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UBC Theses and Dissertations

Studies on the combination of haptoglobin with hemoglobin Chan, Gwendolyn Faye Quen 1968

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STUDIES ON THE COMBINATION OF HAPTOGLOBIN WITH HEMOGLOBIN by GWENDOLYN FAYE QUEN CHAN B.S.P. University of B r i t i s h Columbia, 1956 M.S.P. University of B r i t i s h Columbia, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Biochemistry Faculty of Medicine We accept this thesis as conforming to the required standard January, 196 8 University of B r i t i s h Columbia In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f 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 Columbia, I agree t h a 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 agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p urposes 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 . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Biochemistry The U n i v e r s i t y o f B r i t i s h 'Columbia Vancouver 8, Canada D a t e March l h t 1968 i ABSTRACT H a p t o g l o b i n i s a g e n e t i c a l l y p o l y m o r p h i c p l a s m a p r o t e i n w h i c h p o s s e s s e s t h e p r o p e r t y o f c o m b i n i n g s p e c i f i c a l l y w i t h h e m o g l o b i n . H a p t o g l o b i n b i n d s h e m o g l o b i n s t o i c h i o m e t r i c a l l y i n a s t a b l e and e s s e n t i a l l y i r r e v e r s i b l e c o mplex. The p r e s e n t s t u d i e s a r e c o n c e r n e d w i t h t h e n a t u r e o f t h e hemo-g l o b i n c o m b i n i n g s i t e on h a p t o g l o b i n i n r e l a t i o n t o i t s f u n c t i o n . L a r g e q u a n t i t i e s o f homogeneous h a p t o g l o b i n have b e e n p r e p a r e d f r o m a s c i t e s f l u i d by a newly d e v e l o p e d method i n v o l v i n g f i r s t , ammonium s u l f a t e p r e c i p i t a t i o n , f o l l o w e d by D E A E - c e l l u l o s e c h r o m a t o g r a p h y and f i n a l l y p u r i f i c a t i o n by Sephadex G-200 g e l f i l t r a t i o n . A Sephadex G-200 a s s a y w h i c h s e p a r a t e s t h e f r e e h e m o g l o b i n f r o m t h e Hb-Hp complex and e n a b l e s d i r e c t measurement o f t h e amount o f h e m o g l o b i n bound t o h a p t o g l o b i n has b een d e v e l o p e d . I n o r d e r t o d e t e r m i n e w h e t h e r t h e s i t e o f p o l y m e r i z a t i o n o f h a p t o g l o b i n p o l y m e r s i s d i s t i n c t f r o m t h e h e m o g l o b i n b i n d -i n g s i t e , h a p t o g l o b i n p o l y m e r s p a r t i a l l y r e s o l v e d on Sephadex G-200, were e x a m i n e d f o r t h e i r a b i l i t y t o b i n d h e m o g l o b i n b o t h by t h e p e r o x i d a s e a s s a y and t h e Sephadex a s s a y . L o c a l i -z a t i o n o f t h e h e m o g l o b i n b i n d i n g s i t e on t h e component h a p t o -g l o b i n c h a i n s was a c h i e v e d by d e t e r m i n a t i o n o f t h e a b i l i t y o f i s o l a t e d a 1 , a 2 and 3 c h a i n s t o b i n d h e m o g l o b i n by means o f t h e Sephadex a s s a y . The b i n d i n g o f g l o b i n , m y o g l o b i n and v a r i o u s v e r t e b r a t e h e m o g l o b i n s were a l s o e x amined by t h e Sephadex a s s a y . The e f f e c t s o f e n v i r o n m e n t a l f a c t o r s on t h e c o m b i n a t i o n o f h a p t o g l o b i n w i t h h e m o g l o b i n has b e e n s t u d i e d by a l t e r a t i o n o f i o n i c s t r e n g t h and o f pH and by a d d i t i o n o f p h e n o l as a t y r o s i n e a n a l o g u e . The i n v o l v e m e n t o f amino g r o u p s o f h a p t o g l o b i n i n b i n d i n g w i t h h e m o g l o b i n has b e e n s t u d i e d by s e l e c t i v e c h e m i c a l m o d i f i c a t i o n o f h a p t o g l o b i n amino g r o u p s w i t h t h r e e r e a g e n t s o f i n c r e a s i n g s e v e r i t y f o l l o w e d by measurement o f t h e a b i l i t y o f t h e c h e m i c a l l y m o d i -f i e d p r o t e i n s t o b i n d h e m o g l o b i n . An a p p r o x i m a t e d e t e r m i n a -t i o n o f t h e a r e a o f t h e b i n d i n g s i t e i n t h e Hb-Hp complex has b e e n made by c o m p a r i s o n o f t h e e x t e n t o f g u a n i d i n a t i o n o f amino g r o u p s o f t h e i n d i v i d u a l components w i t h t h a t o f t h e complex and a l s o by c o m p a r i s o n w i t h t h e e x t e n t o f m o d i -f i c a t i o n i n d i s s o c i a t e d h e m o g l o b i n and h a p t o g l o b i n f r o m t h e g u a n i d i n a t e d complex. The r e s u l t s o f t h e s e s t u d i e s show t h a t t h e h e m o g l o b i n c o m b i n i n g s i t e i s d i s t i n c t f r o m t h e s i t e o f p o l y m e r i z a t i o n and t h a t t h e 6 h a p t o g l o b i n c h a i n c a r r i e s t h e h e m o g l o b i n b i n d i n g s i t e . H a p t o g l o b i n combines w i t h g l o b i n b u t n o t w i t h m y o g l o b i n and i t b i n d s s t o i c h i o m e t r i c a l l y w i t h a l l a n i m a l h e m o g l o b i n s e x a m i n e d e x c e p t t h o s e o f t h e f r o g and f i s h , w h i c h may be t h e r e s u l t o f a c o n f o r m a t i o n a l change i n t h e Hb-Hp complex w h i c h c a u s e s e x p u l s i o n o f t h e heme g r o u p . I t i s shown t h a t e l e c t r o s t a t i c f o r c e s c a n n o t be t h e s o l e i n t e r r n o -l e c u l a r f o r c e s i n v o l v e d i n t h e b i n d i n g . C h e m i c a l m o d i f i c a t i o n s t u d i e s show t h a t t h e a r e a o f c o n t a c t between t h e h e m o g l o b i n and t h e h a p t o g l o b i n i n t h e complex i n v o l v e s o n l y a s m a l l a r e a o f t h e h a p t o g l o b i n m o l e c u l e o f e l s e t h e a r e a i s p a r t i c u l a r l y d e f i c i e n t i n l y s y l - s i d e c h a i n s . I t i s a l s o shown t h a t amino groups are not d i r e c t l y involved i n the binding s i t e and that acylation of amino groups p a r t i c u l a r l y with succinyl group cause a profound change i n the haptoglobin molecule and i t s a b i l i t y to bind hemoglobin i s very much reduced. i v TABLE OF CONTENTS ABSTRACT TABLE OF CONTENTS L I S T OF TABLES L I S T OF FIGURES L I S T OF ABBREVIATIONS ACKNOWLEDGEMENT DEDICATION INTRODUCTION B i o c h e m i s t r y o f H a p t o g l o b i n s G e n e t i c s P h y s i o l o g y a n d P a t h o l o g y o f H a p t o g l o b i n A s p e c t s o f H a p t o g l o b i n S t r u c t u r e a n d F u n c t i o n I n v e s t i g a t e d i n t h i s Work EXPERIMENTAL GENERAL METHODS 1. S t a r c h - G e l E l e c t r o p h o r e s i s 2. U r e a S t a r c h - G e l E l e c t r o p h o r e s i s 3. P o l y a c r y l a m i d e G e l E l e c t r o p h o r e s i s 4. P r e p a r a t i o n o f H e m o g l o b i n PART I ISOLATION OF HAPTOGLOBIN AND PARTIAL SEPARATION OF HAPTOGLOBIN POLYMERS INTRODUCTION P U R I F I C A T I O N OF HAPTOGLOBIN AND PARTIAL SEPARATION OF POLYMERS 1. P r e p a r a t i o n o f H a p t o g l o b i n by C o n n e l l a n d Shaw M e t h o d V 2. Preparation of Haptoglobin by Modification of <. • Smith, Edman and Owen, and Connell and Shaw Methods 49 3. P u r i f i c a t i o n of Haptoglobin by a New Method and P a r t i a l Separation of Polymers 52 PART II SEPHADEX G-200 ASSAY 69 INTRODUCTION 69 1. Peroxidase Assay 70 2. Electrophoretic Methods 74 3. Sephadex Assay 75 SEPHADEX ASSAY 76 ASSAY OF Hp 2-1 POLYMERS 82 ASSAY OF a1'," a 2 AND 3 HAPTOGLOBIN CHAINS 89 BINDING OF GLOBIN AND MYOGLOBIN TO HAPTOGLOBIN 95 1. Globin 95 2. Myoglobin 99 ASSAY OF HAPTOGLOBIN WITH HEMOGLOBIN FROM OTHER SPECIES 102 PART III EFFECT OF ENVIRONMENTAL FACTORS UPON THE BINDING OF HEMOGLOBIN AND HAPTOGLOBIN 114 INTRODUCTION 114 EFFECT OF IONIC STRENGTH AND pH UPON HEMOGLOBIN-HAPTOGLOBIN BINDING 116 PART IV CHEMICAL MODIFICATION OF AMINO GROUPS IN HAPTOGLOBIN 125 INTRODUCTION . 125 GUANIDINATION 128 Experimental 130 Results 132 v i ACETYLATION 137 Experimental 137 Results 142 SUCCINYLATION 142 Experimental 14 4 Results 145 DISSOCIATION OF THE Hb-Hp COMPLEX BY SUCCINYLATION 157 Experimental 16 2 Results 162 DISCUSSIONS OF CHEMICAL MODIFICATION 168 BIBLIOGRAPHY 173 v i i LIST OF TABLES Table Page I P u r i f i c a t i o n of Haptoglobin 1-1 66 II Hemoglobin Binding by Haptoglobin Polymers 8 5 III Haptoglobin Binding with Animal Hemoglobins 107 IV Haptoglobin Binding with Varying Concentrations of Trout Hemoglobin 109 V E f f e c t of Salt Concentrations on Hb/Hp Combination (Sephadex G-200 Method) 117 VI E f f e c t of pH on Hb/Hp Combination (Sephadex G-200 Method) 119 VII Guanidination 133 VIII Acetylation of Haptoglobin 1-1 143 IX Succinylation of Haptoglobin 1-1 147 X Succinylation of Hp 2-1, Hp 2-2 and Hp-Hb Complex 158 XI Guanidination of Hb-Hp Complex and Dissociation of the Guanidinated Complex by Subsequent Succinylation 169 v i i i LIST OF FIGURES Figure Page 1. SG-electropherogram of the three common hapto-globin phenotypes 4 2. The amino acid sequence of the N-, C- and J-peptides of haptoglobin a chains resulting from partial duplication in the Hp a 2 chains 8 3. SG-electropherograms of the haptoglobin pheno-types and their respective subtypes 21 1 4. World Map of Hp gene frequencies 24 5. DEAE-cellulose chromatography of crude Hp 1-1 on a 5.0 x 9.5 cm column in 0.01 M sodium acetate buffer, pH 4.7 54 6. DEAE-cellulose chromatography of crude Hp 2-1 on a 5.0 x 9.5 cm column in 0.01 M sodium acetate buffer, pH 4.7 55 7. SG-electropherogram of the DEAE-cellulose chromatography column effluent fractions of crude Hp 1-1 commencing at 250 ml through to 440 ml 56 8. Purification of Hp 1-1 on Sephadex G-200 (2.5 cm x 186 cm) in 0.05 M ammonium acetate, pH 8.5 58 9. SG-electropherogram of fractions following Sephadex G-200 chromatography (Fig. 8) of Hp 1-1 60 10. Purification of crude Hp 2-1, peak A and B from DEAE-cellulose chromatography (Fig. 6) on Sephadex GS200 (2.5 cm x 186 cm) in 0.05 M ammonium acetate, pH 8.5 61 11. SG-electropherogram of Hp 2-1 polymer fractions from Sephadex G-200 chromatography commencing at fraction number 28 through to fraction number 4 6 6 2 12. SG-electropherogram of Hp 2-1 during the i n i t i a l stages of i t s preparation 64 13. Assay of Hp 1-1 in 0.02 M Tris-HCl buffer, pH 7.42 (frame A) and Hp 2-1 polymers on Sephadex G-200 (1 cm x 50 cm) in 0.1 M phosphate buffer pH 7.0 (frames B, C and D). 78 ix 14. Assay of B, a 1 and a 2 haptoglobin chains on Sephadex G-200 (1 cm x 50 cm). (A) 3 chain i n 0.2 M ammonium acetate, pH 7.0 (acetic acid), (B) a 2 chain i n 0.1 M phosphate buffer, pH 7.0, (C) a 1 chain i n 0.1 M phosphate buffer, pH 7.0 93 15. Binding of globin to haptoglobin on Sephadex G-200 (1 cm x 50 cm) i n 0.2 M ammonium hydroxide, pH 9.0 (acetic acid) 98 16. Binding of sperm whale myoglobin to haptoglobin on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phos-phate buffer, pH 7.0 103 17. Assay of Hp 1-1 on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0 containing 0.1 M phenol 122 18. Separation of known basic amino acids on the Beckman/Spinco 120 C amino acid analyzer 129 19. Assay of Hp 1-1 guanidinated with 0.1 GDMP (B), 0.2 M GDMP (C), 0.5 M GDMP (D) compared with a control (A) on Sephadex G-200 i n 0.1 M phosphate buffer, pH 7.0 134 20. Assay of Hp 1-1 after acetylation with acetic anhydride. Ratios of reagent to haptoglobin of (A) 10:1, (B) 40:1, (C) 80:1, (D) 400:1, on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer 140 21. Pa Hp r t i a l separation of two species of acetylated -jj 1-1 following reaction with a r a t i o of 400:1 acetic anhydride to. protein on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0 141 22. Assay of Hp 1-1 succinylated for various times on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phos-phate buffer, pH 7.0. Reaction times are: (A) 10 minutes, (B) 30 minutes, (C) 1 hour, (D) 2 hours, with a f i f t e e n - f o l d excess of succinic anhydride 146 23. SG-electropherogram of 10 minutes and 30 minutes succinylated Hp 1-1 and th e i r controls 149 24. SG-electropherogram of 1 hour and 2 hours succinylated Hp 1-1 and th e i r controls 150 X 25. Ultracentrifuge patterns of 2 hours succinylated Hp 1-1 i n 0.05 M KC1, 0.01 M phosphate buffer, pH 7.0 during the Archibald approach to sedimen-ta t i o n equilibrium 152 26. Sedimentation patterns of 10 minutes succinylated haptoglobin i n 0.1 M NaCl, 0.1 M phosphate buffer, pH 7.0 i n a synthetic boundary c e l l 155 27. Sedimentation patterns of 2 hours succinylated haptoglobin i n 0.0 5 M KC1, 0.01 M phosphate buffer, pH 7.0 i n a synthetic boundary c e l l 156 28. Assay of 10 minutes and 2 hours succinylated Hp 2-1 and the i r controls on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0 159 29. Assay of 10 minutes and 2 hours succinylated Hp 2-2 and the i r controls on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0 160 30. SG-electropherogram of 10 minutes and 2 hours succinylated Hp 2-1 and th e i r controls 161 31. Assay of 10 minutes and 2 hours succinylated Hb-Hp complexes and th e i r controls on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0 163 32. Modified Hb-Hp on Sephadex GS200 i n 0.1 M phosphate buffer, pH 7.0 166 xi LIST OF ABBREVIATIONS 1. BME: bis(N-maleimidomethyl)ether 2. DEAE-cellulose: Diethylaminoethyl-cellulose 3. GDMP: l-guanyl-3,4-dimethyl pyrazole nitrate 4. Hb: hemoglobin 5. Hb-Hp: hemoglobin-haptoglobin 6. Hp: haptoglobin 7. H.I.: Haptoglobin Index 8. O.D.: optical density 9. SG-electropherogram: starch-gel electropherogram 10. TNBS: 2,4,6-trinitrobenzene-l-sulfonic acid 11. TNP: trinitrophenyl 12. Tris: Tris (hydroxymethyl). aminomethane x i i ACKNOWLEDGEMENTS The author is deeply indebted to Dr. G.H. Dixon for his inspiring guidance, encouragement and patience during the course of this study and sincerely appreciates the constructive criticisms during the preparation of this thesis. Special thanks are extended to Dr. Francisco Eng for the discussions and his assistance during our years of association. The author acknowledges the assistance of Mr. J.A. Durgo in the amino acid analyses and the ultracentrifugal studies. The kind cooperation of the Canadian Red Cross Blood Bank, Vancouver, B.C., in supplying human blood and of Dr. D.K. Ford, Faculty of Medicine, University of British Columbia, in arranging for the supply of ascites fluid, is greatly appreciated. National Research Council Studentships and University Graduate Fellowships have enabled this study to be carried out and Medical Research Council has supported the research. x i i i DEDICATION To my husband Edward - 1 -INTRODUCTION BIOCHEMISTRY OF HAPTOGLOBINS Haptoglobins are a group of plasma glycoproteins which possess the property of combining with hemoglobin specifi-cally and irreversibly; this property formed the basis of their discovery and nomenclature (from the Greek haptein, to hold fast) (1). In 1938, Polonovski and Jayle (2) ob-served that the peroxidase activity of hemoglobin is greatly enhanced by the addition of serum. Further studies by these investigators (1,3) revealed that the combination of hemo-globin with a plasma component, to which the name haptoglobin was given, was responsible for this peroxidase activation phenomenon, which later served as a basis of assay of hapto-globin in the serum (4). The existence of more than one type of haptoglobin originated from the observation by Jayle and Gillard (5) that in 10 out of 12 samples of human sera, haptoglobin was salted out at much lower concentrations of ammonium sulfate than was required for the precipitation of the same protein from the remaining two sera. One type of haptoglobin, originally referred to as Hp II, was f i r s t iso-lated from the urine of a nephrotic child (6) and possessed a molecular weight of about 85,000 (7), half the molecular size of another more frequently found haptoglobin type, named Hp I, isolated from human sera (8). It was concluded that this latter type of haptoglobin is a dimer of Hp II (8). A significant advance in the f i e l d of haptoglobin research - 2 -came from the introduction of starch-gel electrophoresis by Smithies (9). The greater resolving power of t h i s method of zone electrophoresis over free-boundary electrophoresis and ex i s t i n g methods of zone electrophoresis enabled Smithies to show that human sera f e l l into three groups on the basis of the occurrence of some component which had the common property of binding hemoglobin (10). Subsequently, Smithies and Walker (11) established the i d e n t i t y of these serum protein groups with the haptoglobins discovered by Jayle; furthermore, they showed that the observed differences of the haptoglobins could be explained by postulating the existence of a l l e l e s 1 2 Hp and Hp of the autosomal haptoglobin locus, Hp, which 1 1 2 1 show incomplete dominance. Each genotype Hp /Hp Hp /Hp 2 2 and Hp /Hp can be distinguished phenotypically as Hp 1-1, Hp 2-1 and Hp 2-2 respectively (11,12). Jayle's group con-firmed the i d e n t i t y of t h e i r proteins with those of Smithies and reversed t h e i r notation i n describing the haptoglobins (13) . 2 In individuals homozygous for the Hp a l l e l e , a single protein band i s v i s i b l e on starch-gel electrophoresis, where-2 as the phenotype of homozygotes for the Hp a l l e l e i s a series of more than ten bands of decreasing concentration and mobility. The phenotype of heterozygotes shows a fas t migra-ting band with mobility s i m i l a r to the Hp 1-1 band i n addition to a series of bands of s l i g h t l y greater mobility than the series seen i n Hp 2-2; t h i s electrophoretic pattern i s d i s t i n c t - 3 -from that obtained with a mixture of the haptoglobins of the two homozygous types (14). The characteristic patterns of these haptoglobin phenotypes which distinguishes them on starch-gel electrophoresis (Fig. 1), cannot be achieved by two-dimensional electrophoresis on paper, the series of zones in Hp 2-2 and Hp 2-1 a l l migrate as a single band at the same rate as the Hp 1-1 protein. Since starch-gel elec-trophoresis exerts a molecular sieving effect on the protein components whereas in filter-paper electrophoresis the greater fr i c t i o n a l retardation force of the large-sized proteins is balanced by a greater net charge (16), the observed differ-ences in the series of protein components appear to l i e in a difference in molecular size (14). The homogeneity of the Hp 1-1 protein and the heterogeneity of the Hp 2-2 protein were confirmed by ultracentrifugal studies (14,17,18), in which the asymmetric patterns both of free Hp 2-2 and i t s complex with hemoglobin were consistent with the presence of a series of components of higher molecular weight than the Hp 1-1 molecule, which appeared as a narrow symmetrical peak. On removal of the s i a l i c acid of these haptoglobin components with neuraminidase (19,20), the anodic migration of each component was markedly reduced in starch gels, nevertheless, the characteristic patterns persisted; therefore, the dif-ference in molecular size could not be related to s i a l i c acid content. Independently, Smithies and Connell (14) and Allison (21) - 4 -• Figure 1. SG-electropherogram of the three common haptoglobin phenotypes (15). - 5 -proposed a mechanism of polymer formation to explain the hap-toglobin polymorphism. Allison suggested the existence of complementary binding sites through which aggregation could occur. Hp 1 subunits would have only a single combining site and thus only monomers would be present in the Hp 1-1 homo-zygotes. The Hp 2 subunit however, would possess two com-plementary binding sites and thus could combine with other Hp 2 subunits to form a series of polymers of increasing size. The heterozygote, Hp 2-1, having equal amounts of Hp 1 and Hp 2 subunits, would form smaller polymers, :;.each terminated by an Hp 1 subunit. Smithies and Connell (14) proposed that the haptoglobins of type 2-2 include a series of polymers (n=l,2,3,4,etc.) varying in amounts inversely as the degree of polymerization and that a minor molecular difference such as the introduction of a cysteine residue into the molecule could allow formation of a complex series of polymers which is not possible in the Hp 1-1 molecule. Treatment of hapto-globin with 0.01 M thioglycollate and electrophoresis in-starchhgels containing 8 M urea and thioglycollate produced a simplified haptoglobin pattern and showed a common cleavage product from a l l the haptoglobin types. Thus disulphide bonds are probably involved in the formation of the complex haptoglobin phenotypes. Further characterization of the haptoglobin molecule by Connell, Dixon and Smithies (22) showed that reductive cleavage of the three haptoglobin types with mercaptoethanol - 6 -in the .presence of urea yields two groups of products separ-able in acidic starch gels containing 8 M urea. One group of product, called the 3 chains, are common to haptoglobins of a l l genetic types, while the other product, called a chains differ according to genetic type. The a chain from Hp 2-2, herein to be called the a 2 chain, migrates behind the a chain in Hp 1-1, which w i l l be called the a 1 chain. In Hp 2-1 there are almost equal proportions of a 2 and a 1 chains. The a polypeptide chains from Hp 1-1 are further subdivided into faster and slower migrating zones, which w i l l be called IF IS herein Hp a and Hp a respectively, the difference in mobility being due to substitution of a single lysine r e s i -1F IS due in a by a glutamic acid residue in a (23,24). It is evident then, that only the a chains are controlled by the Hp locus, the 3 chains being determined at a separate locus (25, 26). Thus, six common haptoglobin phenotypes are obtained on subtyping: IF-IF, 1F-1S, 1S-1S, 2-1F, 2-1S and 2-2. Fingerprint analyses of chymotryptic digests of the a polypeptide chains revealed that the a 2 chain contained a l l IF IS the peptides found in the Hp a or Hp a chains with a relatively decreased amount of the two peptides termed isl-and C- together with an additional peptide, called the J peptide. This extra peptide has the same N-terminal residue (isoleucine) as peptide C and the same C-terminal residue (tyrosine) as the N peptide. Data from amino analyses of peptides N, C and J and further enzymatic digests of these - 7 -peptides suggest that J peptide consists of a fusion of C and N i n a peptide bond with the loss of 22 residues at the 2 terminal regions involved (27). The Hp a chain may then be IF IS regarded as a fusion of the Hp a and Hp a chains, which agrees with a determined molecular weight of 17,300 ± 1,400 2 IF for the Hp a chain, almost twice that of the Hp a and Hp IS a chain molecular weights, 8,860 ± 400. Smithies, Connell and Dixon (23) proposed a mechanism of unequal crossing-over IF IS between asymmetrically paired a l l e l e s occurring i n a Hp /Hp heterozygote, with a resultant p a r t i a l gene duplication i n one chromosome and a corresponding deletion i n the other to 2 explain the evolution of the Hp gene; t h i s i s depicted i n F i g . 2. Further, i t was predicted that by unequal but homolo-gous crossing-over i n the o r i g i n a l Hp gene, new gene 2FF 2SS products, Hp a and Hp a , could be formed. Nance and Smithies (29) subsequently found these products and confirmed the prediction. Also by crossing over i n an asymmetrically paired chromosome i n the Hp 2-2 homozygote, a t r i p l i c a t i o n could r e s u l t ; this appears to be the case i n the Johnson type haptoglobin discovered by G i b l e t t (30) i n a Negro mother and her c h i l d . Preliminary evidence of the molecular sizes of a 1, a 2 and a J (Johnson) on Sephadex G-75 columns indicate molecular weights of 8,000, 17,400 and 24,000 res-pectively, thus representing single, double and t r i p l e length chains (24). .The" N-terminal amino acid analysis of the a chain was - 8 -• - v « l - A » n - A » p - * r ^ l y - A » n - A « p - V « J - m - A i ^ U « - A l ^ ^ ctt. • •I IT 7T " • I ^Ilt^sn~l^t-Jkla-Tal~el^~A»p-ly»-ltu-Pro-ely-Cyt-Olu-Ala-Valiely-i^t-Pre-Lyt-A§ri.Pro-*la-Atn-Pro-Val-Clri.COO!i I 1* C paptid* *j ch. oh. I I I H1 Junction poptide — * I eh- ch. Figure 2. The amino acid sequence of the N-, C- and J-peptide of haptoglobin a chains resulting from partial duplication in Hp a 2 chains (28). - 9 -shown to be valine by Smithies, Connell and Dixon (23) and . Smith, Edman and Owen (31) reported equal amounts of N-termi-nal valine and isoleucine in haptoglobin preparations of a l l genetic types. These results indicate that the 3 chain N-terminal amino acid is isoleucine and that there is an equal number of a and 3 chains in each genetic type of haptoglobin. To account for the three types of haptoglobin, Hp 1F-1F, Hp 1F-1S and Hp 1S-1S, the Hp 1-1 molecule must contain a pair of a chains and also because of the equivalence of N-terminal groups, a pair of 3 chains. Thus, Shim and Beam (32) proposed a model of the secondary structure of Hp 1-1 composed of each of the two a chains joined by disulfide bonds to a 3 chain with the 3 chains also connected by d i -sulfide bridges. This model of haptoglobin is similar to that of the immunoglobulins, which also possess two light and two heavy chains joined by disulphide linkages. In the case of the Hp 2-2 molecule, Shim, Lee and Kang (33) suggested that the series of bands might be represented by the following composition: {a2$)i+, ( a 2 3 ) 6 / ( a z3)e, (a 2 3 ) I o • • • • i n contrast to ( a 1 3 ) 2 of the Hp 1-1 type. The Hp 2-1 series of bands might then be ( a 1 3) 2 , ( a 1 3 ) 2 ( a 2 3 ) 2 / f^a1 3) 2 (a 23) 4 , (a^) 2 (a 28) 6 The fact that Hp 2-2 homo-zygotes produce a series of polymers of limited size and in definite proportions, in approximately the ratio of 11:35: 25:17:9:2, is evidence that the mechanism of polymerization is limited by some mechanism of cyclization (34), such as disulphide linkage between the a 2 chain at the growing end of a polymer chain and the i n i t i a l 8 chain to form a circular Hp 2-2 molecule. The Hp 2-1 polymers, present in a ratio of 17:29:24:19:10:1 (34) would be terminated when two a 1 chains combine with 8 units (35). Experimental support for the pos-tulate advanced by Shim and coworkers comes from the results of Moretti, Cheftel and Cloarec (36), who found that the molecular weights of Hp 2-1 polymers, partially separated by starch-gel electrophoresis and by Sephadex chromatography, agreed well with the proposed polymeric formulae. The molecular structure underlying the polymorphism of each genetic haptoglobin type has also been investigated by immunological techniques. Burtin et al. (37) found that haptoglobin was a potent antigen since antisera obtained from animals immunized with pooled normal human serum usually con-tained antibodies against haptoglobin in appreciable t i t e r s . The existence of three genetically determined haptoglobins that differ in physico-chemical properties might indicate that the haptoglobins would d i f f e r : in antigenic structure. However, Beam and Franklin (18), using antisera against Hb-Hp complexes of each gentic type, which had been absorbed with umbilical cord serum known to contain very l i t t l e hapto-globin (38), and Fine and Battistini (39), employing anti-sera against whole human serum of each haptoglobin type, were unable to detect any immunological differences. However, Korngold (40) using antisera against purified Hp 2-2 - 11 -preparations, demonstrated that the antisera could dis t i n -guish between Hp 2-2 and Hp 1-1 when sera of these hapto-globin types reacted with the antisera. A large number of antigenic determinants characteristic of Hp 2-2 were lacking in Hp 1-1. Starch-gel Immunoelectrophoresis of Hp 2-1 showed that i t was antigenically heterogeneous; one anti-genic determinant was identical with Hp 1-1, while the other was closely related to Hp 2-r2. Korngold (41) also found hemoglobin could block some of these antigenic determinants; the blocking was maximal when one mole of hemoglobin was bound per mole of haptoglobin. Thus, the hemoglobin com-bining sites of haptoglobin are involved in some manner in the combination of haptoglobin with i t s antibodies. With antiserum to purified Hp 1F-1S, Shim and Beam (32) demon-strated that antigenic determinants of the haptoglobin mole-cule reside in both the a and 3 chains. They observed that anti-8-chain antiserum reacted only with free haptoglobin but not with the Hb-Hp complex, whereas anti-a 2-chain anti-serum reacted with both free and complexed haptoglobin. The conclusion was that hemoglobin binds with haptoglobin at the 3 chains and thereby covers most of the 8 chain antigenic determinants. The fact that anti-2-2-serum absorbed with type 1-1 s t i l l reacted with both free and bound 2-1 and 2-2 types but not with free 1-1 suggests that the antigenic differences among normal genetic types of haptoglobin reside mainly in the a chains. - 12 -Haptoglobin migrates as a single boundary with the a 2 fraction on electrophoresis in the Tiselius apparatus and on filter-paper electrophoresis (42), thus indicating that the Hp 2-1 and 2-2 polymers a l l possess identical charges. Hemo-globin has a mobility corresponding to the 8 globulin fraction. However, when hemoglobin is added to serum, i t forms the Hb-Hp complex which migrates between a 2 and 8 i globulins .