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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. U n i v e r s i t y o f B r i t i s h Columbia, 1956 M.S.P. U n i v e r s i t y o f 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 B i o c h e m i s t r y F a c u l t y of Medicine  We accept t h i s to the r e q u i r e d  t h e s i s as conforming 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  this  thesis  in partial  advanced degree at the U n i v e r s i t y  Library  shall  agree that  make  i tfreely  permission  available  I t i s understood  financial  gain  that  te  March l h 1968 t  copying of t h i s  thesis  n o t be a l l o w e d w i t h o u t my w r i t t e n  The U n i v e r s i t y o f B r i t i s h ' C o l u m b i a V a n c o u v e r 8, C a n a d a  D a  f o r r e f e r e n c e and s t u d y .  copying o r p u b l i c a t i o n  Biochemistry  Department o f  I agree that the  I further  f o r scholarly  by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n -  tatives.  shall  o f B r i t i s h Columbia,  f o rextensive  p u r p o s e s may be g r a n t e d  f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an  of this  thesis f o r  permission.  i ABSTRACT  Haptoglobin which  possesses  hemoglobin. in  a  stable  present globin  is a the  genetically  property  Haptoglobin and  binds  essentially  studies are combining  of  polymorphic  combining  concerned  with  the  protein  specifically  hemoglobin  irreversible  plasma  with  stoichiometrically complex.  nature  of  The the  hemo-  site  on  haptoglobin  in relation  to i t s  quantities  of  homogeneous  haptoglobin  have  function. Large prepared  from  involving by  enables  the  In  order  has to  haptoglobin  ing  site,  filtration. hemoglobin  been  polymers  haptoglobin  both  peroxidase  zation  of  the  globin  chains  of  isolated  of  the  and  a , 1  Sephadex  various  Sephadex  a  the  and  assay.  The  method followed  purification  G-200  assay  Hb-Hp c o m p l e x  amount  of  the  assay  3  binding  by  which  and  hemoglobin  the  bound  effects  r e s o l v e d on  to  on  binding  of  bind of were  polymerization  hemoglobin  bind  Sephadex  site  to  of  the  the  determination  chains The  from  ability  and  by  site  partially  v e r t e b r a t e hemoglobins  assay.  the  whether  for their  achieved 2  finally  Sephadex  is distinct  hemoglobin was  and  from  of  developed  precipitation,  A  polymers  examined  the  newly  developed.  determine  G-200, w e r e by  a  sulfate  measurement  haptoglobin  by  chromatography  free  direct  fluid  ammonium  G-200 g e l  separates  of  first,  DEAE-cellulose  Sephadex  to  ascites  been  Sephadex  hemoglobin assay.  Locali-  component of  the  hemoglobin globin, also  environmental  bind-  hapto-  ability by  means  myoglobin  examined  by  the  factors  on  the  combination  of haptoglobin  by  alteration  of  phenol  groups  by s e l e c t i v e  groups  followed  with  tion  of the area  of the  been  made  amino  groups  complex  fication  site  that  binding  site.  may  shown  Haptoglobin  examined  which  that  studies  show  and h a p t o g l o b i n  from t h e  from  that  the site  chain  carries  combines  with  except  those  that  the haptoglobin  the hemoglobin  of polymerization the hemoglobin globin but not with with  of the frog change  a l l animal and f i s h ,  forces  cannot  the area  of contact  i n t h e complex molecule  i n lysyl-side  of else  chains.  