t e s of s y n t h e s i s ( l i v e r and lymphoid t i s s u e r e s p e c t i v e l y ) , i s o e l e c t r i c p o i n t s , and molecular weights of the l i g h t chains (97,98) as w e l l as the absence of complement f i x a t i o n by the hemoglobin-haptoglobin complexes (99). The 1:1 s t o i c h i o m e t r y of the hemoglobin-haptoglobin complex would, at f i r s t s i g h t , suggest that h a p t o g l o b i n might possess only a s i n g l e b i n d i n g s i t e f o r hemoglobin, a c l e a r d i f f e r e n c e from antibody molecules such as IgG which are b i v a l e n t towards antigens. However, L a u r e l l (69) found upon adding l e s s than s t o i c h o m e t r i c amounts of hemoglobin to h a p t o g l o b i n that a d i s t i n c t i n termediate could be observed upon e l e c t r o p h o r e s i s and p o s t u l a t e d that t h i s complex con-s i s t e d of a h a l f molecule of hemoglobin bound to one molecule of h a p t o g l o b i n (100). In more recent s t u d i e s Hamaguchi (70) has p u r i f i e d t h i s intermediate and has found that i t s molecular weight i s 140,000 and i t s s t o i c h i o m e t r y indeed 1/2 hemoglobin to 1 h a p t o g l o b i n . In a d d i t i o n , Nagel and Gibson (71), i n s t u d i e s of the k i n e t i c s of the hemoglobin-haptoglobin r e a c t i o n have found evidence that the combination of h a p t o g l o b i n i s not w i t h i n t a c t hemoglobin tetramers but w i t h e i t h e r (a3) dimers or w i t h f i r s t a and then 3 hemoglobin chain monomers. In order to gain f u r t h e r i n s i g h t i n t o the number of b i n d i n g s i t e s i n h a p t o g l o b i n , the r e a c t i o n between c o v a l e n t l y - l i n k e d double 38 hemoglobin molecues (having e i g h t chains) and ha p t o g l o b i n has been s t u d i e d . Riggs (93) has shown that when the hemoglobin of c e r t a i n s t r a i n s of mice i s allowed to stand i n a i r , d i s u l p h i d e bonds can form between f r e e s u l p h y d r y l groups of c y s t e i n e residues i n the 8 chains g i v i n g r i s e to hemoglobin octamers (a^ 81+) i n which each p a i r of B chains i s connected by a s i n g l e , i n t e r m o l e c u l a r symmetrical d i s u l p h i d e bond. In t h i s s e c t i o n , such hemoglobin octamers have been found to combine r e a d i l y w i t h h a p t o g l o b i n to produce a s e r i e s of aggregates of i n c r e a s i n g s i z e which p r e c i t i p a t e at low i o n i c s t r e n g t h i n a manner analogous to the p r e c i p i t a t i o n of antigen-antibody complexes. The formation of these complexes can most e a s i l y be expl a i n e d i f hapto-g l o b i n i s b i v a l e n t i n i t s combination w i t h hemoglobin. Treatment of these molecular aggregates of ha p t o g l o b i n and octameric hemoglobin w i t h mercaptoethanol converts them to the usu a l s i n g l e hemoglobin-h a p t o g l o b i n complexes. P r e c i p i t a t i o n Studies When s o l u t i o n s of mouse double hemoglobin (Hb.Hb) were mixed w i t h each of the three major ha p t o g l o b i n phenotypes (Hp 1-1, Hp 2-1, Hp 2-2) p r e c i p i t a t e s formed when the input r a t i o s of the two p r o t e i n s were w i t h i n c e r t a i n l i m i t s , a c l e a r d i f f e r e n c e from the r e a c t i o n of hap t o g l o b i n w i t h s i n g l e hemoglobin molecules which y i e l d s o n ly s o l u b l e complexes (55). Two s e r i e s of experiments were conducted; i n the f i r s t , ( s e r i e s A ) , the c o n c e n t r a t i o n of Hb.Hb remained constant and the hapto-g l o b i n c o n c e n t r a t i o n was v a r i e d , w h i l e i n the second, ( s e r i e s B), 39 in c r e a s i n g concentrations of Hb.Hb were added to a constant amount of haptoglobin. These mixtures were allowed to stand f o r several hours at 4° C. The extent of p r e c i p i t a t i o n i n se r i e s A was followed by measuring the decrease of absorbance due to Hb.Hb at 407 nm i n the super-natant a f t e r c e n t r i f u g a t i o n of the p r e c i p i t a t e . The extent of p r e c i p i -t a t i o n was dependent upon i o n i c strength, there being l i t t l e p r e c i p i t a t e i n 0.2 M NaCl. However i n the b u f f e r that was r o u t i n e l y employed, 4 mM sodium phosphate, pH 6.2 there was extensive p r e c i p i t a t i o n . In Figure 5, the p r e c i p i t a t i o n curves i n s e r i e s A of the three common haptoglobin phenotypes with Hb.Hb show a close resemblance to c l a s s i c a l antibody-antigen p r e c i p i t a t i o n curves. In c a l c u l a t i n g the molar input r a t i o s of the three haptoglobin phenotypes and Hb.Hb i t i s necessary to take account of the f a c t that while Hp 1-1 i s a monomeric p r o t e i n of mole-cular weight (98,000), Hp 2-1 and Hp 2-2 both e x i s t as a s e r i e s of stable polymers of increasing s i z e (98). However i t has been shown that the binding capacity per gram of each phenotype of haptoglobin i s v i r t u a l l y i d e n t i c a l (4) and that 1 mole of hemoglobin i s bound per 98,000 gms of haptoglobin of any of the three phenotypes. In the case of Hb.Hb, whose molecular weight i s 130,000, i t appears that the species combining with haptoglobin i s not the f u l l double molecule but rather, as with the si n g l e molecules of human hemoglobin, there i s f i r s t cleavage. For Hb.Hb t h i s would produce disulphide l i n k e d h a l f hemoglobin molecules (aB-8a) as shown i n Figure 6. In accordance with t h i s scheme of cleav-age the haptoglobin-hemoglobin r a t i o s are cal c u l a t e d i n every case on the basis of the 'monomeric u n i t ' of haptoglobin and the (aB-ga) molecules 40 F i g u r e 5 P r e c i p i t a t i o n curves w i t h the c o n c e n t r a t i o n of Hb.Hb maintained constant. The o r d i n a t e i s one-half the absorbance of the o r i g i n a l Hb.Hb s o l u t i o n minus the absorbance of the supernatant s o l u t i o n and t h e r e f o r e represents the amount of Hb.Hb p r e c i p i t a t e d . Hapto-g l o b i n s o l u t i o n s were prepared by d i l u t i o n s of a concentrated h a p t o g l o b i n s o l u t i o n to f i n a l volumes of 0.5 ml; to each of these s o l u t i o n s 0.5 ml of the Hb.Hb s o l u t i o n was added. In curve (a) the f i n a l c o n c e n t r a t i o n s are as f o l l o w s : Hb.Hb 0.16 mg/ml, Hp 1-1 v a r i e d from a h i g h c o n c e n t r a t i o n of 1.7 mg/ml down to 0.027 mg/ml. In curve (b) Hb.Hb 0.20 mg/ml, Hp 2-1 v a r i e d from 1.1 mg/ml to 0.018 mg/ml. In curve (c) Hb.Hb 0.20 mg/ml, Hp 2-2 1.4 mg/ml to 0.022 mg/ml. 0 1.0 2.0 3.0 4.0 5.0 Hp/a(3-pa MOLAR RATIO 42 F i g u r e 6 A scheme f o r the cleavage of Hb and Hb.Hb i n t o h a l v e s ; the d i s u l p h i d e bond i s between c y s t e i n e s at p o s i t i o n 13 i n the 3-chain i n BALB/cJ mice. 43 having molecular weights of 98,000 and 65,000 r e s p e c t i v e l y . The r a t i o s 1% are also c a l c u l a t e d on the basis of an E ° _ of 12.0 for the hapto-280nm globins and 17.5 for the mouse hemoglobin. In the s e r i e s B experiments, the haptoglobin concentration was kept constant and increasing concentrations of Hb.Hb added. P r e c i p i t a -t i o n was again observed and, as may be seen i n the photograph, (Figure 7), was dependent on the input r a t i o s of the combining pr o t e i n s . At both high (0.96) and low (0.015) r a t i o s of haptoglobin to a3-Ba, there was l i t t l e p r e c i p i t a t e but i n the range 0.06-0.48, p r e c i p i t a t i o n was exten-s i v e . Acrylamide Gel E l e c t r o p h o r e s i s When solutions of Hb.Hb and haptoglobin 1-1 i n 0.112 M T r i s , 0.062 M b o r i c a c i d and 2.5 mM disodium EDTA at pH 8.6 were mixed no p r e c i p i t a t i o n was observed. These soluble complexes were then examined by acrylamide d i s c gel electrophoresis i n the above b u f f e r . In Figure 8a, i t may be seen that a s e r i e s of hemoglobin-haptoglobin complexes appears with the r e l a t i v e concentration of each complex w i t h i n the s e r i e s depending upon the input r a t i o of the two p r o t e i n s . At high r a t i o s of haptoglobin/ag-ga (3.0-6.0) the major complexes migrated into the gels but as the r a t i o approaches that at which maximal p r e c i p i t a t i o n occurred at lower i o n i c strength, an increasing proportion of the com-plexes barely entered the g e l s , thus behaving as i f they were very l a r g e . In Figure 8b, an enlarged photograph of the f i r s t s i x gels shows that up to s i x separate complexes of decreasing m o b i l i t y are resolved. 44 F i g u r e 7 A photograph (by r e f l e c t e d l i g h t ) of the t u r b i d i t y observed s h o r t l y a f t e r mixing Hb.Hb and Hp. To o b t a i n the p r e c i p i t a t e s a concentrated Hb.Hb s o l u t i o n was d i l u t e d to a f i n a l volume of 0.5 ml and added to 0.5 ml of a hap t o g l o b i n s o l u t i o n . The f i n a l c o n c e n t r a t i o n s of Hb.Hb v a r i e d from 2.3 mg/ml to 0.036 mg/ml w h i l e h a p t o g l o b i n 1-1 was maintained constant a t 0.05 mg/ml. The white areas a t the bottom of the tubes are caused by r e f l e c t i o n from the g l a s s and are not i n d i c a t i v e of p r e c i p i t a t i o n . 46 F i g u r e 8a D i s c g e l e l e c t r o p h o r e s i s of mixtures of Hp and Hb.Hb. S o l u t i o n s were prepared by mixing v a r y i n g p r o p o r t i o n s of an 11.5 mg/ml s o l u t i o n of Hb.Hb and a 10 mg/ml s o l u t i o n of Hp. The g e l s contained 5% acrylamide and 0.25% N,N' methylene-b i s a c r y l a m i d e and were 0.5 x 7.0 cm. E l e c t r o p h o r e s i s was performed f o r 1.5 hours at 200 v o l t s at 4° C. MOLAR RATIO Hp/a0-/3a oo 6 0 4-2 3 0 1-8 1-2 72 6 5 -3 2 -12 09 06 0 9 w # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 F i g u r e 8b Enlarged photograph of the f i r s t 6 g e l s i n F i g u r e 8a. 1 2 3 Q u a l i t a t i v e l y , i t may be seen that as the p r o p o r t i o n of Hb.Hb i s increased each complex reaches a maximum c o n c e n t r a t i o n and then i s r e p l a c e d by a more s l o w l y running complex u n t i l the m a j o r i t y of the complexes are too l a r g e to migrate a p p r e c i a b l y i n t o the g e l . A s e r i e s of complexes c h a r a c t e r i z e d by i n c r e a s i n g sedimentation co-e f f i c i e n t s has a l s o been observed by u l t r a c e n t r i f u g a t i o n of mixtures of h a p t o g l o b i n and Hb.Hb. The best evidence that the l a r g e s t species are found at r a t i o s of h a p t o g l o b i n to aB-Ba near 1.0 was obtained from 3.5% acrylamide d i s c g e l s of l a r g e r pore s i z e . F i g u r e 9 shows that at a h a p t o g l o b i n to ag-gct r a t i o of 0.74 to 1.1 the major p r o t e i n band i s at the o r i g i n w h i l e at r a t i o s of 0.50 and 1.5 a g r e a t e r p r o p o r t i o n of the complexes runs w e l l i n t o the g e l s . Using 2.5% g e l s i n p l e x i -g l a s s tubes (101) s i m i l a r r e s u l t s have a l s o been obtained. Although the slow-running m a t e r i a l at the o r i g i n now enters the g e l , a s e r i e s of d i s c r e t e bands proceeding from the o r i g i n of the g e l s out to the p o s i t i o n of h a p t o g l o b i n or Hb.Hb cannot be observed because of s t r e a k i n g . This s t r e a k i n g a l s o prevents the o b s e r v a t i o n of a s e r i e s of bands i n the g e l s where Hb.Hb i s i n excess. However at r a t i o s near 1.0 there i s a d e f i n i t e i n c r e a s e i n s t a i n i n g of complexes, which although s t r e a k y , run much c l o s e r to the o r i g i n of the g e l and hence behave as i f they are much l a r g e r . I t would be expected that i n any complex formation between two p r o t e i n s each possessing two b i n d i n g s i t e s that the l a r g e s t complexes would be formed when an equal number of moles of each b i v a l e n t r e a c t i n g s p e c i e s i s present. In the present case i t has been assumed that the 51 Figure 9 Disc g e l electrophoresis of solutions prepared by mixing a 12.5 mg/ml s o l u t i o n of Hb.Hb with a 14 mg/ml s o l u t i o n of Hp 1-1. The gels con-tained 3.5% acrylamide and 0.18% N,N' methylene-bisacrylamide and electrophoresis was performed at 4° C for 1.3 hours at 200 v o l t s and 3.5 ma per g e l . M O L A R oo ] . 