(38, 43,44). The haptoglobin components in the starch gel system migrate in the a8 region and on addition of hemoglobin, these components are retarded in their migration (9). The relatively high carbohydrate content of haptoglobin and i t s precipitation behaviour in the presence of 0.6 M perchloric acid places i t in the category of a seromucoid, a term proposed by Winzler (45) to designate a group of serum glycoproteins with very high carbohydrate content which re-main in solution when serum is exposed to a 0.6 M solution of perchloric acid. -Haptoglobin is reported to consist of 74-76.5% protein, the remainder of the molecule being made up of the same content of carbohydrates in a l l haptoglobin types: 5.1% s i a l i c acid, 5.4% glucosamine, 1% fucose, 0.5% glucose and 8.5% galactose and mannose (46,47). A l l of the carbohydrate portion of the haptoglobin is contained in the 8 chain (32,48). Gerbeck, Rafelson and Bezkarovainy (49) provided evidence that the carbohydrate of intact haptoglobin exists as more than one oligosaccharide chain, since pronase digestion yielded a major glycopeptide with an apparent - 13 -molecular weight of 10,000 based on the sedimentation co-efficient of approximately 1 and a minimum molecular weight based on serine and threonine composition. The proportions of hexose, glucosamine and s i a l i c acid were 1.9:1.2:1, essen-t i a l l y the same as in the native haptoglobin, 1.9:1.5:1. Later isolation of the pure glycopeptides by this group of investigators (50) led to the characterization of two major glycopeptides from Hp 2-1 differing in amino acid composition, but containing essentially the same carbohydrate composition: 5-6 moles hexose, 3-4 moles N-acetylglucosamine and 0-3 moles of s i a l i c acid with fucose present in some units but absent in others. On the basis of the size (molecular weights ranging from 2000 to 3000) and the composition of the isolated glycopeptides, i t was estimated that an intact molecule of Hp 2-1 of molecular weight 200,000 would contain approximately 13 such units. Tryptic digestion of haptoglobin has been less revealing, Jayle 1s group (47) obtained two types of carbohy-drate chains, the smaller is detached by trypsin in the form of dialyzable glycopeptides and contain galactose, glucosamine and the part of s i a l i c acid not released by neuraminidase (51), the other fragment is. non-dialyzable and contains five kinds of carbohydrates and a l l the s i a l i c acid (70%) removed by neuraminidase (51). Tryptic digestion by Dobryszycka and Lisowska (52) also yielded a number of glycopeptides with varying carbohydrate content, one of these glycopeptides, however, was shown to retain the property of activating the - 14 -peroxidase activity of hemoglobin. The complex formed between hemoglobin and haptoglobin is of considerable interest. This combination is extremely tight and stable and is highly specific, reminiscent of the antigen-antibody reaction, with the exception that the Hb-Hp complex remains soluble. That the hemoglobin-haptoglobin reaction is essentially i r r e v e r s i b l e i s shown by the fact that once the complex i s formed there i s no exchange of the bound hemoglobin with Asotopically labelled hemoglobin (53). Also, attempts at dissociation of the Hb-Hp bond have proven unsuccessful. Electrophoretic investigations have shown that haptoglobin binds hemoglobin over a wide range of pH, 4.4 to more than 10.0 (54,55). Haptoglobin from human or various species of animal also combine with human or animal hemo-globins with the specificity of binding residing in the haptoglobin molecule (44,46,55,56). The common phenotypes of haptoglobin a l l combine st o i -chiometrically with hemoglobin and i t has been established by Jayle (46) and confirmed by others (18,57) that 1 mg of hemoglobin combines with 1.3 mg of haptoglobin, which corres-ponds to an equimolar combination of hemoglobin with an Hp 1-1 unit. However, i f a haptoglobin solution is partially saturated with hemoglobin, an intermediate complex appears to be formed consisting of one molecule of haptoglobin bound to one-half molecule of hemoglobin (33,54,58). The linkage between hemoglobin and haptoglobin appears - 15 -to be through the globin moiety (55) and unaffected by various ligands of the heme group, since haptoglobin combines with carbon monoxyhemoglobin, methemoglobin and cyanohemo-globih to the same extent as oxyhemoglobin (54,59). Hapto-globin also binds fetal hemoglobin and several abnormal hemo-globins tested (17,60,61), except hemoglobins composed of tetramers of 6 chains, Hb H, and tetramers of y chains, Hb Barts (61). This lack of binding may be ascribed to either an absence of necessary a chains in the hemoglobin molecule for binding or to an altered configuration, which has been observed for Hb H by Perutz and Mazzarella (62) in their crystallographic studies, and which may be presumed for Hb Barts. As to the a chain's a b i l i t y to bind haptoglobin, Chiancone et al. (63) find that haptoglobin combines with isolated a hemoglobin chains and suggests as a consequence that combination of hemoglobin with haptoglobin is due to the presence of exposed sites peculiar to the a chains. Studies on the effect of altered configuration of the hemoglobin mole-cule on the haptoglobin binding have also been pursued with deoxyhemoglobin. Perutz and coworkers noted that both the deoxy- and -.oxyhemoglobin H have a similar crystal lattice to deoxyhemoglobin A (6 2) and that deoxygenation of oxyhemoglobin A results in a markedly different quaternary structure, the contacts between the a i a 2 chains undergoing a change in dis-tance and angle of contact, while the 8 i $ 2 contacts are broken (64). To test the importance of the oxyhemoglobin configuration - 16 -for binding, Nagel et al. (65) determined the hemoglobin binding capacity of a l l types of haptoglobin which had been exposed to deoxyhemoglobin, by addition of labeled cyano-methemoglobin and measurement of the s p e c i f i c a c t i v i t y of the Hb-Hp complex. In a l l cases greatly decreased binding of deoxyhemogJjobh was observed and i n the presence of d i t h i o n i t e binding of deoxyhemoglobin to haptoglobin was almost absent. Since the 3 chains are reported to undergo the greatest con-formational change i n deoxygenation of the hemoglobin mole-cule (66), and since i t was found that iso l a t e d a chains were unable to form a complex with haptoglobin (61), i t was con-cluded that 3 chains i n the conformation found i n tetrameric oxyhemoglobin A pa r t i c i p a t e i n complex formation (65). The f a i l u r e of deoxyhemoglobin to bind haptoglobin was confirmed by Chiancone and collaborators (63) by sedimentation measure-ments i n the ultrac e n t r i f u g e . Furthermore, these workers demonstrated the i r r e v e r s i b i l i t y of the binding of haptoglobin to oxyhemoglobin, for once attached to haptoglobin, the hemo-globin could not be removed by deoxygenation. Further i n f o r -mation on the k i n e t i c mechanism of the Hb-Hp reaction has been provided by the recent study of Nagel and Gibson (59) using stopped flow measurements of the quenching of the aromatic amino acid fluorescence of haptoglobin upon binding of hemoglobin. Haptoglobin was found to bind a hemoglobin chains s p e c i f i c a l l y ; once a chains have interacted, rapid binding of 3 chains.follows. Their previous i n a b i l i t y to demonstrate binding with a chains - 17 -was attributed to d i l u t e solutions prepared by a less s a t i s -factory method. The reaction with i n t a c t hemoglobin may proceed v i a a hemoglobin subunit since the rate of the reac-t i o n does not increase i n a lin e a r manner with hemoglobin concentration and becomes r e l a t i v e l y slower at higher hemo-globin concentrations. Thus i t was postulated that the binding reaction proceeds either by consecutive binding of a and 8 hemoglobin monomers or by attachment of aB dimers through a chains. The l a t t e r scheme would be i n accordance with t h e i r findings that i n 2 M s a l t solutions the reac-tants show rates of binding i d e n t i c a l with the rate i n solu-tions of low io n i c strength, for i n 2 M s a l t hemoglobin exists largely i n forms of aB dimers (67). Consistent with these data would be a molecule of haptoglobin with two iden-t i c a l but independent binding s i t e s , which would explain the presence of the Hb-Hp intermediate complex observed on under-saturation of a haptoglobin solution with hemoglobin. The model of the haptoglobin molecule thus bears a remarkable resemblance to that of the immunoglobulin molecule. It i s apparent that oxygen or another similar ligand i s required to induce some form or state of hemoglobin which i s s p e c i f i c a l l y reactive to haptoglobin (63). Bunn (68) proposes that i t i s the reduced d i s s o c i a t i o n into a8 dimers i n the deoxy-form which i s the prime factor i n i t s lack of binding to haptoglobin, a conclusion a r i s i n g from his binding studies with a chemically modified hemoglobin, BME-Hb, which has cer t a i n physical and chemical properties i n common with deoxyhemoglobin. Simon and Konigsberg (69) reacted Hb A with a b i f u n c t i o n a l reagent, bis(N-maleimidomethyl)ether, (BME) to y i e l d a molecule with 2 moles of BME per Hb tetramer. One molecule of BME binds covalently with each g-93 cysteine-SH group and the second maleimide ri n g of each BME molecule under-goes reaction at an unknown s i t e with the same g chain (70). BME-Hb shows no co-operative i n t e r a c t i o n , i t s oxygen e q u i l i -brium curve i s hyperbolic and i s independent of pH. On the basis of various physical and chemical c r i t e r i a (69) and the X-ray analysis (70) , i t was established that horse BME-Hb does not change i t s conformation upon reaction with a ligand such as oxygen since i t i s already locked into the conforma-ti o n of the normal horse oxyhemoglobin. Human BME-Hb, on the other hand, resembled native deoxyhemoglobin i n i t s re-duced extent of di s s o c i a t i o n into dimers r e l a t i v e to normal oxyhemoglobin. When human hemoglobin was treated with six d i f f e r e n t s u l f h y d r y l reagents only the derivative with BME showed impaired binding to haptoglobin and only when a large excess of BME human Hb was used did the binding approach that with normal Hb. Moreover, once the modified hemoglobin 0 HC-C C-CH BME - 19 -was bound, a considerable amount of i t could be displaced by normal hemoglobin contrary to the stability exhibited by haptoglobin bound with unmodified hemoglobin. Since horse BME-Hb crystals are isomorphous with those of normal horse oxyhemoglobin (69), Bunn feels that i t is not conformational alteration in the molecule which precludes formation of a stable complex but rather i t is due to a reduced degree of reversible dissociation into symmetrical dimers in both BME-and deoxyhemoglobin which is responsible for impaired binding. This conclusion is based on the assumption of a haptoglobin molecule with two identical binding sites for two a$ dimers. However, although human BME-oxyhemoglobin shows limited dissociation under conditions which cause normal oxyhemo-globin to dissociate, a subsequent report (70) showed that horse BME oxyhemoglobin dissociated to the same extent as normal horse hemoglobin and indicates the need for further binding studies with horse BME-Hb. GENETICS The inheritance of the a chain of haptoglobins based on 1 2 a pair of autosomal alleles, Hp and Hp advanced by Smithies and Walker (12) has been shown to be essentially correct by a number of extensive pedigree studies by Galatius-Jensen (71, 72), Allison (73), Harris, Robson and Siniscalco (74), and Sutton and coworkers (7 5). Further subdivision of the hapto-globin phenotypes on the basis of the electrOphoretic di f -ferences in their a polypeptide chains shows that six common - 20 -IF IS haptoglobin types are determined by three allele s , Hp } Hp 2 and Hp (25). In the absence of reductive cleavage these d i f -ferences in the a chains are indistinguishable. Aside from the three common haptoglobin phenotypes 1-1, 2-1 and 2-2, a number of unusual phenotypes has been found, most of them occurring with very low frequency (76,77). Galatius-Jensen (78) and Harris et al. (79) both encountered a rare Hp phenotype, subsequently called Hp Caflberg (77), in which some bands corresponded in mobility with those found in the Hp 2-1 pattern while other bands were characteristic of the 2-2 phenotype. This pattern appears to result from a relatively decreased production of Hp a"*" polypeptides (77). The most frequent variant Hp phenotype is Hp 2-1M (Fig.3), designated thus by Connell and Smithies (81) because the rate of migration of the bands is the same as those in type 2-1 but the relative concentration of the f i r s t two bands are different and the remaining bands are either absent or scarcely discernible (82). Hp 2-1M occurs predominantly in Negro populations, the frequency of occurrence being 10% in American Negroes (82), but this phenotype is found at a very low frequency in a l l other racial groups (83,84). Subtyping IF studies of Hp 2-1M show that a normal amount of Hp a or IS 2 a is associated with a reduced amount of the usual Hp a polypeptide (77) (Fig. 3). Another variant phenotype which is much rarer than the Hp 2-1M, is Hp Johnson (Hp J) (Fig. 3) found originally in a Negro mother and her daughter by Giblett - 21 -a-Polypeptides of P u r i f i e d hapto- Hp haptoglobin. Urea-mercap-Hp globin. Starch- sub- toethanol starch-gel types gel electrophore^-types electrophoresis s i s Figure 3. SG-electropherograms of the haptoglobin pheno-types and t h e i r respective subtypes (80). - 22 -(30) but subsequently in widely separated racial groups (23, 85). The original phenotype appears to be a triplicated a polypeptide in heterozygous combination with an Hp IS chain (23) . Many other rare Hp phenotypes have been reported. Hp 1-P, Hp 2-P, Hp 1-H, Hp 2-H and Hp 2-L are believed to be heterozygous combinations of three rare alleles, Hp P3 IS Hp H and Hp L with one or another of the common Hp genes Hp , IF 2 Hp and Hp (76). Hp 1-B and Hp 2-B are reported to be a B 1 2 combination of a rare al l e l e Hp with the Hp and Hp genes (86). A B chain mutant is reputedly responsible for the phenotype, Hp 2-1 Bellevue (87). Hp Marburg is a variant which has the unusual property of not being able to combine with the normal proportions of hemoglobin (88). Hp 2-1D is 1D a phenotype ascribed to the expression of a new all e l e Hp which determines Hp a"*"0 which on subtyping migrates slightly IF ahead of the Hp a chain (89). Gene frequencies for the three common haptoglobin types assuming the mode of inheritance proposed by Smithies and Walker (12)), were estimated to be approximately 0.4 for the 1 2 Hp gene and about 0.6 for the Hp gene in various Caucasian populations assuming a Hardy-Weinberg distribution (12,72,90, 91,92,93). In addition, Sutton's group (90) observed signi-ficant differences between Caucasians and Africans of Liberia and the Ivory Coast, the frequency of the Hp gene in the latter case being 0.70 as compared to 0.42 for the former - 23 -group. They suggested that this t r a i t may be of value in anthropological studies. Sutton and coworkers (94) estimate 2 that the Hp frequencies range from a low of 0.09 for Malaya Indians to a high of 0.87 for Nigerian Negroes and 0.93 for 2 Lacandon Indians in Central America. A world map of the Hp gene frequencies (Fig. 4) has been computed by Parker and Beam (9 5) from a l l the published data and shows that the 2 Hp gene frequency rises in areas progresively farther re-moved from a particular area of India, and increases across Europe from east to west, and from south-east Asia through Alaska and North America to Central and South America. The 9 partial gene duplication theory of the evolution of the Hp IF IS a l l e l e from Hp and Hp (23) combined with the high fre-9 quency of the Hp gene in India led Ingram (96) to suggest that this gene arose in India and since presumably i t pos-7 sesses a selective advantage, is displacing the Hp gene. o In support of the recent evolution of the Hp gene is the finding that in certain primates and other mammals, only a single Hp band corresponding to human Hp 1-1 is seen on starch-gel electrophoresis (97). Thus the multiple bands 9 characteristic of the Hp gene appears to be a feature pe-culiar to humans since haptoglobin exists only in the monomeric form (55) in a l l other mammals studied so far. 1F Giblett and Brooks (8 0) reported that whereas the Hp gene 2 s is found at almost half the frequency of the Hp gene in Caucasians and occurs with almost equal frequency in Negro - 2 4 -Figure 4y\ World map of Hp' gene frequencies ( 9 5 ) . populations, the Hp gene i s extremely rare i n the Oriental population. This was confirmed by Shim and Beam (97), who extended the study and found that a l l Mongoloid populations and Australian aborigines (with the exception of c e r t a i n 1S Aborigines from Western Australia) have the Hp gene and do IF not possess the Hp gene. The occurrence of several haptoglobin types side by side i n several human populations represents a true polymorphism, but there i s no known reason for such v a r i a t i o n s , though i t 1 2 i s suspected that the Hp and Hp genes are now or have been i n the past influenced by one or more se l e c t i v e factors. E v i -dence for t h i s hypothesis has been presented i n a study (9 8) 2 i n which the Hp gene frequency differences between the West Afri c a n and the American Negro populations could not be accounted for on the basis of gene migration alone. They suggest that the Hp genes have d i f f e r e n t adaptive values i n the two populations, consequently the polymorphism may pos-s i b l y be unstable i n the d i f f e r e n t regions. A possible s e l e c t i v e advantage (24) may be i n f e r r e d from the studies on a recently discovered l i v e r enzyme, heme a-methenyl oxygenase. Nakajima and coworkers (99,100) reported the i s o l a t i o n and characterization of t h i s enzyme which i s present predominantly i n the l i v e r and kidney. The enzyme catalyzes the conversion of pyridine hemichromogen into f o r m y l b i l i v e r d i n ,(10 1 ) " by oxidative cleavage of the por-phyrin r i n g at the a-methenyl bridge, i n the presence of NADPH, - 26 -ferrous iron and an activator. The substrate s p e c i f i c i t y of this enzyme system i s of p a r t i c u l a r i n t e r e s t . Data indicate that the Hb-Hp complex rather than free hemoglobin i s the sub-strate i n this enzymatic reaction and suggest that under phys i o l o g i c a l conditions hemoglobin i s not metabolized i n i t s free form but requires combination with haptoglobin for con-version to f o r m y l b i l i v e r d i n , which i s then hydrolyzed by a second enzyme, heme a-methenyl formylase to b i l i v e r d i n . Study of the conversion of complexes of methemoglobin with the three genetic types of haptoglobin showed that the i n i t i a l v e l o c i t y of conversion i s greater i n the complex with Hp 2-2 than i n that with Hp 2-1 or Hp 1-1 (99). Thus i t i s possible o that the s e l e c t i v e advantage i n the Hp gene l i e s i n a more e f f i c i e n t metabolism of the hemoglobin complex into b i l e pig-ments. Consistent with t h i s hypothesis i s the established r o l e of the r e t i c u l o e n d o t h e l i a l system i n the catabolism of the complex (102,103). PHYSIOLOGY AND PATHOLOGY OF HAPTOGLOBIN Jayle, on observing the absence of haptoglobin i n sub-jects with hemolytic jaundice, suggested that haptoglobin plays a r o l e i n the elimination of extracorpuscular hemo-globin (104). Studies by numerous investigators had esta-blished that when the concentration of hemoglobin i n the plasma i s below a threshold l e v e l no hemoglobin can be detec-ted i n the urine. This l e v e l , c a l l e d the renal threshold, - 27 -was based on the assumption that hemoglobin circulates in plasma in the free state and passes through the glomeruli even at low plasma concentrations, only to be reabsorbed by the renal tubules. If the plasma concentration exceeds the threshold level, the concentration of hemoglobin in the glomerular f i l t r a t e exceeds the capacity of the renal tubules for reabsorption and hemoglobinuria results. In man, the hemoglobin threshold varies from 100-140 mg/100 ml according to the individual, being about 135 mg/100 ml in the majority of subjects (58). But on repeated daily injections of hemo-globin, the threshold level is lowered to less than half the original value and in pernicious or hemolytic states, less hemoglobin was required to produce hemoglobinuria. A re-markable coincidence between the hemoglobin binding capacity of haptoglobins and the renal threshold for hemoglobin led Laurell and Nyman (105) and Allison and ap Rees (58) to suggest independently a new explanation for this phenomenon. The normal hemoglobin binding capacity of haptoglobin varies widely in individuals 30-190 mg/100 ml (35) with an average value of about 100 mg/100 ml (73), that i s , just below the level claimed to represent the threshold level of the kidney. It was suggested that hemoglobin bound to haptoglobin does not traverse the glomeruli at a l l , as free hemoglobin does. If hemoglobin is present in plasma in amounts exceeding the binding capacity of haptoglobin, i t circulates in the free state and readily passes into the glomerular f i l t r a t e . Thus - 28 -when the intravascular release of hemoglobin i s continuous, a so in hemolytic anemias, or on repeated injections of hemo-globin, the rate of removal of the-complex may exceed the rate of replacement of haptoglobin, so that the haptoglobin l e v e l i n the plasma may f a l l to very low or undetectable l e v e l s , and therefore show a diminished threshold. In v i v o studies of the d i f f e r e n t i a l renal transport of free and pro-tein-bound hemoglobin by Lathem and Worley (106,107) and the studies of hemoglobin clearance with l a b e l l e d iron by Murray and coworkers (10 8) supported the determination of the renal threshold by haptoglobins. Thus one physiological function for haptoglobin may be to prevent the loss of hemoglobin from the body through the kidneys and hence to play a ro l e i n iron retention and to protect the kidney from siderosis produced by chronic entry of hemoglobin into the kidneys. The haptoglobin l e v e l , although i t exhibits a wide range i n the healthy population, i s s t r i k i n g l y constant i n the i n -d i v i d u a l . There appears to be a genetic mechanism involved i n regulating the quantity of haptoglobin, for the mean value i n sera of healthy indiv i d u a l s of type 1-1 was 136, type 2-1, 108 and type 2-2,- 82 mg„(54). Analysis of haptoglobin lev e l s i n monozygotic and dizygotic twins showed a lesser variance of the mean difference between i d e n t i c a l as contrasted to fr a t e r n a l twins (109) and supports the suggested r o l e of gene-t i c factors i n the regulation of haptoglobin l e v e l of the various types. Nyman (54) suggested that hormones influence - 29 -haptoglobin levels since men have a higher mean haptoglobin level than women. It has also been found that androgen ad-ministration to women increases haptoglobin levels (110) and estrogen administration decreases the level (111). However, extensive studies by other investigators (72,112) have re-vealed no significant difference in levels between males and females. There has been suggestive evidence of an increase in haptoglobin with age in adults-(112,113). In newborn: infants, a well established haptoglobin pattern is exceptional (71), about 90% of cord blood samples and samples collected during the f i r s t " 14;tdays of l i f e contain scarcely any hapto-globin (114). However, the haptoglobin level soon rises gradually during the f i r s t months of l i f e and reaches the mean level of healthy adults at 4 months (55). This absence or low concentration of haptoglobin was attributed to a low rate of synthesis in the newborn (114,115) rather than to an accelerated rate of erythrocyte destruction (116). Extensive investigations have been carried out on the variation of haptoglobin levels in pathological conditions, since the determination of haptoglobin may aid in the dif-ferential diagnosis of certain diseases and aid in following the prognosis of the disease. In a l l hemolytic disorders, the plasma haptoglobin level is either very low or absent (73). Haptoglobin is absent in pernicious anemia (73,117) but reappears within a few days of the beginning of treatment. Low levels of haptoglobin are also found in hepatocellular - 30 -f a i l u r e (54,104); this decrease was reported (54) to be due to an increased hemoglobin catabolism rather than to decreased haptoglobin. However, Owen, McKay and Got (118) believe reduced synthesis of haptoglobin to be the prime factor since they obtained some co r r e l a t i o n between haptoglobin and serum albumin l e v e l . Haptoglobin lev e l s increase with acute or chronic inflammation, tissue destruction or under d i f f e r e n t forms of stress and i n various malignancies (46,119). Hapto-globin belongs to the serum mucoprotein f r a c t i o n which be-haves as an acute phase reactant, i t s serum l e v e l r e f l e c t i n g the a c t i v i t y of the disease process (120). Thus changes i n serum haptoglobin l e v e l are analogous to other non-specific changes i n serum mucoproteins i n disease. However, in d i c a -tions are that although there i s a p a r a l l e l i s m i n the i n -crease of haptoglobin and orosomucoid, there i s no s t r i c t c o r r e l a t i o n (46,54,121). The close c o r r e l a t i o n between haptoglobin, fibrinogen and orosomucoid found i n patients with infectious diseases, based on the s t r i k i n g c o r r e l a t i o n between degradative changes i n connective tissue and high levels of c i r c u l a t i n g glyco-proteins (122), gave r i s e to Jayle's hypothesis on the o r i g i n and metabolism .of haptoglobin (104). Jayle postulated that haptoglobin i s synthesized by the fibrocytes i n the connec-t i v e tissue and i s depolymerized by the action of neuramini-dase and released. This hypothesis was no longer tenable when Murray and Connell (123) reported that the haptoglobin - 31 -level of the exudate withdrawn from the site of subcutaneous turpentine injections which had the effect of increasing the haptoglobin level in rabbits, was much lower than the hapto-globin level of the serum during the height of the rise. It is unlikely then that haptoglobin is released into the blood at the site of injection. The haptoglobin rise represents de novo synthesis rather than liberation from a tissue pool of the protein, since haptoglobin response to multiple in-jections is more intense and longer lasting (121,124) and puromycin and other inhibitors of protein synthesis prevented the elevation of plasma haptoglobin level (125). Several lines of evidence places the