I ti s  be t h e s o l e Chemical  between  I t i s also  interrnomodification  the hemoglobin  involves only the area  which  i n t h e Hb-Hp  e x p u l s i o n o f t h e heme g r o u p .  involved i n the binding.  the haptoglobin  deficient  that of o f modi-  stoichiometrically  electrostatic  forces  of  with  the extent  of a conformational  causes  lecular  and  complex  of guanidination  components with  modi-  determina-  i n t h e Hb-Hp  s t u d i e s show  i s distinct  be t h e r e s u l t  complex  of the chemically  of the extent  hemoglobin  of these  and i tbinds  hemoglobins  site  by comparison  the 6 haptoglobin  myoglobin  severity  complex.  results  combining  has been  An approximate  of the individual  i n dissociated  The  and  hemoglobin.  of the binding  and a l s o  guanidinated  of increasing  of the a b i l i t y  by comparison  o f amino  modification of haptoglobin  reagents  by measurement  hemoglobin  studied  addition  The i n v o l v e m e n t  i n binding with  three  proteins to bind  has been  s t r e n g t h a n d o f pH a n d b y  chemical  fied  has  hemoglobin  as a t y r o s i n e analogue.  of haptoglobin  studied amino  of ionic  with  a small  area  i s particularly shown  that  amino  groups are not d i r e c t l y i n v o l v e d i n the b i n d i n g s i t e and t h a t a c y l a t i o n of amino groups p a r t i c u l a r l y w i t h s u c c i n y l group cause a profound change i n the h a p t o g l o b i n molecule and i t s a b i l i t y to b i n d hemoglobin i s very much reduced.  iv T A B L E OF CONTENTS  ABSTRACT T A B L E OF  CONTENTS  L I S T OF T A B L E S L I S T OF F I G U R E S L I S T OF A B B R E V I A T I O N S ACKNOWLEDGEMENT DEDICATION INTRODUCTION Biochemistry  of  Haptoglobins  Genetics Physiology  and Pathology  of  Haptoglobin  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 and 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 G E N E R A L METHODS 1.  Starch-Gel Electrophoresis  2. U r e a 3.  Starch-Gel Electrophoresis  Polyacrylamide  4. P r e p a r a t i o n o f PART  I  GelElectrophoresis Hemoglobin  I S O L A T I O N OF H A P T O G L O B I N AND P A R T I A L S E P A R A T I O N OF H A P T O G L O B I N POLYMERS  INTRODUCTION P U R I F I C A T I O N OF H A P T O G L O B I N AND S E P A R A T I O N 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 Shaw M e t h o d  PARTIAL by C o n n e l l and  V  2. P r e p a r a t i o n o f Haptoglobin by M o d i f i c a t i o n o f Smith, Edman and Owen, and C o n n e l l and Shaw Methods 3. P u r i f i c a t i o n  49  of Haptoglobin by a New Method  and P a r t i a l S e p a r a t i o n o f Polymers PART I I  <. •  52  SEPHADEX G-200 ASSAY  69  INTRODUCTION  69  1. Peroxidase Assay  70  2. E l e c t r o p h o r e t i c Methods  74  3. Sephadex Assay  75  SEPHADEX ASSAY  76  ASSAY OF Hp 2-1 POLYMERS  82  ASSAY OF a'," a  89  1  2  AND 3 HAPTOGLOBIN CHAINS  BINDING OF GLOBIN AND MYOGLOBIN TO HAPTOGLOBIN  95  1. G l o b i n  95  2. Myoglobin  99  ASSAY OF HAPTOGLOBIN WITH HEMOGLOBIN FROM OTHER SPECIES PART I I I EFFECT OF ENVIRONMENTAL FACTORS UPON THE BINDING OF HEMOGLOBIN AND HAPTOGLOBIN  102 114  INTRODUCTION  114  EFFECT OF IONIC STRENGTH AND pH UPON HEMOGLOBINHAPTOGLOBIN BINDING  116  PART IV  CHEMICAL MODIFICATION OF AMINO GROUPS IN HAPTOGLOBIN  INTRODUCTION GUANIDINATION  125 .  