5 RATIO HpAxP-poc •37 0 r e a c t i n g species from Hb.Hb i s a p a i r of d i s u l p h i d e l i n k e d ct3 u n i t s , otB-Bot, of molecular weight 65,000. Thus the l a r g e s t complexes should occur at a r a t i o of 1.0 and t h i s appears c o n s i s t e n t w i t h the f a c t that the complexes formed at t h i s r a t i o cannot enter even the l a r g e pored g e l s . In c o n t r a s t to these f i n d i n g s , maximal p r e c i p i t a t i o n was seen at r a t i o s of 0.2-0.6. Thus i t appears that complexes r i c h e r i n aB-ga tetramers are more i n s o l u b l e i n 4 mM phosphate at pH 6.2 than those l a r g e r complexes i n which the amount of hap t o g l o b i n and ag -3a i s more n e a r l y equal. The behaviour observed upon mixing h a p t o g l o b i n w i t h Hb.Hb i s i n strong c o n t r a s t to i t s r e a c t i o n w i t h normal hemoglobin where only two s o l u b l e complexes are formed, an intermediate of s t o i c h i o m e t r y 1/2 Hb/1 Hp and the f u l l complex 1 Hb/1 Hp. Since the i n t e r m o l e c u l a r d i s u l p h i d e s of Hb.Hb can be cleaved under m i l d c o n d i t i o n s by mercaptoethanol (93), a 20 y l a l i q u o t of haptoglobin/Hb.Hb mixtures i n Tris-borate-EDTA, pH 8.6, was reacted w i t h 2 y l of 0.5 M mercaptoetha-n o l f o r 1 hour at' 25° C fo l l o w e d by the a d d i t i o n of 2 y l of 0.6 M iodocetamide f o r 30 minutes at 25° C. In F i g u r e 10,the s e r i e s of ha p t o g l o b i n (aB-Ba) complexes formed at r a t i o s of 3.4 to 1.0 (Gel 6) and 1.1 to 1.0 (Gel 5) gave r i s e upon treatment w i t h mercaptoethanol to the p a t t e r n s seen i n Gels 2 and 1 r e s p e c t i v e l y . At the higher input r a t i o of 3.4 (Gel 6) there i s s t i l l excess uncombined haptoglobin as w e l l as a s e r i e s of complexes which move w e l l i n t o the g e l . I t i s l i k e l y t hat these complexes are of the type depicted i n Fi g u r e 11a as Complex 1 and Complex 2. As i n d i c a t e d 54 Figure 10 Disc gels (5%) showing the e f f e c t of mercaptoethanol on Hp-(a$-Ba) complexes. Solutions were prepared by mixing an 8 mg/ml s o l u t i o n of Hb.Hb and a 14 mg/ml s o l u t i o n of Hp. Al i q u o t s were removed and then mercaptoethanol and iodoacetamide were added (see t e x t ) . E l e c t r o p h o r e s i s was performed f o r 2.3 hours at 200 v o l t s . Gel 3 shows the bands produced by mixing s i n g l e mouse hemoglobin molecules with excess haptoglobin. Gel 4 shows s i n g l e mouse hemoglobin. (a) Haptoglobin/(ot,P—s—s—P,a) input ratio = 3.4/1.0 Free ) Hp ^  ) Hp ) Hp ^ ) Hp ) Hp ( Free (4) :(4) Complex 1 ) Hp a , p _ S - s - P , a ) Hp ( P-meroaptoethanol> H S - P , o t ) Hp ( i complex ( Complex 2 j Hp <x,p—s—s—P,a j Hp ^ a ,P— s —s—p ,a j Hp ( HS—p,oc ^ Hp ( a,P—SH Full complex (1) (6) Haptoglobin/^,P—s—s—P,«) input ratio =1.1/1.0 Large complexes ...,p,a j Hp P,a—s—s—P,a ) » P ( a , P — s — s — P , a j Hp a ,P—s— s—P,a J Hp ( a , P — s — s — P , a J Hp ^  a > p \ . P-mercaptoethanol HS—a,P ) Hp ( ot,P—SH Figure 11 A scheme to illustrate the possible complexes of haptoglobin with double hemoglobin at (a). High input ratio (b). Equi-molar input ratio. i n F i g u r e 11a, a mixture of f r e e h a p t o g l o b i n , Complex 1 and Complex 2 would give r i s e , upon treatment w i t h 3-mercaptoethanol predominantly to the 1/2 HbrlHp complex w i t h a smaller amount of the f u l l complex. This p r e d i c t i o n i s c o n s i s t e n t w i t h the p a t t e r n observed i n Fi g u r e 10, Gel 2. In c o n t r a s t , at the input r a t i o of 1.1/1.0, the complexes are very much l a r g e r (Figure 10, Gel 5) and upon t h i o l treatment (Figure 10, G e l 1) gi v e r i s e almost q u a n t i t a t i v e l y to the f u l l Hb.Hp complex. Since the 1/2 Hb:Hp complex comes only from the ends of polymeric chains of Hb.Hb/Hp complexes, the absence of t h i s species i n Fi g u r e 10, Gel 1 and the predominance of the f u l l Hb:Hp complex i n d i c a t e s that the slow-running polymeric complexes of Hb.Hb/Hp formed at equimolar input r a t i o s of Hb.Hb and Hp are e i t h e r very l o n g , as de p i c t e d i n Fi g u r e l i b or a l t e r n a t i v e l y these complexes could be c i r c u l a r i n which case, the f u l l Hb:Hp complex would be the s o l e product. At the moment i t i s not p o s s i b l e to choose between these a l t e r n a t i v e s . In most antigen-antibody r e a c t i o n s , both antige n and antibody possess at l e a s t two b i n d i n g s i t e s . This leads to the formation of l a r g e , three-dimensional complexes which are o f t e n i n s o l u b l e . The i n t e r a c t i o n of hemoglobin w i t h h a p t o g l o b i n at f i r s t s i g h t appears not to be of t h i s type s i n c e the complex i s s o l u b l e and i t s o v e r a l l s t o i -chiometry i s c o n s i s t e n t w i t h a s i n g l e b i n d i n g s i t e on each p r o t e i n . However, the f a c t that at r a t i o s of Hb to Hp of l e s s than one an inter m e d i a t e complex of s t o i c h i o m e t r y 1/2 Hb/1 Hp i s seen suggests that only one of the two s i t e s i s occupied by a h a l f molecule of hemo-g l o b i n and that the f u l l complex would comprise two h a l f molecules of hemoglobin combined at two separate s i t e s . Thus hemoglobin, a symmetrical and b i v a l e n t molecule, d i s s o c i a t e s i n t o two monovalent halves upon combination w i t h h a p t o g l o b i n . There i s , t h e r e f o r e , no p o s s i b i l i t y of forming l a r g e complexes as i n the case of an an t i g e n -antibody r e a c t i o n . In the present study, a hemoglobin has been examined i n which the two halves of the molecule are c o v a l e n t l y l i n k e d by a d i s u l p h i d e bond so that i t behaves as a b i v a l e n t molecule. When ha p t o g l o b i n combines w i t h the b i v a l e n t hemoglobin a n o t i c e a b l y d i f f e r e n t behavior i s seen. Large complexes are formed which p r e c i p i t a t e at low i o n i c s t r e n g t h i n a manner very s i m i l a r to that of antigen-antibody r e a c t i o n s . As soon as the covalent l i n k a g e between the 8 chains of Hb.Hb i s broken the l a r g e complexes disappear and are replaced by the simple ones. IV THE DISULPHIDES OF HAPTOGLOBINS - STUDIES ON CYANOGEN BROMIDE REACTIONS WITH HAPTOGLOBIN AND ON HALF-HAPTOGLOBIN I n t r o d u c t i o n The a and B chains of the human haptoglobins are h e l d together by d i s u l p h i d e bonds (12). T h i s f a c t was based on the observations that h a p t o g l o b i n 1-1 migrated as a s i n g l e band on g e l e l e c t r o p h o r e s i s i n 8M urea but when very low concentrations of B-mercaptoethanol, a reagent which i s known to break d i s u l p h i d e bonds, were added the h a p t o g l o b i n was s p l i t i n t o i t s a and B chains. S i m i l a r l y the polymeric hapto-g l o b i n s (2-1 and 2-2) maintained t h e i r s t r u c t u r e i n 8M urea but were s p l i t i n t o a and B chains by mercaptoethanol. As discussed p r e v i o u s l y Kauffman and Dixon i s o l a t e d an a-a' d i -s u l p h i d e peptide a f t e r pepsin d i g e s t s of h a p t o g l o b i n 2-1. The s t r u c t u r e of t h i s peptide i s shown i n F i g u r e 2 (page 8). In a d d i t i o n , Smithies, C o n n e l l and Dixon (76) have presented evidence f o r the e x i s t e n c e of an i n t r a c h a i n d i s u l p h i d e i n the a chain of h a p t o g l o b i n . A f t e r r e d u c t i o n of h a p t o g l o b i n 1-1 w i t h very low concentrations of mercaptoethanol, they detected a band a f t e r e l e c t r o p h o r e s i s which migrated s l i g h t l y f a s t e r than the completely-reduced a c h a i n . Thus t h i s p o l y p e p t i d e appeared to migrate f a s t e r than the f u l l y - r e d u c e d a c h a i n of h a p t o g l o b i n because i t contained an i n t r a c h a i n d i s u l p h i d e and thus had a more compact conformation. However, s i n c e i n these experiments no mercaptoethanol was i n c l u d e d i n the g e l b u f f e r , such a loop d i s u l p h i d e might have been formed by o x i d a t i o n during e l e c t r o p h o r e s i s and so need not n e c e s s a r i l y be present i n n a t i v e h a p t o g l o b i n . The b i n d i n g of the hapt o g l o b i n chains s o l e l y by noncovalent f o r c e s appears very u n l i k e l y because of the s t a b i l i t y of the molecule under a v a r i e t y of denaturing c o n d i t i o n s . In a d d i t i o n to i t s m i g r a t i o n as a s i n g l e band i n 8M urea, h a p t o g l o b i n 1-1 maintains i t s molecular weight a f t e r complete s u c c i h y i a t i o n (54), a very powerful method f o r d i s r u p t i n g n on-covalently bonded subunits (102), and i t i s not d i s s o c -i a t e d i n 0.1% sodium dodecyl sulphate (SDS) (96). A l s o the ha p t o g l o b i n polymers main t a i n t h e i r polymeric s t r u c t u r e i n 8M urea, a f t e r s u c c i n y l a -t i o h (54) or i n 0.1% SDS. In 1961 (103) Dixon and C b n n e l l showed that when hapt o g l o b i n was t r e a t e d w i t h s u l p h i t e and parahydroxymercuribenzoate (pHMB) a l i m i t e d cleavage of d i s u l p h i d e bonds occurred. The product migrated i d e n t i c a l l y w i t h h a p t o g l o b i n i n borate s t a r c h g e l s but i n the a c i d i c 8M urea g e l s i t moved s l i g h t l y more r a p i d l y and e x h i b i t e d a broader band than hapto-g l b b i i n Since there was only one product formed i t appeared that h a p t o g l o b i n was being s p l i t i n t o symmetrical h a l v e s . This i n t e r p r e t a -t i o n has been confirmed by molecular weight s t u d i e s oh the " h a l f - h a p t o -g l o b i n molecule" u s i n g g e l e l e c t r o p h o r e s i s i n sodium dodecyl sulphate (104,96). A l s o the very i n t e r e s t i n g o b s e r v a t i o n was made that i f the m u l t i p l e h a p t o g l o b i n polymers were t r e a t e d w i t h s u l p h i t e they were converted to a s i n g l e product which a l s o appeared to c o n t a i n a hapto-g l o b i n l i g h t and heavy c h a i n (103). Since the bond cleaved by s u l p h i t e and p-chloromercurisulphonate (pCMS) must be symmetrical to give r i s e to symmetrical h a l v e s , i t seemed l o g i c a l that the s u l p h i t e must be s p l i t t i n g the ct-a' d i s u l p h i d e and thus breaking h a p t o g l o b i n i n t o h a l v e s . I t a l s o appeared that t h i s p a r t i c u l a r d i s u l p h i d e was r e s p o n s i b l e f o r ha p t o g l o b i n p o l y m e r i z a t i o n . Thus i t was p o s s i b l e to i n v e s t i g a t e the d i s u l p h i d e s of h a p t o g l o b i n 35 f u r t h e r by u s i n g S - s u l p h i t e f o r cleavage and then determining i n which p a r t of the molecule the r a d i o a c t i v i t y was l o c a t e d (96). This approach was attempted by t r e a t i n g the h a l f - h a p t o g l o b i n molecule, which 35 had been prepared w i t h S - s u l p h i t e , under more r i g o r o u s c o n d i t i o n s w i t h 8M urea and u n l a b e l l e d s u l p h i t e . In t h i s way, h a p t o g l o b i n was s p l i t i n t o i t s heavy and l i g h t chains which were subsequently separated on Sephadex G-75 (28). The r i g o r o u s c o n d i t i o n s l e f t o n l y a s m a l l amount 35 of the S - s u l p h i t e i n the ha p t o g l o b i n and t h i s was found i n the 3 c h a i n f r a c t i o n . Thus at f i r s t s i g h t i t seemed that the a-a' d i s u l p h i d e 35 was not being s p l i t by S - s u l p h i t e . I t appeared p o s s i b l e to analyze the nature of the a-3 l i n k a g e s and a-a' l i n k a g e s f u r t h e r because the a ch a i n of h a p t o g l o b i n contained no methionine (28) . Thus i f one cleaved the ha p t o g l o b i n molecule w i t h a reagent which s p l i t peptide bonds at methionine r e s i d u e s the 3 c h a i n of h a p t o g l o b i n would be cleaved i n t o a s e r i e s of s m a l l e r fragments but the a c h a i n would remain i n t a c t . A l s o , s i n c e the d i s u l p h i d e bonds i n ha p t o g l o b i n would s t i l l be i n t a c t , one could study the 3 c h a i n peptide fragment(s) to which the a ch a i n was attached s i n c e the a chain could be c h a r a c t e r i z e d a f t e r the fragment c o n t a i n i n g i t was reduced w i t h 61 mercaptoethanol and a l k y l a t e d w i t h iodoacetamide (12). In f a c t , a w e l l c h a r a c t e r i z e d reagent f o r cleavage of methionyl p e p t i d e s , cyanogen bromide, appears to be w e l l s u i t e d f o r these s t u d i e s (105). Cyanogen bromide i s a u s e f u l reagent f o r p r o t e i n s t u d i e s . A l -though i t r e a c t s w i t h b a s i c groups i n p r o t e i n s i n a l k a l i , i n a c i d i t r e a c t s only w i t h c y s t e i n e and methionine. The r e a c t i o n w i t h c y s t e i n e i s a slow o x i d a t i o n to c y s t e i c a c i d and the reagent w i l l not r e a c t w i t h carboxymethylcysteine or S-benzylcysteine (106). The r e a c t i o n w i t h methionine i n a methionyl peptide r e s u l t s i n the cleavage of a peptide bond (Figure 12) and the methionyl r e s i d u e i s converted i n t o an e q u i l i -brium mixture of homoseryl and homoseryl l a c t o n e r e s i d u e s at the c a r b o x y l - t e r m i n a l p o r t i o n of the cleaved p e p t i d e . The mixture can be converted to homoserine la c t o n e by heating i n a c i d or can be opened to homoserine by treatment w i t h a l k a l i at room temperature. The reagent has now been used w i t h success on over 20 p r o t e i n s and f r e q u e n t l y i s the reagent of choice f o r l i m i t e d cleavage of peptide bonds. During t h i s i n v e s t i g a t i o n of the r e a c t i o n of cyanogen bromide w i t h haptoglobins i t was not known that the peptide Th3A i s o l a t e d by Kauffman and Dixon was i n f a c t a peptide from the a chain of hapto-g l o b i n (see I n t r o d u c t i o n , page 8 ) , and that t h i s peptide was l i n k e d to the 6 chain of h a p t o g l o b i n . However, the s t u d i e s to be d e s c r i b e d are i n complete agreement w i t h t h i s l i n k a g e and, as w i l l be shown, do con f i r m the d i s u l p h i d e s presented i n the I n t r o d u c t i o n . NH-CHR'COOH H / R N H - C - C :S- } C = N Methionyl peptide Cyanogen bromide R N H - C -H Q NH— CHR'COOH Cs^—C=N I © C H 3 B r u [Cyanosulfonium bromide] HjN—CHR'COOH Amino acid (or aminoacyl peptide) + H // R N H - C - C I \ O I / Peptidyl homoserine lactone „ NH—CHR'COOH RNH—C—C > H j C - C H j Br © Iminolactone bromide + : S - C = N I C H 3 Methyl thiocyanate F i g u r e 12 The r e a c t i o n of cyanogen bromide w i t h a methionyl peptide. 