site of haptoglobin biosynthesis in the li v e r . After perfusion of rabbit liver with blood containing labelled amino acids (126) or radioactive leucine and galactose (127), the predominant incorporation of radio-activity is in the haptoglobin fraction. Carbon tetrachlor-ide-induced liver damage completely prevents incorporation of labelled amino acids into the haptoglobin fraction and greatly decreased the specific radioactivity of other proteins synthe-sized in the liver but did not affect the radioactivity of y-globulin which is known to be synthesized outside the liver (128). Partial hepatectomy also resulted in a striking re-duction in serum haptoglobin levels produced in response to injury (129). The factors responsible for stimulation of haptoglobin production are not known. Increased haptoglobin catabolism in connection with hemolysis is not an adequate - 32 -stimulus for haptoglobin synthesis, since the o r i g i n a l hapto-globin l e v e l i s not recovered u n t i l 5-7 days afte r t o t a l e l i -mination of haptoglobin (54). Krauss (130) has shown that the plasma haptoglobin l e v e l of adrenalectomized rats increases a f t e r turpentine i n j e c t i o n but to a lesser extent than that of i n t a c t r a t s , so that factors other than s o l e l y adrenocorti-c a l stimulation must be involved i n the stimulation of hapto-globin synthesis. The Hb-Hp complex i s cleared as a unit much more rapidl y than free haptoglobin from the plasma (131); the h a l f - l i f e of the former i s 88-120 minutes (132) and of the l a t t e r i s 4.5 days (133). As long as the concentration of the complex remains above 0.4 gm/1., i t i s removed from the blood stream at a constant rate; a f t e r t h i s , i t i s eliminated as an expo-nentia l function of time (134). I t i s established that the r e t i c u l o e n d o t h e l i a l system plays a major role i n the re-moval of the complex. Both Murray, Connell and Pert (10 2) and Keene and Jandl (103):. reported that the l i v e r accounted for most of the complex sequestered i n the r e t i c u l o e n d o t h e l i a l system. C e l l u l a r l o c a l i z a t i o n studies by means of s p e c i f i c immunofluorescence (135) and autohistography (134) indicate that the Kupffer c e l l s of the sinusoids of the l i v e r and the splenic c e l l s are the s i t e of catabolism of the Hb-Hp complex. Studies on the fate of uncomplexed haptoglobin by Krauss and Sarcione (132) showed that 4 days afte r i n j e c t i o n , the protein had lar g e l y degraded. Mouray et a l . (128) found that - 33 -1 3 1 the h a l f - l i f e of I-Hp was not altered by carbon tetra-chloride-induced liver damage. Therefore the liver also does not appear to play a major role in the degradation of uncomplexed haptoglobin. ASPECTS OF HAPTOGLOBIN STRUCTURE AND FUNCTION INVESTIGATED IN THIS WORK The present studies are concentrated on the reaction of haptoglobin with hemoglobin. In order to determine whether the site of polymerization of haptoglobin polymers is dis-tinct from the site of hemoglobin binding, partially resolved haptoglobin polymers are examined for their a b i l i t y to bind hemoglobin.both by the peroxidase assay and a newly developed Sephadex assay. Localization of the hemoglobin binding site on the component haptoglobin chains is achieved by determina-tion of the a b i l i t y of isolated a 1 , a 2 and 6 chains to bind hemoglobin by means of the Sephadex assay. Also, measurement of the binding of globin by haptoglobin confirms that the linkage between hemoglobin and haptoglobin is through the globin moiety. Furthermore, study of the reaction of hapto-globin with myoglobin, a protein similar in function and conformation to hemoglobin, and with various vertebrate hemo-globins provide additional information on the hemoglobin combining site. The effects of environmental factors on the combination of haptoglobin with hemoglobin has been studied by alteration of physical parameters, ionic strength and pH, and addition of a tyrosine analogue. The involvement of - 34 -amino groups of haptoglobin i n binding with hemoglobin has been studied by selec t i v e chemical modification of haptoglo-bin amino groups with three reagents of increasing severity followed by measurement of the a b i l i t y of the chemically modified proteins to bind hemoglobin. An approximate deter-mination of the area of the binding s i t e i n the Hb-Hp com-plex has been made by comparison of the extent of guanidina-ti o n of the amino groups of the i n d i v i d u a l components with that of the complex and also by comparison with the extent of modification i n dissociated hemoglobin and haptoglobin from the guanidinated complex. - 35 -EXPERIMENTAL GENERAL METHODS 1. STARCH-GEL ELECTROPHORESIS Starch gels, comprising 15 gram percent of concentrated starch-hydrolyzed (Connaught Medical Research Laboratories) in 0.0270 M boric acid, 0.0108 M NaOH buffer, pH 8.92, were prepared according to the procedure described by Smithies (9) employing plexiglas trays and covers whose dimensions are detailed diagramatically by Smithies (136). The plastic covers were inserted with either an 8 or 20 slot-former, then the cover and tray were lightly coated with Dow Corning 70 4 silicone f l u i d . After adding 500 ml of the borate buffer to the starch-hydrolyzed in a 1 1. suction flask, the flask was immediately swirled and then heated with continual swirling in a water-bath until the starch suspension had passed a sudden rise in viscosity and then showed a marked decrease in viscosity. Negative pressure was then applied to the starch solution for a few minutes. The degassed solution is immediately poured into the gel tray, the cover carefully lowered into place without trapping air bubbles and weights placed on the cover at the perimeter of the gel tray unti l the gel is cool. In preparation for the electrophoretic run, the cover is l i f t e d carefully and the samples inserted into the slots with Pasteur pipettes. Melted Hartz white petro-latum is poured into thin film over the gel surface. After the petrolatun had sol i d i f i e d , the end plates of the gel tray were removed and the gel is assembled in a vertical - 36 -position with the apparatus illustrated by Smithies (136) . The outer compartments of each electrode vessel were approxi-mately h a l f - f i l l e d with 10% NaCl solution and the inner com-partments f i l l e d to a slightly higher level than that of the salt solution with a bridge buffer of ten times the concentra-tion of the gel buffer. After electrical contacts between the two solutions in the electrode vessel and between the bridge solution and the gel were made with several thick-nesses of f i l t e r paper, the Ag/AgCl electrodes were immersed in the salt solutions and connected to a Heathkit IP-32 cohstantcvoltag.e power supply. Electrophoresis at a poten-t i a l gradient of 7 v/cm was applied for 16-18 hours at room temperature. A double staining technique (15) was used on each half of the gel after slic i n g longitudinally with a fine tungsten wire. The top half of the gel was stained about 5 minutes with a saturated solution of Amido Black 10B in a solvent of composition, methanol-.distilled water-.acetic acid in ratios of 50:50:10 v/v (9). The stained gel was washed once with water and several times with the dye solvent. Pro-tein components of the applied sample are revealed by a dark blue band. The bottom slice of the gel was stained with a benzidine reagent by mixing 1 ml of a saturated solution of benzidine in ethanol with 1 ml of glacial acetic acid, then adding 100 ml of d i s t i l l e d water and just prior to use, 0.5 ml of 30% w/v/ H2O2 (137). After several minutes the gel was washed a few times with water. Heme-containing compon-ents of the sample are detected by zones of blue color, which - 37 -is unstable and gradually fades to green and then to brown. 2. UREA STARCH-GEL ELECTROPHORESIS Starch-gels which contained 8 M urea were prepared in 0.05 M acetate buffer, pH 5.0 by the method of Smithies, Connell and Dixon (25). A starch gel in a concentration of 15 gram percent was prepared by thoroughly mixing 75 g starch-hydrolyzed with 240 gm of urea and the mixture gradu-ally added with vigorous mechanical stirring to 300 ml of 0.05 M acetate buffer contained in a metal beaker. With the beaker immersed in a water-bath and while under continual vigorous stir r i n g , the mixture was heated at 70° for about 7 minutes, after which time, the resulting viscous solution was poured into a previously warmed 2 or 3 1. suction flask and degassed with a good water aspirator. The gel was then poured into a gel tray and used within 48 hours. 3. POLYACRYLAMIDE GEL ELECTROPHORESIS Polyacrylamide gels were prepared according to the pro-cedure of Cruft (138) with slight modifications. A solution was made of 50 gm (10%) of acrylamide (Matheson, Coleman and Bell) and 1 gm of N,N'-methylenebisacrylamide (0.2%) in 500 ml of 0.0270 M boric acid, 0.0108 M NaOH buffer, pH 8.92. After the solution was degassed, 5 ml of 10% v/v tetramethylethylene-diamine in ethanol and 5 ml of 10% w/v of ammonium persulfate in d i s t i l l e d water were added to the solution which is rapidly swirled and immediately poured into the gel tray. The cover - 38 -was carefully lowered into place without trapping air bubbles and weights placed on the cover at the perimeter of the gel tray. The cover adhered to the gel tray through a thin film of petrolatum on the upper surface of the perimeter of the gel tray. After the gel has set i t was ready for use. 4. PREPARATION OF HEMOGLOBIN (a) The hemoglobin solutions employed in the electro-phoretic runs were prepared from sedimented erythrocytes of human blood treated with ACD (sodium citrate, c i t r i c acid) anti-coagulant (by the kind cooperation of the Canadian Red Cross Blood Bank, Vancouver, B.C.). The red blood cells were washed at least twice with a physiological saline solution and separated from the saline solution by centrifugation after each washing. The washed pellet which remained after centrifugation was lysed with 9 times i t s volume of dis-t i l l e d water. The stroma was removed by centrifugation and 2 drops of toluene was added to the supernatant solution to discourage bacterial growth. A small portion of this stock hemoglobin solution was stored at 4 ° for current use, the remaining solution was stored frozen in several small bottles. The hemoglobin control solutions used in electrophoresis was prepared from the stock hemoglobin solution by adding 1 drop of the stock solution to 5 drops of d i s t i l l e d water. When hemoglobin was added to column effluents or to purified haptoglobin preparations, 1 drop of the stock hemoglobin - 39 -solution was added to 5 drops of the column eluate or to a 1% haptoglobin solution. (b) Hemoglobin used in the peroxidase activity assays or in the Sephadex assays was in the form of the carbonmonoxy derivative, which was prepared from twice-crystallized hemo-globin according to the technique of Drabkin (139) for human blood treated with ACD anti-coagulant. A stroma-free solu-tion of about 8-10 mmoles Hb/1. concentration, referred to a molecular weight equivalent of 16,700, was obtained in the following manner (140): The red blood cells were separated from the plasma and washed once with a physiological saline solution, then washed three times with a mixture of 1.2% saline and 0.0025 M A1C13. The packed cells after dilution with an equal volume of d i s t i l l e d water were thoroughly mixed with 0.4 volume of toluene. After refrigeration overnight the mixture was centrifuged and the clear hemoglobin solution was siphoned off. Concentration of this hemoglobin solution was accomplished by dialysis in a 2.8 M phosphate buffer, pH 8.0 composed of 371 gm of K2HPCv3H20 and 160 gm of KH2P04 made up to 1 1., with a ratio of 1:3 volume within and out-side the dialysis sac. Thus, 498 ml of phosphate buffer previously warmed to 37° was used to dialyze 166 ml of hemoglobin solution in the cold room for a period of 6-10 hours. The dialyzate was then replaced with an equal volume (820 ml) of fresh unwarmed 2.8 M phosphate buffer and dia-lysis continued for a further 24 hours. At the end of this - 40 -period of time, more 2.8 M phosphate buffer is added slowly to the dialyzate until microscopic examination of the contents of the dialysis sac revealed that crystallization had occurred. The material was then l e f t in the cold room to dialyze a further 24 hours. Recrystallization of the hemoglobin was carried out in a similar manner on a solution of the hemoglobin crystals collected by suction f i l t r a t i o n . The crystals ob-tained were a mixture of small and large sized crystals of irregular shapes and sometimes in the typical bipyramidal shape. The twice-crystallized hemoglobin crystals were dissolved in 55 ml of d i s t i l l e d water and dialyzed against four changes of glass-distilled water. The carbonmonoxy derivative was prepared by saturating 230 ml of the dialyzed hemoglobin solu-tion with carbon monoxide gas. The solution was then lyo-philized to yield 13.7 g carbonmonoxyhemoglobin, which was stored in the frozen state. (c) Hemoglobin solutions of various species of animal were prepared from heparinized blood, the concentration of heparin being lower than the amount which is said to i n f l u -ence the hemoglobin binding capacity or peroxidase activity (54), with the exception of horse blood which was collected in Becton-Dickdnson vacuotubules containing EDTA. The red blood cells .were sedimented by centrifugation and washed 5 times with cold 0.9% sodium chloride solution. In the case of the dog, rabbit and rat hemoglobin, 5 volumes of d i s t i l l e d - 41 -water was added to the-sedimented erythrocytes, i n the other animal hemoglobins, 9 volumes of d i s t i l l e d water was added. After allowing the material to stand overnight at 4°, the hemoglobin solution was separated from the stroma by c e n t r i -fugation. - 42 -PART I ISOLATION OF HAPTOGLOBIN AND PARTIAL SEPARATION OF HAPTOGLOBIN POLYMERS INTRODUCTION Haptoglobin was f i r s t isolated in the form of the Hb-Hp complex by van Royen (141) on the basis of the observation by Jayle and Gillard (5) that the complex is more soluble in ammonium sulfate than haptoglobin. But a l l attempts to re-cover haptoglobin from i t s complex were unsuccessful. Purified haptoglobin was f i r s t isolated-, by Jayle and Boussier (6) in 19 54 from the urine of a nephrotic child by successive ammonium sulfate fractionation of the urinary proteins to yield a Hp 1-1 preparation which was homogen-eous by electrophoresis and sedimentation (6,7) and by Im-munoelectrophoresis (37). In 1958, Boussier (142) succeeded in isolating a haptoglobin of type 2-2 from human serum by successive ammonium sulfate fractionation, the f i n a l hapto-globin-rich fraction being precipitated with 50% ammonium sulfate. An additional step of purification of the protein by an improved method of zone electrophoresis was subsequently introduced (143). By this method haptoglobin can be prepared with 80-100% purity, but the yields are extremely low, being from 10-15% (144). The technique of Laurell (57) is also based on ammonium sulfate fractionation. His source of haptoglobin was ascites f l u i d obtained from cancer patients with a high level of - 43 -haptoglobin. The ascites f l u i d was f i r s t precipitated twice with ammonium sulphate, and followed by acetone precipitation in the cold. Further precipitation with ethanol at low ionic strength was used to remove impurities, notably Hb-Hp complex. A f i n a l two-step salt precipitation produces a 70-90% pure Hp 1-1 while Hp 2-2 preparations are usually contaminated with a component with higher mobility, which can be removed by zone electrophoresis. Cloarec and Moretti (144) found that the successive precipitations with acetone in Laurell's method risked denaturation of the protein and that the ethanol precipitation is unnecessary. By modification of Laurell's method, Cloarec and Moretti obtained- 2-5% of 90-100% pure Hp 2-1 or Hp 2-2. Purified haptoglobin has also been isolated by Steinbuch and Pejaudier (145) during the routine fractionation of human plasma. Conn's fraction IV, the only fraction contain-ing haptoglobin in significant quantities, was obtained at pH 5.8 by precipitation with ethanol. Successive additions of 0.5% rivanol (a cationic detergent) to this fraction IV removed the ceruloplasmin, and albumin was removed by precipitation with 3.5% ethanol of the supernatant at pH 5.9, followed by rivanol fractionation at pH 8.5. Haptoglobin could then be separated from transferrin by alcoholic pre-cipitation at pH 4.4-4.6. A preferable method of removing the transferrin contaminant is by chromatography on DEAE-cellulose in 0.03 M acetate buffer, pH 5.0. At this pH hapto-- 44 -globin is adsorbed whereas transferrin is not. Elution is effected with 0.5 M acetate buffer at the same pH and a haptoglobin preparation of about 80% purity is obtained (146). Steinbuch and Quentin (147) subsequently used DEAE-cellulose chromatography in a rapid method of isolation of haptoglobin from whole plasma. After dilution of the plasma and adjusting the pH to 5.0 with 0.3 M acetic acid, i t is chromatographed on DEAE-cellulose in 0.0 3 M acetate buffer, pH 5.0. Adsorp-tion of haptoglobin at this pH is less selective than at lower pH values but by development with 0.1 M acetate buffer, pH 5.0, haptoglobin is eluted whereas ceruloplasmin and other proteins are retained. By this rapid technique 20% of Hp 1-1 in 90-100% purity and 8-30% of Hp 2-1 in 90-95% purity could be obtained (144). This method is applicable to samples as small as 2-4 ml as well as to volume of 1 1. or more. Connell and Smithies I n 1959 (81) introduced the f i r s t anion-exchange chromatographic technique for the isolation of haptoglobin. In this elegant method, serum was dialyzed against a pH 4.2 buffer comprised of 0.2 M acetic acid and 0.04 M NaOH and then chromatographed on Dowex 2-X10 at pH 4.2. At this pH, haptoglobin whose isoelectric point is about 4.2 (46) is the main serum protein bearing a net nega-tive charge while most other serum proteins have a net posi-tive charge. Thus i t is observed that haptoglobin of pro-gressively greater purity is obtained as the pH of adsorption is decreased from 5.5 to 4.3. However, adsorption below - 45 -pH 3.9 yields no haptoglobin. By this single step procedure of adsorption and desorption haptoglobin in 48% yield is obtained. However, under conditions of such an acidic pH, haptoglobin in whole serum is denatured rapidly (148) . Connell and Shaw (148) improved the preparation of hapto-globin and made the method more convenient on a large scale by using DEAE-cellulose. DEAE-cellulose is employed as the adsorbent because i t s capacity for protein is several hundred-fold greater than Dowex 2. A pH of 4.6 was chosen for ad-sorption since below pH 4.4 haptoglobin is rapidly denatured as indicated by loss of a b i l i t y to form a complex, while above pH 4.8 the selectivity of adsorption decreases and less pure preparations are obtained. After the serum is adjusted to pH 4.6 with 1.0 M acetic acid, i t is desalted on a Sepha-dex G-25 column. The desalted serum is successively treated with batches of 0.5-2.0 gm:of DEAE-cellulose. The absorbent had been previously washed with 1 N NaOH, followed by several washings with water and titrated to pH 4.6 with 1 N HC1. The DEAE-cellulose is then packed in a small column and eluted with 0.2 M NaCl. The eluates which are considered s u f f i -ciently rich in haptoglobin as determined by measuring the hemoglobin binding capacity (81) are diluted with water to give an optical density of 10.0 at 280 my. Haptoglobin is then precipitated by treatment with ammonium sulfate at 55% saturation and a solution of the precipitate is dialyzed and lyophilized. Haptoglobin of approximately 94% purity is - 46 -obtained in 32% yield. Smith, Edman and Owen (31) have modified the method of Connell and Shaw. Pooled human plasma is dialyzed against 0.01 M acetate buffer, pH 4.7 and then chromatographed on a DEAE-cellulose column equilibrated with the same buffer. The column is washed with 0.01 M acetate buffer containing 0.01 M NaCl, after which the haptoglobin is eluted with ace-tate buffer containing 0.04 M NaCl adjusted to pH 4.7. The effluent is brought to pH 7.0 and the volume reduced on a rotary evaporator. The solution is dialyzed against water and then lyophilized. A further fractionation with ammonium sulfate is necessary to purify the protein preparation. Hp 2-1 and 2-2 preparations appeared pure on starch gel electro-phoresis; however, Hp 1-1 preparations contained traces of impurities. With a slight modification in the elution tech-nique, Cloarec and Moretti (144) reported obtaining pure Hp 1-1 in excellent yields, 75%, but the Hp 2-1 was partially denatured. It is apparent that the method of isolation of hapto-globin by the methods of Jayle or Laurell are too laborious and the yields of protein entirely too low. Also certain of these methods (31,57,81) may yield partially denatured products. Connell and Shaw's method appeared to be the method of choice since a f a i r l y good yield of protein of high purity is obtained. Therefore at the start of this study, haptoglobin was isolated from pooled human plasma of the same - 47 -phenotype determined by at least two typings. However, the haptoglobin was easily denatured during preparation by this technique and when native haptoglobin was obtained, the yield was extremely low. A procedure was then followed which was adapted from'both the methods of Connell and Shaw and Smith, Edman and Owen. Again extremely small amounts of pro-tein was obtained from the pooled plasma. Thus a method was developed in which ascites f l u i d was used as the source of the protein, a method which could be easily adapted to pre-paration of large amounts of genetically homogeneous hapto-globin in high purity. Ascites f l u i d , which was f i r s t used by Laurell (57), proved to be an excellent source of hapto-globin since large quantities (up to 25 1.) can be obtained from a single patient, thus ensuring genetic homogeneity. It has been reported that in certain malignancies the hapto-globin level is elevated (54,104). It was observed during this study that ascites f l u i d associated with abdominal carcinoma generally had a high level of haptoglobin when examined by starch-gel electrophoresis and thus this source of ascites f l u i d was used for isolation of haptoglobins. PURIFICATION OF HAPTOGLOBIN AND PARTIAL SEPARATION OF POLYMERS 1. PREPARATION OF HAPTOGLOBIN BY CONNELL AND SHAW METHOD Plasma was aspirated from human ACD-treated blood in which the cells had settled after standing in the cold room. A sample of the plasma was gentically typed once on a 20 slot - 48 -starch gel to enable a large number of samples to be examined and once on an 8 slot gel to confirm the typing. Plasma from each donor was stored in the frozen state either individually or pooled according to phenotype and allowed to thaw prior to a preparation. Clotting of the plasma was promoted by the addition of an amount of 3.0 M CaCl 2 equivalent to the amount of citrate present. After standing overnight at 4° the clot was removed by centrifugation. Approximately 350 ml of pooled serum of type 1-1 was adjusted to pH 4.6 with 1.0 M acetic acid and then desalted on a 60 cm x 9 cm Sephadex G-25 (coarse) column. The ad-sorbent was prepared by suspending 25 g of DEAE-cellulose, 0.94 meq/g capacity (Selecta-cel, Schleicher and Schuell) in 1 1. of NaOH and stirring, for 30 minutes. After suction f i l t r a t i o n , the f i l t e r cake was washed with 2-3 1. of water and then suspended in water and titrated to pH 4.6 with 1.0 N HC1. The concentration was then adjusted to 2 5 mg/ml. The desalted serum was treated with 6 successive 1 g.v lots of DEAE-cellulose suspension. After stirring for 15 minutes, each lot of adsorbent was f i l t e r e d by suction, using Eaton Dikeman coarse f i l t e r paper and then washed several times with d i s t i l l e d water. A suspension of the DEAE-cellulose in water was poured into a column and elution carried out with 0.2 M NaCl. A faint green band moves with the sodium chloride front and haptoglobin was eluted practically at the salt front. A starch-gel electropherogram of the 6 eluate fractions indicated - 49 -that the f i r s t three eluates had the highest haptoglobin content. Combination of these haptoglobin-rich eluates gave an O.D. of 18 at 280 my. The solutions were diluted to an O.D. of 10 followed by precipitation of the haptoglobin i with ammonium sulfate to 55% saturation. The precipitates-was collected by suction f i l t r a t i o n . It was dissolved in 4.5 ml of water and the solution dialyzed exhaustively against d i s t i l l e d water and then lyophilized. Yield of hapto-globin was 132.6 mg. Electrophoresis on starch gel indicated that the protein combined readily with hemoglobin to give the typical Hb-Hp complex and that there were only traces of impurities. Although this particular preparation of haptoglobin yielded native product, often the preparation was found to be partially or wholly denatured as demonstrated by the lack or the very low binding of hemoglobin. Also the yield of haptoglobin was too low and i t was f e l t that this could be improved upon. Therefore, a method of preparing haptoglobin by modification of the Smith, Edman and Owen method and the Connell and Shaw method was attempted in order to overcome these disadvantages. 