125 128  Experimental  130  Results  132  vi 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 BIBLIOGRAPHY  168 173  vii LIST OF TABLES Table I II III IV V VI  VII VIII IX X XI  Page P u r i f i c a t i o n of Haptoglobin  1-1  Hemoglobin B i n d i n g by Haptoglobin  66 Polymers  85  Haptoglobin B i n d i n g with Animal Hemoglobins  107  Haptoglobin B i n d i n g w i t h V a r y i n g of Trout Hemoglobin  109  Concentrations  E f f e c t o f S a l t C o n c e n t r a t i o n s on Hb/Hp Combination (Sephadex G-200 Method) E f f e c t o f pH on Hb/Hp Combination  117  (Sephadex  G-200 Method)  119  Guanidination  133  A c e t y l a t i o n of Haptoglobin  1-1  S u c c i n y l a t i o n of Haptoglobin  1-1  S u c c i n y l a t i o n of Hp 2-1, Hp 2-2 and Hp-Hb Complex G u a n i d i n a t i o n of Hb-Hp Complex and D i s s o c i a t i o n of the Guanidinated Complex by Subsequent Succinylation  143 147 158 169  viii LIST OF FIGURES Figure  Page  1.  SG-electropherogram of the three common haptoglobin phenotypes  4  2.  The amino acid sequence of the N-, C- and J peptides of haptoglobin a chains resulting from p a r t i a l duplication i n the Hp a chains  8  SG-electropherograms of the haptoglobin phenotypes and their respective subtypes  21  2  3.  1  4.  World Map of Hp  5.  DEAE-cellulose chromatography of crude Hp 1-1 on a 5.0 x 9.5 cm column i n 0.01 M sodium acetate buffer, pH 4.7  54  DEAE-cellulose chromatography of crude Hp 2-1 on a 5.0 x 9.5 cm column i n 0.01 M sodium acetate buffer, pH 4.7  55  SG-electropherogram of the DEAE-cellulose chromatography column effluent fractions of crude Hp 1-1 commencing at 250 ml through to 440 ml  56  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  58  SG-electropherogram of fractions following Sephadex G-200 chromatography (Fig. 8) of Hp 1-1  60  P u r i f i c a t i o n of crude Hp 2-1, peak A and B from DEAE-cellulose chromatography (Fig. 6) on Sephadex GS200 (2.5 cm x 186 cm) i n 0.05 M ammonium acetate, pH 8.5  61  6.  7.  8. 9. 10.  11.  gene frequencies  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 through to f r a c t i o n number 4 6  24  62  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 i n 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) i n 0.1 M phosphate buffer pH 7.0 (frames B, C and D).  78  ix 14.  Assay o f B, a and a h a p t o g l o b i n chains on Sephadex G-200 (1 cm x 50 cm). (A) 3 c h a i n i n 0.2 M ammonium a c e t a t e , pH 7.0 ( a c e t i c a c i d ) , (B) a c h a i n i n 0.1 M phosphate b u f f e r , pH 7.0, (C) a c h a i n i n 0.1 M phosphate b u f f e r , pH 7.0 1  2  2  1  93  15.  Binding o f g l o b i n t o h a p t o g l o b i n on Sephadex G-200 (1 cm x 50 cm) i n 0.2 M ammonium hydroxide, pH 9.0 ( a c e t i c acid) 98  16.  Binding o f sperm whale myoglobin t o h a p t o g l o b i n on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0  103  17.  Assay o f Hp 1-1 on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0 c o n t a i n i n g 0.1 M phenol 122  18.  S e p a r a t i o n o f known b a s i c amino a c i d s on the Beckman/Spinco 120 C amino a c i d a n a l y z e r  129  19.  Assay o f Hp 1-1 g u a n i d i n a t e d w i t h 0.1 GDMP (B), 0.2 M GDMP (C), 0.5 M GDMP (D) compared w i t h a c o n t r o l (A) on Sephadex G-200 i n 0.1 M phosphate b u f f e r , pH 7.0  134  20.  Assay o f Hp 1-1 a f t e r a c e t y l a t i o n with a c e t i c anhydride. R a t i o s o f reagent t o h a p t o g l o b i n o f (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 r t i a l s e p a r a t i o n of two s p e c i e s o f a c e t y l a t e d j j 1-1 f o l l o w i n g r e a c t i o n w i t h a r a t i o o f 400:1 Hp a c e t i c anhydride to. p r o t e i n on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0 141  22.  