63 Nature of the Reaction as Examined by Disc Gels Cyanogen bromide cleavages were attempted i n 70% formic acid and i n 0.01 or 0.1M sodium acetate buffers pH 4.7. The former has been used s u c c e s s f u l l y on IgG by Edelman (107) and the l a t t e r was attempted because i t was thought p o s s i b l e to get a more l i m i t e d cleavage under conditions where the pro t e i n maintains a more compact three-dimensional s t r u c t u r e . Also haptoglobin r e t a i n s hemoglobin-binding a b i l i t y at pH 4.7 and so i t might be po s s i b l e to obtain a fragment with hemo-globin-binding a b i l i t y . The reactions at pH 4.7 were done with 0.01M or 0.1M sodium acetate, using Hp concentrations of 1% and CNBr concentrations of 0.5 or 5%. In both cases the re a c t i o n s o l u t i o n developed a p r e c i p i t a t e a f t e r a short time and was not studied f u r t h e r . Reactions i n 70% formic a c i d were analyzed by d i s c gel e l e c t r o -phoresis as described e a r l i e r . Samples were dissolved i n T r i s - g l y c i n e 4 to 5 times more concentrated than the gel b u f f e r . The r e s u l t s shown iri Figure 13 demonstrate that one main slow-running band and two f a s t -running bands are formed by the r e a c t i o n and the pattern of bands appears s i m i l a r i n d i f f e r e n t haptoglobin types (Fig. 13a Gels 1 and 2). When electrophoresis i s c a r r i e d out f o r a longer time the slow-running band resolves into a s e r i e s of mult i p l e bands (Figure 13b Gels 3,4,5). These m u l t i p l e bands form a c h a r a c t e r i s t i c pattern with darker bands at the center and f a i n t e r bands at the outside. Because of t h i s v a r i a t i o n i n i n t e n s i t y of the bands they form a gaussian d i s t r i b u t i o n and f o r s i m p l i c i t y w i l l subsequently be r e f e r r e d to as 'gaussian' bands. 64 Figure 13 Acrylamide d i s c gel electrophoresis (7.5%) of the re a c t i o n products of cyanogen bromide with haptoglobins. Figure 13a shows the r e s u l t s a f t e r e l e c t r o -phoresis f o r 10 minutes at 300 v o l t s i n 0.05M gly c i n e , 0.01M T r i s pH 8.5. Figure 13b shows the r e s u l t s when e l e c t r o -phoresis i s c a r r i e d out for 75 minutes, Figure 13c - the acrylamide gels contain 8M urea and electrophoresis i s f o r 40 minutes at 300 v o l t s . Gels 1, 5, and 6 show CNBr Hp 1-1; 2, 3, and 8 show CNBr Hp 2-2; and 4 and 7 show CNBr Hp 2-1. Gel length 5 cm. 13a 1 2 13b 3 4 5 66 A s e r i e s of bands w i t h a s i m i l a r d i s t r i b u t i o n has been obtained a f t e r s t a r c h g e l e l e c t r o p h o r e s i s of the Hp 8 chain (76) and i t appears that a s i m i l a r phenomenon i s r e s p o n s i b l e f o r the p a t t e r n of banding i n both cases. Other s t u d i e s i n t h i s l a b o r a t o r y suggest that the p a t t e r n may be caused by a v a r i a b l e degree of attachment of s i a l i c a c i d r esidues to the h a p t o g l o b i n 3 chain (32). The CNBr r e a c t i o n products were a l s o analyzed by d i s c g e l e l e c t r o p h o r e s i s i n T r i s - g l y c i n e b u f f e r c o n t a i n i n g 8M urea (Figure 13c). Ten m i c r o l i t e r s of 2% (v/w) s o l u t i o n s of the r e a c t i o n mixture were a p p l i e d to the g e l s . The r e s o l u t i o n of bands i n the 8M urea g e l s appeared to be b e t t e r than i n the g e l s which contained no urea. I n a l l of the d i s c g e l s observed the p a t t e r n s obtained from Hp 1-1, 2-1, and 2-2 were s i m i l a r , but the r e g i o n of maximum s t a i n i n g , although composed of a complex of bands, was i n d i f f e r e n t p o s i t i o n s i n the three h a p t o g l o b i n types (Figure 13c). However much c l e a r e r d i f f e r -ences were seen i n l a t e r s t u d i e s u s i n g s t a r c h g e l s to r e s o l v e the cyanogen bromide fragments (as discussed below). Since the a h a p t o g l o b i n chains should not be cleaved by cyanogen bromide and s i n c e there are only 4 methionines i n the 8 chain of hapto-g l o b i n (28), i t was l o g i c a l to i n v e s t i g a t e more c l o s e l y the r e a c t i o n of cyanogen bromide w i t h the h a p t o g l o b i n polymers to see whether or not a polymeric s e r i e s was present a f t e r the r e a c t i o n of the h a p t o g l o b i n polymers w i t h cyanogen bromide. In order to examine the e f f e c t of cyanogen bromide on h a p t o g l o b i n polymers more c l o s e l y and to c h a r a c t e r i z e the r e a c t i o n f u r t h e r , a time study of the r e a c t i o n w i t h h a p t o g l o b i n 2-1 was performed (Figure 14). The r e a c t i o n was stopped at v a r i o u s times by a l Q - f o l d d i l u t i o n of the r e a c t i o n mixture and subsequent f r e e z i n g and l y o p h i l i z a t i o n . With the 5 minute sample a s m a l l amount of r e a c t i o n could be observed (Figure 14 Gel 2). New bands appeared running s l i g h t l y ahead of each of the polymers and these appeared a l s o to form a polymeric s e r i e s . A l s o a s m a l l amount of a f a s t - r u n n i n g band appeared. At 20 minutes (Figure 14 Gel 3) the appearance of t h i s new s e r i e s and a l s o of the f a s t - r u n n i n g band was more pronounced. A l s o the m u l t i p l e "gaussian" bands began to appear. A f t e r 24 hours t h i s "new s e r i e s " was no longer present w h i l e the f a s t - r u n n i n g band remained and a new slow-running d i f f u s e r e g i o n was present (Figure 14 Gel 4 ) . Starch-Urea Gel Analyses of the R e a c t i o n Products Improved r e s o l u t i o n of the CNBr fragments i n pH4 s t a r c h g e l s r e l a t i v e to the s e p a r a t i o n i n acrylamde gels was obtained, p a r t l y be-cause of the g r e a t e r l e n g t h of the s l a b g e l s and p a r t l y because of the d i f f e r e n t r e l a t i v e m o b i l i t i e s of the polypeptides at the lower pH. CNBr Hp 1-1 (Figure 15 s l o t 3) showed a d a r k - s t a i n i n g f a s t - r u n n i n g band not present i n the other haptoglobins (Figure 15 s l o t s 4,5). The l a t t e r appeared to have a g r e a t e r p r o p o r t i o n of slower-running bands. A f t e r r e d u c t i o n and a l k y l a t i o n , the CNBr Hp 1-1 produced the hapto-g l o b i n chain (Figure 15 s l o t 6) w h i l e the CNBr Hp 2-1 produced both 2 2 and a chains (Figure 15 s l o t 7 ) , and CNBr Hp 2-2 produced the a chain (Figure 15 s l o t 8 ) . The p r e d i c t i o n that CNBr would not a t t a c k the a chains of h a p t o g l o b i n was thus confirmed. 68 Figure 14 Acrylamide d i s c gel a n a l y s i s of the r e a c t i o n of CNBr with Hp 2-1 as a fu n c t i o n of time. Figure 14a shows the r e s u l t s of elect r o p h o r e s i s i n a 4% g e l at 300 v f o r 150 minutes using T r i s -g l ycine b u f f e r . Figure 14b shows an a n a l y s i s of the same samples using 7.5% gels at about 300 v f o r 85 minutes. For the 7.5% gels a good separa-t i o n was achieved by running the elect r o p h o r e s i s twice as long as i t takes a marker of bromophenol blue to migrate from the top to the bottom of the g e l . Gels l a l b , 2a 2b, 3a 3b, 4 a 4b, show the r e s u l t s a f t e r r e a c t i o n times of 0 minutes, 5 min-utes, 20 minutes, and 24 hours r e s p e c t i v e l y . Gel length 7 cm. 70 F i g u r e 15 A n a l y s i s of the CNBr Hp r e a c t i o n s u s i n g s t a r c h g e l e l e c t r o p h o r e s i s i n 8M urea-formate b u f f e r pH 4.0. Gels 1 and 2 show haptoglobins 1-1, and 2-1 r e s p e c t i v e l y which have been reduced and a l k y l a t e d . Gels 3, 4, and 5 show the r e s u l t s of cyanogen bromide r e a c t i o n w i t h haptoglobins 1-1, 2-1, and 2-2 r e s p e c t i v e l y . Gels 6, 7, and 8 show the CNBr r e a c t i o n products of 1-1, 2-1, and 2-2 r e s p e c t i v e l y a f t e r they have been reduced and a l k y l a t e d . P r o t e i n con-c e n t r a t i o n s were; samples 1 and 3, 6 to 8, 2% samples 3 to 5, 3% Samples 6 to 8 were reduced w i t h 0.04M 6 -mercaptoethanol f o r 30 minutes and a l k y l a t e d w i t h O.IM iodoacetamide. 72 In the hope of l o c a t i n g which band(s) contained the a c h a i n s , two-dimensional s t a r c h g e l e l e c t r o p h o r e s i s was performed. As shown i n F i g u r e 15 (page 70) a s e r i e s of bands was r e s o l v e d i n the CNBr haptoglobins and, a f t e r r e d u c t i o n and a l k y l a t i o n , h a p t o g l o b i n a chains were obtained. I f a s l i c e of g e l c o n t a i n i n g these CNBr-produced bands was removed and t r a n s f e r r e d to another g e l c o n t a i n i n g mercaptoethanol w i t h subsequent e l e c t r o p h o r e s i s at r i g h t angles to the f i r s t d i r e c t i o n , then any bands which contained the a chains should be reduced by the mercaptoethanol i n the second g e l and a chains should appear. By the use of a h a p t o g l o b i n marker i n the second dimension i t should be p o s s i b l e to a s c e r t a i n which bands were a chain bands and thus which band i n the f i r s t dimension contained the h a p t o g l o b i n a ch a i n s . A l l of the p o l y p e p t i d e s which c o n t a i n no d i s u l p h i d e s should r e t a i n the same m o b i l i t y i n the second dimension as i n the f i r s t . The method i s a di a g o n a l technique i n which d i s u l p h i d e - c o n t a i n i n g p o l y p e p t i d e s w i l l run o f f the d i a g o n a l . f In the case of CNBr ha p t o g l o b i n 1-1, a peptide running o f f the di a g o n a l w i t h the same m o b i l i t y as the a 1 c h a i n (Figure 16) was observed. This a 1 chain was produced by the f a s t - r u n n i n g band i n CNBr Hp 1-1 which d i d not appear i n the other h a p t o g l o b i n s . Using h a p t o g l o b i n 2-2 (Figure 17) a s e r i e s of a 2 chains running o f f the d i a g o n a l was obtained. Thus a polymeric s e r i e s of bands, w i t h each band i n the s e r i e s c o n t a i n -i n g an a c h a i n , appeared to be present i n h a p t o g l o b i n 2-2 a f t e r cyanogen bromide cleavage of t h i s polymeric h a p t o g l o b i n . Figure 16 Two dimensional urea-formate e l e c t r o p h o r e s i s of CNBr Hp 1-1. In the second dimension the g e l con t a i n s 0.1M mercaptoethanol. 74 F i g u r e 17 Two dimensional s t a r c h g e l a n a l y s i s of CNBr Hp 2-2. In the f i r s t dimension the g e l c o n t a i n s aluminum l a c t a t e b u f f e r and 8M urea. In the second dimension the g e l contains formate b u f f e r , 8M urea and 0.2M mercaptoethanol. *• CD 8 M Urea-Aluminum L a c t a t e Studies of the Reaction of CNBr w i t h the H a l f - H a p t o g l o b i n Molecule As discussed p r e v i o u s l y , h a l f - h a p t o g l o b i n (1/2 Hp) can be formed by l i m i t e d cleavage of h a p t o g l o b i n w i t h sodium s u l p h i t e and p-chloromercurisulphonate and appears to r e s u l t from the s c i s s i o n of a l i m i t e d number of d i s u l p h i d e bonds as s u l p h i t e i s known to cleave d i s u l p h i d e bonds. Thus the h a l f - h a p t o g l o b i n i s a u s e f u l d e r i v a t i v e f o r the study of the p a r t i c u l a r d i s u l p h i d e ( s ) b i n d i n g the two halves of the h a p t o g l o b i n molecule. F i g u r e 18 shows the r e s u l t s of a s t a r c h - u r e a a n a l y s i s of the products obtained a f t e r h a p t o g l o b i n was cleaved w i t h s u l p h i t e and p-chloromercurisulphonate (pCMS) according to the method of Rorstad and Dixon (96). In agreement w i t h the previous r e s u l t s , the product of the r e a c t i o n ( h a l f - h a p t o g l o b i n ) i s seen to migrate more r a p i d l y i n the g e l s than h a p t o g l o b i n (Figure 18a). The nature of the s p l i t t i n g of h a p t o g l o b i n by sodium s u l p h i t e and pCMS has been examined by f u r t h e r c l e a v i n g the h a l f - h a p t o g l o b i n w i t h cyanogen bromide i n 70% formic a c i d . H a l f - h a p t o g l o b i n produced by the sulphite-pCMS r e a c t i o n w i t h haptoglobin must have e i t h e r a s u l p h i t e group or a p-mercurisulphonate group attached to one of i t s c y s t e i n e s . The s t a b i l i t y of these two groups when attached to c y s t e i n e s i n p r o t e i n s has not been s t u d i e d e x t e n s i v e l y . However, the S-sulpho-c y s t e i n e group i n S-sulphokeratin i s s t a b l e from pH 1 to pH 9 (108). -18b 77 new bands 1 2 1 2 3 Fig u r e 18 a) Demonstration of the formation of Hp/2 by s t a r c h - u r e a g e l e l e c t r o p h o r e s i s . S l o t 1, h a l f - h a p t o g l o b i n ; s l o t 2, ha p t o g l o b i n . b) Comparison of the r e a c t i o n products of Hp 1-1 and Hp 12 w i t h CNBr by s t a r c h g e l e l e c t r o p h o r e s i s i n formate-urea. S l o t 1, CNBr ha p t o g l o b i n ; s l o t s 2 and 3, CNBr h a l f -h a p t o g l o b i n . 78 F i g u r e 18b compares the peptides produced by the r e a c t i o n of cyanogen bromide w i t h half-Hp 1-1 ( s l o t s 2 and 3) w i t h the peptides produced by the r e a c t i o n w i t h Hp 1-1 ( s l o t 1 ) . A l l of the fragments i n the ha p t o g l o b i n 1-1 r e a c t i o n mixture appeared to be present i n the h a l f - h a p t o g l o b i n mixture. However, the f a s t - r u n n i n g , d a r k - s t a i n i n g band appeared to be much f a i n t e r i n the h a l f - h a p t o g l o b i n r e a c t i o n mixture than i n the Hp 1-1 r e a c t i o n mixture. In a d d i t i o n two new very f a s t -running bands appeared i n the C N B r - s p l i t h a l f - h a p t o g l o b i n s l o t which were not present i n the C N B r - s p l i t Hp 1-1 s l o t . The r e a c t i o n of CNBr w i t h h a l f - h a p t o g l o b i n was terminated a f t e r 10 hours whereas that w i t h h a p t o g l o b i n 1-1 was terminated a f t e r 18 hours. However, i t i s not l i k e l y that t h i s d i f f e r e n c e i n the d u r a t i o n of the r e a c t i o n s would account f o r the d i f f e r e n c e s i n g e l p a t t e r n s . The d i f f e r e n c e i n pa t t e r n s between C N B r - s p l i t Hp 1-1 and C N B r - s p l i t h a l f - h a p t o g l o b i n must have been caused by the previous s p l i t t i n g of ha p t o g l o b i n 1-1 by sodium s u l p h i t e and pCMS. This p r e d i c t i o n has been confirmed by g e l a n a l y s i s of a 20-hour CNBr h a l f - h a p t o g l o b i n r e a c t i o n . The f a s t - r u n n i n g d a r k - s t a i n i n g p e p t i d e , v i r t u a l l y absent i n C N B r - s p l i t h a l f - h a p t o g l o b i n , corresponded to the CNBr fragment shown by two-dimensional g e l e l e c t r o p h o r e s i s to c o n t a i n the a chain of hapto-g l o b i n . A l s o , s t u d i e s of the p u r i f i e d CNBr fragment PC I I I (see s e c t i o n on p u r i f i c a t i o n and p r o p e r t i e s of fragments, p. 80) confirmed that t h i s f a s t - r u n n i n g d a r k - s t a i n i n g peptide d i d c o n t a i n the a c h a i n . Thus the a c h a i n - c o n t a i n i n g peptide i n C N B r - s p l i t Hp 1-1 was almost absent i n C N B r - s p l i t h a l f - h a p t o g l o b i n . However, s i n c e the h a l f - m o l e c u l e of 79 h a p t o g l o b i n s t i l l contained the a chains and s i n c e only two new bands appeared i n the C N B r - s p l i t h a l f - h a p t o g l o b i n , i t f o l l o w s that these new f a s t - r u n n i n g bands i n the h a l f - h a p t o g l o b i n must c o n t a i n the a chains. H a l f - h a p t o g l o b i n c o n s i s t s of two e s s e n t i a l l y i d e n t i c a l species d i f f e r i n g by only the presence of e i t h e r a S-sulpho-cysteine or p-mercurisulphonate mercaptide i n the molecule. Treatment of the two e s s e n t i a l l y i d e n t i c a l h a l f - h a p t o g l o b i n s w i t h CNBr, should produce two a l t e r e d p e p t i d e s , one c o n t a i n i n g a S-sulpho-cysteine and the other a p-mercurisulphonate mercaptide, which p r e v i o u s l y had formed a symmetrical bond. These new CNBr fragments would be expected to have molecular weights one-half that of the corresponding fragment from n a t i v e hapto-g l o b i n . In C N B r - s p l i t h a l f - h a p t o g l o b i n two new bands appeared, one which ran s l i g h t l y f a s t e r than the a chain of h a p t o g l o b i n and one s l i g h t l y slower. A p o s s i b l e e x p l a n a t i o n f o r the e x i s t e n c e of the two new bands i n C N B r - s p l i t h a l f - h a p t o g l o b i n i s that under the c o n d i t i o n s of cyanogen bromide cleavage, the s u l p h i t e was hydrolyzed from the S-sulpho-c y s t e i n y l peptide thus removing a negative charge from t h i s peptide. 