2. PREPARATION OF HAPTOGLOBIN BY MODIFICATION OF SMITH, EDMAN AND OWEN, AND CONNELL AND SHAW METHODS. Serum from 2 pints of whole ACD-treated human blood was dialyzed against a buffer of 0.01 M acetic acid buffer, ad-- 50 -justed to pH 4.7 with NaOH referred to as 0.01 M sodium acetate buffer, pH 4.7 hereafter. The dialyzate was changed several times. During dialysis an insoluble precipitate formed in the dialysis sac, this was removed by centritfugation at the end of the dialysis. The pH of the serum after the dialysis was 4.8; this was adjusted to pH 4.7 with 1.0 M acetic acid. The serum was then applied to a DEAE-cellulose column (5 cm x 7.5 cm) equilibrated in 0.01 M sodium acetate buffer, pH 4.7. The column was washed with 14 0 ml of the same buffer. A narrow pale green band could be seen at the uppermost part of the column. Elution was carried out with a linear salt gradient made with 100 ml of 0.01 M NaCl in 0.01 M sodium acetate buffer, pH 4.7 and 100 ml of 0.1 M NaCl in 0.01 M sodium acetate buffer, pH 4.7. The f i n a l concentration of NaCl proved to be insufficient to elute the proteins, there-fore the column was eluted with an additional 50 ml of buffered :0..I MlNaCl followed by 0.2 M NaCl in the acetate buffer until the elution was complete. The protein-rich effluents were pooled into three fractions and the pH adjusted to approxi-mately 7.0 for frozen storage since in this particular case i t was inconvenient to continue work on the preparation. After four days of storage, the fractions were thawed and dialyzed against several changes of d i s t i l l e d water, then lyophilized. Starch-gel electrophoretic pattern of the frac-tions revealed that the preparations were rich in haptoglobin, but albumin and post-albumin impurities were present. Further - 51 -purification of the protein was carried out by saturation with 55% ammonium sulfate. However, this procedure caused considerable denaturation of the protein as demonstrated by the considerable amount of insoluble material after dialy-sis of a solution of the precipitate against d i s t i l l e d water and by the large amount of dark brown heavy precipitate after lyophilization. The starch gel of the undenatured haptoglo-bin showed that most impurities had been removed, but a fast-migrating contaminant was s t i l l present. Therefore, an additional purification step was carried out on an analytical DEAE-cellulose column (2 cm x 95.5 cml equilibrated with 0.005 M sodium acetate buffer, pH 5.5. The column was eluted with a gradient system of 300 ml of 0.005 M acetate buffer, pH 5.5 and 300 ml of the same buffer containing 0.1 M NaCl. Elution was completed with 300 ml of 0.2 M NaCl in 0.005 M acetate buffer. The haptoglobin fractions were combined, dialyzed and lyophilized. This method of preparation of haptoglobin is unsuitable for a number of reasons. Besides the low yield, the method is tedious and the ammonium sulfate purification step causes partial denaturation of the product. Also, the impurities are not completely removed even after the second purification step. Therefore another procedure was developed and ascites f l u i d was used as the source of material with the objective of increasing the yield of protein by choosing ascites f l u i d from cancer patients with a high haptoglobin level. - 52 -3. PURIFICATION OF HAPTOGLOBIN BY A NEW METHOD AND PARTIAL SEPARATION OF POLYMERS Ascites f l u i d , which had been previously classified according to haptoglobin type by i t s SG electropherogram, was decanted from clots and frozen for storage and thawed as needed for preparation of haptoglobin. After centrifugation of any remaining clots from freshly thawed f l u i d , haptoglobin was precipitated by addition of solid ammonium sulfate to 470 ml of ascites f l u i d of type 1-1 and to 505 ml of type 2-1 ascites f l u i d to give a f i n a l salt concentration of 55% saturation. Each haptoglobin type was subsequently treated in parallel. After 1 hour of stirring in an ice-bath, the precipitate was collected by centrifugation. A 100 ml solution of the precipitate in 0.01 M acetic acid, pH 4.7 (with NaOH), was dialyzed against the same buffer in the cold room. The dialyzate was changed 3-4 times. At the end of dialysis, some precipitated protein, proven to not contain haptoglobin by starch-gel electrophore-sis, was removed by centrifugation. The supernatant solution was applied to a preparative DEAE-cellulose column (5 x 9.5 cm) which was equilibrated with 0.01 M sodium acetate buffer, pH 4.7. The column was washed with the same buffer until the washings showed an absorbance at 280 my lower than 0.02 O.D. units.. A faint green band near the top of the column could be observed. Haptoglobin was eluted with a linear gradient consisting of 300 ml of 0.01 M NaCl in 0.01 M sodium acetate - 53 -buffer, pH 4.7 and 300 ml.of 0.3 M NaCl in 0.01 M sodium acetate buffer, pH 4.7. The absorption at 280 my, the pH and the con-ductivity at 0° of the effluent fractions were measured. Ver-t i c a l starch-gel electrophoresis of the column eluates was also carried out to monitor the appearance and the purity of the haptoglobin peak. In the DEAE-chromatography of Hp 1-1 (Fig. 5), fractions from the f i r s t half and those from the last half of the optical density peak were combined in separate pools. In the Hp 2-1 chromatography (Fig. 6), fractions from the peak designated A and those from the peak designated B were separately pooled. These combined eluates were dialyzed against several changes of d i s t i l l e d water and then lyophil-ized. From 470 ml of Hp 1-1 ascites f l u i d , 1.618 g of crude haptoglobin was obtained, and 505 ml of Hp 2-1 ascites f l u i d yielded 0.9692 g from peak A and 1.066 g from peak B. In the elution profile of the preparative DEAE column of Hp 1-1 ascites f l u i d , (Fig. 5), a single 280 my absorbing peak is observed. The SG electropherogram of the DEAE column effluent fractions indicated that the haptoglobin-rich frac-tion corresponded to the optical density peak, as revealed by the benzidine-stained half of the gel. The amido black-stained section of the gel showed that at this stage of i t s preparation, haptoglobin is contaminated with mainly albumin, some pre- and post-albumins, transferrin and minute amounts of a slow-migrating impurity (Fig. 7). In the DEAE chromatography of the ascites, f l u i d of type - 54 -Figure 5. DEAE-cellulose chromatography of crude Hp 1-1 on a 5.0 x 9.5 cm column in 0.01 M sodium acetate buffer, pH 4.7. - 55 -Effluent Volume , ml Figure 6. DEAE-celiluiese chromatography of crude Hp 2-1 on a 5.0 x 9.5 cm column in 0.01 M sodium acetate buffer, pH 4.7. - 56 -Figure 7. SG-electropherogram of the DEAE-cellulose chromato-graphy column e f f l u e n t f r a c t i o n s of crude Hp 1-1 commencing at 250 ml through to 440 ml. (Slots are numbered consecutively l e f t to r i g h t for each 10 ml. f r a c t i o n ) . Figure 7. SG-electropherogram of the DEAE-cellulose chromato-graphy column effluent fractions of crude Hp 1-1 commencing at 250 ml through to 440 ml. (Slots are numbered consecutively l e f t to r i g h t for each 10 ml f r a c t i o n ) . - 57 -2-1, the elution diagram (Fig. 6) shows 2 major peaks, a larger front-running peak merging with a heterogeneous slow-running peak. The SG electropherogram of these effluent fractions shows that the foremost optical density peak has a higher haptoglobin content with the haptoglobin reaching a maximum corresponding to the maximum 280 my absorption of peak A and gradually declining through peak B. This crude haptoglobin preparation also contains some albumin and post-albumin impurities. Further purification of the haptoglobin was achieved by gel f i l t r a t i o n on Sephadex G-200. Approximately half the crude Hp 1-1, fraction A Hp 2-1, or fraction B Hp 2-1 were each treated in the following manner: the haptoglobin was dissolved in 5 ml of 0.05 M ammonium acetate buffer, pH 8.5 and chromatographed on a Sephadex G-200 column (2.5 cm x 186 cm) in 0.05 M ammonium acetate, pH 8.5 buffer. Column effluents were monitored at 280 my and analyzed for hapto-globin content by starch-gel electrophoresis and the Hp 1-1 fractions combined and lyophilized. In the case of the Hp 2-1 chromatography, the eluates were individually lyophilized to preserve the partial resolution of haptoglobin polymers obtained on gel f i l t r a t i o n . Final yield of purified Hp 1-1 is 988.9 mg from 470 ml of ascites f l u i d and 740.7 mg of Hp 2-1 polymers from 505 ml of ascites f l u i d . The Sephadex G-200 profile (Fig. 8) of Hp 1-1 shows a small peak running ahead of the main protein fraction, Figure 8. P u r i f i c a t i o n of Hp 1-1 on Sephadex G-200 (2.5 cm x 186 cm) i n 0.05'M ammonium acetate, pH 8.5. - 59 -followed by some small molecular weight components. It may be seen that good resolution of haptoglobin from i t s contam-inants can be achieved by gel f i l t r a t i o n and by omitting the overlapping protein fractions when i s o l a t i n g the haptoglobin peak, a highly p u r i f i e d haptoglobin preparation i s obtained. A SG-electropherogram (Fig. 9) of representative fractions of the Sephadex G-20 0 column shows that the main peak i s very r i c h i n haptoglobin while the small peak ahead of i t i s a high molecular weight impurity and the slow eluting proteins are albumin and post-albumins. A SG-electropherogram of the f i n a l Hp 1-1 product af t e r l y o p h i l i z a t i o n of combined f r a c -tions of the main peak shows that i t combines with hemoglobin to give a sharp and intense Hb-Hp band with only a minute amount of a slow-migrating impurity being present. Peroxi-dase and Sephadex assays (to be described i n a subsequent section) indicate that the haptoglobin combines i n an approxi-mately equimolar r a t i o with hemoglobin. U l t r a c e n t r i f u g a l analysis of another Hp 1-1 prepared by the same method con-firms the e s s e n t i a l homogeneity of the product, there being only a trace of impurity. Thus the haptoglobin i s of a very high degree of purity and i s f u l l y native. The Sephadex G-200 elut i o n diagram of the proteins of both peaks A and B following DEAE-cellulose chromatography of the Hp 2-1 preparation may be seen i n Figure 10. The i n i t i a l peaks i n both cases consist of haptoglobin. A starch gel (Figure 11) of consecutive fractions throughout the hapto-- 60 -+ 1 1 2 3 4 5 6 7 8 Figure 9. SG-electropherogram of fracti o n s following Sepha-dex G-200 chromatography (Fig. 8) of Hp 1-1. Protein f r a c -tions at the following e l u t i o n volumes: il) 280 ml, (2) 420 ml, (3) 450 ml, (4) 490 ml, (5) 530 ml, (6) 560 ml, (7) 590 ml, (8) hemoglobin co n t r o l . - 60 -+ 0 I 1 2 3 4 5 6 7 8 Figure 9. SG-electropherogram of fractions following Sepha-dex G-200 chromatography (Fig. 8) of Hp 1-1. Protein f r a c -tions at the following e l u t i o n volumes: $1) 280 ml, (2) 420 ml, (3) 450 ml, (4) 490 ml, (5) 530 ml, (6) 560 ml, (7) 590 ml, (8) hemoglobin control. - 61 -T 1 —r 1 1 1 1 r-\ Fraction Number Figure 10. Purification of crude Hp 2-1, peak A and B from DEAE-cellulose chromatogrphy (Fig. 6) on Sephadex G-200 (2.5 cm x 18 6 cm) in 0.05 M ammonium acetate, pH 8.5. Figure 11. SG-electropherogram of Hp 2-1 polymer fractions from Sephadex G-200 chromatography commencing at f r a c t i o n number 28 consecutively through to f r a c t i o n number 46. (Slots are numbered l e f t to r i g h t ) . - 63 -globin peaksshows a p a r t i a l resolution of polymers ranging from predominantly slowly migrating ones to predominantly rapid l y migrating ones. There are no d i s c e r n i b l e impurities i n the SG electropherogram of the polymer f r a c t i o n s . Sepha-dex assays of the polymer fractions gives an average value of 1.05 moles hemoglobin bound per 9 5,00 0 gm haptoglobin. This indicates that the haptoglobin preparation i s close to 100% purity. A SG electropherogram of the i n i t i a l stages of the Hp 2-1 preparation i s shown i n Figure 12. The s l o t s numbered from the extreme l e f t contain the following samples to each of which hemoglobin had been added and the r e s u l t s are as follows: (1) the untreated Hp 2-1 ascites f l u i d which shows a high haptoglobin l e v e l , (2) the insoluble p r e c i p i t a t e formed during d i a l y s i s of 55% ammonium sulfate p r e c i p i t a t e i n which no haptoglobin i s d i s c e r n i b l e , (3) the 5,5% ammonium sulfate p r e c i p i t a t e , r i c h i n haptoglobin, (4) the solution a f t e r DEAE-cellulose adsorption, depleted of haptoglobin, (5) the DEAE-cellulose at the height of peak A (Fig. 6), the f r a c t i o n r i c h e s t i n haptoglobin which has bound a l l the added hemoglobin and i s contaminated with some impurities, (6) the DEAE-cellulose f r a c t i o n midway on the declining slope of peak A (Fig. 6), the haptoglobin l e v e l i s decreasing - 6 4 -WW 1 2 3 4 5 6 7 8 Figure 1 2 . SG-electropherogram of Hp 2 - 1 during the i n i t i a l stages of i t s preparation (for i d e n t i f i c a t i o n of s l o t s r e f e r to text, p. 57). Figure 12. SG-electropherogram of Hp 2-1 during the i n i t i a l stages of i t s preparation (for i d e n t i f i c a t i o n of slo t s refer to text, p. 5 7 ) . - 65 -as judged by a small amount of excess hemoglobin, (7) the foremost part of peak B, DEAE-cellulose column (Fig. 6), haptoglobin level is decreasing and amount of im-purities increasing, (8) the latter part of peak B, DEAE-cellulose column (Fig. 6), a large excess of hemoglobin and a lower level of haptoglobin present. A purification table based on the preparation of Hp 1-1 from ascites f l u i d obtained from another carcinoma patient by the same method is shown in Table I. The molecular weight of Hp 1-1 was f i r s t reported as 85,000 by Jayle and Boussier (104) but later studies suggested a molecular weight of 100,000 (149). The most recent value reported by this group (150) is 98,770 ± 2270. Since both the molecular weight determination by Mr. Choy Hew and the value obtained with succinylated Hp 1-1 in these studies (see p. 151) suggest a molecular weight of approximately 95,000, the calculations here are based on this value. A molecular weight of 66,800 is used for hemoglobin. It can be seen that ammonium sul-fate fractionation of the ascites f l u i d gave almost a 3-fold purification. The single DEAE-cellulose peak was divided into three approximately equal fractions, with fraction C representing the t a i l end of the peak, so that fractions A and B containing the bulk of the haptoglobin, showed approxi-mately 5-fold enrichment. After Sephadex purification, the main haptoglobin fraction B more than doubled in specific TABLE I P u r i f i c a t i o n of H a p t o g l o b i n 1-1 STEP TOTAL VOLUME (ML) PROTEIN OD 280 my TOTAL PROTEIN VOL. x OD 280 my TOTAL 4 07 UNITS OD 40 7 x VOL. x DIL'N TOTAL Hb BOUND (MG) TOTAL Hp (MG) TOTAL 40 7 TOTAL 28 0 PURIF. YIELD 1. Crude a s c i t e s f l u i d 600 15.8 9480 3039 445 586.6 0.320 1 100 2. 55% ammon-ium s u l -f a t e p r e -c i p i t a t e 242. 5 12. 2 2959 2683 392.8 517.6 0.907 2 . 8 3 8 8 . 3 3 . DEAE-cellu-l o s e Peak: A 185 1.73 320 .1 518.6 1 . 6 2 5 . 0 6 B 170 3.94 6 6 9 . 8 1013.0 233.6 1307.8 1 . 5 1 4 .73 } 52. 5 C 201. 5 0 . 5 2 104.8 6 3 . 25 } 0. 60 1 . 89 5 4. Sephadex G-200 A 22. 7 1 . 32 29.96 9 . 4 3 0.315 0 . 9 8 } B 8 9 . 7 4 .44 398. 3 1392 2 5 8 . 1 •340.1 3.49 10.92 158.0 C 9 2 . 5 2. 69 248.8 361. 7 1 1 . 4 5 4. 53 I 5. ' A & C r e - r u n 21.4 1 .94 41. 52 154.7 ( 3 . 7 3 11.66 1 6. B r e - r u n 116.5 2.68 312.2 1241 204.4 •269.3 3 . 9 8 12.42 145.9 - 67 -a c t i v i t y . Fractions A and C are the front and back overlap sections containing considerable impurities and therefore these fractions were r e p u f i f i e d . Fraction B was also re-p u r i f i e d to remove a small amount of a high molecular weight impurity detected by the Sephadex G-200 assay. The f i n a l haptoglobin preparation from r e p u r i f i e d fractions A, B and C are of a high degree of purity since the Sephadex assay indicated that 1 mole of haptoglobin has bound with almost 1 mole of hemoglobin. A f i n a l p u r i f i c a t f o n of about 12-fold was achieved and a y i e l d of 45.9% of pure haptoglobin was obtained. The difference i n the p u r i f i c a t i o n r a t i o obtained in this method and the 28-fold p u r i f i c a t i o n i n the Connell and Shaw method (148) i s due to ascites f l u i d having a lower t o t a l protein concentration than plasma and to haptoglobin forming a far greater p r o p o r t i o n , of the ascites f l u i d protein than the plasma proteins. The lower amount, 269.3 mg from 600 ml of thi s ascites f l u i d compared to 988.9 mg from 4 70 ml i n the previous preparation of haptoglobin from a d i f f e r e n t sample of ascites f l u i d , i s due to the consider-able v a r i a t i o n i n haptoglobin lev e l s i n d i f f e r e n t patients and at d i f f e r e n t stages of the disease i n a single patient. It i s evident that t h i s simple method of preparation of haptoglobin from ascites f l u i d i s a far superior method to those previously used. The technique i s rapidl y executed, and the product i s pure and not denatured. A s i g n i f i c a n t advantage to thi s procedure i s that large quantities of - 6 8 -haptoglobin can be readily obtained from a single patient and thus in a genetically homogeneous state. Haptoglobin of a single phenotype proved invaluable in amino acid sequence studies of the a 1 , a 2 and 3 chains, which are uncomplicated by heterogeneity due to the presence of other possible hapto-globins which might vary at unrecognized l o c i . As might be expected, i f Hp 2-2 consists of a heterogen-eous populations of polymeric molecules as postulated by Smithies and Connell (14) and by Allison (21), resolution of Hp 2-2 and Hp 2-1 should be possible on Sephadex G-200. This has been observed by Killander (151) and by Javid (152) and confirmed here,, (for further discussion see p. 82). - 69 -PART II SEPHADEX G-200 ASSAY INTRODUCTION Quantitation of the haptoglobin level of a biological fl u i d or of a purified haptoglobin solution is based on the abi l i t y of the protein to combine stoichiometrically with hemoglobin to form a complex. Measurement of the complex can be achieved by an indirect method in which the peroxidase activity of the Hb-Hp complex is measured by iodometry (153) or spectrophotometry (81,154,155). On the other hand, the amount of hemoglobin bound has been estimated by tit r a t i o n of a haptoglobin solution with a series of hemoglobin solu-tions of increasing concentration. The resulting complexes were then separated from excess hemoglobin by paper electro-phoresis and the end point determined as the f i r s t appearance of excess hemoglobin (105,118). Alternatively, the determin-ation of the amount of hemoglobin present in the complex can be achieved by densitometry (156), photometry (10 6) or tannin turbidometry (157), and excess hemoglobin can be separated from the complex by means of starch electrophoresis (131), agar electrophoresis (158,159), cellulose-acetate electro-phoresis (160,161), gel f i l t r a t i o n (162,163), Immunoelectro-phoresis (164). The determination of serum haptoglobin has also been automated (165,166). The multitude of methods avai-lable for the quantitation of haptoglobin is a reflection of the c l i n i c a l significance of this serum protein, which has - 70 -necessitated the development of rapid and precise means of determination of haptoglobin content of b i o l o g i c a l f l u i d s . 1. PEROXIDASE ASSAY It had been known for some time that blood could catalyze oxidation of aromatic chromogens i n the presence of hydrogen peroxide, Wu (167) showed that t h i s a c t i v i t y i s a l l account-ed for by the hemoglobin content and that the peroxidase a c t i v i t y of hemoglobin and i t s derivatives i s linked to the presence of the heme component. Although hemoglobin can behave l i k e a peroxidase i n ce r t a i n systems, i t does not sat-i s f y ' a l l the c r i t e r i a recognized for c l a s s i f i c a t i o n as a "true peroxidase", therefore i t i s termed a "pseudo-peroxi-dase" (54). Polonovski and Jayle (2) f i r s t observed that the peroxidase a c t i v i t y of hemoglobin i s markedly enhanced by the addition of serum or plasma; furthermore, the pH optimum of the reac-ti o n was displaced from 5.6 to 4.4 (3). Subsequently, these investigators showed (153^8) that i t was the stoichiometric combination of hemoglobin to the haptoglobin i n the serum or plasma which markedly increased the peroxidase a c t i v i t y of free hemoglobin and transformed i t into a true peroxidase (168). I t i s not understood how haptoglobin when bound to hemoglobin influences the peroxidase a c t i v i t y of the heme groups. Bajic (169) has suggested that haptoglobin s t a b i l i z e s the hemoglobin molecule; t h i s may be through protection of the hemoglobin from the denaturing e f f e c t of acid solutions, - 71 -since Connell and Smithies (81) reported that as the pH increases the peroxidase activity of hemoglobin is progres-sively less affected by the presence of haptoglobin. Moretti and Yon (170) suggests that haptoglobin acts as an activator of hemoglobin and not as a protector. As a result of his i n i t i a l observation, Jayle (4) developed a method of determining the haptoglobin index in serum by measuring the participation of the Hb-Hp complex in an oxi-dation reaction system in which ethyl hydrogen peroxide was the oxidizing substrate and iodide the electron donor. The stoichiometry of this system is as follows: C2H5OOH + 2I~ + 2H+ >• C2H5OH + I 2 + H20 The extent of the reaction may be followed by titra t i o n with thiosulfate of the iodine liberated in 5 minutes at pH 4.4 and 32°. The velocity of the reaction has been established to be proportional to the concentration of the Hb-Hp complex (168). The method consists of adding increasing volumes of serum to a fixed amount of hemoglobin so that the peroxidase activity in the system is proportional to the amount of serum added until a saturation point is reached. The haptoglobin content of the sample is then equal to the smallest volume of serum for which maximum activity can be obtained; this value is expressed as the Haptoglobin Index (H.I.), the concentration of haptoglobin in a serum which saturates a M/20,000 hemo-globin solution. One H.I. corresponds to 105.4 mg haptoglobin per 100 ml. - 72 -I t i s generally recognized that t h i s method gives precise values but i t involves a t i t r a t i o n carried out with very care-f u l timing and uses ethyl hydrogen peroxide, a p o t e n t i a l l y explosive reagent. For routine c l i n i c a l work, Jayle (4) has proposed a more rapid method, the "activation method", i n which hemoglobin i n excess of that required for binding to haptoglobin i s added to the serum and af t e r the excess hemo-globin has been p a r t i a l l y inactivated by the addition of iodine, the peroxidase a c t i v i t y of the complex i s measured by the same reaction system. This a c t i v a t i o n method gives r e s u l t s s u f f i -cient for routine analysis (168). Connell and Smithies (81) confirmed the p r i n c i p l e of the peroxidase method i n t h e i r thorough study of the reaction con-d i t i o n s , that is, the pH of the reaction, concentration of hydrogen peroxide and concentration of guaiacol, under which the peroxidase a c t i v i t y of the free hemoglobin i s e s s e n t i a l l y zero while that of i t s complex i s maximal. Guaiacol was chosen as the reducing substrate since as the concentration used i n the assay, i t proved to be a powerful i n h i b i t o r of the peroxi-dase a c t i v i t y of free hemoglobin while the complexes were in h i b i t e d to a much lesser extent. Methemoglobin, which i s more stable during storage, i s the standard. The reaction at a pH of 4.0,is c a r r i e d out i n a cuvette and the formation of tetraguaiacol during the reaction i s followed at 470 my i n a spectrophotometer thermostatted at 30°. .'.ignoring the i n i t i a l lag phase of a few seconds, the slope of the l i n e a r portion - 73 -of the plot of the observed extinction against time is used as a measure of the peroxidase activity. The haptoglobin level of the sample is obtained from this activity by refer-ence to a calibration curve prepared with standard haptoglobin solutions of increasing dilution. This quantitative method has been found to be sensitive and reliable (168). The re-quirement of a spectrophotometer f i t t e d with a thermostati-cally controlled chamber may present an obstacle, and s k i l l is required in this technique since i t requires the taking of several readings at short intervals unless the spectro-photometer is f i t t e d with a recording unit. Owen and coworkers (154) proposed a simplified method based on the same reaction used by Connell and Smithies. A lower concentration of hydrogen peroxide and a reaction time of 25° were used to slow down the reaction. The linear re-lationship between the complex and the optical density at 470 my reaches a maximum before 10 minutes after which the color fades. Subtraction of a serum blank corrects for any peroxidase activity of the serum i t s e l f , which is presumably due to the presence of verdoperoxidase from leucocytes or to the presence of methemalbumin. Precise values are obtained by this commonly used method of haptoglobin determination. However, objections have been raised to measuring the developed color immediately after the reaction has ended and the color has begun to fade (168,171). - 74 -2. ELECTROPHORETIC METHODS A different principle underlies the electrophoretic methods of quantitation of haptoglobin which utilizes the difference in migration between the Hb-Hp complex and free hemoglobin. Laurell and Nyman (105) developed a technique which measures the maximum amount of hemoglobin that can be bound by the haptoglobin in the biological f l u i d before any free hemoglobin is demonstrable by paper electrophoresis at pH 7.0. Near the isoelectric point of hemoglobin, a good separation between the complex and free hemoglobin is achieved, the complex migrates to the anode and the hemoglobin which is close to its isoelectric point is carried towards the cathode due to endosmotic flow. Increasing amounts of hemo-globin are added to a standard amount of serum with the di f -ference between two successive samples being 10 mg Hb/100 ml serum. At the end of electrophoresis, the heme-containing proteins were developed with an acetic acid solution of leukomalachite green. This electrophoretic method is both reproducible and sensitive, even at low hemoglobin binding capacity values (54). Although a "heme-binding B i protein", which migrates at pH 7.0 with the a2-globulins, as the complex does, is present in sera, i t s aff i n i t y for hemoglobin is much lower than that of haptoglobin, even when haptoglobin is ful l y saturated with hemoglobin, and therefore, this compon-ent does not significantly affect the results. Although an error of ± 10 mg/100. ml is indicated by the increments of - 75 -hemoglobin solutions used, with experience this error can be reduced to ± 5 mg/100 ml. This assay method, although i t yields reproducible results, is time-consuming, necessitating several electrophoreses for a single haptoglobin determination. The three principle methods of haptoglobin quantitation, Jayle's saturation method, Connell and Smithies' spectro-photometric assay and Laurell and Nyman's electrophoretic method, are a l l capable of giving precise and reproducible values and results obtained by these three procedures have been found to be in perfect agreement (168).. For c l i n i c a l analysis, Jayle's activation method and Owen's modification of Connell and Smithies' technique are less laborious and exacting. As for the many other simplified electrophoretic methods, their precision is believed to be less satisfactory (168). 