Assay o f Hp 1-1 s u c c i n y l a t e d f o r v a r i o u s times on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0. R e a c t i o n times are: (A) 10 minutes, (B) 30 minutes, (C) 1 hour, (D) 2 hours, w i t h a f i f t e e n - f o l d excess o f s u c c i n i c anhydride  146  SG-electropherogram o f 10 minutes and 30 minutes s u c c i n y l a t e d Hp 1-1 and t h e i r c o n t r o l s  149  SG-electropherogram o f 1 hour and 2 hours s u c c i n y l a t e d Hp 1-1 and t h e i r c o n t r o l s  150  23. 24.  X 25.  U l t r a c e n t r i f u g e p a t t e r n s of 2 hours s u c c i n y l a t e d Hp 1-1 i n 0.05 M KC1, 0.01 M phosphate b u f f e r , pH 7.0 d u r i n g the A r c h i b a l d approach t o sedimentation equilibrium 152  26.  Sedimentation p a t t e r n s of 10 minutes s u c c i n y l a t e d h a p t o g l o b i n i n 0.1 M NaCl, 0.1 M phosphate b u f f e r , pH 7.0 i n a s y n t h e t i c boundary c e l l 155  27.  Sedimentation p a t t e r n s of 2 hours s u c c i n y l a t e d h a p t o g l o b i n i n 0.0 5 M KC1, 0.01 M phosphate b u f f e r , pH 7.0 i n a s y n t h e t i c boundary c e l l  156  Assay of 10 minutes and 2 hours s u c c i n y l a t e d Hp 2-1 and t h e i r c o n t r o l s on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0  159  Assay o f 10 minutes and 2 hours s u c c i n y l a t e d Hp 2-2 and t h e i r c o n t r o l s on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0  160  30.  SG-electropherogram of 10 minutes and 2 hours s u c c i n y l a t e d Hp 2-1 and t h e i r c o n t r o l s  161  31.  Assay o f 10 minutes and 2 hours s u c c i n y l a t e d Hb-Hp complexes and t h e i r c o n t r o l s on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0  163  M o d i f i e d Hb-Hp on Sephadex GS200 i n 0.1 M phosphate b u f f e r , pH 7.0  166  28.  29.  32.  xi LIST OF ABBREVIATIONS 1.  BME:  bis(N-maleimidomethyl)ether  2.  DEAE-cellulose: Diethylaminoethyl-cellulose  3.  GDMP:  4.  Hb:  5.  Hb-Hp:  6.  Hp:  7.  H.I.:  Haptoglobin Index  8.  O.D.:  o p t i c a l density  9.  SG-electropherogram:  l-guanyl-3,4-dimethyl pyrazole n i t r a t e  hemoglobin hemoglobin-haptoglobin  haptoglobin  10.  TNBS:  11.  TNP:  12.  Tris:  starch-gel electropherogram  2,4,6-trinitrobenzene-l-sulfonic acid trinitrophenyl T r i s (hydroxymethyl). aminomethane  xii  ACKNOWLEDGEMENTS The author i s deeply indebted to Dr. G.H. Dixon for his i n s p i r i n g guidance, encouragement and patience during the course of this study and sincerely appreciates the constructive c r i t i c i s m s 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 i n the amino acid analyses and the u l t r a c e n t r i f u g a l studies. The kind cooperation of the Canadian Red Cross Blood Bank, Vancouver, B.C., i n supplying human blood and of Dr. D.K. Ford, Faculty of Medicine, University of B r i t i s h Columbia, i n arranging for the supply of ascites f l u i d , i s 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.  xiii  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 s p e c i f i c a l l y and i r r e v e r s i b l y ; this property formed the basis of their discovery and nomenclature (from the Greek  haptein,  to hold fast) (1). In 1938, Polonovski and Jayle (2) observed that the peroxidase a c t i v i t y of hemoglobin i s 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 f o r this peroxidase a c t i v a t i o n phenomenon, which l a t e r served as a basis of assay of haptoglobin i n the serum (4).  