35 This p o s t u l a t e has been confirmed by Rostad and Dixon (96) using S-s u l p h i t e - l a b e l l e d h a l f - h a p t o g l o b i n . They have shown that 70 to 80% of the r a d i o a c t i v i t y was r e l e a s e d from the h a l f - h a p t o g l o b i n under the c o n d i t i o n s of CNBr cleavage. The slower-running of the two new bands probably has the mercurisulphonate mercaptide group. Since the two new bands d e r i v e d from h a l f - h a p t o g l o b i n ran f a s t e r than the a c h a i n - c o n t a i n -i n g band d e r i v e d from h a p t o g l o b i n , they must be of s m a l l e r s i z e because 80 the i n t r o d u c t i o n of a negative charge by s u l p h i t e or mercurisulphonate would decrease r a t h e r than i n c r e a s e the m o b i l i t y of these c a t i o n i c p o l y p e p t i d e s . P u r f i c i a t i o n and P r o p e r t i e s of Cyanogen Bromide Fragments In order to prepare s u f f i c i e n t m a t e r i a l f o r f u r t h e r c h a r a c t e r i -z a t i o n of the a c h a i n c o n t a i n i n g fragments from the CNBr-treated hapto-g l o b i n s , the CNBr r e a c t i o n mixture was f r a c t i o n a t e d by i o n exchange chromotography on phosphocellulose. Ion exchange was used i n preference to g e l f i l t r a t i o n because the two-dimentional s t a r c h g e l (Figure 17, p.75) i n d i c a t e d that the alpha chains from Hp 2-1 and 2-2 were s t i l l present as a polymeric s e r i e s and so would not appear as a s i n g l e peak upon g e l f i l t r a t i o n but would be e l u t e d as a broad peak or s e r i e s of peaks. However, i t was known th a t the i s o e l e c t r i c p o i n t s of the polymers were a l l s i m i l a r (4) so that a s i n g l e peak f o r these a c h a i n - c o n t a i n i n g fragments might be obtained upon i o n exchange chromatography. Chro-matography was performed at pH 4.0 i n 8M d e i o n i z e d urea s i n c e i t was known from the s t a r c h g e l s t h a t a l l the CNBr fragments were p o s i t i v e l y charged under these c o n d i t i o n s . Phosphocellulose was chosen i n p r e f e r -ence to c a r b o x y m e t h y l c e l l u l o s e s i n c e each phosphoryl group would possess a f u l l n egative charge at t h i s pH and would r e t a i n h i g h c a p a c i t y f o r absorbing c a t i o n i c p r o t e i n s . As shown i n F i g u r e 19, three peaks were obtained a f t e r chro-matography of a l l three h a p t o g l o b i n types. In the case of h a p t o g l o b i n 1-1 and h a p t o g l o b i n 2-2 there was a s a l t and a pH gradient (Figure 19a,c) 81 F i g u r e 19 Phosphocellulose (Bio-Rad L a b o r a t o r i e s Lot #6049) chromatography of the fragments obtained a f t e r the r e a c t i o n of haptoglobin w i t h cyanogen bromide. Top F i g u r e CNBr Hp 1-1; column 0.9 cm; g r a d i e n t , 500 ml 0.05M formic a c i d , 0.01N NaOH, 8M d e i o n i z e d urea, pH 4.0 to 500 ml 0.03M formic a c i d , 0.016M NaOH, 8M urea, 2M NaCl pH 4.6, (3.5 ml f r a c t i o n s ) ; 20 mg of sample were d i s s o l v e d i n 1.0 ml of s t a r t i n g b u f f e r and then a p p l i e d to the column. Middle F i g u r e CNBr Hp 2-1; column 0.9 by 50 cm; gradient 500 ml 0.05M formic a c i d , 0.01M NaOH, 7.2M urea pH 3.8 to 500 ml 0.05M formic a c i d , 0.01M NaOH, 7.2M urea^O.SM NaCl. The sample contained 120 mg and 12 to 15 ml f r a c t i o n s were c o l l e c t e d . Bottom F i g u r e CNBr Hp 2-2; column 0.9 by 25 cm; g r a d i e n t , 200 ml 0.05M formic a c i d , 0.01M NaOH, 8M urea, pH 4.0 to 200 ml 0.03M formic a c i d , 0.016M NaOH, 8M urea, 0.8M NaCl pH 4.6; sample 40 mg. For l a r g e s c a l e p r e p a r a t i o n s of cyanogen bromide fragments 600 mg of CNBr 1-1 was chromatographed on a phosphocellulose column 2 cm by 40 cm w i t h a grad i e n t of 1 l i t r e of 0.05M NaCl, 0.05M formic a c i d , 0.01M NaOH, 7.2M deionize d urea pH 3.9 to 1 l i t r e of 0.4M NaCl, 0.05M formic a c i d , 0.01M NaOH, 7.2M urea. 82 but i t was subsequently found that an e q u a l l y good s e p a r a t i o n w i t h Hp 2-1 could be obtained using only a s a l t g r a d i e n t . The tubes corresponding to the o p t i c a l d e n s i t y peaks were pooled, and d e s a l t e d by chromatography on Sephadex G-25 coarse using 0.2N a c e t i c a c i d , and then l y o p h i l i z e d . The p u r i f i e d peptides were then analysed by starch-urea g e l e l e c t r o p h o r e s i s i n formate b u f f e r . The g e l (Figure 20) showed that the f i r s t peak e l u t e d from phosphocellulose represented the p a r t of the hap t o g l o b i n molecule which ran e l e c t r o p h o r e t i c a l l y as a s e r i e s of bands w i t h a gaussian d i s t r i b u t i o n (Figure 20 s l o t 13). The g e l a l s o showed that these bands were very s i m i l a r i n Hp 1-1 and Hp 2-2 (Figure 20, s l o t s 13 and 7 r e s p e c t i v e l y ) and t h a t the m o b i l i t y of the bands w i t h i n t h i s s e r i e s d i d not change a f t e r r e d u c t i o n and a l k y l a t i o n (Figure 20, compare s l o t 4 w i t h s l o t 7 ). A s i m i l a r s e r i e s of bands was present i n the C N B r - s p l i t g c h a i n of h a p t o g l o b i n as shown by Hew (32). When examined e l e c t r o p h o r e t i c a l l y the second peak (PC I I ) from phosphocellulose showed s t a i n e d m a t e r i a l only i n the case of hapto-g l o b i n 2-2 (Figure 20, s l o t 6 ) . The t h i r d peak (PC I I I ) i n the case of h a p t o g l o b i n 1-1 c o n s i s t e d of a major band which had a m o b i l i t y 2 almost the same as the a chain of haptoglobin and a f a i n t minor band (Figure 20, s l o t 11). A f t e r r e d u c t i o n and a l k y l a t i o n t h i s major pept i d e has a m o b i l i t y i d e n t i c a l w i t h that of the a"*" ch a i n ( s l o t 8) . In the case of CNBr Hp 2-2 t h i s same peak (PC I I I ) was a slow-running s t r e a k y band ( s l o t 5) which upon r e d u c t i o n and a l k y l a t i o n gave r i s e to 2 the a chain of hap t o g l o b i n ( s l o t 2 ) . 84 Fig u r e 20 Starch urea g e l e l e c t r o p h o r e s i s i n formate and aluminum l a c t a t e b u f f e r s of CNBr peptides a f t e r p u r i f i c a t i o n on phosphocellulose. 1. CNBr Hp 2-2 2. CNBr Hp 2-2, PC I I I , reduced and a l k y l a t e d 3. CNBr Hp 2-2, PC I I , reduced and a l k y l a t e d 4. CNBr Hp 2-2, PC I , reduced and a l k y l a t e d 5. CNBr Hp 2-2, PC I I I 6. CNBr Hp 2-2, PC I I 7. CNBr Hp 2-2, PC I 8. CNBr Hp 1-1, PC I I I , reduced and a l k y l a t e d 9. Hp 2-1, reduced and a l k y l a t e d 10. CNBr Hp 1-1, PC I reduced and a l k y l a t e d 11. CNBr Hp 1-1. PC I I I 12. CNBr Hp 1-1, PC I I 13. CNBr Hp 1-1, PC I 14. CNBr Hp 1-1 15. CNBr Hp 2-2, PC I I I 16. Hp 2-1, reduced and a l k y l a t e d 17. CNBr Hp 2-1, PC I I I 18. CNBr Hp 2-1, PC I I I , reduced and a l k y l a t e d Formate Aluminum L a c t a t e 86 I t was expected that another p o l y p e p t i d e i n a d d i t i o n to the a chain would be observed a f t e r r e d u c t i o n and a l k y l a t i o n of peak I I I . None was observed. However another p o l y p e p t i d e could have escaped d e t e c t i o n i f i t migrated so r a p i d l y as to move completely through the g e l under the e l e c t r o p h o r e t i c c o n d i t i o n s or i f i t washed out of the g e l d u r i n g s t a i n i n g and d e s t a i n i n g . The r e s u l t s of the two-dimensional e l e c t r o p h o r e s i s u s i n g aluminum l a c t a t e b u f f e r i n the f i r s t dimension and sodium formate b u f f e r c o n t a i n -i n g mercaptoethanol i n the second dimension have shown that the a chains were present i n a polymeric s e r i e s i n CNBr Hp 2-2. A f t e r f r a c t i o n a t i o n of CNBr 2-2 on phosphocellulose the t h i r d peak (PC I I I ) ran as a broad band during e l e c t r o p h o r e s i s . However when the PC I I I fragments from CNBr Hp 2-1 and CNBr Hp 2-2 were run us i n g aluminum l a c t a t e b u f f e r (74) i n 8M urea the bands which p r e v i o u s l y streaked were now r e s o l v a b l e i n t o a polymeric s e r i e s (compare s l o t s 15 and 17 i n the aluminum l a c t a t e g e l w i t h s l o t 5 i n the formate g e l ) . In f a c t , the s e r i e s bears a strong resemblance to the polymeric s e r i e s of Hp 2-1 and Hp 2-2 except t h a t the bands run f a s t e r . This r e s u l t was confirmed by d i s c g e l e l e c t r o -p h o r e s i s i n 0.1% SDS. Again a f a i n t s e r i e s of polymers was observed w i t h phosphocellulose peak I I I from 2-1 and 2-2 and a s i n g l e band was observed from 1-1. The polymers were seen to run c o n s i d e r a b l y f a s t e r than Hp 2-1 i n these d i s c g e l s . A f t e r r e d u c t i o n and a l k y l a t i o n of PC I I I from CNBr 2-1 (Figure 20, s l o t 18) a f a i n t f a s t - r u n n i n g band appeared which may represent a piece of the 3 chain attached to the a c h a i n by a d i s u l p h i d e bond. One problem i n the study of d i s u l p h i d e bonds i n p r o t e i n s i s the p o s s i b i l i t y of d i s u l p h i d e interchange which can r e s u l t i n the i n c o r r e c t assignment of d i s u l p h i d e s . The d i s u l p h i d e interchange r e a c t i o n was f i r s t c h a r a c t e r i z e d by Ryle and Sanger (109) who found an interchange i n s t r ong a c i d (6N HCl and ION ^SO^) and i n weak base pH 8.0 and above. These s t u d i e s were subsequently extended by Spackman, S t e i n and Moore who confirmed that the i d e a l pH f o r studying d i s u l p h i d e s was around pH 2(110). The occurrence of d i s u l p h i d e interchange i n a c t u a l p r o t e i n d i s u l p h i d e s t u d i e s was f i r s t observed f o r i n s u l i n (111) and may have c o n t r i b u t e d to i n c o r r e c t d i s u l p h i d e assignments f o r r i b o n u c l e a s e (110) and carboxypeptidase A (112). The c o n d i t i o n s used f o r the p r e p a r a t i o n of fragment PC I I I were r e a c t i o n i n 70% formic a c i d and chromatography at pH 4.0. Both of these c o n d i t i o n s are u n l i k e l y to cause d i s u l p h i d e interchange. A l s o , Edelman (107) has s t u d i e d the d i s u l p h i d e s of a myeloma p r o t e i n a f t e r r e a c t i o n i n 70% formic a c i d and has obtained no evidence f o r d i s u l p h i d e i n t e r -change. F i g u r e 15 s l o t 3 and F i g u r e 20 s l o t 14 show g e l s of Hp 1-1 a f t e r r e a c t i o n w i t h cyanogen bromide and i n both cases that the PC I I I fragment i s one of the major bands. I f d i s u l p h i d e interchange had occurred d u r i n g the r e a c t i o n w i t h cyanogen bromide i t might be expected th a t the a c h a i n would not be present s o l e l y i n a major band i n PC I I I but i n a s e r i e s of minor bands. S i m i l a r l y the PC I I I fragment produced a major peak a f t e r i o n exchange chromatography i n d i c a t i n g that d i -s u l p h i d e exchange had not occurred. Further Studies on Fragment PC I I I Further c h a r a c t e r i z a t i o n of fragment PC I I I was c a r r i e d out by molecular weight determinations u s i n g 7.5% acrylamide g e l s which con-t a i n SDS. The r e s u l t s (Figure 21) showed that fragment PC I I I ( g e l 5) 2 d e f i n i t e l y had a molecular weight not only g r e a t e r than the a c h a i n of h a p t o g l o b i n ( g e l 3) but a l s o g r e a t e r than chymotrypsinogen ( g e l 4 ) . The molecular weight of t h i s PC I I I fragment from h a p t o g l o b i n 1-1 was c a l c u l a t e d to be near 30,000 by the dodecylsulphate g e l technique (104). Fragment PC I I I must s t i l l have two a"*" chains and two p i e c e s of 8 c h a i n s i n c e no d i s u l p h i d e s have been s p l i t . One can deduce t h a t the molecular weight of the 8 chain p i e c e i s approximately 6,000, i . e . , (M.W. PC I I I - 2 x M.W. of a 1) (30,000 - 2 x 9,000) , n n n 2 = 2 = o » u u u ' Gel 7 shows that i n a d d i t i o n to the a 1 chain of h a p t o g l o b i n a band w i t h approximately the same m o b i l i t y as i n s u l i n (see arrow) i s obtained a f t e r r e d u c t i o n and a l k y l a t i o n of