3. SEPHADEX ASSAY It is apparent that the above procedures depend either directly, as in Jayle's or Connell and Smithies' peroxidase assay, or indirectly, as in the electrophoretic method, on the peroxidase activity of the heme-containing proteins. It is known that many substances in serum influence: the oxida-tion of benzidine and similar substrates. Nyman (54) reported that the peroxidase activity appears to depend not only on the haptoglobin concentration in the sample, but also on the com-position of the serum, since she found evidence for the presence of serum components other than haptoglobin, capable - 76 -of activating the peroxidase activity of hemoglobin. Also, there were indications of factors inhibiting the same reaction. Thus errors may arise in assessing the level by the peroxidase activity of the serum. Although, a catalase is present in variable quantitites in serum which is sensitive to hydrogen peroxide but not to ethyl hydroperoxide and serum can exhibit a peroxidase due to the presence of leucocytes, these are pre-sumably taken care of by a serum blank. Heparin is reported not only to decrease the a f f i n i t y of haptoglobin for hemoglobin but also to inhibit the peroxidase activity of the complex, although the amount of heparin used as an anticoagulent has no influence (54). Evidently, there is need for a direct assay of the haptoglobin content which would preclude the many influences on the peroxidase reaction. Another factor leading to the development of the direct Sephadex assay was the re-peated observation that the peroxidase assay on haptoglobin polymer solutions which had been frozen and thawed several times gave rise to erratic results and a steady inactivation of the hemoglobin binding, indicating that haptoglobin is rather easily denatured by this procedure, contrary to the observation reported by Nyman (54). SEPHADEX ASSAY The assay method for the binding of hemoglobin to hapto-globin used in this study takes advantage of the difference in molecular weight between free hemoglobin, molecular weight 66,800, and the Hb-Hp complex, which is at least molecular - 77 -weight 161,800 for the Hp 1-1 complex and higher for the polymeric species in Hp 2-1 and 2-2. An amount of hemoglobin in excess of that required to fu l l y saturate the binding sites on haptoglobin, is added to a sample and the mixture subjected to gel f i l t r a t i o n on Sephadex G-200. Since the rate of elu-tion of proteins on Sephadex is dependent on molecular size (172), the Hb-Hp complex is separated from the uncomplexed free hemoglobin. The stoichiometry of binding is obtained by measuring the ratio of the absorption due to the Soret band of heme at 407 my to the absorption at 280 my, which represents the protein portion of hemoglobin together with a substantial contribution from haptoglobin. A Sephadex G-200 column, 1 cm x 50 cm equilibrated with 0.1 M phosphate buffer, pH 7.0 is used for the assay, ex-cepting in a few preliminary runs and in those cases where a different buffer is indicated, for example, in the study of the effect of pH upon the binding. In the control Hp 1-1 assay shown in Figure 13, 0.5 ml of 1.2 mg hemoglobin solu-tion in 0.02 M Tris-HCl buffer, pH 7.42 was added to 1 mg Hp 1-1 in 0.5 ml of the same buffer. The solutions are mixed and then chromatographed on the Sephadex G-200 column. Eluates of approximately 1 ml volume are collected and the absorption of each fraction at 280 my and 407 my are measured. The elution diagram (Fig. 13, frame A) shows the clear separ-ation of hemoglobin from the front-funning Hb-Hp complex. The ratio of the absorbance at 40 7 my due to the heme to - 78 -F i g u r e 13. Assay of Hp 1-1 i n 0.02 M T r i s - H C l b u f f e r , pH 7.42 (frame A) and Hp 2-1 p o l y m e r s o h Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0 (frames B,CC and D). - 79 -the absorbance at 2 80 my due to the protein portions is approxr imately 3.9 in free hemoglobin, while in the complex with pure Hp 1-1 the ratio f a l l s to 2.0. This latter value corresponds closely to that calculated for a 1:1 complex. Based on the extinction coefficients of 1.19 for 1 mqfol of Hp 1-1 at 280 my (46) and 1.75 for 1 mg/ml of hemoglobin "(personal data) and molecular weights of 66,800 and 95,000 for hemoglobin and Hp 1-1 respectively, the theoretical ratio of the absorban-cies at 407 my to 280 my for stoichiometric binding is 1.99. This method of assay of haptoglobin thus gives a direct measure of the amount of hemoglobin bound by the haptoglobin molecule and is therefore not subject to the influence of known and unknown factors on the peroxidase reaction. In addition, the elution profile resulting from this assay gives an immediate indication of any change in the conformation of the protein molecule since besides molecular size, the shape of the molecule also affects i t s a b i l i t y to penetrate the pores of the dextran gel (172). Thus an unfolding of the three-dimensional configuration of a molecule w i l l hinder i t s penetration into the molecular sieve and cause the protein to be eluted ahead of the elution volume for the native protein. This was observed by Habeeb (173) who utili z e d the sensitivity of the elution volume of a protein molecule on a column of Sephadex G-200 to i t s Stokes radius to evaluate the conforma-tional changes associated with chemical modification of bovine serum albumin. Also, the presence of any impurities which - 80 -differ in molecular weight from the complex or the free hemo-globin is immediately detected. This assay method besides being applicable to pure haptoglobin solutions is also suit-able for assay of serum, ascites or other biological fluids, although additional calculations are required for these assays, since haptoglobins are the only class of serum pro-teins shown conclusively to combine significantly with hemo-globin. Other proteins are able to form weak complexes with dissociated heme, for example, the heme-binding globulin and serum albumin. These heme-binding proteins can only be shown to bind heme after haptoglobin is fu l l y saturated and there is an excess of free hemoglobin, thus they do not influence the haptoglobin assay (174). In addition, hemoly-sis in the serum samples does not vitia t e the analysis. As many as four assays have been performed simultaneously and the technique is simple.and quick. If, on the other hand, only one assay is required to be carried out, this is easily done without necessitating fresh reagents to be made up and titrated each time an assay is performed, as would be the case ihi the-peroxidase assays. It was subsequently noted that a somewhat similar gel f i l t r a t i o n method had been performed by Ratcliffe and Hardwicke (162) and by Lionetti, Valeri, Bond and Fortier (163). In both cases, excess hemoglobin was separated from the Hb-Hp complex on Sephadex G-100, followed by elution with 2% sodium chloride solution (162) or with a solution of 0.15 M sodium - 81 -chloride (163). Ratcliffe and Hardwicke computed the hemo-globin binding capacity of the sample by pooling the compon-ent fractions of the f i r s t peak, the Hb-Hp peak, and by mea-suring the 415 my absorption. The hemoglobin concentration in this peak is read off a calibration chart and extrapolated to the amount of hemoglobin present in 100 ml of the serum. Lionetti and coworkers (163) in a similar manner combined the fractions corresponding to bound hemoglobin and those corresponding to free hemoglobin, and determined the bound hemoglobin from i t s absorbancy at 418 my relative to the total 418 my absorption of the effluent. Since these methods rely on the quantitative recovery of the fractions, the Sephadex assay proposed in this study is more accurate. Ratcliffe and Hardwicke preferred to use G-100, since G-200 partially re-solved the haptoglobin complexes and produced a broader band of elution of bound hemoglobin. Partial resolution of hapto-globin polymer complexes has also been observed in the assays reported here, however, since as w i l l be shown later, the polymers bind the same amount of hemoglobin regardless of the size of the polymers, this partial resolution of the com-plex does not invalidate the assay with Sephadex G-200. Also, there was only a slight broadening of the peaks observed with the complexes with haptoglobin polymers, much less than that observed by Ratcliffe and Hardwicke, and the peak was well separated from the free uncomplexed hemoglobin peak. These authors report reproducible results which correlated well - 82 -with Rowe1s agar gel method (162) and with the peroxidase and electrophoretic methods (163). Immediately aft e r the Sephadex G-200 assay method had been developed, Gordon and Beam (175) presented the re s u l t s on the binding of the component a and 3 chains of haptoglobin with hemoglobin i n which these experi-ments were conducted on Sephadex G-100 and the amount of bin-ding determined by measurement of the absorbancies at 418 my and at 280 my of each f r a c t i o n . These measurements were used q u a l i t a t i v e l y to determine the extent of binding and the absorbancies were not used qu a n t i t a t i v e l y as has been done i n the Sephadex G-200 assay here. Also at the same time, Cloarec and Moretti (176) reported on the preparation of a Hb-Hp 2-1 complex and subsequent f r a c t i o n a t i o n of this com-plex on a Sephadex G-200 column. They also found that the r a t i o of the 406 my to the 278 my absorbancies of each ef f l u e n t f r a c t i o n i s a constant figure, that i s , i n the v i c i n i t y of 2.0. ASSAY OF Hp 2-1 POLYMERS Recently, Javid (152,177) concluded that the hemoglobin binding of p a r t i a l l y resolved polymers of Hp 2-1 and 2-2 varies inversely as the size of the polymer. After gel f i l t r a t i o n on Sephadex G-200 of Hp 2-1 and 2-2, Javid ob-tained a series of fractions representing decreasing average polymer si z e . The protein concentration was determined on equally spaced fractions throughout the haptoglobin peak by the biuret value and--the hemoglobin binding capacity was - 83 -measured by Smith and Owen's peroxidase assay (178). From these values the amount of hemoglobin bound to each milligram of haptoglobin was calculated. The res u l t s showed that the larger the polymer, the smaller the amount of hemoglobin bound. The conclusion reached was that the s i t e through which poly-merization occurs must also be involved i n the hemoglobin binding. However, Jayle (4 6) had reported that 1 mg of hemo-globin combines with 1.3 mg of haptoglobin of a l l genetic types. I t i s thus necessary to c l a r i f y the binding between hemoglobin and the various haptoglobin polymers. For this reason, the p a r t i a l resolution of Hp 2-1 polymers obtained during i t s p u r i f i c a t i o n on Sephadex G-200 was preserved and polymeric species representing the large polymers, the medium polymers and the small polymers were tested for th e i r binding capacity by the Sephadex assay. The results obtained were i n d e f i n i t e disagreement with those of Javid (177); however, since Javid used the peroxidase assay for hemoglobin binding to haptoglobin, i t was possible that t h i s method could be measuring a d i f f e r e n t aspect of the binding from the d i r e c t observation of. binding with the Sephadex assay. Therefore, peroxidase assays using the method of Connell and Smithies (81) were also c a r r i e d out on representative polymeric f r a c -tions. For the assay, an early f r a c t i o n from the Sephadex G-200 column of Hp 2-1, f r a c t i o n A, (Fig. 10), tube 29, was taken to represent the large polymers, tube 37, at the height of - 84 -the haptoglobin peak was considered representative of medium polymers, and tube 4 3, a slower eluting f r a c t i o n , was taken to represent the small polymers. The SG electropherogram of these fractions (Fig. 11) shows that tube 29 consists of several species of high molecular weight slowly^nigrating polymers. Tube 37 has 3 p r i n c i p l e components along with a minor component^all migrating with medium speed. Tube 43 con-s i s t s p r i n c i p a l l y of molecules of a size similar to Hp 1-1. In each case, the Sephadex assay was ca r r i e d out i n 0.1 M phosphate buffer, pH 7.0. An excess of hemoglobin, 1.2 mg, was added to 1 ml solution of phosphate buffer containing 1 mg of the polymer and the mixture subjected to the Sephadex G-200 assay. The e l u t i o n diagrams of the large polymers, medium poly-mers and small polymers are shown i n frames B, C and D of Figure 13 respectively. The r a t i o s of the absorbancies at 407 my to 2 80 my are 1.81 for the large polymers, 2.0 for medium polymers and 2.10 for small polymers (Table I I ) . These values are equivalent to 0.85, 1.04 and 1.26 moles of hemoglobin bound per mole of haptoglobin (Table I I ) . Thus, there i s l i t t l e difference i n binding between the polymeric species of the same haptoglobin type. This d i r e c t l y contra-d i c t s the findings of Javid, who reported that the largest Hp 2-1 polymers, that i s , the least retarded on Sephadex G-200, possessed only 8.7% of the hemoglobin binding capa-c i t y of the non-retarded or smallest polymers. Peroxidase assays were performed on the same polymeric fractions according to the method of Connell and Smithies - 85 -TABLE II Hemoglobin Binding By Haptoglobin Polymers Mole Hb Mole Hb 95,000 g Hp Preparation Fraction OP 407 my 95,000 g Hp Peroxidase OP 2 80 my G-200 assay assay Hp 1-1 whole 1.92 0.99 0.94 Hp 2-1 whole 0.94 large, fraction 29 1.81 0.85: large, fraction 30 0.67 medium, fraction 37 2.00 1.04 0.98 small, fraction 43 2.10 1.26 1.00 Hp 2-2 whole 0.54 large, fraction 41+42+43 1.21 0.44 large, fraction 42+43 . 0.46 large, fraction 44 0.53 medium, fraction 49 1.47 0.60 medium, fraction 50 0.61 medium, fraction 51 0.68 small, fraction 65+66 1.50 0.63 0.53 small, fraction 67 0.46 - 86 -(81). The reagents were prepared as follows: 0.600 M H 20 2 solution i s prepared immediately before use by d i l u t i o n with cold d i s t i l l e d water of an approximately 30% w/v solution, which i s standardized (179) about once a week by t i t r a t i o n with 0.100 N KMn04. The 0.600 M H 20 2 reagent i s kept i n the re f r i g e r a t o r i n a t i g h t l y capped bottle during the assay and a fresh aliquot used for the assay approximately every 15-20 minutes. A 0.03 M buffered solution of guaiacol i s made up by di s s o l v i n g 1.86 g;a of guaiacol i n 50 ml of 1.0 M acetic acid, adjusting the volume to 400 ml with water, then t i t r a -t ing to pH 4.0 with aqueous NaOH and a f i n a l adjusting of the volume to 50 0 ml with water. The methemoglobin reagent i s prepared according to the procedure of Smith and Owen (178) in which 10 ml of potassium ferricyanide i s added to a solu-t i o n of 250 mg of hemoglobin i n 25 ml of d i s t i l l e d water to oxidize the hemoglobin to methemoglobin. After 10 minutes, the volume was made up to 450 ml with water. Prio r to the assay the methemoglobin solution was d i l u t e d to a concentra-ti o n of 18.6 mg Hb/100 ml, according to a millimolar extinc-t i o n of 38.2 at 500 my (58). C a l i b r a t i o n curves were con-structed with pure Hp 1-1, 2-1 and 2-2. A solution of 3 mg of haptoglobin i n 3 ml of 0.15 M sodium chloride was made, from which 22 to 25 solutions of increasing d i l u t i o n s were made. The H 20 2 reagent, guaiacol reagent and methemoglobin solutions were each placed i n a water bath thermostatted at 30°. Each of the di l u t e d haptoglobin solutions and the stock haptoglobin solution was mixed with an equal volume of methemoglobin reagent and placed in the water bath. In a cuvette, 0.20 ml of the mixture is placed and 2.75 ml of the guaiacol reagent is added. Immediately after the addition of 0.05 ml of H 20 2 reagent to the mixture, the cuvette is capped with a ground-glass stopper, quickly inverted 2 to 3 times, then inserted along with a blank in a recording Beckman DB-spectrophotometer which is thermostatted at 30° and zeroed at 470 my with blanks Simultaneously, the automatic recorder is started. On com-pletion of the assay for the series of haptoglobin solutions, the slope in extinction units x 10 3/sec, that i s , the peroxi-dase activity for each solution, is calculated from the pro-gress curve of the reaction ignoring the i n i t i a l time lag of a few seconds. A plot of the peroxidase activity of each solution versus the haptoglobin concentration yields the calibration curve. The hemoglobin binding of fractions 30, 37 and 43 of the Hp 2-1, of the Sephadex G-2 0 0 column were assayed in the above manner. Due., to insufficient material, fraction 30 was used in place of fraction 29 to represent the large polymers. A concentration range of 0.168 mg/ml to 0.331 mg/ml hapto-globin polymers was used in the peroxidase assay and the peroxidase activity was obtained by reference to the calibra-tion curve of unfractionated Hp 2-1. The results in Table II are expressed as the fraction of a mole of hemoglobin (molecular weight 66,800) bound to 95,000 - 88 -g haptoglobin and are averages of four determinations. The binding data by the two methods show a f a i r l y good agreement and the peroxidase assay confirms the fact that there i s no ess e n t i a l difference i n binding of hemoglobin by the large and small polymeric species. Although the large polymers appear to show s l i g h t l y lower binding than the medium and small mole-cular weight polymers, i t i s i n no way the large decrease reported by Javid. A s i m i l a r series of Sephadex and peroxidase assays on heavy, medium and small polymer fract i o n s of Hp 2-2, which were is o l a t e d by Dr. G.H. Dixon, further substantiates th i s f i nding. The hemoglobin binding of Hp 2-2 i s s l i g h t l y low. However, i n this case, resolution was not as e f f e c t i v e and there was evidence of a high molecular weight non-hapto-globin contaminant i n the large polymer f r a c t i o n . Correcting for this material, i t i s clear that the large Hp 2-2 poly-mers also show strong hemoglobin binding. I t has been re-peatedly observed during a very extensive series of assays by the peroxidase method that repeated freezing and thawing of haptoglobin solutions give r i s e to e r r a t i c assay r e s u l t s and a steady i n a c t i v a t i o n of hemoglobin binding, i n d i c a t i n g that haptoglobin i s rather e a s i l y denatured by t h i s procedure. It i s possible that the larger polymers are more susceptible to t h i s treatment and would therefore become p r e f e r e n t i a l l y i n -activated, thus accounting for the lack of binding observed i n Javid's work. Moreover, Cloarec and Moretti (176), i n agreement with our r e s u l t s , were also unable to confirm - 89 -Javid's findings. These investigators reported that t h e i r Hb-Hp 2-1 fractions from a G-200 column gave r a t i o s of the absorbancies of 407 my to 280 my of about 2.0 throughout the peak and thus the same amount of hemoglobin i s bound to haptoglobin regardless of the size of the polymers. ASSAY GF a 1 , a 2 AND 8 HAPTOGLOBIN CHAINS The i n t e r a c t i o n between hemoglobin and haptoglobin to form the Hb-Hp complex i s very rapid as indicated by the k i n e t i c measurements of Nagel and Gibson by the stopped flow technique (59). Also the reaction between the two proteins i s an ex-tremely s p e c i f i c one which can be likened to the s p e c i f i c i t y of an antigen for i t s s p e c i f i c antibody. In order to gain more information on the molecular architecture of the binding s i t e so as to correlate i t s structure with i t s function, i t was of int e r e s t to investigate the binding capacities of the component chains of the haptoglobin molecule. The binding s i t e on the haptoglobin molecule may comprise amino acid residues from both the a and the 8 chains of the haptoglobin molecule or sol e l y from the a or the 8 chain. These amino acid residues may be located on both chains or on only one type of chains; i n any event, they are probably remote from each other when considered as a lin e a r sequence, but are i n close juxt-a position i n space. Studies on the active s i t e s of enzyme molecules have indicated that the c a t a l y t i c ac-t i v i t y i s a function of the d e l i c a t e orientation of c r i t i c a l groups. - 90 -An inherent d i f f i c u l t y i n studying the hemoglobin binding capacity of the in d i v i d u a l chains i s that conditions neces-sary to separate the constituent chains usually also decrease the binding a b i l i t y of the molecule as a whole. This pro-blem has also been encountered i n the many attempts to determine the involvement of i s o l a t e d heavy and l i g h t chains of the antibody molecule i n the formation of the combining s i t e . Whatever technique i s used to dissociate the peptide chains, considerable loss of a f f i n i t y for antigen follows (180). Thus, Gordon and Beam (175) found that conditions si m i l a r to those used i n d i s s o c i a t i o n of immunoglobulins causes considerable loss of binding capacity i n the hapto-globin molecule. Hp 2-2 was reduced with 0.2 M mercapto-ethanol for 1 hour at room temperature, alkylated with 0.24 M iddoacetic acid for 30 minutes at 4° for 15 hours. Gel f i l t r a t i o n through Sephadex G-100 i n 0.01 N propionic acid, pH 3.5, yielded a front-running peak comprised of both 3 and a 2chains, a middle peak of v i r t u a l l y pure 3 chains and a small slow-eluting peak of a 2 chains which were probably contaminated with small amounts of 3 chains. In th e i r binding experiments with Sephadex G-100 chromatography and measure-ment of the 418 my and 280 my absorbancies, i t was observed that the 3 chains had bound a substantial amount of hemoglobin, in. spite of the harsh conditions used i n d i s s o c i a t i o n of the chains. The iso l a t e d a 2chains showed a trace of binding a b i l i t y which was probably due.to contamination by the small - 91 -amount of 8 chains. The mixture of undissociated 3 and a 2 chains i n the front-running peak of the column had lower binding capacity than pure 3 chain. These re s u l t s agree with the findings by Shim, Lee and Kang (33) that anti-3-chain antiserum reacted only with free haptoglobin but not with the Hb-Hp complex while i n contrast, anti-a 2-chain antiserum reacted with both free and bound haptoglobin. I t appears then that haptoglobin binds with hemoglobin at the 3 chain, while i n the complex the antigenic determinants"of 7 the 3 chain are covered by the hemoglobin molecule and are therefore unavailable to the antibody. I t was f e l t advisable to confirm the finding of Gordon and Beam since i n the i r experiments the chains were incompletely dissociated. Hp a 1 , a 2 and 3 chains were isola t e d from p u r i f i e d Hp 2-1 by Dr. J.A. Black for amino acid sequence analysis after i t was reduced with 0.5 M mercaptoethanol i n 8 M urea and the free t h i o l s alkylated with 1.0 M iodoacetamide (26). The polypeptide chains were separated by Sephadex G-7 5 chro-matography . i n 1.0 M acetic acid. The haptoglobin chains i s o l a t e d under these conditions were probably p a r t i a l l y de-natured since the 8 and the a 2 chains showed limited solu-b i l i t y i n 0.1 M phosphate buffer, pH 7.0. I t had been observed by Mr. Choy Hew that the 3 chain could be brought into solution by f i r s t d issolving i t i n 0.2 M.NH^ OH and then lowering the pH with acetic acid. Since under the proper conditions these chains can be s o l u b i l i z e d , determination - 92 -of the hemoglobin binding capacity of these and the a 1 chains could be car ried'out. The Sephadex assay was carried out on 2 mg of the 8 chain which was dissolved in 1 ml of 0.2 M NH^ OH and the pH then lowered to 7.0 with 0.2 M acetic acid. An ammonium acetate buffer of pH 7.0 was prepared in the same manner and used for the Sephadex G-200 assay of the 8 chain. The 8 chain solution was added to 2 mg of hemoglobin in 0.25 ml of the ammonium acetate buffer and the mixture assayed on Sepha-dex. The earlier elution of the 8 chain (Figure 14, frame A), that i s , in the void volume instead of in the vi c i n i t y of 20 ml, indicates possible change in the shape of the molecule such as an unfolding phenomenon. This is likely since the 8 chain appeared partially denatured. Another possibility is that the 8 chain is aggregated, as has been observed by Mr. C. Hew during ultracentrifuge studies of 8 chain. In spite of possible changes,in conformational state or aggre-gation in the molecule, the 8 chain binds a significant amount of hemoglobin, the 407 my/280 my ratio being 0.268, approximately one-eighth of the normal binding. The isolated a 1 chain was soluble in the 0.1 M phos-phate, pH 7.0 buffer usually used in the Sephadex G-200 assay. A solution of 2 mg of the a 1 chain in 1 ml of the 0.1 M phosphate buffer was added to 5.5 mg of hemoglobin in 0.25 ml of the same buffer. The mixture was then assayed 0.9 6 O <• 05 O CO >-u < O »-D L O 0.6 0.3 0 280 rr.u 0 10 20 30 40 EFFLUENT VOLUME, mi Figure 14. Assay of 3 , a 1 and a 2 haptoglobin chains on Sephadex G-200 (1 cm x 50 cm). (A) g chain i n 0.2 M ammonium acetate, pH 7.0 (acetic a c i d ) , (B) a 2 chain i n 0.1 M phosphate buffer, pH 7.0, (C) a 1 chain i n 0.1 M phosphate buffer, pH 7.0. - 94 -on Sephadex G-200. The elution profile (Fig. 14, frame C) of the assay of the a 1 chain did not show a Hb-la complex, which would be expected to be eluted ahead of the free hemoglobin peak. The uncombined a 1 chain emerges just slightly behind the hemo-globin peak as is evidenced by a slight t a i l i n g of the hemo-globin peak. The a 2 chain was partially soluble in 0.10 M phosphate buffer, pH 7.0, therefore a 0.9 5 ml supernatant solution in this buffer with an absorption at 280 my of 1.55 was used. The a 2 chain solution was combined with 5.5 mg hemoglobin in 0.25 ml of 0.1.0 M phosphate buffer and then subjected to gel f i l t r a t i o n assay. In this case, a small peak is observed in the elution pattern (Fig. 14, frame B) which is eluted in the void volume. This protein peak has some hemoglobin bound to i t . However, this small peak does not account for a l l the material applied, the remaining is eluted just behind the hemoglobin peak. The a 2 chain is likely contaminated.with some 3 chain since in the isolation of the chains by gel f i l t r a t i o n , the a 2 chain is eluted immediately after the 3 chain. This would also be in agreement with the observation that the a 1 chain shows no hemoglobin binding capacity. In spite of the severe conditions used in dissociation of the polypeptide chains, the 8 chain bound an appreciable amount of hemoglobin, as has been observed by Gordon and Beam. - 95 -The finding that the a 1 chain shows no a b i l i t y to bind hemo-globin i s more conclusive evidence than that presented by Gordon and Beam that the a chain i s not d i r e c t l y involved i n the binding s i t e , since t h e i r a 2 chain was contaminated by 8 chain, as wa<s: the a 2 chain used here. Thus the evidence here confirms the finding that the binding s i t e for hemoglobin i s located on the haptoglobin 8 chains as suggested by Gordon and Beam and by Shim, Lee and Kang. The question as to whether the a chain plays any ro l e i n the binding, such as a semispecific one i n maintaining the correct conformation of the molecule and thereby enhancing the binding capacity of the 3 chains, as has been suggested for the heavy and l i g h t chains of the antibody molecule (1801 remains to be answered. BINDING OF GLOBIN AND MYOGLOBIN TO HAPTOGLOBIN 1. GLOBIN Turning to the hemoglobin moiety of the complex, i t appears that reactions involving the iron atom or neighboring atoms do not i n t e r f e r e with complex formation with haptoglobin since carbonmonoxy-, cyano-, and methemoglobin as well as the H 2S-derivative are bound by haptoglobin to the same extent as oxyhemoglobin (54,168). I t has been reasoned that since the ligand on the heme does not influence complex formation the heme group probably does not p a r t i c i p a t e i n the int e r a c t i o n between hemoglobin and haptoglobin. That th i s is/likely,-eomes from paper electrophoretic experiments of Nyman (174) and of Neale, Aber and Northam (181), i n which i t was shown that haptoglobin did not interac t with pyridine hemichrome or hematin. In the early studies on haptoglobin, i t was re-ported by Jayle (18 2) and by van Royen (141) that native globin binds haptoglobin; however, no published evidence was presented. Competition experiments by Nyman (54) with hemo-globin and globin did show that globin can block the hemo-globin binding groups of the haptoglobin. Also Rowe and S o o t h i l l (183), using antiserum against globin and mixtures of globin and serum as antigen, demonstrated an arc of pre-c i p i t a t e of i d e n t i c a l :'m-obility to the p r e c i p i t i n l i n e of haptoglobin bound hemoglobin and suggested that haptoglobin also binds globin. Electrophoretic investigations (55) also showed formation of a globin-Hp complex. Nevertheless, i t was considered of in t e r e s t to determine by the d i r e c t Sepha-dex assay method that globin binds with haptoglobin. Globin was prepared i n the usual manner by acid acetone treatment of hemoglobin (18,4) . A solution of 100 mg of twice c y s t a l i z e d hemoglobin i n 1 ml of d i s t i l l e d water was added to 20 ml of 20% v/v cone. HC1 i n acetone, previously c h i l l e d i n a dry ice-acetone mixture (-30°). After shaking the f l a s k r a p i d l y , the pr e c i p i t a t e was immediately c o l l e c t e d by suction f i l t r a t i o n and washed several times with cold acetone (-30°) and f i n a l l y washed with cold ether. The pre-c i p i t a t e was dried under a nitrogen atmosphere. Globin pre-pared i n t h i s manner i s e s s e n t i a l l y free of heme, since the absorption at 407 my was less than 0.03% of the normal ab-- 97 -s o r p t i o n . The p r e p a r a t i o n was i n s o l u b l e i n 0.1 M p h o s p h a t e b u f f e r , pH 7.0 and was, t h e r e f o r e , p r o b a b l y a g g r e g a t e d . However, by a p p l y i n g t h e same t e c h n i q u e as was u s e d f o r s o l u -t i o n of- t h e 8 c h a i n s , g l o b i n was b r o u g h t i n t o s o l u t i o n . I n t h i s c a s e , however, a f t e r s o l u t i o n o f g l o b i n i n 0.2 M NH^OH, on t i t r a t i o n w i t h a c e t i c a c i d , g l o b i n p r e c i p i t a t e s b e f o r e a pH o f 9 i s r e a c h e d , a t a p p r o x i m a t e l y pH 9.2. I f i n s t e a d , t h e g l o b i n i n 0.2 M N H 4 O H s o l u t i o n i s c o m b i n e d w i t h a 0.2 M NHi*OH s o l u t i o n o f h a p t o g l o b i n , t h e pH o f t h e m i x t u r e c o u l d be l o w e r e d t o 9 w i t h o u t any s i g n o f p r e c i p i t a t i o n . T h u s , 1.5 mg o f g l o -b i n i n 1 ml 0.2 M N H 4 O H was a d d e d t o 2 mg o f h a p t o g l o b i n i n 05 ml 0.2 M N H 4 O H and t h e m i x t u r e , a f t e r t i t r a t i n g t o a pH o f 9.0 w i t h 1.0 M a c e t i c a c i d , was s u b j e c t e d t o t h e Sephadex a s s a y i n an ammonium a c e t a t e b u f f e r p r e p a r e d i n t h e same manner. G e l f i l t r a t i o n o f a 1.5 mg g l o b i n c o n t r o l was a l s o c a r r i e d o u t . The e l u t i o n d i a g r a m o f t h e g l o b i n and h a p t o g l o b i n mix-t u r e ( F i g . 