PC I I I , thus c o n f i r m i n g the a p p r o x i -mation of 6,000 f o r the molecular weight f o r the B chain p i e c e . The fragment of the 8 c h a i n of h a p t o g l o b i n which i s attached to the a c h a i n i n the PC I I I cyanogen bromide fragment has been i s o l a t e d by g e l e x c l u s i o n chromatography on Sephadex G-75 a f t e r r e d u c t i o n and a l k y l a t i o n of PC I I I . Figure 22 shows that a f t e r r e d u c t i o n and a l k y -14 l a t i o n w i t h C-iodacetamide two main bands of o p t i c a l d e n s i t y and r a d i o a c t i v i t y were seen (peaks 1 and 2) i n a d d i t i o n to the peak c o r r e s -ponding to the reagents. A s m a l l amount of other m a t e r i a l running near the v o i d volume was a l s o observed. A s i m i l a r s e p a r a t i o n of peaks 1 and 89 1 2 3 4 5 6 7 Fi g u r e 21 E l e c t r o p h o r e s i s i n 0.1M sodium phosphate pH 7.0, 0.1% SDS. The g e l s were 10 cm long and contained 7.5% acrylamide, and 0.38% bismethylene acrylamide. The samples were d i s s o l v e d i n 8M urea before e l e c t r o p h o r e s i s . Gels were s t a i n e d overnight w i t h 1% Amido b l a c k i n 10% a c e t i c a c i d . Gel 1, i n s u l i n ; g e l 2, a 1 c h a i n ; g e l 3, a 2 c h a i n ; g e l 4, chymotrypsinogen; g e l 5, bovine serum albumin; g e l 6, CNBr Hp 1-1 PC I I I ; g e l 7, g e l 6 sample a f t e r r e d u c t i o n and a l k y l a t i o n . E l e c t r o p h o r e s i s was performed at approximately 50 v o l t s u n t i l a marker of hemoglobin moved 7.5 cm. 90 F i g u r e 22 Separation of Fragment I I I Polypeptides a f t e r Reduction and A l k y l a t i o n Fragment I I I (7.0 mg) was d i s s o l v e d i n 100 y l 0.16M b o r i c a c i d , 0.06M NaOH, 8.0M urea, 0.029M B-mercapto-ethanol pH 8.8. A f t e r 15 to 20 minutes 20 y l of 0.6M 1 I +C-iodoacetamide (.34 mci/mMole) were added. The pH of the s o l u t i o n was maintained w i t h 0.5M NH^OH and a f t e r 30 to 40 minutes 20 y l of 1.4M B-mercaptoethanol was added and again the pH was maintained f o r 5 to 10 min-ute s . The pH was brought down to about pH 2 using 1.0M HCl and a ml of 0.01M HCl was added to the s o l u t i o n . The s o l u t i o n was chromatographed on Sephadex G-75 (2 cm by 65 cm) using 0.01M HCl. F r a c t i o n s of 3 ml were c o l l e c t e d and from these f r a c t i o n s 100 y l a l i q u o t s were taken f o r s c i n t i l l a t i o n counting (see methods). The PC I I I sample had been chromatographed 2 times on phosphocellulose. 91 2 was a l s o obtained by chromatography on Sephadex G-50. The s e p a r a t i o n on G-50 showed a t h i r d peak of o p t i c a l d e n s i t y which appeared i n the v o i d volume. However, g e l e l e c t r o p h o r e s i s i n 0.1% SDS showed that t h i s peak c o n s i s t e d of three minor bands which were i m p u r i t i e s i n the PC I I I p r e p a r a t i o n . Gel e l e c t r o p h o r e s i s of peaks 1 and 2 i n 0.1% SDS confirmed that these peaks corresponded to the p o l y p e p t i d e s which had been observed p r e v i o u s l y upon g e l e l e c t r o p h o r e s i s . The s e p a r a t i o n s were performed i n 10% acrylamide g e l s and i t was found more s u i t a b l e to use 0.25% Coomassie Blue to s t a i n the g e l s i n s t e a d of 1% Amido Black (113). As can be seen i n F i g u r e 23, peak 1 corresponds to the a c h a i n of hapto-g l o b i n and peak 2 corresponds to the fragment of the 6 c h a i n l i n k e d by d i s u l p h i d e s to the a c h a i n . Amino a c i d a n a l y s i s of the 3 c h a i n fragment i s shown i n Table I . When the number of amino a c i d r e s i d u e s i n the peptide were determined on the b a s i s of the value of carboxymethylcysteine being .95 the values f o r the other amino a c i d s were c l o s e to whole number values w i t h the only two exceptions being those values f o r homoserine and v a l i n e . However i n the case of homoserine i t i s known th a t h y d r o l y t i c l o s s e s amount to about 20% (113) and i n the case of v a l i n e there i s incomplete recovery during short times of h y d r o l y s i s . From s t u d i e s on the r e a c t i o n of cyanogen bromide w i t h the i s o l a t e d 3 c h a i n of h a p t o g l o b i n , a peptide has been obtained which has a very s i m i l a r amino a c i d composition to the 3 c h a i n fragment obtained from the PC I I I fragment (Table I) (32). A l s o , t r y p t i c f i n g e r p r i n t s of the two s • Figure 23 Electrophoresis i n 0.1M sodium phosphate pH 7.0, 0.1% SDS. Gels contained 10% acrylamide, 0.5% bismethylene acrylamide and were 10 cm i n length. Samples were dissolv e d i n 0.1M borate NaOH buffer pH 8.8 containing 8M urea. E l e c t r o p h o r e s i s was performed u n t i l a marker of bromophenol blue moved approximately 9/10 of the length of the g e l . Gel 1, 20 yg PC I I I reduced and a l k y l a t e d ; g e l 2, G50 peak 1 10 yg; g e l 3, G50 peak 2, 10 yg; gel 4, G50 peak 2, 20 yg; g e l 5 i n s u l i n , 10 yg. 93 TABLE I AMINO ACID ANALYSIS OF @ CHAIN FRAGMENT ISOLATED FROM FRAGMENT PC I I I AFTER REDUCTION AND ALKYLATION The r e s u l t s presented are an average of two determinations. uM Residues Fragment Isolated from 8 chain by Hew and Dixon Carboxymethylcysteine 0.0232 .95 -A s p a r t i c Acid 0.105 4.3 3.5 Threonine .0253 1.0 .85 Serine .0492 2.0 1.6 Glutamic Acid .0305 1.25 1.0 P r o l i n e .0525 2.1 1.8 Glycine .0985 4.0 3.8 Alanine .0520 2.1 1.8 Va l i n e .0828 3.4 3.8 Methionine .00 Isoleucine .024 .97 ;85 Leucine .051 2.1 2.0 Tyrosine .0726 3.0 2.6 Phenylalanine .0463 1.9 1.8 Homoserine .0183 .75 .8 Lysine - - 2.5 H i s t i d i n e .0292 1.2 .8 Arginine .0480 1.95 1.6 3 c h a i n fragments i n d i c a t e that these fragments have i d e n t i c a l amino a c i d sequences. The only apparent d i f f e r e n c e between the amino a c i d compositions of the fragment obtained from PC I I I and that obtained from the 3 chain i s the low amount of carboxymethylcysteine present i n the fragment obtained from the 3 c h a i n . However, subsequent analyses of peptides obtained from the 3 chain fragment (fragment E) showed t h a t , i n s t e a d of carboxymethylcysteine, c y s t i n e was present (32). In f a c t , the sequence of t h i s fragment has now been completed by Hew, Kauffman and Dixon and i s shown i n F i g u r e 24. The sequence demonstrates th a t the fragment contains 4 v a l i n e s and thus confirms t h a t the h y d r o l y -t i c c o n d i t i o n s used i n determining the amino a c i d a n a l y s i s r e s u l t e d i n incomplete r e l e a s e of v a l i n e . The sequence and amino a c i d a n a l y s i s of fragment E both demon-s t r a t e that there i s only 1 h a l f - c y s t i n e i n t h i s p e p t i d e . Since t h i s fragment i s j o i n e d to the a chain by a d i s u l p h i d e bond, fragment E must be j o i n e d to the a chain by t h i s p a r t i c u l a r h a l f - c y s t i n e . From the r e s u l t s of Kauffman and Dixon i t can be seen that the sequence of the 3 chain c y s t e i c a c i d peptide (Th3B) which i s j o i n e d to the a chain i s i d e n t i c a l to a sequence present i n fragment E. Therefore the f a c t t h a t peptide Th3B contains a sequence i d e n t i c a l to that present i n fragment E demonstrates independently that the h a l f - c y s t i n e present i n Th3B i s attached to the a c h a i n . The presence of a 3 chain polypeptide i n the PC I I I fragment which was i d e n t i c a l i n sequence to the fragment i s o l a t e d by Hew and Dixon (fragment E) was f u r t h e r confirmed by amino-terminal and Pro-Ile-Cys-Pro-Leu-Ser-Lys-Asp-Tyr-Ala-Glu-Val-Gly-Arg-Val Gly-Tyr-Val-Ser-Gly-Try-Gly-Arg-Asp-Ala-Asn-Phe-Lys-Phe-Thr Asp-His-Leu-Lys-Tyr-Val-Hst F i g u r e 24 The sequence of fragment E J 96 carboxy-terminal a n a l y s i s . The r e s u l t s of amino-terminal a n a l y s i s are shown i n F i g u r e 25. In F i g u r e 25a a n a l y s i s of the dansyl-amino a c i d s shows that from PC I I I a f l u o r e s c e n t spot w i t h a m o b i l i t y corresponding to that of e i t h e r d a n s y l - v a l i n e or d a n s y l - p r o l i n e i s obtained. However only a s m a l l s e p a r a t i o n of d a n s y l - v a l i n e and d a n s y l - p r o l i n e i s obtained i n t h i s system. A b e t t e r s e p a r a t i o n of these two dansyl-amino a c i d s was obtained a f t e r t h i n l a y e r chromatography i n a so l v e n t system con-t a i n i n g chloroform 90 mis, isoamyl a l c o h o l 10 mis, and a c e t i c a c i d 0.5 mis (Figure 25b). F i g u r e 25b demonstrates the s e p a r a t i o n of d a n s y l -v a l i n e from d a n s y l - p r o l i n e i n t h i s system and shows that fragment PC I I I contai n s both p r o l i n e and v a l i n e as amino-terminal amino a c i d s . Since the amino-terminal of the a ha p t o g l o b i n chain i s known to be v a l i n e and the amino-terminal of fragment E i s known to be p r o l i n e the r e s u l t confirms the assignment of the 3 chain and fragment E as c o n s t i t u e n t s of fragment PC I I I . The r e s u l t s of carboxypeptidase A d i g e s t i o n of p e r f o r m i c - o x i d i z e d PC I I I are shown i n Table I I . These r e s u l t s showed that equimolar amounts of glutamine, homoserine, v a l i n e , t y r o s i n e , and l e u c i n e are r e l e a s e d from fragment PC I I I . L y s i n e was not determined i n t h i s a n a l y s i s because of the l a r g e amount of ammonia which s t i l l remained a f t e r the ammonia e x t r a c t i o n of the r e l e a s e d amino a c i d s from Dowex 50. Previous s t u d i e s on the carboxy-terminal of the a chain of ha p t o g l o b i n have shown that only glutamine was r e l e a s e d by carboxypeptidase A. Thus the remaining amino a c i d s must have been r e l e a s e d from the fragment attached to the a c h a i n . The other four amino a c i d s are found at four 25a 25b 97 1 2 3 4 5 1 2 3 4 5 F i g u r e 25 A n a l y s i s by t h i n l a y e r chromatography of the d a n s y l -amino acid s obtained from the amino-terminal r e s i d u e s of fragment P C I I I . 1.0 mg of fragment was r e a c t e d , hydrolysed, d r i e d and then r e d i s s o l v e d i n 25 u l of 2N ammonia. 1/5 to 1/8 of t h i s sample was used per spot f o r a n a l y s i s . 25a A n a l y s i s u s i n g the system of Black and Dixon (89). Samples 1 and 5, d a n s y l - v a l i n e ; samples 2 and 4, d a n s y l - p r o l i n e ; sample 3, fragment PC I I I . 25b A n a l y s i s using chloroform 90 ml, isoamyl a l c o h o l 10 ml, and a c e t i c a c i d 0.5 ml as s o l v e n t . Samples are the same as i n 25a. TABLE I I AMINO ACID ANALYSIS OF THE AMINO ACIDS RELEASED BY CARBOXYPEPTIDASE A DIGESTION OF FRAGMENT PC I I I AFTER THE FRAGMENT HAD BEEN OXIDIZED USING PERFORMIC ACID uM Residues Homoserine 0.021 1.0 Glutamine 0.023 1.1 V a l i n e 0.018 0.88 Leucine 0.017 0.84 Tyrosine 0.017 0.84 99 of the f i v e carboxy-terminal p o s i t i o n s of fragment E, the other amino a c i d being l y s i n e which was not determined. Thus the data are c o n s i s -t e n t w i t h carboxypeptidase removing the f i r s t f i v e amino a c i d s from the carboxy-terminal of fragment E and not removing the h i s t i d i n e at the s i x t h p o s i t i o n . This h i s t i d i n e does not appear to be removed because i t i s next to an a s p a r t i c a c i d (a re s i d u e which carboxypeptidase A removes only very slowly) and i t i s known t h a t the penultimate r e s i d u e a f f e c t s the removal of the carboxy-terminal r e s i d u e (114). A l s o , a l -though l y s i n e i s not normally considered to be r e l e a s e d by carboxy-peptidase A, i t i s one of the residues which i s r e l e a s e d s l o w l y by t h i s enzyme (114). Diagonal Analyses on Fragment PC I I I The d i s u l p h i d e s of fragment PC I I I were i n v e s t i g a t e d f o l l o w i n g the diagonal technique described by Brown and H a r t l e y (115). Using t h i s technique the pol y p e p t i d e under i n v e s t i g a t i o n i s d i g e s t e d w i t h an enzyme and the r e s u l t i n g peptides are separated by hig h v o l t a g e e l e c t r o p h o r e s i s . Then a s t r i p of paper c o n t a i n i n g the separated peptides i s p e r f o r m i c - o x i d i z e d , sewn onto another sheet of hig h v o l t a g e paper, and then, as described by Brown and H a r t l e y (115), e l e c t r o -p h o r e s i s i s again performed at the same pH as the f i r s t e l e c t r o p h o r e s i s but at r i g h t angles to the d i r e c t i o n of the f i r s t e l e c t r o p h o r e s i s . Using t h i s technique the peptides which do not c o n t a i n d i s u l p h i d e s form a di a g o n a l on the paper and d i s u l p h i d e peptides u s u a l l y migrate o f f the diagonal and so can be i d e n t i f i e d . In a d d i t i o n Brown and 100 H a r t l e y (115) have shown that t h i s method can be used f o r i s o l a t i n g and i d e n t i f y i n g d i s u l p h i d e peptides. In a n a l y s i n g d i s u l p h i d e - c o n t a i n i n g p r o t e i n s or polypeptides the enzyme or enzymes used f o r d i g e s t i o n i s somewhat c r i t i c a l because of two phenomena. On one hand many p r o t e i n s cannot be d i g e s t e d by some enzymes when t h e i r d i s u l p h i d e s are i n t a c t (110) and on the other hand when d i g e s t i o n i s allowed to proceed at s l i g h t l y a l k a l i n e pH d i s u l p h i d e interchange can occur (109). As a r e s u l t of these problems most pro-t e i n s are i n i t i a l l y d i g e s t e d w i t h pepsin both because i t a t t a c k s n a t i v e p r o t e i n s and because i t a t t a c k s at low pH where d i s u l p h i d e interchange i s not favoured. Then, i f necessary, a f u r t h e r d i g e s t i o n w i t h another enzyme has o f t e n been used (110,115). In the present study on the d i s u l p h i d e s of fragment PC I I I d i a g o n a l analyses have been