15) shows t h a t t h e m a i n peak i s e l u t e d a t a volume c o r r e s p o n d i n g t o t h a t u s u a l l y f o u n d f o r a c omplex between h e m o g l o b i n and h a p t o g l o b i n . A hump a t t h e t a i l i n g edge o f t h e m a i n peak i n d i c a t e s a v e r y s m a l l e x c e s s o f g l o b i n . The q u a n t i t i e s o f g l o b i n and h a p t o g l o b i n u s e d i n t h i s e x p e r i -ment were c h o s e n so t h a t g l o b i n w o u l d be j u s t s l i g h t l y i n e x c e s s , s i n c e 407 my a b s o r p t i o n i s n o t a p p l i c a b l e i n t h i s p a r t i c u l a r a s s a y . T h u s , t h e f o r m a t i o n o f one m a i n peak a t t h e p o i n t o f e l u t i o n o f t h e complex, i n s t e a d o f two l a r g e p e a k s - 98 -EFFLUENT VOLUME (ML) Figure 15. Binding of globin to haptoglobin on Sephadex G-200 (1 cm x 50 cm) i n 0.2 M ammonium hydroxide, pH 9.0 (acetic a c i d ) . - 99 -of uncomplexed haptoglobin followed by uncombined globin, indicates complex formation. This i s further substantiated by the globin control p r o f i l e . Another l i n e of evidence i s that a solution of globin i n the presence of haptoglobin does not p r e c i p i t a t e as r e a d i l y during lowering of the pH of the solution as a globin solutionr.in the absence of haptoglobin. This may be taken to mean that a complex has formed and that haptoglobin on combining with globin has s t a b i l i z e d the globin molecule i n some way. Thus the res u l t s of the globin assay confirms the previous suggestions that haptoglobin binds to hemoglobin through the globin moiety. 2. MYOGLOBIN Myoglobin has a similar function to hemoglobin i n that i t i s the oxygen carrying pigment found i n muscles of verte-brates and certa i n invertebrates (185). While both hemo-globin and myoglobin contain an i d e n t i c a l prosthetic group, the heme, myoglobin i s a r e l a t i v e l y simple structure, com-posed of a single polypeptide chain containing 153 amino acids and a single heme moiety, i n contrast to the four pep-tide chains of 574 amino acids and four heme molecules i n the hemoglobin molecule. A comparison of the known primary struc-ture of sperm whale myoglobin and of human hemoglobin (186) shows that although there are many differences i n amino acid c o n s t i t u t i o n i n the a and 8 chains of hemoglobin and the myoglobin polypeptide chain, many s i m i l a r i t i e s are also ap-parent between the three types of peptide chains (186,187), - 1 0 0 -there being 68 amino acid residues i n i d e n t i c a l positions i n the a and 3 chains (186), and about 37 i d e n t i c a l residues between myoglobin and the a and-8 chains (188). A compari-son of a l l three chains shows that they are c e r t a i n l y s t i l l homologous but there are only 21 amino acids i n i d e n t i c a l positions. Moreover, the X-ray crystallographic studies by Perutz (188) and by Kendrew and coworkers (189) revealed that sperm whale myoglobin and the a and 8 hemoglobin chains were s t r i k i n g l y similar i n th e i r t e r t i a r y structure. Each polypeptide chain i s folded i n a tetrahedral arrangement with the heme group ly i n g on the surface of the molecule i n pockets formed by the folds i n the polypeptide chain. This polypep-tide c h a i n - f o l d , f i r s t discovered i n sperm whale myoglobin, has since been found also i n seal myoglobin and i t s appearance i n horse hemoglobin suggests that a l l hemoglobins and myoglobins of vertabrates follow the same pattern C188,190,191). Extensive investigation of vertebrate hemoglobin shows a basic s i m i l a r i t y i n chemical structure, the molecule i n each case i s composed of 4 polypeptide chains of approximately 17,000 molecular weight with one heme group each, except i n the case of the hemoglobin of lamprey, Lampetra f l u v i a t i l i s } generally accepted as the most primitive l i v i n g vertebrate, which i s characterized by a single polypeptide chain of molecular weight 17,500 and the presence of a single heme group. Hagfishl (Myxine glutinosa) hemoglobin may be similar or possibly a dimer of 34,000 molecular weight (192). - 101 -Since myoglobin appears to be clo s e l y related to hemo-globin, i t i s of considerable i n t e r e s t to determine whether haptoglobin w i l l bind to this oxygen-carrying protein. Javid, Fischer and Spaet (193) reported no binding of myoglobin to haptoglobin and i n fa c t could not demonstrate binding of myo-globin by any serum protein i n th e i r starch gel electrophore-s i s . Myoglobin when added i n varying concentrations to serum always moved as a single band with the same mobility of myo-globin alone and caused no change in. mobility of haptoglobin bands i n contrast to the re s u l t s shown by the addition of hemoglobin to serum. However, Lathem (194) l a t e r presented evidence of protein binding of myoglobin to form.a complex which has electrophoretic c h a r a c t e r i s t i c s s i m i l a r but not i d e n t i c a l with what he terms "protein-bound hemoglobin" which appears to be the Hb-Hp compex. Subsequently, Wheby, Barrett and Crosby (195) used :;.ahaptoglobinemic serum and demonstrated that hemoglobin, myoglobin and hematin a l l formed complexes which migrated i d e n t i c a l l y with each other and with the non-haptoglobin protein-heme complexes seen with normal serum on starch gel electrophoresis. The protein believed to be responsible for th i s binding i s what i s termed the heme binding 8-globulin by Nyman (54). Since the above evidence i s not clear-cut, i t appeared advisable to show more d i r e c t -l y whether or not myoglobin i s bound by haptoglobin. Two mg of sperm whale myoglobin (Mann Research Labora-tories) was dissolved i n 0.25 ml of 0.1 M phosphate buffer, - 102 -pH 7.0 and added to a 0.25 ml solution of 2 mg of haptoglobin. The mixture was then subjected to the Sephadex assay. The elution diagram (Fig. 16) of the myoglobin assay shows that haptoglobin is eluted free, not combined with any myo-globin, since the front-running peak shows no 407 my absorp-tion due to the heme group of the myoglobin. A l l the myoglo-bin is observed as a single peak eluting late in the gel f i l t r a t i o n in accordance with i t s smaller molecular weight. This result then confirms the observations of Wheby and associates that the myoglobin binding noted by Lathem is not due to haptoglobin. It is evident that the single poly-peptide chain in myoglobin does not carry a binding site for haptoglobin in contrast to the results observed with iso-lated a hemoglobin chains C59). This result further i l l u s -trates the great specificity of binding of haptoglobin with hemoglobin and may be inherent in what is described as the biological role of haptoglobin, that of combining specifi-cally with extracorpuscular (160) hemoglobin to conserve iron. ASSAY OF HAPTOGLOBIN WITH HEMOGLOBIN FROM OTHER SPECIES Tryptic hydrolyzates of hemoglobin of various animals give fingerprint patterns in which most of the peptide fragments are very similar. The patterns from fish hemo-globin were most different from mammals, consistent with their separation in the phylogenetic scale (196). The ob-servation that hybridization can be achieved between hemo-globins from distantly related animals under a variety of - 103 -Figure 16. Binding of sperm whale myoglobin to hapto-globin on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phos-phate buffer, pH 7.0. - 104 -e x p e r i m e n t a l c o n d i t i o n s ( 1 9 7 ) , f u r t h e r s u g g e s t s t h a t t h e g r o s s a r c h i t e c t u r e o f s u b u n i t s i s s i m i l a r i n a l l h e m o g l o b i n s . H o w e v e r , i t i s r e a d i l y a p p a r e n t t h a t t h e p r i m a r y s t r u c t u r e o f h e m o g l o b i n v a r i e s c o n s i d e r a b l y f r o m one s p e c i e s t o a n o t h e r . P r e s e n t e v i d e n c e s u g g e s t s t h a t t h e number o f d i f f e r e n c e s b e -t w e e n a g i v e n p o l y p e p t i d e c h a i n ( e g . a o r 8) f o u n d i n d i f f e r e n t a n i m a l s i s r o u g h l y p r o p o r t i o n a l t o t h e r e l a t e d n e s s o f t h e s e a n i m a l s as e s t a b l i s h e d b y s t a n d a r d m e t h o d s o f p h y l o g e n e t i c c l a s s i f i c a t i o n . The g r e a t e r t h e number o f d i f f e r e n c e s t h e more d i s t a n t t h e r e l a t i o n s h i p . S i n c e m y o g l o b i n , a m o l e c u l e w i t h s i m i l a r f u n c t i o n a n d t h r e e - d i m e n s i o n a l c o n f i g u r a t i o n t o t h e h e m o g l o b i n m o l e c u l e and w h i c h i s r e l a t e d t o h e m o g l o b i n on t h e e v o l u t i o n a r y s c a l e , d o e s n o t p o s s e s s t h e c a p a b i l i t y o f b i n d i n g w i t h h a p t o g l o b i n , i t i s o f i n t e r e s t t o a s c e r t a i n w h e t h e r h e m o g l o b i n s f r o m o t h e r s p e c i e s o f a n i m a l s , whose h e m o g l o b i n , a l t h o u g h i t h a s u n d e r g o n e i n d e p e n d e n t n a t u r a l s e l e c t i o n a n d a d a p t i o n t o t h e e n v i r o n m e n t h a s n e v e r t h e l e s s r e t a i n e d t h e b a s i c f u n c t i o n o f o x y g e n t r a n s p o r t , w o u l d a l s o b i n d t o h a p t o g l o b i n i n a s i m i -l a r way. I t i s known t h a t h a p t o g l o b i n b i n d s h o r s e h e m o g l o b i n f o r J a y l e ' s p e r o x i d a s e a s s a y e m p l o y s h o r s e h e m o g l o b i n ( 1 5 3 ) . A l l h a p t o g l o b i n s t h u s f a r e x a m i n e d h a v e b e e n r e p o r t e d t o c o m b i n e w i t h numerous a n i m a l h e m o g l o b i n s , h o r s e , cow, d o g , monkey, r a b b i t , r a t and mousey ( 4 4 , 4 6 , 5 6 ) . The b i n d i n g o f h e m o g l o b i n a p p e a r s t o be d e t e r m i n e d by t h e h a p t o g l o b i n a n d n o t by t h e g l o b i n p a r t o f t h e h e m o g l o b i n m o l e c u l e ( 4 4 , 5 6 ) . - 105 -However, the peroxidase a c t i v i t y of a complex with horse hemoglobin i s twice as great as the corresponding complex with human hemoglobin, although the binding capacity of human haptoglobin i s the same for these two types of hemo-globin as determined by.the paper electrophoretic method (54). The complex with dog hemoglobin has l i t t l e peroxi-dase a c t i v i t y and that with rat hemoglobin has none (46). The peroxidase a c t i v i t y is variable depending on the o r i g i n of the hemoglobin and therefore.the a b i l i t y of haptoglobin to enhance the peroxidase, a c t i v i t y .is c l e a r l y dissociated from i t s a b i l i t y to combine with hemoglobin. The peroxidase assay i s i n e f f e c t measuring a d i f f e r e n t property .of ..the. system and i s , therefore, more equivocal as a measure of. binding than the direct' Sephadex assay. No quantitative, studies of the binding of d i f f e r e n t animal hemoglobins by pure haptoglobin have been reported previously. Investigations have been carried out with serum or plasma (44,56) but these have been q u a l i t a t i v e ; Since haptoglobin binds to the globin moiety of the hemoglobin molecule, the e f f e c t of the v a r i a t i o n i n structure of the hemoglobin molecule i n d i f f e r e n t species on i t s a b i l i t y to combine with haptoglobin may provide some information on the nature of the binding between hemoglobin and haptoglobin. To this end, a series of Sephadex assays were carried out with hemoglobin from a series of vertebrates ranging from the c l o s e l y related domestic mammals to the more d i s t a n t l y related .bird, amphibia and fishes. - 106 -Hemoglobin solutions were prepared from heparinized blood of the following animals: cow, sheep, pig, dog, rabbit, cat, rat, mouse, chicken, frog and rainbow trout. The amount of heparin used to prevent coagulation of the blood is an amount which is less than that which is said to inhibit binding of haptoglobin to hemoglobin, that i s , less than 40 mg heparin per 100 ml plasma (54). Horse blood col-lected in Becton-Dickinson EDTA vacuotubules was used to prepare the horse hemoglobin solution. In each case, the Sephadex assay with each kind of animal hemoglobin was carried out with 0.1 M phosphate buffer, pH 7.0. An amount of hemoglobin in excess of that required for equimolar haptoglobin binding was added to 1.5 mg or 2.0 mg of pure haptoglobin dissolved in 0.5 ml of 0.1 M phosphate buffer. The mixture was then separated by the Sephadex G-200 method. With the hemoglobins of rat, rabbit, dog, trout and frog, controls of the hemoglobin solution alone were also performed. The results of these assays are summarized in Table III. In every case a Hb-Hp complex has formed between human hapto-globin and the vertebrate hemoglobin, confirming the observa-tions by previous investigators. These complexes have approxi-mately the same molecular weights as the human Hb-Hp complex, since the point of elution on the column is the same as that in the latter case. The observed ratio of the absorbancies at 407 my to 280 my i s , in the horse, cow, sheep, pig and cat TABLE I I I H a p t o g l o b i n B i n d i n g w i t h A n i m a l Hemoglobins High mol. wt. peak H b - H p peak F r e e Hb Assay 407 my/280 my 407 my/280 myr-obs. 407 my/280 my corrected" 1" 407 my/280 my 1. Rat Hb + Hp 0.99 1.62 2. 00 2.67 2. R a b b i t Hb + Hp 1.24 1.68 1.83 3.29 3. Dog Hb + Hp 1.22 1.80 1.91 3.47 4. Cat Hb + Hp 0.92 1.95 1.86 4.32 5. Sheep Hb + Hp 0.59 2.02 1.92 4.34 6. Cow Hb + Hp - 1.99 1.96 4.03 7. P i g Hb + Hp - 2.00 2.05 3.71 8. . Mouse Hb + Hp 0.81 1.84 2.00 3.33 9. C h i c k e n Hb + Hp - 1.62 1.90 2.89 10. F r o g Hb + Hp - 0.74 0.76 3.39 11. T r o u t Hb + Hp - 0.65 0.68 2.91 12. Horse Hb + Hp - 2.16 2.01 4.49 f V a l u e s c o r r e c t e d f o r a l t e r e d 407/280 r a t i o s i n the hemoglobin. - 108 -hemoglobins, very similar to that found with human hemoglobin. In hemoglobins of the dog, rabbit, chicken, rat and mouse, the observed ratio is slightly lower and is due to a lower 407/280 ratio in the native hemoglobin. Correcting for this deviation from the observed value of 3.9 for human haptoglobin, the 407/280 ratio of a l l the complexes is very close to the value of 2.0 except in two cases. Fish and trout, the two more distantly related species, show a marked variation in the 407/280 ratio, the corrected values being 0.68 and 0.76 respectively. The stoichiometry of binding appears to be one molecule of haptoglobin to one-quarter molecule of hemoglobin. If the lower absorbancy ratios found with the more dis-tantly related vertebrate hemoglobins are a reflection of the greater degree of difference of these hemoglobins from mammalian hemoglobins, then the lower; binding observed might be due to a looser linkage and a state of reversible equili-brium in the complex. To test this possibility, increasing amounts of trout hemoglobin solution were added to the same amount of pure haptoglobin and subjected to the Sephadex assay. The highest absorbing fractions of the trout Hb-Hp peak in experiment 1 (Table IV) were combined and re-run on the Sephadex G-200 assay column for experiment 6. The results summarized in Table IV shows that the 407/280 absorbancy ratio remains essentially constant regardless of the quantity of hemoglobin, whether i t is just slightly over the stoichio-metric amount required for binding as in experiment 1 or - 109 -TABLE IV Haptoglobin Binding-with Varying Concentrations of Trout Hemoglobin OD 407 Experiment Trout Hb Human Hp Hb/Hp OD 28 0 my mg/ml mg/ml 1 5.80 7.64 0.76 0.93 2 1.60 1.91 0.84 0.63 3 1.74 1.91 0.91 0.67 4 2.03 1.91 1.06 0.64 5 2.32 1.91 1.22 0.76 6 re-run of Hb-Hp complex 0.59 - n o -whether a large excess of hemoglobin is present as in experi-ment 5. The slightly higher ratio in experiment 1 compared to the other values in the remaining experiments is due to a larger scale separation to provide sufficient Hb-Hp complex for a re-run in experiment 6 and as a consequence, there is a slight overlap of the free hemoglobin peak. The re-run of the trout Hb-Hp complex 2 days later in experiment 6, shows that the complex is not readily reversible for no free hemo-globin is observed, although there is a very slight skewing of the peak at the t a i l end. The conclusion arrived at from these experiments is that the binding between fish hemoglo-bin and human haptoglobin is not readily reversible and the lowered ratio observed in the binding i s not due to a loose combination between the two proteins. The lower ratio in the complexes with frog and fish hemoglobin may be related to the reported heterogeneity of trout hemoglobin (198) and frog hemoglobin (199^,200), so that haptoglobin could be selecting for a particular hemo-globin in the mixture of components. Another more likely possibility is that the heme is bound more loosely in fish and frog hemoglobins, particularly when these hemoglobins are bound by haptoglobin. There is evidence of an altered conformation in the mammalian Hb-Hp complex (201) and the heme in the complex becomes accessible to the enzyme, heme a-methenyl oxygenase (99). Direct evidence for looser bind-ing of the heme comes from the observation that when fish - I l l -hemoglobin i s added to human serum, a strong heme-albumin band appears on a SG-electropherogram. Since there i s very l i t t l e heme-albumin complex formed with similar amounts of human hemoglobin, this suggests that f i s h hemoglobin i s i n equilibrium with appreciable concentration of free heme and thus the complex of heme with globin i s much weaker than i n human hemoglobin. Reaction of f i s h or frog hemoglobin with human haptoglobin may cause.some loss of the heme groups, analogous to the reaction of anti-globin (myoglobin) with native myoglobin observed by Sela (202)'. Myoglobin reacts with the antibody directed against the globin moiety of the myoglobin to produce a col o r l e s s p r e c i p i t a t e ; this implies that a conformational change i n the myoglobin molecule has resulted i n expulsion of the heme group. I t was also observed from the eluti o n p r o f i l e s i n assays of c e r t a i n mammalian hemoglobins that a heme-containing peak was often eluted at the void volume (Table I I I ) . This peak when present i n the Hb-Hp assay was also present i n control samples of the hemoglobin alone. In most cases, i t was a minor component of the solution; however, when dog hemoglo-bin was allowed to stand for a period of 5 days at 4°, the high molecular weight pigment increased considerably and on further storage, a pr e c i p i t a t e appeared. In only one case out of twelve species of hemoglobin studied, namely r a t , this peak formed an i n i t i a l l y s i g n i f i c a n t f r a c t i o n 19.4% of the t o t a l heme proteins i n the blood. In mouse this value - 112 -was 3.8%. Hemoglobin from the particular strain of mouse used, DBA/2J (Roscoe B. Jackson Memorial Laboratories, Bar Harbor, Maine), has been shown to possess in addition to i t s normal tetrameric hemoglobin, a polymer with a sedimentation coefficient of 6.32, formed by disulfide interaction between chains of 2 hemoglobin molecules (203). Polymerization of hemoglobin molecules has also.been observed in other strains of mice (204) termed "diffuse" (205,206), in which the approxi-mately 7S component increases upon storage, as contrasted to the strains of mice termed "single" in which the hemoglobin never exhibits polymerization either fresh or in storage (206). This phenomenon has been explained as being due to a single mutation (207).which facil i t a t e s polymerization to dimers and higher polymers. Polymerization of hemoglobin was f i r s t noted by Svedberg and Hedenius (208) in reptiles and amphibian hemoglobin hemolyzates, and studied in the frog (209) and turtle (210) by other investigators. The appearance of the larger molecular weight component in dog hemoglobin upon storage, which has not so far been reported, is similar to the phenomenon found with frog, mice and turtle hemoglobin. It is also interesting that storage of an abnormal human hemo-globin, Hb Porte Alegre, also results in dimerization (211). The above series of Sephadex assays on haptoglobin polymers, isolated a 1 , a 2 and 8 haptoglobin chains, globin, myoglobin and with animal hemoglobins demonstrate the use-fulness of this method of quantitation. The procedure is - 113 -rapid and simple and several assays may be carr i e d out simul-taneously. A d i r e c t measure of the amount of hemoglobin bound to the component under investigation i s given by the heme absorption at 407 my i n r e l a t i o n to the absorption due to protein i n the Hb-Hp complex. Also, the assay appears not to be influenced by any unknown factors as has been observed by Nyman (54) i n the peroxidase assay and the e f f e c t of species v a r i a t i o n of hemoglobin on the peroxidase reaction i s avoided. Furthermore, the eluti o n p r o f i l e s provides an immediate p i c -ture of the process, thus i n the assay of a p u r i f i e d hapto-globin preparation impurities possessing molecular weights d i f f e r e n t from those of the complex or free hemoglobin may be immediately detected and any conformational change or polymerization i n either hemoglobin or haptoglobin i s evident from a change i n the point of elution of the molecule. - 114 -PART III EFFECT OF ENVIRONMENTAL FACTORS UPON THE BINDING OF HEMOGLOBIN AND HAPTOGLOBIN INTRODUCTION The binding assay using Sephadex G-200 columns lends i t s e l f well to a study of the environmental factors upon the combination of hemoglobin and haptoglobin. Parameters such as ionic strength and pH can be varied over wide limits without affecting the assay. This provides consid-erable advantage over gel electrophoresis or ion-exhange chromatography which are possible only within very narrow limits of salt concentration and pH. In the peroxidase reaction, the pH of the reaction is a c r i t i c a l factor and cannot be varied to any great extent without adversely affecting the reaction (81), similarly, in paper electro-phoresis the pH cannot be varied greatly without affecting the separation between the Hb-Hp complex and excess hemo-globin. It has been observed repeatedly (53,54,63,153) that the binding between hemoglobin and haptoglobin is tight and essentially irreversible. For instance, there is almost no exchange between labeled components in the complex with non-radioactive molecules in the system (53,131) and although haptoglobin does not bind deoxyhemoglobin, once i t is bound to oxyhemoglobin, deoxygenation does not remove the hapto-globin (63). The great stability of binding between hemo-- 115 -globin and haptoglobin is not due to the formation of a disulfide bond for haptoglobin contains no free sulfhydryl groups (212,213) and treatment of hemoglobin with Hg + +, Ag +, Cu +, p-mercuribenzoate (212) or iodoacetamide, N-ethyl-maleimide, cystine and cystamine (68) did not inhibit hapto-globin binding. Thus blocking of the reactive sulfhydryl groups of hemoglobin has no effect on i t s a b i l i t y to bind to haptoglobin. Polyelectrolytes such as heparin, protamine and to a lesser extent, certain carbohydrates, such as galac-turonic acid and glucosamine inhibit the reaction between hemoglobin and haptoglobin (214). These observations led to the suggestion that the interaction is of an electrostatic nature with the intervention of the carbohydrate moiety of haptoglobin in the combination with hemoglobin (214). Sub-sequently, however, neuraminidase action on haptoglobin and on the Hb-Hp complex (19,51) was shown to affect neither complex formation nor the peroxidase activity of the com-plex, therefore the carbohydrates themselves appear not to be essential in binding. Bajic (169) claims that the stabi-l i t y of the Hb-Hp complex is ascribed to electrostatic forces and the action of van der Waal's forces between surfaces with pronounced complementarity, since the formation of the complex can be inhibited equally by electric a l l y positive, negative and neutral polymeric substances (protamine, heparin and sodium alginate). The st a b i l i t y of the complex is very high and i t has - 116 -resisted various attempts at dissociation (104). In vitro, the complex is said to be formed within a wide pH range, from 4.4 to more than 10, but at a pH less than 4.4 the hemoglobin binding groups of haptoglobin are irreversibly destroyed (54). The inab i l i t y of haptoglobin in acid media to bind hemoglobin was confirmed by Pavlicek and Kalous (215) whose titration curves, differential absorption spectra and optical rotation measurements indicated that in the region below pH 5.0 the haptoglobin molecule undergoes far-reaching structural changes. In the work described here, the effect of increasing salt concentrations to produce very high ionic strength and the effect of varying the pH over a wide range on the forma-tion of the complex was studied in order to cl a r i f y the nature of the linkage between hemoglobin and haptoglobin. Also a single experiment was conducted to test the importance of an analogue of tyrosyl-residues in complex formation. EFFECT OF IONIC STRENGTH AND pH UPON HEMOGLOBIN-HAPTOGLOBIN BINDING A series of Sephadex G-200 assays was performed in 0.2 M Tris-HCl buffer, pH 7.5 containing 0, 0.5, 1.0 and 2.0 M ( N H O a S O i * . In each case, 1 mg haptoglobin was dissolved in 0.5 ml of the buffer and 1.2 mg hemoglobin was dissolved in a separate 0.5 ml of the same buffer. After combining the two solutions, the mixture was assayed for hemoglobin binding capacity by the Sephadex G-200 method. The results of the assays with increasing salt concen-trations are summarized in Table V. Even at 1 M ammonium - 117 -TABLE V E f f e c t of Sa l t Concentration on Hb/Hp Combination (Sephadex G-200 Method) 0.0 2 M Tris-HCl pH 7.5 (NHQzSCU (M) OP 407 my/OP 28 0 my 0 1.92 0.5 1.98 1.0 1.93 2.0 1.61* * began to p r e c i p i t a t e - 118 -sulphate and probably above, combination occurs at the usual stoichiometry. This result was observed both in the presence of the divalent anion, sulfate as well as in the monovalent anion, chloride. At the highest salt concentration, there was evidence of precipitation and the elution profile was somewhat erratic, which may account for the slightly lower binding observed. In another series of binding experiments, buffers rang-ing from pH 3.0-pH 11.0 of composition listed in Table VI, were used. A solution of 1.2 mg of hemoglobin in 0.5 ml of the buffer was added to 1.0 mg of haptoglobin in 0.5 ml of the same buffer. The mixture was then assayed for hemo-globin binding capacity on Sephadex. The results of the experiments in which the pH was varied are summarized in Table VI. It is evident that stoichiometric complex formation occurred between pH 4.0 and pH 11.0. The apparent stoichiometry appears to change above pH 9.0 but this was found due to specific absorption changes at the higher pH1s in which the absorption spectra of hemoglobin at higher pH1s show a decrease in 407 my absorption and an increase in 280 my absorption. When corrected for these changes the 407/280 ratio from pH 4.0 to 11.0 is close to the value of 2.0. At the extreme acidic pH1s, pH 3.0 and 3.5, an instant darkening of the hemoglobin solution indicated an irreversible denaturation of the molecule, which has been reported by Field and - 119 -TABLE VI Effect of pH on Hb/Hp Combination (Sephadex G-200 Method) Buffer (0 .1 M) OD 407 my/OD 280 3.0 ci t r i c - c i t r a t e (Na) 0.61* 3.5 formic-formate (Na) 0.50* 4.0 formic-formate (Na) 1.83 4.5 formic-formate (Na) 2.12 5.0 acetic-acetate (Na) 1.89 7.0 phosphate (Na) 2.05 9.0 Tris-HCl 1.81** 11.0 bicarbonate-carbonate (Na) 1.95** * hemoglobin instantly denatured ** corrected for absorption changes at these pH's - 120 -O'Brien to occur at pH below 3.5 (216). At these acidic pH's, there are also conformational changes in the haptoglobin molecule (215). Therefore, an altered stoichiometry of bind-ing is to be expected and a significant lower amount of hemo-globin is observed to be bound to the haptoglobin. Thus, both the results of the experiments with increasing salt concentration and. varying pH indicate that electrostatic interactions cannot be the sole intermolecular forces involved in binding, since masking the electrica l l y charged groups by a high concentration of ions does not affect the interaction nor does wide variation in the ionic state of charged groups on the two proteins produce any significant effect on the binding. These results minimize the importance.of electro-static binding in complex formation, contrary to the obser-vations of Robert, Bajic and Jayle (214). Although electro-static forces may not be the sole factor involved in the binding, there is s t i l l the possibility that electrostatic forces together with other types of interactions acting in a co-operative manner may be responsible for the sta b i l i t y of the Hb-Hp complex. Anfinsen (217) has found that the tertiary folding of reduced RNase is severely inhibited by analogu'es of tyro-sine. In a single experiment to test the effect of a tyro-syl-analogue, phenol, in the binding, solutions of 1.2 mg hemoglobin and 1 mg haptoglobin each in 0.