performed a f t e r pepsin d i g e s t s , p e p s i n -t r y p s i n d i g e s t s , p e p s i n - t r y p s i n - c h y m o t r y p s i n d i g e s t s , p e p s i n - s u b t i l i s i n d i g e s t s , p e p s i n - t h e r m o l y s i n d i g e s t s , p a r t i a l a c i d h y d r o l y s e s , and p e p s i n - p a r t i a l a c i d d i g e s t s . The d i g e s t which appeared to be most s u i t a b l e f o r f u r t h e r study was the p e p s i n - t r y p s i n d i g e s t . The r e s u l t of a p e p s i n - t r y p s i n d i a g o n a l a n a l y s i s of PC I I I from h a p t o g l o b i n 1-1 i s shown i n F i g u r e 26. The peptide AA had i d e n t i c a l m o b i l i t i e s i n the f i r s t dimension to the 21 a d i s u l p h i d e peptide ob-t a i n e d by Kauffman and Dixon from a pepsin d i g e s t of h a p t o g l o b i n 1-1 and i n the second dimension to the 21 a c y s t e i c a c i d peptide obtained from h a p t o g l o b i n 1-1. A l s o , i t s i d e n t i t y w i t h the 21 a peptide was confirmed by the f a c t that i t gave a p o s i t i v e h i s t i d i n e r e a c t i o n w i t h 101 Figure 26 pH 6.5 di a g o n a l map of 1.5 mg of a p e p t i c - t r y p t i c d i g e s t of fragment PC I I I . BA1 i s the i s a t i n p o s i t i v e spot. E l e c t r o p h o r e s i s was performed i n the f i r s t dimension u n t i l a marker of c r y s t a l v i o l e t moved 13 cm. 102 the Pauly s t a i n and that i t reacted s l o w l y w i t h n i n h y d r i n . Peptide BA1 was found to s t a i n blue w i t h i s a t i n reagent thus demonstrating that i t contained p r o l i n e as amino-terminal amino a c i d . I t was suspected that peptide BA1 came from the amino-terminal of fragment E which does have p r o l i n e at i t s amino-terminal. A l s o i t was reasoned that t h i s p r o l i n e -amino- t e r m i n a l peptide would c o n t a i n h a l f - c y s t i n e and thus would run o f f the d i a g o n a l s i n c e i t i s only two r e s i d u e s removed from the only h a l f - c y s t i n e r e s i d u e which i s present i n fragment E. Peptides BA1, BA2, and BA3 have been i s o l a t e d from p e p t i c -t r y p t i c d i g e s t s of fragment PC I I I i n s u f f i c i e n t amount and p u r i t y f o r t h e i r p o s i t i o n i n the h a p t o g l o b i n sequence to be e s t a b l i s h e d . The amino a c i d analyses and amino-terminal amino a c i d s of these peptides are shown i n Table I I I . Peptide BA1 as expected corresponded to the amino-terminal peptide from fragment E. This p e p t i d e would have a net charge of -1 at pH 6.5 and s i n c e i t contains one c y s t e i c a c i d , i t s charge must have been zero before o x i d a t i o n . Peptides BA2 and BA3 are d e r i v e d from a p a r t of the a chain corresponding to residues 61 to 77 and 64 to 77 r e s p e c t i v e l y . These two peptides have a net charge of -2 at pH 6.5 and s i n c e they c o n t a i n two c y s t e i c a c i d s t h e i r charge must a l s o have been zero before o x i d a t i o n . The d i a g o n a l map shown i n Figure 26 i n d i c a t e s that peptides BA2 and BA3 are both attached by a d i s u l p h i d e to BA1 before performic o x i -d a t i o n because a l l three peptides had the same e l e c t r o p h o r e t i c m o b i l i t y before o x i d a t i o n . To f u r t h e r c o n f i r m t h i s p o i n t d i a g o n a l a n a l y s i s of p e p t i c - t r y p t i c peptides from PC I I I was performed at pH 4.0. As shown 103 TABLE I I I AMINO ACID ANALYSES AND AMINO-TERMINAL ANALYSES OF PEPTIDES BA1, BA2, AND BA3 The peptides were obtained from 25 mg of a p e p t i c - t r y p t i c d i g e s t of fragment PC I I I as des c r i b e d i n methods. The peptides were e l u t e d from pre-washed Whatmann 3 MM paper w i t h 30% a c e t i c a c i d . 22% of each peptide was d r i e d and hydrolyzed f o r amino a c i d a n a l y s i s . Then 2/3 of the h y d r o l y z a t e was used f o r a n a l y s i s . BA1 BA2 BA3 yM yM* c o r r . Residues yM Residues yM Residues L y s i n e .0703 .052 0.9 .113 3.0 .063 3.0 C y s t e i c .0534 .046 0.8 .0631 1.7 .0349 1.6 A s p a r t i c .0800 .073 1.25 .0466 1.25 .0268 1.25 Serine .0639 .063 1.1 0 0 0 Glutamic .0181 .004 0 .0845 2.25 .0502 2.3 P r o l i n e .132 .118 2.0 .0840 2.25 .0470 2.15 G l y c i n e .0147 0 0 .0808 2.2 .0274 1.25 A l a n i n e .0129 0 0 .0748 2.0 .0253 1.2 V a l i n e .0115 0 0 .0726 2.0 .0228 1.05 I s o l e u c i n e .0613 .061 1.0 0 0 0 Leucine .0710 .064 1.0 .0416 1.1 .0239 1.1 Amino Terminal P r o l i n e A l a n i n e A s p a r t i c A c i d *The micromolar values of peptide BA1 are c o r r e c t e d f o r a 10% contamin-a t i o n by peptide BA2. 104 i n F i g ure 27 the i s a t i n - p o s i t i v e peptide was seen to be mated to three other peptides. The question remained as to whether two of these three peptides were i d e n t i c a l w i t h peptides BA2 and BA3. To answer t h i s q u e s t i o n a p e r f o r m i c - o x i d i z e d pH 4.0 electrophorogram of a p e p t i c -t r y p t i c h y d r o l y s a t e of fragment PC I I I was run i n the second dimension at pH 6.5. Peptides BA1, BA2, and BA3 could be i d e n t i f i e d and so con-firmed that the 8 chain must be attached e i t h e r to a h a l f - c y s t i n e at p o s i t i o n 69 or 73 i n the a c h a i n of h a p t o g l o b i n . Since peptides BA1, BA2, and BA3 are each present as doublets i n the pH 6.5 diagonals they must a r i s e from two d i s u l p h i d e peptides which are separated by e l e c t r o p h o r e s i s i n the f i r s t dimension. Thus there must be two otB d i s u l p h i d e peptides present i n p e p t i c - t r y p t i c d i g e s t s of PC I I I . The d i a g o n a l a n a l y s i s of PC I I I shows that peptide BN1 i s a l s o mated to BA1, BA2, and BA3. I f peptide BN1 contains 1 c y s t e i c a c i d i t would have a net charge of +1 before o x i d a t i o n and s i n c e BA1, BA2, and BA3 a l l have charges of zero before o x i d a t i o n , t h e r e f o r e the net charge on the d i s u l p h i d e peptide would be +1. This i s c o n s i s t e n t w i t h the s l i g h t l y b a s i c m o b i l i t y of the d i s u l p h i d e peptide c o n t a i n i n g BN1, BA1 and BA2 or BA3. Kauffman and Dixon have shown that the 6 chain of h a p t o g l o b i n i s j o i n e d to a h a l f - c y s t i n y l group at p o s i t i o n 73 i n the a chain of hapto-g l o b i n and that the h a l f - c y s t i n y l group at p o s i t i o n 69 i s j o i n e d to p o s i t i o n 35. The r e s u l t s of the p e p t i c - t r y p t i c diagonals of fragment PC I I I are c o n s i s t e n t w i t h t h i s s t r u c t u r e . Proposed s t r u c t u r e s f o r the two aB d i s u l p h i d e peptides i s o l a t e d from the p e p t i c - t r y p t i c d i g e s t s of 105 Figure 27 pH 4.0 diagonal map of 2.0 mg of a p e p t i c - t r y p t i c digest of fragment PCIII. The hatched spot i s i s a t i n p o s i t i v e . Electrophoresis was performed i n the f i r s t dimension u n t i l a marker of c r y s t a l v i o l e t moved 13 cm. fragment PC I I I are shown i n Figure 28. These two d i s u l p h i d e peptides d i f f e r only i n the s t r u c t u r e of one of t h e i r component h a l f - c y s t i n e peptides (from the a35 r e g i o n ) , the other two peptides (from the 6 chain and from the a69 region) being i d e n t i c a l . As the h a l f - c y s t i n e peptides from the a35 r e g i o n are presumed to have d i f f e r e n t charges the presence of two d i s u l p h i d e peptides before o x i d a t i o n i s exp l a i n e d . Further Studies on H a l f Haptoglobin The r e s u l t s of Kauffman and Dixon (35) suggested that the two halves of the ha p t o g l o b i n molecule are l i n k e d together by a symmetrical d i s u l p h i d e bond l o c a t e d at the h a l f - c y s t i n y l r e s i d u e 21 of the hapto-g l o b i n a ch a i n . Thus one would p r e d i c t that when haptoglobin i s s p l i t i n t o symmetrical halves by sodium s u l p h i t e and pCMS that the a21 d i s u l p h i d e would be s p l i t and thus the c*21 h a l f - c y s t i n y l group would be present i n the h a l f - h a p t o g l o b i n as a mixture of S - s u l f o c y s t e i n e and the p-mercurisulphonate mercaptide d e r i v a t i v e . This p r e d i c t i o n has been examined by a n a l y s i n g the p e p t i c peptides produced when hapt o g l o b i n 35 which had been s p l i t by S-sodium s u l p h i t e and pCMS, was digested w i t h pepsin. F i g u r e 29 shows the r e s u l t s of an autoradiogram of a p e p t i c 35 d i g e s t of S - l a b e l l e d h a l f - h a p t o g l o b i n 1-1 a f t e r h i g h v o l t a g e e l e c t r o -p h o resis at pH 6.5. One major a c i d i c r a d i o a c t i v e band i s obtained from the p e p t i c d i g e s t . Previous r e s u l t s (35) had shown that (among the d i s u l p h i d e peptides) under the c o n d i t i o n s of p e p t i c d i g e s t i o n only the a-a' d i s u l p h i d e peptide was obtained i n good y i e l d . Thus t h i s r e s u l t 107 charge=0 (Ala-Val-Gly)-Asp-Lys-Leu-Pro-Glu-C /ys-Glu-Ala-Val-Cys-Gly-Lys-Pro-Lys / S Pro-Ile-Cys-Pro-Leu-Ser-Lys-Asp charge=0 a35 peptide charge=+l charge=0 (Ala-Val-Gly)-Asp-Lys-Leu-Pro-Glu-Cys-Glu-Ala-Val-C.ys-Gly-Lys-Pro-Lys Pro-Ile-Cys-Pro-Leu-Ser-Lys-Asp charge=0 a35 peptide charge=+2 Fi g u r e 28 S t r u c t u r e of a6 d i s u l p h i d e peptides obtained from p e p t i c t r y p t i c d i g e s t s of fragment P C I I I . 108 + -Figure 29 Autoradiogram of the a c i d i c peptides a f t e r high v o l t a g e e l e c t r o p h o r e s i s at pH 6.5 of a p e p t i c d i g e s t of 3 5 s-half-haptoglobin. suggested that the S-peptide was the a c h a i n p e p t i d e . 35 To f u r t h e r c h a r a c t e r i z e t h i s S - l a b e l l e d peptide 40 mgs. of the p e p t i c d i g e s t of l a b e l l e d h a l f - h a p t o g l o b i n was a p p l i e d to 20 cm of Whatmann 3 MM paper and electrophoresed at pH 6.5. The p o s i t i o n of the r a d i o a c t i v e peptide was determined by autoradiography and then the area corresponding to the r a d i o a c t i v i t y was cut out and sewed onto another p i e c e of 3 MM paper f o r pH 3.6 high v o l t a g e e l e c t r o p h o r e s i s . In order to o b t a i n pure p e p t i d e , e l e c t r o p h o r e s i s was performed f i r s t at pH 6.5 then at pH 3.6 and f i n a l l y at pH 1.9. This peptide at pH 6.5 had e x a c t l y the same m o b i l i t y as the dye XCFF and thus XCFF was a u s e f u l marker f o r l o c a t i n g the peptide at t h i s pH. At pH 3.6 the peptide ran f a s t e r than e - D N P l y s i n e but slower than c r y s t a l v i o l e t and at pH 1.9 i t had about 2/3 the m o b i l i t y of c r y s t a l v i o l e t . In order to p u r i f y the peptide i t was electrophoresed u n t i l i t had run 27 cm at pH 6.5. Some minor slower running r a d i o a c t i v e bands were observed but were very f a i n t . At pH 3.6 two peptides were observed. The minor peptide which c o n s i s t e d of about 1/4 to 1/3 of the amount of the major peptide was not s t u d i e d f u r t h e r . The major peptide was run u n t i l i t migrated 16 cm r e l a t i v e to a m i g r a t i o n of 10 cm f o r E - D N P l y s i n e . At pH 1.9 a s i n g l e r a d i o a c t i v e band was observed which ran 20 cm r e l a t i v e to a m o b i l i t y of 29 cm f o r c r y s t a l v i o l e t . Ten cm of the s t r i p was then cut out and the r a d i o a c t i v e peptide e l u t e d chromatographically from the paper w i t h 0.5 ml d i s t i l l e d water. A f t e r d r y i n g , the peptide was hydrolysed i n vacuo f o r 15 hours at 110° C and 3/4 of the sample was used f o r amino a c i d a n a l y s i s . 110 Table IV shows a comparison between the amino a c i d a n a l y s i s of 35 the S-peptide and the amino a c i d composition of the c y s t e i c a c i d pep-t i d e d e r i v e d from the p e p t i c 21ot-21a' d i s u l p h i d e peptide. The f a c t that these peptides had the same amino a c i d composition showed that the 35 S-peptide and the 2 1 a - c y s t e i c a c i d peptide represented the same sequence. 35 As mentioned p r e v i o u s l y , when S - l a b e l l e d h a l f - h a p t o g l o b i n was f u r t h e r cleaved using u n l a b e l l e d sodium s u l p h i t e i n the presence of 8M urea and the h a p t o g l o b i n chains were subsequently separated, the m a j o r i t y of the r a d i o a c t i v i t y was found i n the 3 c h a i n . This r e s u l t i s not c o n t r a d i c t o r y to the r e s u l t s obtained here s i n c e i t has been shown that under the c o n d i t i o n s of f u r t h e r cleavage w i t h u n l a b e l l e d 35 s u l p h i t e more than 80 per cent of the S - s u l p h i t e was l o s t from the 35 h a l f - h a p t o g l o b i n molecule (96). Thus i t can be argued that the S l a b e l on the a c h a i n was very l a b i l e and a l l of the s u l p h i t e was l o s t from i t under the above c o n d i t i o n s . The reason why counts were found i n the heavy chain r e g i o n i s not known at present but could have been 35 due to exchange of the S - s u l p h i t e , cleaved from the 21a p o s i t i o n , w i t h the f i v e h a l f - c y s t i n y l residues of the 3 c h a i n . The f a c t that the 21a-21a' d i s u l p h i d e was s p l i t i n the formation of h a l f - h a p t o g l o b i n has a l s o been demonstrated by d i a g o n a l analyses. F i g u r e 30a shows a P a u l y - s t a i n e d , p e r f o r m i c - a c i d - d i a g o n a l a n a l y s i s of h a p t o g l o b i n which has been digested w i t h pepsin. Only one Pauly-p o s i t i v e spot was off. the d i a g o n a l . The m o b i l i t y of t h i s spot i n the I l l TABLE IV COMPARISON OF THE AMINO ACID COMPOSITION OF THE a-a' DISULPHIDE PEPTIDE OBTAINED AFTER PEPSIN DIGESTION OF Hp 1-1 WITH THE 35s-PEPTIDE OBTAINED AFTER PEPSIN DIGESTION OF 35S-Hp/2 y M 3 5 S - P e p t i d e from Half-Hap to glob i n Amino Acid Composition of Cy s t e i c Acid Peptide from 21 -21 1 Disulphide Peptide.* (Kauffman and Dixon). A s p a r t i c Acid 0.023 2.2 2 Threonine 0.0 0.0 0 Serine 0.001 0.1 0 Glutamic Acid 0.013 1.2 1 P r o l i n e 0.031 3.0 3 Glycine 0.023 2.1 2 Alanine 0.021 2.0 2 V a l i n e 0.0 0.0 0 Methionine 0.0 0.0 0 Isoleucine 0.020 1.9 2 Leucine 0.0 0.0 0 Tyrosine 0.0 0.0 0 Phenylalanine 0.0 0.0 0 Lysine 0.0093 0.9 1 H i s t i d i n e 0.0094 0.9 1 Arginine 0.0 0.0 0 * The amino acid composition was deduced from the sequence of the peptide. 112 Fi g u r e 30 Diagonal map of p e p t i c d i g e s t s of h a p t o g l o b i n and h a l f - h a p t o g l o b i n . Haptoglobin or h a l f -h a p t o g l o b i n (4 mg) were d i s s o l v e d i n 0.4 mis of 5% formic a c i d . To t h i s s o l u t i o n 40 y l of a 1% pepsin s o l u t i o n were added and d i g e s t i o n proceeded f o r 17 hours at 37° C. Then the samples were d r i e d , r e d i s s o l v e d i n a s m a l l volume of pH 6.5 p y r i d i n e - a c e t a t e b u f f e r , a p p l i e d to 2.5 cm of Whatmann 3 mM paper, and diag o n a l a n a l y s i s was performed as des c r i b e d by H a r t l e y (115). A f t e r d i a g o n a l a n a l y s i s the paper was sprayed w i t h Pauly reagent. HAPTOGLOBIN 113 HALF-HAPTOGLOBIN b f i r s t dimension was about 1/2 that of the marker XCFF and was equal to the marker i n the second dimension. In the second dimension t h i s spot corresponded i n m o b i l i t y to the 21a c y s t e i c - a c i d - c o n t a i n i n g peptide i s o l a t e d by Kauffman and Dixon. I t a l s o had the same m o b i l i t y as the 35 S - s u l p h i t e - l a b e l l e d peptide obtained from s u l p h i t e - l a b e l l e d h a l f -h a p t o g l o b i n . A f t e r p e r f o r m i c - a c i d o x i d a t i o n of the 21a-21a' d i s u l -phide peptide the c y s t i n y l moiety which was present i n the peptide would be converted to c y s t e i c a c i d and, a f t e r s p l i t t i n g w i t h s u l p h i t e , the moiety would be converted to S-sulphocysteine. In both of these cases t h i s sulphur c o n t a i n i n g amino a c i d would be n e g a t i v e l y charged and so the peptide a f t e r o x i d a t i o n or a f t e r s p l i t t i n g w i t h s u l p h i t e would have the same change as w e l l as e s s e n t i a l l y the same s i z e . In the P a u l y - s t a i n e d d i a g o n a l of h a l f - h a p t o g l o b i n (Figure 30) the Pauly-p o s i t i v e band which was o f f the dia g o n a l i n the case of Hp 1-1 i s not seen and i n s t e a d a new Pauly p o s i t i v e spot i s seen on the d i a g o n a l which has the same m o b i l i t y as XCFF i n both the f i r s t and second dim-ensions. Again the r e s u l t i s c o n s i s t e n t w i t h the p r e d i c t i o n that the S-sulpho-peptide and the c y s t e i c peptide have the same m o b i l i t i e s . A schematic r e p r e s e n t a t i o n of the comparative diagonals i s shown i n Fi g u r e 31. The s p l i t t i n g of the 21a d i s u l p h i d e i n hapt o g l o b i n w i t h the r e s u l t i n g formation of h a l f - h a p t o g l o b i n can be seen i n two d i f f e r e n t 35 ways. In one case u s i n g S - l a b e l l e d s u l p h i t e , the peptide correspon-ding to the sequence around the 21a p o s i t i o n has been i s o l a t e d , and i n the other case u s i n g comparative diagonals i t i s seen that the 21a 115 HAPTOGLOBIN s aB f so: pepsin pepsin y^0.5 XCFF 21a p e p t i c peptide i f + s 21a p e p t i c peptide other peptides performic o x i d a t i o n 21a p e p t i c peptide 3 y=XCFF HALF-HAPTOGLOBIN other peptides y - XCFF 21a p e p t i c peptide so-performic o x i d a t i o n other peptides 21a p e p t i c peptide ^ other ^3 peptides y = XCFF Fig u r e 31 Scheme to e x p l a i n the comparitive diagonals of haptoglobin and h a l f - h a p t o g l o b i n . d i s u l p h i d e peptide i s m i s s i n g i n h a l f - h a p t o g l o b i n but i s r e p l a c e d by another peptide which has the p r o p e r t i e s of a 21a-peptide i n the S-s u l p h o c y s t e i n y l form. In a d d i t i o n i t was a l s o shown p r e v i o u s l y that a comparison of the cyanogen bromide fragments of h a p t o g l o b i n and h a l f -h a p t o g l o b i n showed that the a c h a i n c o n t a i n i n g fragment (PC I I I ) was a l t e r e d i n the h a l f - h a p t o g l o b i n . Since PC I I I contains a p a r t of the 3 c h a i n l i n k e d by a d i s u l p h i d e to the a c h a i n , and t h i s p a r t of the 3 c h a i n (fragment E) contains only 1 h a l f - c y s t i n e i t i s apparent t h a t only a a-a' d i s u l p h i d e could have been broken i n the conversion of h a p t o g l o b i n to h a l f - h a p t o g l o b i n . D i s c u s s i o n and Conclusions The h a p t o g l o b i n 1-1 molecule con t a i n s nine d i s u l p h i d e bonds as shown i n F i g u r e 32. S i x of these d i s u l p h i d e s are i n t r a c h a i n d i s u l p h i d e s and the other three are i n t e r c h a i n . Four of the i n t r a c h a i n d i s u l p h i d e s are i n the 3 chains and two are i n the a chains connecting the h a l f -c y s t i n y l 35 r e s i d u e s w i t h the h a l f - c y s t i n y l r e s i d u e s at p o s i t i o n 69. Of the three i n t e r c h a i n d i s u l p h i d e s one i s a symmetrical i n t e r c h a i n d i s u l p h i d e between the h a l f - c y s t i n y l r e s i d u e s at p o s i t i o n 21 i n the a c h a i n and the others are the a3 l i n k a g e d i s u l p h i d e s which j o i n the 3 chains to the c*73 h a l f - c y s t i n y l r e s i d u e s . The e x i s t e n c e of only one unique ct3 d i s u l p h i d e i n h a p t o g l o b i n has been confirmed by s t u d i e s on the PC I I I fragment. From t h i s PC I I I fragment another fragment (E) has been i s o l a t e d which i s a p a r t of the 3 c h a i n of h a p t o g l o b i n . This fragment E which i s the only 3 c h a i n Q chains N N 2135 69 . L S _ S J 7 3 21 35 69 c c s I s r s ~ s n B chain F i g u r e 32 St r u c t u r e of haptoglobin 1-1 showing the d i s u l p h i d e bonds. The p o s i t i o n ' of a l l f i v e h a l f - c y s t i n e s i n the B chain i s unknown. 118 fragment l i n k e d to the a chain contains only one h a l f - c y s t i n y l group and so there can only be one d i s u l p h i d e between the a and 6 chains i n h a p t o g l o b i n . The r e s u l t s of p e p t i c - t r y p t i c d i g e s t s are c o n s i s t e n t w i t h a s t r u c t u r e i n which there are two h a l f - c y s t i n y l groups at p o s i -t i o n s 69 and 73 and i n which one of these h a l f - c y s t i n y l groups i s attached to the B chain. I t i s i n t e r e s t i n g to observe that as a r e s u l t of the p o s t u l a t e d p a r t i a l gene d u p l i c a t i o n which gives r i s e to the 2a chain of hapto-g l o b i n , the r e g i o n of the DNA corresponding to a sequence c o n t a i n i n g the h a l f - c y s t i n e at p o s i t i o n 73a has been d e l e t e d . Although the h a l f -c y s t i n e which would be at 73a i f the a chain were completely d u p l i -cated has been de l e t e d i n the a 2 chain of hap t o g l o b i n there i s s t i l l a l i n k a g e of the a chain to the 8 c h a i n by the h a l f - c y s t i n e at p o s i -t i o n 132 a 2 . However, because one of the l i n k a g e h a l f - c y s t i n e s which would be present i f the a chain were completely d u p l i c a t e d i s missing i n a 2 , i n the case of both the a 1 and a 2 h a p t o g l o b i n c h a i n s , each a chain can be j o i n e d to only 1 B chain. The gammaglobulins are t e t r a c h a i n molecules w i t h two p a i r s of i d e n t i c a l chains and so s t r u c t u r a l l y resemble the haptoglobins. The d i s u l p h i d e s of the YG subclass y G l have been s t u d i e d i n d e t a i l (107, 116,117). I t has been shown that y G l myeloma p r o t e i n s have two i n t r a -c h ain d i s u l p h i d e s i n each of the l i g h t chains and four i n each of the heavy chains. There i s only one unique l i g h t - h e a v y d i s u l p h i d e and there are two symmetrical d i s u l p h i d e s l i n k i n g the heavy chains. The d i s u l p h i d e s of the gammaglobulins d i f f e r markedly between the subclasses (117) and i t has been shown that i n subclass yG3 there are f i v e symmetrical i n t e r c h a i n d i s u l p h i d e s between the heavy chains. A l s o i t has been shown i n one antibody subclass yA2 that there i s no d i s u l p h i d e l i n k i n g the l i g h t and heavy chains (118). In comparing the d i s u l p h i d e s of yGl and hapt o g l o b i n 1-1 many d i f f e r e n c e s are apparent. In both the l i g h t and heavy chains (a and 3 chains r e s p e c t i v e l y f o r haptoglobins) there are twice as many i n t r a -c h a i n d i s u l p h i d e s i n y G l as i n Hp 1-1. In a d d i t i o n there are twice as many symmetrical i n t e r c h a i n d i s u l p h i d e s i n YGl as there are i n Hp 1-1 and the symmetrical i n t e r c h a i n d i s u l p h i d e s a t t a c h the heavy chains together i n yGl w h i l e they a t t a c h the a ( l i g h t ) chains together i n Hp 1-1. In both molecules there i s one unique i n t e r c h a i n d i s u l p h i d e which i s l o c a t e d towards the carboxy-terminal end of each of the l i g h t chains. Recently a very i n t e r e s t i n g study on the r e f o l d i n g of hapto-g l o b i n has been performed by B e r n i n i and B o r r i - V o l t a t t o r n i (119). These workers have been able to completely reduce ha p t o g l o b i n w i t h mercaptoethanol i n the presence of 8M urea and then by c a r e f u l l y r e o x i d i z i n g a mixture of a and 8 chains i n the absence of urea they have been to reform h a p t o g l o b i n . In the case of ha p t o g l o b i n 1-1 they have been able to o b t a i n a recovery of 85 to 90 per cent. In the case of h a p t o g l o b i n 2-2 they have been able to reform the ha p t o g l o b i n p o l y -mers w i t h a y i e l d of 60 to 70 per cent. A l s o these workers have been able to separate the a and 8 chains of h a p t o g l o b i n , remix them, and o x i d i z e the remixed chains to form h a p t o g l o b i n . In t h i s way they have been able to make Hp 2-1 from the chains d e r i v e d from Hp 1-1 and Hp 120 2-2. Studies on the r e o x i d a t i o n of the i n d i v i d u a l chains showed that a chains formed dimers w h i l e a formed polymers and 3 chains d i d not polymerize. The r e s u l t s of the I t a l i a n workers are completely c o n s i s t e n t w i t h those presented here. In the f i r s t i n s t a n c e the only symmetrical i n t e r c h a i n d i s u l p h i d e s that we have found i s between a chains. This e x p l a i n s why only a chains form dimers. S i m i l a r l y the cleavage of t h i s a-a' d i s u l p h i d e breaks down the h a p t o g l o b i n polymers and i t can be seen th a t the a-a' d i s u l p h i d e alone can be r e s p o n s i b l e f o r h a p t o g l o b i n p o l y m e r i z a t i o n . Thus the a 2 chain by i t s e l f should be able to p o l y -merize and t h i s has been found by B e r n i n i and B o r r i - V o l t a t t o r n i . F i n a l l y s i n c e there are no 3.-3' d i s u l p h i d e s the 3 chain should remain monomeric a f t e r o x i d a t i o n as found by B e r n i n i and B o r r i - V o l t a t t o r n i . V THE INFLUENCE OF HAPTOGLOBIN ON THE REACTIVITY ON THE -SH GROUPS OF HEMOGLOBIN This s e c t i o n of the t h e s i s examines the r e a c t i v i t y of the 893 c y s t e i n y l r e s i d u e i n f r e e hemoglobin and i n the hemoglobin-haptoglobin i complex toward three s u l p h y d r y l reagents, iodoacetamide, 2,2 i d i t h i o d i p y r i d i n e (2-PDS) and 4,4 d i t h i o d i p y r i d i n e (4-PDS) (46,94). Iodoacetamide i s one of a group of compounds c o n t a i n i n g a c t i v e halogen atoms which are used as reagents f o r s u l p h y d r y l groups. G e n e r a l l y the r e a c t i o n takes place w i t h the mercaptide i o n . The reagents w i l l a l s o r e a c t w i t h amino groups but the r a t e of r e a c t i o n w i t h s u l p h y d r y l groups i s much f a s t e r . P y r i d i n e d i s u l p h i d e s r e a c t w i t h s u l p h y d r y l groups by the d i s u l p h i d e interchange r e a c t i o n . The thi o p y r i d o n e products of the r e a c t i o n are almost e x c l u s i v e l y i n the tautomeric t h i o form and as a r e s u l t the u l t r a v i o l e t a b s o r p t i o n s p e c t r a of the th i o p y r i d o n e s i s q u i t e d i f f e r e n t from the corresponding d i s u l p h i d e s . P y r i d i n e D i s u l p h i d e and Iodoacetamide Reactions The d i t h i o d i p y r i d i n e s are e s p e c i a l l y s u i t e d f o r r e a c t i o n w i t h hemoglobin s i n c e the products of the r e a c t i o n absorb at 324 or 343 nm (94) where hemoglobin has l i t t l e absorbance. In c o n t r a s t , other reagents such as para-hydroxymercuribenzoate (PMB) and Ellman's reagent (121) absorb i n regions where hemoglobin a l s o absorbs s t r o n g l y . 