-5 ml of 0.1 M phosphate buffer, pH 7.0 containing 0.1 M phenol were combined - 121 -and assayed on G-200 previously equilibrated with the same buffer. Measurement of 280 my absorption was unsuccessful even after prolonged dialysis to remove the aromatic pheno-l i c groups. However, the heme absorption of the fractions (Fig. 17) shows that complex formation had indeed occurred, since an i n i t i a l heme-absorbing peak was observed in the elution volume of the Hb-Hp complex, ahead of a second peak whose elution volume corresponds to that of free hemoglobin. Further experiments with other compounds which might be thought of loosely as tyrosine analogues (217) were not pur-sued since the flow rate in this assay was considerably re-duced to render the method unpractical for further studies, the presence of the aromatic compound tending to affect the porosity of the dextran gel. However, the phenol experi-ment tends to question the involvement of tyrosine in the Hb-Hp complex as postulated by Kalous and Pavlie ;ek (218) . A further aspect of the Hb-Hp binding is revealed by the experiments in which salt concentration and pH are varied. Several investigators have shown that hemoglobin dissociates readily into half molecules at high salt concentrations and at both acid and alkaline pH1s (219). The osmotic pressure data by Gutfreund (220), the sedimentation and diffusion studies by Benbamou and coworkers (221) and the light scattering and sedimentation studies by Rossi-Fanelli, Antonihi and Caputo (222), a l l indicate that a reversible dissociation of hemo-globin into subunits occurs above 0.5 M NaCl (219), there being - 122 -Figure 17. Assay of Hp 1-1 on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0 containing 0.1 M phenol. - 123 -similar effects with other salts (222). At the highest salt concentration studied by Rossi-Fanelli and associates, 3 M NaCl, the weight average molecular weight indicated that the great majority of the molecules exist in a dissociated form. Similarly at the extreme pH's reversible dissociation occurs, from pH 6.0 to 3.5 a dissociation into subunits takes place and the molecular weight of the protein approaches half of i t s normal value at about pH 4.5 (216,219). Below pH 4.5, hemoglobin further dissociates into one-quarter molecules and the dissociation is no longer reversible (219). In the alkaline direction, at a pH just beyond 10 dissociation begins until at pH 11.0 the molecule is completely dissociated into 2 subunits (223). At pH 11.6, the changes in the hemoglobin molecule are no longer reversible (223). Thus, at the higher salt concentrations used in the present study, as well as at the extreme pH1s, hemoglobin would certainly exist largely as half molecules and since binding by Sephadex G-200 appears unchanged, i t would seem likely that the binding site on haptoglobin is able to combine with these half molecules. This is a point of considerable interest, since compari-son of haptoglobin with the 7S immunoglobulins shows a simi-l a r i t y in basic structure in that both molecules possess two pairs of dissimilar (two light and two heavy) chains' connected by disulphide bonds. Furthermore, the combination of hapto-globin with hemoglobin is similar to the antigen-antibody reaction in that the combination is extremely tight and very specific. One major difference, however, is that the complex between hemoglobin and haptoglobin remains soluble. An inter-esting question is whether haptoglobin, like the IS antibody, is bivalent, but since the stoichiometry of binding is one hemoglobin to one haptoglobin, there are two possibilities in the binding, either there is one site per haptoglobin mole-cule which could bind the whole hemoglobin or there are two sites per haptoglobin molecule each of which bind one-half molecule of hemoglobin. Laurell (57) and Allison and ap Rees (58) by paper electrophoresis observed the formation of two kinds of Hb-Hp complexes. On starch gel electrophoresis Laurell (55) and Shim, Lee and Kang (33) provided evidence that when haptoglobin is in excess over hemoglobin, an inter-mediate complex is formed which is likely to have the stoi-chiometry of one-half molecule of hemoglobin to one haptoglo-bin, since the intermediate complex migrates in starch gels more slowly than uncombined haptoglobin but faster than f u l l y saturated haptoglobin. These data are consistent with that obtained from kinetic studies by Nagel and Gibson (59) in which a reaction between aB hemoglobin dimers with two simi-lar but independent binding sites on the haptoglobin molecule is proposed. Very recently, such a Hb-Hp intermediate has been isolated by Hamaguchi and Sasazuki (224) by addition of calculated amounts of hemoglobin to haptoglobin followed by separation and purification on DEAE-Sephadex chromatography. From the molecular weight and the heme content of the inter-mediate complex, Hamaguchi (225) concluded that i t consists of one molecule of haptoglobin and one-half a molecule of hemo-globin. - 125 -PART IV CHEMICAL MODIFICATION OF AMINO GROUPS IN HAPTOGLOBIN INTRODUCTION Chemical modification of proteins with specific group reagents plays an important role in elucidating the groups involved in the active site of a biologically active protein. Ideally the reagent under suitable conditions should affect only one type of group or one particular residue of several of the same kind. A method of measuring the extent of reac-tion of the protein and a means for assessing the effect of the modification on the biological activity must also be available so that changes in the biological activity can be interpreted in relation to the modification. It is to be expected that random reaction of a particular chemical group would produce a family of proteins varying in number of sub-stituted groups as well as distribution of these groups through-out the molecule. Correlation of the change in biological activity with the actual modification in the fractionated material as distinct from the overall modification in the heterogeneous protein population would provide more meaning-ful interpretations. Furthermore, i t is necessary to dis-tinguish between loss of biological activity due to chemical modification on a specific group in the active site from an unfolding of the tertiary structure of the molecule from a modification of a group located outside the active site but which is involved in maintaining the molecule in the native - 126 -conformation by participation in tertiary interactions (226). If loss of biological activity can be shown to occur on modi-fication of a specific group without conformational changes in the protein, then i t may be concluded that the amino acid modified is an important component of the active site. On the other hand, i f changes in size and shape of the protein occur after the reaction, no such conclusions may be drawn. Very l i t t l e has been reported on the chemical modifica-tion of haptoglobin. Bajic (169) carried out experiments with formaldehyde and considered that substitution of amino groups of haptoglobin diminished i t s a b i l i t y to react with hemoglobin. However, the reaction of formaldehyde with proteins is complicated and i t s usefulness as a specific reagent i s , therefore, limited. Formaldehyde reacts under gentle conditions almost instantaneously with amino and sulfjhydryl groups. The mono-substituted derivatives of these groups show a high degree of reactivity and further condense with amide, guanidyl, phenolic and heterocyclic groups to yield cross-linking methylene bridges (227). Shinoda (228) labeled the amino groups by their conver-sion into trinitrophenyl-residues (TNP-residues) with 2,4,6-trinitrobenzene-l-sulfonic acid (TNBS). This reagent is reported to react almost quantitatively with amino groups in slightly alkaline solution without detectable substitution of amino groups or hydroxyl groups (229). It was found that with 15.5,-16.4 and 11.8 moles of TNP-residues in Hp 1-1, 2-1 - 127 -and 2-2 molecules respectively, the hemoglobin binding capa-c i t y was 50% of the i n i t i a l capacity and a semilogarithmic plo t showed a lin e a r r e l a t i o n s h i p between increases of TNP-residues i n the protein and loss of hemoglobin binding capae c i t y . I t was concluded from these results that some of the amino groups comprise one of the es s e n t i a l constituents of the active s i t e of haptoglobin. However, the TNP group i s a rather bulky moiety and there was no evidence that the loss of binding capacity could not have been due to a non-s p e c i f i c i n a c t i v a t i o n such as an altered conformational state. Thus, the chemical modification experiments so far repor-ted are inconclusive. Furthermore, i n order to obtain an accurate picture of the changes that may be taking place i n the molecular structure when a protein i s subjected to chemical modification, i t i s necessary to follow the effects of modification by employing as many methods as possible. In the work to be described here modifications with three d i f -ferent reagents of increasing severity have been examined to study the e s s e n t i a l i t y of the amino groups. F i r s t , guanidi-nation with l-guanyl-3,5-dimethyl pyrazole n i t r a t e (GDMP) converts l y s i n e to homoarginine, a chemically d i f f e r e n t r e s i -due but one which remains p o s i t i v e l y charged. The extent of modification i s e a s i l y determined by measuring the homo-arginine content following acid hydrolysis of the guanidinated protein; homoarginine appears on the amino acid analyzer - 128 -clearly separated after arginine (Fig. 18). The second type of modification of amino groups, acetylation with acetic an-hydride, in the presence of sodium acetate, is somewhat more drastic than guanidination, since positively charged e-lysyl side chains are converted to neutral, c-N-acetyl side chains. Lastly, the most drastic modification is succinylation of amino groups with succinic anhydride which converts the positive amino group to the negatively charged N-succinyl compounds. In the last two types of modification, the extent of the reaction may be measured by trinitrophenyl assay of the residual free amino groups (230). GUANIDINATION Guanidination involves the least radical change in the nature of the chemical group, the basic amino group of lysine is replaced by the more basic guanidino group. Application of the reaction to chymotrypsinogen by Chervenka and Wilcox (231) and to ribonuclease by Klee and Richards (.232) resulted in modification of lysine residues without causing major changes in secondary structure. Guanidination is also essen-t i a l l y specific for the e-amino groups of lysine (233) and is therefore a good method for investigating the essential nature of these residues for biological activity. Guanidination was carried out with GDMP instead of with the classic reagent, o-methylisourea, because of i t s lower op-timum pH, 9.5 as compared with 10.5-11 for the latter reagent (234). In the present studies, i t was found that reaction - 129 -A 1 2 3 4 5 Figure 18. Separation of known basic amino acids on the Beckman/Spinco 120 C amino acid analyzer. Peaks (1) lysine (2) h i s t i d i n e (3) ammonia (4) arginine (5) homoarginine - 130 -at a pH of 9.0 for a period of 3 days with 0.5 M GDMP reagent produced sufficiently extensive modification so that the lower pH was used throughout. EXPERIMENTAL Guanidination of haptoglobin, Hb-Hp and hemoglobin was carried out by a procedure similar to the procedure of Habeeb (235) with GDMP. Purified Hb-Hp complex was prepared for the reaction by addition of 118 mg hemoglobin dissolved in 2.5 ml of 0.05 M N H 4 H C O 3 , pH 8.0 buffer to 100 mg Hp 1-1 in 2.5 ml of the same buffer and separation of the complex on a Sepha-dex G-200 reverse flow column, 2.8 cm x 97 cm equilibrated in 0.05 M N H 4 H C O 3 , pH 8.0. After the mixture was applied to the column, about 9 ml of a 15% sucrose solution colored with 2,4-dinitrophenol was applied to displace the protein solu-tion into the bottom of the column. It had been observed pre-viously that in reverse flow chromatography when a dense protein solution was applied to the bottom of a column and then immediately displaced by aqueous buffer, the less dense buffer "broke through" the protein layer and badly distorted i t . When the eluting buffer was made more dense than the protein solution, the protein band remained very sharp and as soon as i t passed into the gel the sucrose buffer could be replaced by the normal buffer. After separation of the complex, the combined fractions were lyophilized. For the guanidination reaction, a 0.5 M GDMP solution was made up - 131 -by adding 2.515 go. GDMP to 7 ml of d i s t i l l e d water and the pH adjusted to 9.0 with 1.0 N NaOH and the volume then made up to 25 ml. Separate 25 mg portions of haptoglobin were each dissolved i n 2.5 ml of 0.1 M GDMP, 0.2 M GDMP and 0.5 M GDMP solutions at pH 9.0 together with a control in 0.1 M phosphate buffer, pH 7.0. In addition, 25 mg of Hb-Hp complex and 25 mg of hemoglobin were each dissolved i n 2.5 ml 0.2 M GDMP. Each of the above solutions were adjusted to pH 9.0 with NaOH and allowed to react at 4° for approxi-mately 72 hours. At the end of the sp e c i f i e d period, each solution was desalted on a Sephadex G-25, 2.5 x 30 cm column, to remove excess reagent and to stop further reaction. The guanidinated hemoglobin mixture had a great deal of p r e c i -p i t a t e which was removed before desalting. The solutions were then l y o p h i l i z e d . Sephadex GfSOO assays were performed on the haptoglobins modified with 0.1 M, 0.2 M and 0.5 M reagent and on the control. In each case, 2 mg hemoglobin i n 0.2 5 ml of 0.1 M phosphate buffer, pH 7.0 was added to 2.5 mg of the modified and control haptoglobin i n 0.25 ml of phosphate buffer. The mixtures were then assayed by the Sephadex method. To determine the extent of modification, 5 mg of each of the modified proteins and the control were separately dissolved i n 1 ml 6 N HC1 and the mixture hydro-lyzed i n sealed ampoules at 105° for 18 hours. At the end of the hydrolysis period, the black p r e c i p i t a t e (humin) i n the hydrolyzates was removed by centrifugation and the super-- 132 -natant dried in vacuo over NaOH pellets. The residue was taken up in 1 ml of pH 2.2 citrate buffer and aliquots of this solution analyzed in duplicate for basic amino acid content on the Beckman/Spinco Model 120C amino acid analyzer. RESULTS The results of the binding assay of the guanidinated proteins and the control are shown in Table VII along with the extent of modification of these compounds. On guanidi-nation with the 0.1 M and 0.2 M GDMP solutions, in which 26.3% and 52.9% of the e-amino groups were guanidinated, there is no effect on the binding capacity, as may be seen by the 407 my/280 my ratio. From the elution profiles in Figure 19, frames B and C representing these two modified proteins respectively, the guanidinated haptoglobins com-bined with hemoglobin to give a symmetrical peak eluting in the same volume for the Hb-Hp complex as that seen in the control (Fig. 20, frame A). However, at the greatest extent of modification with 0.5 M GDMP reagent, there is evidence that a new phenomenon is occurring (Fig. 19, frame D). A portion of the highly substituted haptoglobin is eluted earlier from the Sephadex G-200 column, indicating that a conformational change has occurred. Following this change, the binding is very much decreased, the ratio having dropped to 0.73. However, the greater part of the protein, which is also very highly modified judging from an 82% homoarginine content for the mixture of the two species of modified protein, - 133 -TABLE VII Guanidination (±) HC C-CH 3 NH 2 W I I I CH3-C N C=NH NH 3 ^ N NH (CH 2K + C=NH P H 9 > (CH 2) 4 I I © G> I -NH-CH-CO... NK2 NO3 ..NH-CH-CO.. lysyl residues l-guanyl-3,5- homoarginine dimethyl pyrazole nitrate (GDMP) OD 407 Reaction Conditions % lysine % homoarginine OD 28 0 Hp 1-1, pH 9.0, 0°, 72 hrs Control 0.1 M GDMP 0.2 M GDMP 0.5 M EDMP peak A peak B Hb, pH 9.0,0°, 7 2 hrs 0.2 M GDMP Hb-Hp, pH 9.0, 0°, 72 hrs 0.2 M GDMP 4 7.8 52.2 * Lower elution volume on Sephadex G-200 indicates confor-mational change 100 73.7 47.1 17.8 41. 7 26.3 52.9 82.2 58.3 2.19 2.23 2.12 0.73* 2.07 - 134 -~r i r~~ 1 ™ r j — — i 2—™i EFFLUENT VOLUME, ml Figure 19. Assay of Hp 1-1 guanidinated with 0.1 M GDMP (B), 0.2 M GDMP (C), 0.5 M GDMP (D) compared with a control (AXcon Sephadex G-200 i n 0.1 Mv-phbsphate buffer, pH 7.0. - 135 -s t i l l binds essentially the same amount of hemoglobin, the 407 my/280 my ratio being 2.07. It appears then that binding remains essentially constant until a very large percentage of the amino groups have been converted to guanidino groups when there is an attendant conformational change in the molecule and the binding is greatly reduced. An attempt to ascertain the extent of the area involved in the binding site was made by simultaneous guanidination of haptoglobin, hemoglobin and the Hb-Hp complex with the same reagent, 0.2 M GDMP, on the assumption that there would be a s t a t i s t i c a l l y equal distribution of the lysine residues over the surface of the haptoglobin molecule since lysine is a frequently occurring amino acid residue in proteins and, being highly polar, usually occurs at the surface where i t is able to interact with solvent. Table VII shows that hemoglobin is modified to a slightly greater extent, 58.3% than haptoglobin (52.9%) although i t contains fewer lysines, that i s , 44 lysine residues (219) as compared with 64 lysines in haptoglobin (236). Guanidination of the complex leads to 52.2% of the combined lysine content of hemoglobin and hapto-globin being converted to homoarginine. This result can be compared with a value of 55.2% calculated from the homoarginine content of hemoglobin and haptoglobin when guanidinated separ-ately under the same conditions. The difference between the observed and theoretical values gives an indication of the extent of the area of the binding site. The small difference - 136 of 3% indicates that the extent of involvement of lysine residues in the active site is very small. However, another possibility exists that on binding of hemoglobin by hapto-globin there are alterations in the conformation of the molecule(s) such".that more lysine residues become available for the modification reaction. There are indications that such conformational changes in the hemoglobin molecule do occur on combination of hemoglobin with haptoglobin and affect the heme to globin linkage. The fact that the Hb-Hp complex has a high af f i n i t y for oxygen and lacks a Bohr effect may reflect conformational changes in the hemoglobin molecule that make impossible the slight alterations in quaternary interactions underlying heme-heme interaction and the Bohr effect (237). Alteration of the peroxidase pH optimum (3) and spectral changes in the spectrum due to heme also suggest a change in the heme environment in the complex (201). Nevertheless, in those globular proteins whose detailed three-dimensional structures have been solved, lysyl-side chains are always exposed on the surface of the molecule so that i t would be unlikely that conformational change could expose other "buried" lysines. Iii the case of chymotrypsinogen, Chervenka and Wilcox (231) have shown that a l l the lysyl residues can be substituted by guanidi-nation and the protein remains activatable. The conclusion, therefore, remains that the area of contact between hemo-globin and haptoglobin in the complex involves only a small - 137 -area of the surface of the haptoglobin molecule or else the area is particularly deficient in lysyl-side chains. ACETYLATION Acetylation of amino groups with acetic anhydride con-verts the positively charged ammonium ion to a neutral N-acetyl group and the chemical change is therefore more radi-cal in nature than guanidination. Acetylation of amino groups is one of the most common means employed for the chemical modification of proteins for the lysine residues being located on the surface of the molecule are readily accessible to reac-tion with one of the specific acetylation reagents under mild conditions and the extent of the modification is assessed by relatively simple analytical means. The reagent of choice for the substitution of acetyl groups on amino groups is acetic anhydride, which under certain conditions is a highly specific reagent for amino groups (238,239). The reaction is most often carried out in half-saturated sodium acetate, which functions as a buffer of the reaction mixture and also appears to serve as a catalyst (240). In addition, the high concentrations of acetate ions catalyzes the hydrolysis of O-acetyltyrosyl residues and thereby increases the speci-f i c i t y of the reaction towards amino groups (241). EXPERIMENTAL Acetylated haptoglobins were prepared by the method of Fraenkel-Conrat, Bean and Lineweaver (242). Four portions of 50 mg of haptoglobin _w_er.e. dissolved in 5 ml of half-- 138 -saturated sodium acetate and cooled in an ice-bath during the reaction. A 0.1 M acetic anhydride solution in ace-ton i t r i l e was made up and also kept in the ice-bath. Cold acetic anhydride solution was added to each flask during an interval of 1 hour so that the f i n a l molar ratios of acetic anhydride to haptoglobin is 10:1, 40:1, -80:1 and 400:1 in flasks designated A, B, C, and D respectively. Flask C corresponds to an approximately 1:1 ratio for acetic anhydride to lysine on the basis of 64 lysine r e s i -dues in Hp 1-1. Addition of reagent was as follows: a total of 50 y l of acetic anhydride was added to Flask A by an i n i t i a l addition of 10 y l followed by 10 yl every 15 minutes. An amount of 50 y l was i n i t i a l l y added to Flask B, then 25 y l every 10 minutes to provide a total of 200 y l of reagent. Flask C contained a fi n a l 400 yl of reagent, which was added as an i n i t i a l 100 y l portion f o l -lowed by 50 y l every 10 minutes. To Flask D, 200 yl was added i n i t i a l l y , then 150 y l every 5 minutes, which results in a total of 2 ml of reagent added. After each addition of reagent, the flask was immediat«ly. swirled and returned to the ice-bath. The reaction was allowed to proceed for 4 hours, after which time, the mixture was desalted on a Sephadex G-25, 2.5 x 30 cm column. The solutions were then lyophilized. The binding capacity of each of the modified proteins was deter-mined by the Sephadex G-200 method on a mixture made up by adding a 0.25 ml solution of 1.5 mg of acetylated haptoglobin - 1 3 9 -in 0 . 1 M phosphate buffer, pH 7 . 0 to 1 . 5 mg of hemoglobin in 0 . 2 5 ml of the same buffer. Since the Sephadex assay elution diagram (Fig. 2 0 , frame D) of the haptoglobin modified with a large excess of reagent showed the presence of two species of protein, an unfolded protein which was eluted as a larger peak ahead of a smaller peak of haptoglobin in the native conformation, these two grossly different species of acetylated haptoglobins were partially resolved on a Sephadex G - 2 0 0 , 1 cm x 5 0 cm column in 0 . 1 M phosphate buffer, pH 7 . 0 . Fractions from the peak designated A and those from the peak designated B (Fig. 2 1 ) were pooled and dialyzed against 2 1 . of d i s t i l l e d water with several changes of water. These two solutions were then used in the assay for free amino groups. The extent of the acetylation reaction was determined by reaction with trinitrobenzenesulfonic acid (TNBS) according to the method of Satake and coworkers ( 2 3 0 ) . Solutions of 1 . 5 mg of control and of the acetylated haptoglobins modified with 1 0 : 1 , 4 0 : 1 and 8 0 : 1 reagent to protein were each dis-solved in 2 . 5 ml of d i s t i l l e d water. One ml of these solu-tions and 1 ml of peak A and 1 ml of peak B dialyzed solu-tions representing partially separated species of haptoglobin acetylated with a ratio of 4 0 0 : 1 reagent to protein, were reacted (in duplicate) with TNBS to determine the content of amino groups. To 1 ml of the test solution, 1 ml of 4 % NaHC03 and 1 ml of 0 . 1 % TNBS were added and the mixture incubated - 140 -EFFLUENT VOLUME, m? Figure 20. Assay of Hp 1-1 afte r acetylation with acetic an-hydride. Ratios of reagent to haptoglobin of (A) 10:1, (B) 40:1, (C) 80:1, (D) 400:1 on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0 - 141 -i — — r E F F L U E N T V O L U M E , ml Figure 21. P a r t i a l separation of two species of acetylated Hp 1-1 following reaction with a r a t i o of 400:1 acetic anhydride to protein on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0. - 142 -at 40° for 2 hours in the dark. At the end of this period, 1.0 N HC1 was added to each solution and the absorption at 340 mu measured. A blank of 1 ml of d i s t i l l e d water was used. RESULTS The results from the Sephadex G-200 assays of the acetylated haptoglobins and the degree of modification of these proteins are tabulated in Table VIII. It can be seen that binding of hemoglobin by the modified protein is l i t t l e affected by nearly 50% acylation of the lysine groups. At these levels of acetylation, in which there is no great excess of reagent, the elution profiles (Fig. 20, frames A, B and C) shows that the bulk of the haptoglobin is in the native conformation, although a slight skewing of the peaks suggests the beginning of a change in a very small propor-tion of the proteins. At the highest degree of acetylation, a conformational change in the haptoglobin is clearly apparent-and a new unfolded species appear (Fig. 20, frame D). The peak appearing f i r s t and representing a somewhat unfolded haptoglobin, shows only low binding, about 14% of the usual binding (Table VIII), whereas the acetylated protein appear-ing in the normal elution volume shows almost 40% of the normal binding (Table VIII). This is a further i l l u s t r a t i o n of the profound effect of the conformation of haptoglobin upon i t s a b i l i t y to bind hemoglobin.. SUCCINYLATION Succinylation causes the most drastic change in the - 143 -TABLE VIII Acetylation of Haptoglobin 1-1 CH 3 N H 3 © \ : = o C H 3 I I 0 Na acetate pH 8.2-> C=0 | (half saturated) | C=0 NH / I CH3 N-terminal acetic e-N-acetyl-lysyl-and anhydride e - l y s y l -residues OD 407 Reaction Conditions % Acetylation* OD 28 0 Control 2.25 10:1 acetic anhydride^ 23.4 2.01 to Hp 1-1 40:1 " 39.1 1.97 80:1 " 47.8 1.86 400:1 " peak A 83.9 0.37** peak B 69.9 0.85 * Measured by trinitrobenzenesulfonic acid reaction ' ** Lower elut i o n volume on G-200 indicates conformational change t Dissolved i n a c e t o n i t r i l e and added i n approximately equal aliquots over 60" at 0°.' -- 144 -chemical nature of the molecule in which the positively charged amino group is replaced by a negatively charged N-succinyl group. Succinic anhydride has been reported to attack the lysyl amino groups specifically (243) and being a relatively stable, non-volatile substance is convenient to use. Gounaris and Pe^r.imann (244) found that reaction can also occur with tyrosine and hydroxyamino acid residues although these O-succinyl groups would be rapidly hydro-lyzed under the reaction conditions employed here. EXPERIMENTAL Succinylated haptoglobins were prepared according to the procedure of Habeeb, Cassidy and Singer (245) with a ratio of succinic anhydride to lysine of approximately 15:1 (246). To a solution of 25 mg of Hp 1-1 in 2.5 ml of 0.01 M Tris-HCl buffer, pH 8.0, cooled in an ice-bath throughout the entire reaction, 31.5 mg of solid succinic anhydride was added and the mixture continuously stirred. Another 2.5 ml of a 1.0% haptoglobin solution served as a control. The pH of. the reaction mixture was maintained near 8.0 by addition of 1.0 N NaOH from a syringe. At intervals of 10 minutes, 30 minutes, 1 hour and 2 hours, from the time of addition of succinic anhydride, 0.5 ml aliquots were removed from the reaction mixture and from the control and subjected to chromatography on a Sephadex G-25, 0.9 x 30 cm column. The protein column effluents were used for binding studies and analysis on starch gel electrophoresis. Deter-- 145 -mination of the sedimentation coefficients and the molecular weights by the Archibald sedimentation equilibrium method was carried out on 10 minutes and 2 hours succinylated hapto-globins prepared in a subsequent experiment. Ultracentri-fugation of the 10 minutessuccinylhaptoglobin was in 0.1 M phosphate buffer, pH 7.0 containing 0.1 M NaCl and the 2 hours succiny.l-haptoglobin was in 0.01 M phosphate buffer, pH 7.0 with 0.05 M KC1. Various attempts at fractionating the 10 minutes and 2 hours succinylated haptoglobins by DEAE-cellulose chromatography with different gradient systems proved unsuccessful. RESULTS In Figure 22, frames A, B, C and D are the elution dia-grams from the binding assay of the succinylhaptoglobins pre-pared by 10 minutes, 30 minutes, 1 hour and 2 hours of reaction respectively. It is apparent that at the shortest reaction time, 10 minutes, a conformational change in the molecule is occurring and at the same time the binding capa-city has decreased by 65% (Table i x ) . As the reaction time is allowed to increase there is a progressive decrease in binding a b i l i t y ; by 1 hour and 2 hours, succinylated hapto-globins are essentially devoid of hemoglobin binding capa-city (Table IX). The Sephadex assays of the 10 minutes and 2 hours haptoglobin controls show .no decrease in hemoglobin binding and therefore precludes any influence on the hemo-globin binding by the shearing action of the magnetic i 1 i 1 T — i i — — T 1 — — T — — - r ~ EFFLUENT VOLUME, ml Figure 22. Assay of Hp 1-1 succinylated for various times on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0. Reaction times are: CA) 10 minutes, CB) 30 minutes, (C) 1 hour, (D) 2 hours, with a f i f t e e n - f o l d excess of succinic anhydride. NB - Frame C - due to malfunction of the f r a c t i o n c o l l e c t o r not a l l f ractions were co l l e c t e d . - 147 -TABLE I X S u c c i n y l a t i o n o f H a p t o g l o b i n 1-1 NH a-NH 2 o r e - l y s y l CO— I C H 2 •I C H 2 CO— 0 s u c c i n i c a n h y d r i d e CO-CH 2-CH 2-COO I. NH N - s u c c i n y l -d e r i v a t i v e <3 R e a c t i o n C o n d i t i o n s  Hp 1-1, 0.01 M T r i s - H C l , pH 8.0 1 0 1 c o n t r o l 120' c o n t r o l t 1260:1 s u c c i n i c a n h y d r i d e t o p r o t e i n 10 ' 30 1 60' 120 ' 10' 120' t OP 407  OP 280 2. 55 2.20 % S u c c i n y l a t i o n 0.76** 0.68** 0.24** 0.11** 0.41** 0.07** 85.6 96.9 a d d e d as a s o l i d a t 0° ' w i t h s t i r r i n g a n d pH m a i n t a i n e d c l o s e t o 8 by a d d i t i o n o f NaOH ** l o w e r e l u t i o n v o l u m e on G-200 i n d i c a t e s c o n f o r m a t i o n a l c h a n g e - 148 -stirring for such lengths of time during the reaction (Table IX) . The SG-electropherograms(Fig. 23 and 24) also confirm that the 10 minutes and 30 minutes modified proteins have retained some binding capacity, while the 1 hour and 2 hours reacted proteins show no detectable zone corresponding to the Hb-Hp complex. Figure 23 shows that i n i t i a l l y two more slowly migrating components (slot 2) than the unmodified haptoglobin (slot 4) are formed, the more rapidly migrating of these two modified components having retained the hemo-globin binding capacity while the slower migrating modified component having lost a l l i t s binding a b i l i t y (slot 1). On further reaction, the slower migrating modified protein increases at the expense of the faster running component so that the latter is scarcely discernible in the 30 minutes preparation (slot 6). At the greatest extent of modifica-tion, 1 hour and 2 hours reactions, only one slow migrating band is evident (Fig. 24, slots 2 and 6), which does not bind with hemoglobin (slots 1 and 5). Introducing negative charges into the haptoglobin molecule by succinylation would be expected to increase the anodic migration rate, but this was not observed either in starch gel or polyacrylamide electrophoresis. In 8.0 M urea starch gels in 0.05 M acetate buffer, pH 5 however, the control haptoglobin remained close to the origin as would be expected from i t s isoelectric point of 4.2 (46), and the succinylhaptoglobins migrated at a 149 -free Hp 0 ^--^ CD CZD __ C ) ^ ' c ,-- Hp-Hb .eakage A o 8 7 6 5 4 3 2 1 Figure 23. SG-electropherogram of 10 minutes and 30 minutes succinylated Hp 1-1 and t h e i r controls. The samples are as follows: (1) 10 minutes succinyl-Hp with Hb added, (2) 10 minutes succinyl-Hp, (3) 10 minutes control Hp with Hb added, (4) 10 minutes control Hp, (5) 30 minutes succinyl-Hp with Hb added, (6) 30 minutes succinyl-Hp, (7) 30 minutes control Hp with Hb added, (8) 30 minutes control Hp. - 149 -8 7 6 5 4 3 2 1 Figure 23. SG-electropherogram of 10 minutes and 30 minutes succinylated Hp 1-1 and th e i r controls. The samples are as follows: (1) 10 minutes succinyl-Hp with Hb added, (2) 10 minutes succinyl-Hp, (3) 10 minutes control Hp with Hb added, (4) 10 minutes control Hp, (5) 30 minutes succinyl-Hp with Hb added, (6) 30 minutes succinyl-Hp, (7) 30 minutes control Hp.with Hb added, (8) 30 minutes control Hp. - 150 -8 7 6 5 4 3 2 1 Figure 24. SG-electropherogram of 1 hour and 2 hours succiny-lated Hp 1-1 and t h e i r controls. The solutions are: (1) 1 hour succinyl-Hp with Hb added, (2) 1 hour succinyl-Hp, (3) 1 hour control Hp with Hb added, (4) 1 hour control Hp, (5) 2 hours succinyl-Hp with Hb added, (6) 2 hours succinyl-Hp, (7) 2 hours control Hp with Hb added, (8) 2 hours control Hp. - 150 -+ 0 8 7 6 5 4 3 2 1 Figure 24. SG-electropherogram of 1 hour and 2 hours succiny-lated Hp 1-1 and th e i r controls. The solutions are: (1) 1 hour succinyl-Hp with Hb added, (2) 1 hour succinyl-Hp, (3) 1 hour control Hp with Hb added, (4) 1 hour control Hp, (5) 2 hours succinyl-Hp with Hb added, (6) 2 hours succinyl-Hp, (7) 2 hours control Hp with Hb added, (8) 2 hours control Hp. - 151 -considerably greater rate towards the anode. The slower mi-gration of the succinylated components than the unmodified haptoglobins in borate starch gel could not be attributed to an aggregation phenomenon as a molecular weight of 9 9 , 2 0 0 ( 1 0 3 , 4 7 5 ; 9 7 , 4 8 5 ; 9 6 , 7 9 2 ) was determined for the 2 hours succinylated haptoglobin by the Archibald approach to equili-brium technique (Fig. 2 5 ) , and assuming quantitative reaction a correction factor of 6500 for the 64 lysyl residues and the N-terminal amino group yields 9 2 , 7 0 0 , essentially a single Hp 1-1 unit. A sample calculation based on measurements of the ultracentrifugal patterns in Fig. 25 is the following = 0 . 6 0 cm _ co ~ ~ — 2 - ~w~ 1 xn2zn'' dx m no=0 Cm = 0 . 6 7 - 3 4 1 3 4 ' -y-^  ( 6 3 . 8 4 0 4 ) = 0 . 0 5 2 RT (dc/dx)m A nc-i r i r , 1 * . 0 . 6 0 Mm = /-, w \ T -JIL = 4 . 9 5 1 F X 10 ~p—or n nan m (l-vp)(jo^ Xm°m 5 . 8 6 x 0 . 0 5 2 = 9 7 , 4 8 5 The slower migration of the succinylhaptoglobins in starch gel can be correlated to the unfolding phenomenon observed on the Sephadex G-200 columns. The unfolded nature of these molecules would hinder their migration through the starch gel so that the fr i c t i o n a l retardation outweighs their in-crease in charge ( 172 ). When both the modified and un-modified haptoglobins are subjected to electrophoresis on 8 M urea starch gels, these proteins are now equally un-folded by the denaturing effect of urea and the charge Time 2.7 h r 3.2 h r 3.7 h r 4.3 h r 4.8 h r b a r a n g l e - 70 F i g u r e 25. U l t r a c e n t r i f u g e p a t t e r n s o f 2 h o u r s s u c c i n y l a t e d s p e e d = 12,000 rpm R p 1 _ 1 ± n Q ^ Q 5 M K C 1 ^ o.01 M p h o s p h a t e b u f f e r , pH 7.0 d u r i n g t h e A r c h i b a l d a p p r o a c h t o s e d i m e n t a t i o n e q u i l i b r i u m . - 153 -differences become apparent. In subsequent succinylation reactions, haptoglobin was modified to a slightly greater extent, that i s , the 10 minutes and 2 hours succinylated haptoglobin show ratios of 0.41 and 0.07 respectively in the binding assay (Table IX). The variation in the degree of modification during the same time intervals may be explained by the influences of the pH fluctuations and the effectiveness of the stirring on the reaction since succinic anhydride is only slowly soluble in the aqueous medium. Coincident with the considerable de-crease, almost 80%, in binding a b i l i t y of the 10 minutes succinylated haptoglobin in the second preparation, is an 85.6% reaction of the lysyl groups and binding is completely lost in the quantitatively substituted 2 hours modified protein. In this kind of chemical modification, the conversion of the positively charged e-amino groups of lysine to the negatively charged N-succinyl groups has caused a more rapid and profound conformational change so that conformation re-organization rather than actual modification of functional groups probably is the major influence on the binding activity. A confirmation that there has been considerable unfolding of the molecular structure due to the high charge density of the succinylated protein comes from the lower sedimentation coefficients for these modified proteins. These highly charged succinylated proteins exhibit marked electrostatic effects on sedimentation which causes a decrease in S2 0/ w. - 154 -The 10 minutes succinylhaptoglobin showed a heterogeneous pattern i n a synthetic boundary c e l l , a major component with a 3.70 S and a minor peak with 3.57 S (Fig. 26). The 2 hours modified haptoglobin has a sedimentation constant of 3.18 S (Fig. 27) considerably lower than the 4.4 S of unmodified haptoglobin (46). Since the f r i c t i o n a l c o e f f i c i e n t i s re-lated to volume and shape of the sedimenting unit (247) , an expanded and unfolded molecule i s subject to greater f r i c -t i o n a l force and the sedimentation c o e f f i c i e n t i s expected to be lower. That the lower sedimentation constants ob-served i n the succinylated haptoglobins i s not due to a d i s -sociation into succinylated subunits due to e l e c t r o s t a t i c repulsion between the negatively charged carboxylate groups as has1, been reported for other proteins (248,249), i s evident from the molecular weight of 99,200 for the 2 hours modified protein. These data indicate then that the observed decreased hemoglobin binding by the succinylated haptoglobins i s mainly a r e f l e c t i o n of the great conformational change i n the mole-cule which profoundly a l t e r s the conformation of the active s i t e of the molecule. Chemical modification by succinylation was studied further i n Hp 2-1 and Hp 2-2. In each case the proteins were reacted for 10 minutes and 2 hours and controls were prepared by the same procedure as that for the Hp 1-1. Each preparation was assayed for binding a b i l i t y by the Sephadex G-200 method and analyzed by starch gel electrophoresis. Time 0 4' 8' 12' 16 Bar Angle 25° 50° 50° 50° 50" Speed = 56,000 rpm Temp. = 25.17° Direction of Sedimentation Figure 26. Sedimentation patterns of 10 minutes succinylated hapto-globin i n 0.1 M NaCl, 0.1 M phosphate buffer, pH 7.0 i n a synthetic boundary c e l l . Time 0 4' 8' 12' 16' B ci 3T Angle 30° 70° 50° 50° 50° Speed = 56,000 rpm Figure 27. Sedimentation patterns of 2 hours succinylated haptoglobin Temp. = 21.95° j _ n 0.05 M KC1, 0.01 M phosphate buf f e r , pH 7.0 in a synthetic boundary Dir e c t i o n of -. c e l l . Sedimentation - 157 -A reduced hemoglobin binding is also observed in both 10 minutes succinylated Hp 2-1 and 2-2 (Table X). On longer treatment of the proteins, 2 hours, a further reduction in binding is seen. Again the decreased binding is coincident with a rapid conformational change. In Figure 28, frames B and D, the control Hb-Hp 2-1 peaks and in Figure 29, frames B and D, the control Hb-Hp 2-2 peaks show a gradual t a i l i n g due to the partial resolution of the polymeric complexes on Sephadex G-200. The succinylated protein complexes, on the other hand (Fig. 28, frames A and C, Fig. 29, frames A and C), show a more sharpened peak. The SG-electropherogram of these succinylated proteins also confirms that a conformational change has occurred since each polymer band is slightly retarded although the sharp-ness and relative positions of the bands are maintained (Fig. 30). The succinylated Hp 2-2 polymers are similarly retarded in their migration rate and s t i l l maintain their characteristic series of bands. This means that succinyla-tion of haptoglobin does not produce non-specific struc-tural changes. Rather i t appears that the subunits of each polymer molecule on succinylation must unfold in a highly specific manner with the result that each unfolded polymer molecule is retarded to a similar extent on the starch gels, thereby maintaining the characteristic patterns. DISSOCIATION OF THE Hb-Hp COMPLEX BY 'SUCCINYLATION Since succinylation causes profound conformational - 158 -TABLE X Succinylation of Hp 2-1,. Hp 2-2 and Hb-Hp complex OP 407 Reaction Conditions OP 280 Hp 2-1, 0.01 M Tris-HCl, pH 8.0 10' control 2.16 120' control 2.20 t 1260:1 succinic anhydride to protein 10' 0.83** 120' 0.32** Hp 2-2, 0.01 M Tris-HCl, pH 8.0 10' control 1.69 120' control 1.73 1260:1 succinic anhydride^ to protein 10' 0.86** 120' 0.39** Hb-Hp, 0.01 M Tris-HCl, pH.8^0 10' control 2.32 120' control 2.25 10' • _ 2.15 120' 0.15 + added as a solid at 0°f with stirring and pH maintained close to 8 by addition of NaOH ** lower elution volume on G-2 00 indicates confor-mational change -159 -- i 1 1 1 1 j 1 ~r- 1 i r 0 10 20 30 40 50 0 10 20 30 40 50 EFFLUENT VOLUME, ml Figure 28. Assay of 10 minutes and 2 hours succinylated Hp 2-1 and th e i r controls on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0. (A) 10 minutes succinylated Hp 2-1, (B) 10 minutes control, (C) 2 hours succinylated Hp 2-1, (D) 2 hours control. - 160 -T _ _ 1 1 [ — — — i r — — i — — i — i 1 T EFFLUENT VOLUME , mi Figure 29. Assay of 10 minutes and 2 hours succinylated Hp 2-2 and th e i r controls on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0. (A) 10 minutes succinylated Hp 2-2, (B) 10 minutes control, (C) 2 hours succinylated Hp 2-2, (D) 2 hours control. - 161 -+ o 8 7 6 5 4 3 2 1 Figure 30. SG-electropherogram of 10 minutes and 2 hours succinylated Hp 2-1 and t h e i r controls. (1) 10 minutes succinyl-Hp 2-1 with Hb, (2) 10 minutes succinyl-Hp 2-1, (3) 10 minutes control Hp 2-1 with Hb, (4) 10 minutes control Hp 2-1, (5) 2 hours succinyl-Hp 2-1 with Hb, (6) 2 hours succinyl-Hp 2-1, (7) 2 hours control Hp 2-1 with Hb, (8) 2 hours control Hp 2-1. - 162 -changes in the haptoglobin molecule, the possibility exists that such great changes in conformation may result in a dissociation of the very stable Hb-Hp complex. It was, there-fore, of interest to determine the effect of the succinylation reaction on the Hb-Hp complex. EXPERIMENTAL A large excess of succinic anhydride was reacted with the complex. To a solution of 20 mg of the complex in 2 ml of 0.01 M Tris-HCl buffer, pH 8.0, cooled in an ice-bath, 25.2 mg of solid succinic anhydride was added i n i t i a l l y and the pH of the reaction maintained near 8.0 by the addition of 1.0 N NaOH. After 10 minutes, a 1 ml aliquot of the solution was desalted on a Sephadex G-25, 0.9 cm x 30 cm column. A further 25.2 mg of succinic anhydride was added to the reaction mixture after 20 minutes of reaction, and the succinylation continued for a total of 2 hours, after which time the mixture was desalted on Sephadex G-25. The complex stirred in the same buffer for the same length of time served as a control. The Sephadex G-200 assays were performed on the succinylated complexes and the controls without further addition of hemoglobin. RESULTS The elution profiles of the succinylated complexes (Fig. 31, frames A and C), shows a highly interesting result of the chemical modification. The protein modified for 10 0 10 20 30 40 50 60 0 10 20 30 40 EFFLUENT VOLUME, ml Figure 31. Assay of 10 minutes and 2 hours succinylated Hb-Hp complexes and th e i r controls on Sephadex G-20 0 (1 cm x 50 cm) i n 0.1 M phosphate buffer, pH 7.0. (A) 10 minutes succinylated Hb-Hp, (B) 10 minutes control Hb-Hp, (C) 2 hours succinylated Hb-Hp, (D) 2 hours control Hb-Hp. - 164 -minutes shows a main peak of complex which has essentially retained the same amount of bound hemoglobin as may be seen from the absorbancy ratio of 2.15 (Table X). However, there is evidence of the beginning of a dissociation of the complex since a small peak corresponding to free hemoglobin may be seen along with two other minor components. These disso-ciated proteins do not appear in the elution diagram of the Hb-Hp controls (Fig. 31, frames B and D) and therefore cannot be ascribed to any physical phenomenon during magnetic stirr i n g of the reaction mixture. In ithe 2 hours succinylated complex, the protein is now largely dissociated into a succinylated haptoglobin peak with a very small amount of bound hemoglobin (0.15 absorbancy ratio) and a succinylated hemoglobin peak (Fig. 31, frame C). This dissociation phenomenon is of considerable interest since a l l previous attempts at splitting the very stable Hb-Hp complex had proved unsuccessful (55). In the present studies, i t was found that neither 4.0 M urea in 0.2 M acetic acid, pH 5.0,nor 8.0 M urea in 0.1 M Tris-HCl buffer, pH 8.0, were able to separate the complex into i t s components although strong urea is usually able to dissociate non-covalently bound subunits. The observed dissociation of the succinylated com-plex is analogous to that observed by Klotz and Keresztes-Nagy (250) that electrostatic repulsions introduced by suc-cinylation resulted in complete dissociation of non-covalently linked protein subunits at neutral pH and without addition of - 165 -any other d i s s o c i a t i n g agents such as urea or detergent. That the Hb-Hp complex i s dissociated by succinylation con-firms that covalent bonds are not involved i n complex fo r -mation. Further studies on the nature of the hemoglobin binding s i t e were carr i e d out by the use of t h i s d i s s o c i a t i o n tech-nique on the complex. The Hb^ -Hp complex, which was guani-dinated with 0.2 M GDMP reagent described i n a previous section, and i n which 47.8% lysine residues remained unreac-ted, was succinylated for 2 hours by reaction of 50.4 mg of succinic anhydride with 10 mg of guanidinated Hb-Hp complex i n 1 ml of 0.01 M Tris-HCl buffer, pH 8.0. After desalting on G-25 and l y o p h i l i z a t i o n , an aliquot of the doubly-modi-f i e d complex was applied to a Sephadex G-200 column (1 x 50 cm) column i n 0.1 M phosphate buffer, pH 7.0, to deter-mine the extent of the d i s s o c i a t i o n . The remainder of the doubly-modified complex, 9.0 mg, was succinylated a second time with 10.49 mg of s o l i d reagent for 2 hours. After de-s a l t i n g and l y o p h i l i z a t i o n , the dissociated complex was p a r t i a l l y resolved on a Sephadex G-200 column (1 x 87 cm) i n 0.1 M phosphate buffer, pH 7.0. Fractions comprising peak A (dissociated haptoglobin) and those comprising peak B (dissociated hemoglobin) (Fig. 32, frame C) were separately combined and dialyzed exhaustively against d i s t i l l e d water, then l y o p h i l i z e d . The s p l i t complex, peak A and peak B were hydrolyzed by addition of 0.4 ml of 6.0 N HC1 to 2 mg of each - 166 -T ~1 1 ~T~ T E F F L U E N T VOLUME, ml Figure 32. Modified Hb-Hp on Sephadex G-200 i n 0.1 M phosphate-buffer, pH 7.0. (A) Hb-Hp guanidinated with 0.2 M GDMP, 72 hr., 0°, pH 9.0 chromatographed on Sephadex G-200 (1 x 50 cm column). (B) Guanidinated Hb-Hp complex succinylated 2 hr., pH 8.0 on an 1 cm x 50 cm column, (C) Succinylated and guanidinated Hb-Hp com-plex succinylated a further 2 hr., pH 8.0, on an 1 cm x 87 cm column. - 167 -protein fraction and heated at 105° for 18 hours in evacuated ampoules. After centrifuging to remove the black precipi-tate, the hydrolyzates were dried under vacuum and then taken up in 0.45 ml of pH 2.2 citrate buffer. Aliquots of the hy-drolyzates were analyzed in duplicate on the Beckman/Spinco Model 120C amino acid analyzer for basic amino acid contents. The elution diagram of the guanidinated complex (Fig. 32, frame A) shows that a conformational change is occurring in the molecule since a faster eluting peak is separating from the complex in the native conformation which has retained the bound hemoglobin. However, modification with this rea-gent has not caused any dissociation of the complex. On introduction of negative succinyl groups into this guanidin-ated Hb-Hp complex, considerable dissociation of the hemo-globin and haptoglobin components occurs (Fig. 32, frame B). Succinylation a second time carries this dissociation almost to completion (Fig. 32, frame C). Thus the relatively mild guanidination reaction has produced a conformational change in the Hb-Hp complex molecule but dissociation is not achieved until a high density of negative charges are intro-duced in place of the positive charge. Determination of the homoarginine content of the com-ponents of the dissociated complex should give a further indi-cation of the area of the binding site or any conformational change of the molecules upon binding of hemoglobin by hapto-globin, again assuming a s t a t i s t i c a l distribution of lysyl residues in the molecule. It is expected that in the complex - 168 -the lysine residues i n the binding s i t e would be covered and prevented from reaction with the GDMP reagent, the ex-tent of protection being a measure of the area of contact. Thus the decrease i n homoarginine of the haptoglobin and hemog T.orbin s p l i t from the complex as compared to the haptoglobin and the hemoglobin guanidinated separately should give an i n d i c a t i o n of the approximate area of the binding s i t e assuming equal r e a c t i v i t i e s of the l y s y l groups i n the free proteins and the bound protein. In Table XI, peak A, containing modified haptoglobin dissociated from the complex, has 52.6% homoarginine as compared to the amount of 52.9% i n the free guanidinated haptoglobin. Comparison of the amount of homoarginine i n the modified hemoglobin derived from the complex, peak B, possessing 51.0%, to the value of 58.3%, i n a guanidinated hemoglobin with the binding s i t e exposed, shows a difference of about 7%. This results i n an average decrease of 3.5% i n homoarginine content i n the guanidinated complex and agrees with the r e s u l t s obtained by comparison of the extent of guanidination of the Hb-Hp complex with a t h e o r e t i c a l l y calculated value based on guani-dination of free hemoglobin and free haptoglobin. These r e s u l t s can best be explained by the area of contact between the proteins being quite limited. DISCUSSIONS OF CHEMICAL MODIFICATION Three reagents of increasing severity have been used for the chemical modification of haptoglobin i n order to - 169 -TABLE XI Guanidination of Hb-Hp Complex and Dissociation of the Guanidinated Complex by Subsequent Succinylation Reaction Conditions | Lysine % Homoarginine Hp 1-1 guanidination with 0.2 M 47.1 52.9 GDMP, pH 9.0, 72 hrs, 0° Hb guanidination with 0.2 M 41.7 58.3 GDMP, pH 9.0, 72 hrs, 0° Hb-Hp (1) guanidination with 0.2 M GDMP, pH 9.0, 72 hrs, 0° (2) succinylation, pH 8.0, 2 hrs (3) succinylation, pH 8.0, 2 hrs peak A (Hp)_ 47.4 52.6 peak B (Hb) 49.0 51.0 - 170 -study the r o l e of amino groups i n the protein. It i s advan-tageous i n a study of chemical modification to use several reagents both as a check on the s p e c i f i c i t y of the action toward the desired functional group and to minimize non-s p e c i f i c s t r u c t u r a l changes or denaturation. The molecular conformation of the haptoglobin molecule i s important i n maintaining the proper orientation of the hemoglobin binding s i t e . Chemical modification of the pro-t e i n by introduction of new covalently bound groups may so modify the intramolecular forces as to cause reorganization depending upon the groups introduced. Habeeb (173) used the s e n s i t i v i t y of the elu t i o n volume of a protein molecule on a Sephadex G-20 0 column to i t s Stokes radius to evaluate conformational changes associated with chemical modification of bovine serum albumin. I t was found that s i g n i f i c a n t changes i n asymmetry of the protein molecule due to succiny-l a t i o n and acetylation occurred, whereas the shape changes associated with guanidination were small. In the present studies, Sephadex G-200 assays of the chemically modified haptoglobins for hemoglobin binding capa-c i t y also served to detect any unfolding of the molecules which might a f f e c t the binding a b i l i t y n o n - s p e c i f i c a l l y . In agreement with Habeeb 1s findings and as expected from the mild chemical nature of the substitution, even at an o v e r a l l conversion of about 82% of the amino groups to guanidino groups (Table VII), only a small portion, approximately - 171 -one-quarter, of the molecules show conformational changes (Fig. 19, frame D), these are undoubtedly the more highly modified species, while the remainder of the guanidinated proteins have maintained the native configuration and show no alteration in the hemoglobin binding capacity (Table VII). In the acetylation reaction, in which there is a ratio of acetic anhydride to haptoglobin of 80:1, corresponding to just over an equimolar ratio of reagent to lysine residues, the reaction would be expected to be specifically directed towards the amino groups (238) . In this case, there is no significant conformational change when almost 50% of the posi-tive charges have been replaced with neutral groups and the hemoglobin binding a b i l i t y is reduced only slightly (Table VIII). With a large excess of acetic anhydride reagent, a greater portion of the molecule unfolds (Fig. 20, form D) and in this form, the haptoglobin shows much reduced binding. The remainder of the molecules with normal elution volume are 70% modified overall and now show less than 50% of the bind-ing (Table VIII). The reduced binding observed at the high-est level of acetylation may be due to more extensive reac-tion with groups other than the amino groups or undetected physicochemical changes in the molecule which affect the hemoglobin binding property. It is unlikely that this reduced binding is due to the loss of a functionally important lysine residue since an extensive guanidination of the lysines did not influence the binding. It could not be the loss of - 172. -positively charged groups at the higher level of acetyla-tion that directly influenced the binding capacity since binding experiments in strong salt solutions showed that the electrostatic interactions are not of prime importance. In the succinylation reaction, structural changes in the haptoglobin molecule are immediately evident both from the gel chromatography profiles (Fig. 22) and the SG-electro-pherograms (Fig. 23 and 24) . 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