122 Fig u r e 33 shows a comparison between the r e a c t i o n of 4-PDS w i t h hemo-g l o b i n and i t s r e a c t i o n w i t h the Hb-Hp complex i n which i t can be seen that a f t e r complex formation w i t h h a p t o g l o b i n there was a greater than 90 per cent i n h i b i t i o n of the r e a c t i o n r a t e of hemoglobin. As shown i n F i g u r e 34, the i n h i b i t i o n , i n the case of both 4-PDS and 2-PDS was p r o p o r t i o n a l to the amount of complex formed w i t h a maximum i n h i -b i t i o n at a 1 to 1 molar r a t i o of hemoglobin to hap t o g l o b i n . Although the r e a c t i o n w i t h the Hp-Hb complex was much slower than w i t h f r e e Hb, -4 at a higher c o n c e n t r a t i o n of reagents, i . e . , 8 x 10 M 4-PDS and 9.2 —6 x 10 M Hb, r e a c t i o n w i t h Hp-Hb complex occurred and was e s s e n t i a l l y complete i n 10 minutes. S i m i l a r l y , the r e a c t i o n of iodoacetamide w i t h the complex was slower than that w i t h hemoglobin (Figure 35). The i n h i b i t i o n of the 14 r e a c t i o n of C-iodoacetamide w i t h Hb by Hp was 70 to 80 per cent whether the Hb was i n the form of methemoglobin or oxyhemoglobin whereas w i t h the d i t h i o d i p y r i d i n e s the i n h i b i t i o n was grea t e r than 90 per cent. An e f f e c t of hapt o g l o b i n on the environment of c y s t e i n y l 893 has a l s o been seen (122) by comparing the e l e c t r o n s p i n resonance (E.S.R.) spectrum of N - ( l - o x y l - 2 , 2 , 5 , 5 , - t e t r a m e t h y l - 3 - p y r o l i d o n y l ) iodoacetamide-labelled hemoglobin w i t h the l a b e l l e d Hb-Hp complex Since the r a t e of r e a c t i o n of s u l p h y d r y l reagents w i t h the Hb-Hp complex was markedly d i f f e r e n t from that of Hb, i t was necessary to determine the s i t e of r e a c t i o n on the Hp-Hb complex to be sure t h a t , i n f a c t , the r e a c t i o n was s t i l l w i t h the 893 s u l p h y d r y l group. Auto-radiography (Figure 36), a f t e r complete a c i d h y d r o l y s i s and high 123 < LU t o O. o o x t o I TIME - min F i g u r e 33 The r e a c t i o n of 4-PDS w i t h hemoglobin, the hemoglobin-haptoglobin (Hb-Hp) complex, and f r e e h a p t o g l o b i n . The o r d i n a t e represents -SH groups reacted per mole of hemoglobin tetramer. Thus complete r e a c t i o n would generate 2 moles of th i o p y r i d o n e per mole of hemo-g l o b i n tetramer. 124 F i g u r e 34 The r e a c t i o n of 4-PDS and 2-PDS w i t h methemoglobin i n the presence of i n c r e a s i n g amounts of ha p t o g l o b i n . The i n i t i a l v e l o c i t y of the r e a c t i o n i s p l o t t e d against the molar r a t i o of ha p t o g l o b i n to hemoglobin. 125 TIME , h F i g u r e 35 The r e a c t i o n of C-iodoacetamide w i t h hemoglobin and hemoglobin•haptoglobin mixtures. For the r e a c t i o n of •^C-iodoacetamide w i t h f r e e h a p t o g l o b i n , the r a t e i s p l o t t e d per 0.19 absorbance at 280 nm s i n c e the absor-bance of hemoglobin at 407 nm i s 5.2 times that of h a p t o g l o b i n at 280 nm. 126 Figure 36 High voltage electrophoresis at pH 6.5 a f t e r 16 hour acid h y d r o l y s i s of arboxymethyl-Hb and l^C-carboxymethyl-Hb-Hp. 36B i s a ninhydrin s t a i n and 36A i s an autoradio-graph of the high voltage paper. Numbers 1, 4, and 7 show markers of S-carboxymethylcysteine and 3, 6, and 9 are 3-carboxymethylhistidine. Numbers 2 and 8 show acid hydrolysates of carboxymethyl-Hb and number 5 i s the hydrolysate of carboxymethyl-Hb-Hp. v o l t a g e e l e c t r o p h o r e s i s at pH 6.5 showed that i n the Hb-Hp complex the major r a d i o a c t i v e spot was the same as that w i t h hemoglobin and had the same m o b i l i t y as a u t h e n t i c S-carboxymethylcysteine. Two minor spots which ran more slo w l y toward the anode than 3-carboxymethyl-h i s t i d i n e were a l s o seen. Products w i t h s i m i l a r m o b i l i t i e s to these minor spots have a l s o been described i n a previous a n a l y s i s of the r e a c t i o n of hapt o g l o b i n w i t h iodoacetamide f o l l o w i n g a c i d h y d r o l y s i s (123). Comparison of Residues Reacting i n Hb-Hp Complex With Those i n Hb In order to i d e n t i f y which c y s t e i n y l r e s i d u e i n hemoglobin was r e a c t i n g , the technique of comparative p a r t i a l a c i d h y d r o l y s i s , dev-eloped f o r comparison of the a c t i v e s i t e s of s e r i n e proteases, was 14 used (124). P a r t i a l h y d r o l y s a t e s of C-carboxymethyl-labelled Hb and Hb-Hp complex were separated by high v o l t a g e e l e c t r o p h o r e s i s at pH 3.6 and an autoradiograph of the electrophoretogram i s shown i n Fi g u r e 37. At l e a s t e i g h t r a d i o a c t i v e bands show i d e n t i c a l m o b i l i t i e s and appear i n approximately s i m i l a r p r o p o r t i o n s i n sample 1 ( f r e e Hb) and sample 2 (the Hb-Hp complex). This demonstrates c o n c l u s i v e l y that the s i t e of r e a c t i o n i n the Hp-Hb complex i s s t i l l at the 693 c y s t e i n y l r e s i d u e of hemoglobin. Sample 3 shows the products of p a r t i a l a c i d 14 h y d r o l y s i s of f r e e h a p t o g l o b i n a f t e r treatment w i t h C-iodoacetamide. The s i t e ( s ) of m o d i f i c a t i o n , which leads to the appearance of the two s l i g h t l y cathodic r a d i o a c t i v e bands, has not been c h a r a c t e r i z e d but 128 1 2 3 o r i g i n F i g u r e 37 High v o l t a g e e l e c t r o p h o r e s i s at pH 3.6 a f t e r 20 minute a c i d h y d r o l y s i s of l^C-carboxymethyl-Hb, 14c-carboxymethyl Hb-Hp and l^C-carboxymethyl Hp. E l e c t r o p h o r e s i s was performed at approximately 3,500 v o l t s u n t i l a marker of c r y s t a l v i o l e t had moved 22 cm. 129 f a i n t corresponding bands i n sample 2 i n d i c a t e that a s i m i l a r r e a c t i o n i s probably o c c u r r i n g w i t h the Hb-Hp complex. The e x p l a n a t i o n f o r the decreased r e a c t i v i t y of c y s t e i n y l 393 i n the Hb-Hp complex cannot, at the moment, be unequivocal. Three p o s s i b i l i t i e s e x i s t , (a) There i s a covalent i n t e r a c t i o n between the s u l f h y d r y l group of the c y s t e i n y l residue at 393 and a group i n h a p t o g l o b i n , f o r example, a r e a c t i v e d i s u l f i d e . Two l i n e s of evidence make t h i s u n l i k e l y ; f i r s t , 393 can re a c t completely, a l b e i t more s l o w l y , w i t h iodoacetamide or the d i t h i o d i p y r i d i n e s so that the decreased r e a c t i v i t y i s e s s e n t i a l l y a r a t e phenomenon. Secondly, the Hb-Hp complex can be completely d i s -s o c i a t e d by s u c c i n y l a t i o n of amino groups (54) , a procedure which does not a f f e c t -SH or -S-S- groups but i s known to cause an extensive p h y s i c a l u n f o l d i n g of ha p t o g l o b i n and presumably d e s t r u c t i o n of the con-formation of the hemoglobin b i n d i n g s i t e on haptoglobin. (b) 393 i s a "contact" amino a c i d (125) i n the hemoglobin s i t e bound by haptoglobin. This p o s s i b i l i t y has been explored by Bunn (64) who prepared s e v e r a l d e r i v a t i v e s of hemoglobin i n which 393 was modi-f i e d by groups v a r y i n g i n s i z e from carboxymethylamido- (from iodoacetamide) to p-mercuribenzoate (from p-HMB) and found that there was no e f f e c t on the b i n d i n g of hemoglobin by haptoglobin. This would argue a g a i n s t the decrease i n r e a c t i v i t y of 393 i n the complex being the r e s u l t of a d i r e c t s h i e l d i n g by haptoglobin. Moreover, HbH (3^) i s not bound by haptoglobin (57) and i s o l a t e d 3-chain i s bound only weakly (72), so that i t seems u n l i k e l y that a major p o r t i o n of the 130 binding s i t e i s on the B-chain. (c) The t h i r d and perhaps most l i k e l y p o s s i b i l i t y i s that a conformational change i s induced i n the environment of 393 as a r e s u l t of the re a c t i o n with haptoglobin. The e f f e c t of haptoglobin on 693 i s very s i m i l a r to the decrease i n r e a c t i v i t y of 393 toward iodoacetamide upon deoxygenation of hemoglobin (47). Since t h i s e f f e c t has been inter p r e t e d (47) as r e s u l t i n g from the known conformational d i f f e r e n c e s between oxy- and deoxy-hemoglobin (42,50), the same type of conforma-t i o n a l change upon haptoglobin binding seems reasonable. Detailed studies of the immediate environment of 393 (42,50) have shown that c y s t e i n y l 393 i s close to h i s t i d y l 397, a residue intimately involved i n the (a^,^^) contact area between the d i s s o c i a t i n g halves of the hemoglobin molecule (50). A more recent X-ray c r y s t a l l o g r a p h i c study i n d i c a t e s that upon formation of deoxyhemoglobin, h i s t i d y l 3146 forms a hydrogen bond with the 3 -carboxyl group of a s p a r t y l 894 and r e s t r i c t s access to the sulphydryl group of 893 (51) thus accounting f o r the decreased r e a c t i v i t y of 893 (47). Since the combination of haptoglobin appears to be with d i s s o c i a t e d a8 dimers of hemoglobin (70,71) i t i s poss i b l e that haptoglobin may react with some portion of the exposed 0 . ^ , 8 2 contact area thus i n d i r e c t l y a f f e c t i n g the r e a c t i v i t y of 893 toward sulphydryl reagents. Thus the evidence i n d i c a t e s that haptoglobin induces a confor-mational change i n the v i c i n i t y of the 893 sulphydryl of hemoglobin. I t i s known that haptoglobin combines with a number of hemoglobins modified at 893 (64) but i t i s not known i f haptoglobin induces a 131 s i m i l a r conformational change i n these modified hemoglobins. The question a l s o a r i s e s as to how hemoglobins w i t h a modified conformation at 893 re a c t w i t h hap t o g l o b i n . Deoxyhemoglobin, known to have a d i f f e r e n t conformation at 893, does not combine w i t h hapto-g l o b i n . Human bis(N-maleimidomethyl)ether hemoglobin, which i s modi-f i e d a t 893 and has d i f f e r e n t d i s s o c i a t i o n p r o p e r t i e s than human oxyhemoglobin, r e a c t s l e s s completely w i t h h a p t o g l o b i n than oxy-hemoglobin does. In f a c t t h i s 8ME-hemoglobin has r e c e n t l y been shown to have a d i f f e r e n t conformation i n the FG corner of the hemoglobin 8 chain (the 893 region) (126). Thus two hemoglobins both w i t h d i f f e r -ent conformations i n the c y s t e i n y l 893 r e g i o n both have d i f f e r e n t a f f i n i t i e s f o r haptoglobin than oxyhemoglobin has. As a r e s u l t t h i s r e g i o n of the hemoglobin molecule or an area near t h i s r e g i o n appears to be the l i k e l y s i t e of hapt o g l o b i n a t t a c k . Since the a - j ^ contact r e g i o n i s near the 893 area a conformational change i n the 893 area could change the conformation i n the a-j.^ c o n t a c t area. The f a c t that both human deoxyhemoglobin and human BME-hemoglobin have d i f f e r e n t d i s -s o c i a t i o n p r o p e r t i e s than oxyhemoglobin (49,65) confirms the p r e d i c t i o n that these hemoglobins have d i f f e r e n t conformations i n the a^$2 c o n t a c t area than oxyhemoglobin. In the case of deoxyhemoglobin the d i f f e r e n t conformation i n the a j $ 2 c o n t a c t area has been demonstrated d i r e c t l y by X-ray c r y s t a l l o g r a p h y (43). As mentioned p r e v i o u s l y , t h i s change i n conformation i n the ^ •^'2 c o n t a c t a r e a m a Y D e the reason that hapto-g l o b i n r e a c t s d i f f e r e n t l y w i t h human BME-hemoglobin and human deoxy-hemoglobin than w i t h human oxyhemoglobin. Since haptoglobin does 132 combine w i t h a8 hemoglobin dimers and does r e a c t more r a p i d l y w i t h hemoglobins which are d i s s o c i a t e d i n t o dimers to a greater extent (68), hapto g l o b i n appears to be combining w i t h an area of the hemoglobin molecule which i s exposed a f t e r hemoglobin d i s s o c i a t i o n . This area appears to be the a - i^2 c o n t ; a c t a r e a -The f o l l o w i n g observations are c o n s i s t e n t w i t h the p r e d i c t i o n that haptoglobin combines w i t h the a ] $ 2 c o n t a c t area of hemoglobin; h a p t o g l o b i n combines w i t h the a6 dimer of hemoglobin; haptoglobin causes a conformational change i n a re g i o n of the hemoglobin molecule (893 region) which i s i n contact w i t h the c o n t a c t r e g i o n ; two hemoglobins w i t h modified conformations i n the c o n t a c t a r e a com-b i n e l e s s completely or not at a l l w i t h h a p t o g l o b i n ; and hapt o g l o b i n combines most r a p i d l y w i t h those hemoglobins which are d i s s o c i a t e d to the g r e a t e s t extent. In recent years n i t r o x i d e d e r i v a t i v e s have been discovered which c o n t a i n unpaired e l e c t r o n s and are s t a b l e i n aqueous s o l u t i o n s (127). The attachment of these compounds to macromolecules has pro-v i d e d new and e x c i t i n g i n f o r m a t i o n about the nature of p r o t e i n s t r u c t u r e because the e l e c t r o n s p i n resonance s p e c t r a of these modified p r o t e i n s has been s t u d i e d . Because the 893 c y s t e i n y l group of hemoglobin can r e a d i l y be modified and because i t i s s e n s i t i v e to the conformation of hemoglobin t h i s group has been modified w i t h s e v e r a l s p i n - l a b e l reagents and s t u d i e d i n d e t a i l i n H. M. McConnells l a b o r a t o r y (127,128). 133 One very i n t e r e s t i n g aspect of t h i s work was the demonstration of a new component i n the s p e c t r a of s p i n - l a b e l compounds a f t e r the s p i n - l a b e l reagents were attached to the 893 s u l p h y d r y l i n hemoglobins. In f a c t the s p e c t r a of horse carbonmonoxyhemoglobin and horse methe-moglobin show d i f f e r e n t amounts of t h i s component (129). The explana-t i o n f o r the v a r y i n g amounts of the new component i n the s p i n resonance spectrum of modified carbonmonoxyhemoglobin and methemoglobin was that the conformation of these two p r o t e i n s i n the area of the s p i n - l a b e l s was d i f f e r e n t . X-ray c r y s t a l l o g r a p h y of horse methemoglobin and horse carbonmonoxyhemoglobin had shown that these p r o t e i n s had i d e n t i c a l s t r u c t u r e s (130). However c r y s t a l l i z a t i o n of these two s p i n -l a b e l l e d hemoglobins demonstrated that the d i f f e r e n c e i n s p e c t r a between the two p r o t e i n s remained (131) and thus the two p r o t e i n s appear to have a very s m a l l conformational d i f f e r e n c e which cannot be demonstrated by X-ray c r y s t a l l o g r a p h y . 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