The existence of more than one  type of haptoglobin originated from the observation by Jayle and G i l l a r d  (5) that i n 10 out of 12 samples of human sera,  haptoglobin was salted out at much lower concentrations of ammonium sulfate than was required for the p r e c i p i t a t i o n of the same protein from the remaining two sera.  One type of  haptoglobin, o r i g i n a l l y referred to as Hp I I , was f i r s t lated from the urine of a nephrotic c h i l d  iso-  (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).  I t was concluded  that this l a t t e r type of haptoglobin i s a dimer of Hp II (8). A s i g n i f i c a n t advance i n the f i e l d of haptoglobin research  - 2 came from the i n t r o d u c t i o n of s t a r c h - g e l e l e c t r o p h o r e s i s Smithies  (9).  The  greater  by  r e s o l v i n g power of t h i s method of  zone e l e c t r o p h o r e s i s over free-boundary e l e c t r o p h o r e s i s  and  e x i s t i n g methods of zone e l e c t r o p h o r e s i s enabled Smithies to show t h a t human sera f e l l  i n t o three groups on the b a s i s of  the occurrence of some component which had of b i n d i n g hemoglobin Walker  (11)  (10).  the common  Subsequently, Smithies  discovered  by J a y l e ;  they showed t h a t the observed d i f f e r e n c e s of the c o u l d be e x p l a i n e d 1  and  e s t a b l i s h e d the i d e n t i t y of these serum p r o t e i n  groups w i t h the haptoglobins  Hp  by p o s t u l a t i n g the e x i s t e n c e  furthermore, haptoglobins  of  alleles  2  and Hp  of the autosomal h a p t o g l o b i n  l o c u s , Hp,  which 2  1 1  show incomplete dominance. 2  2-1  firmed and  Each genotype Hp /Hp  Hp  1  /Hp  2  and Hp /Hp Hp  property  and  can be d i s t i n g u i s h e d p h e n o t y p i c a l l y Hp  2-2  r e s p e c t i v e l y (11,12).  as Hp  1-1,  J a y l e ' s group con-  the i d e n t i t y of t h e i r p r o t e i n s with those of Smithies  reversed  their notation  i n d e s c r i b i n g the  haptoglobins  (13) . 2  In i n d i v i d u a l s homozygous f o r the Hp a l l e l e , a single p r o t e i n band i s v i s i b l e on s t a r c h - g e l e l e c t r o p h o r e s i s , where2  as the phenotype of homozygotes f o r the Hp  allele is a  s e r i e s of more than ten bands of decreasing  concentration  mobility.  The  and  phenotype of heterozygotes shows a f a s t migra-  t i n g band w i t h m o b i l i t y s i m i l a r to the Hp  1-1  band i n a d d i t i o n  to a s e r i e s of bands of s l i g h t l y g r e a t e r m o b i l i t y than s e r i e s seen i n Hp  2-2;  the  this 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 c h a r a c t e r i s t i c 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 i n 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 e f f e c t on the protein components whereas i n f i l t e r - p a p e r electrophoresis the greater f r i c t i o n a l retardation force of the large-sized proteins i s balanced by a greater net charge  (16), the observed  differ-  ences i n the series of protein components appear to l i e i n a difference i n molecular size (14).  The homogeneity of the  Hp 1-1 protein and the heterogeneity of the Hp 2-2 protein were confirmed by u l t r a c e n t r i f u g a l studies (14,17,18), i n 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 i n starch gels, nevertheless, the c h a r a c t e r i s t i c patterns persisted; therefore, the d i f ference i n molecular size could not be related to s i a l i c acid content. Independently, Smithies and Connell (14) and A l l i s o n  (21)  - 4 -  •  F i g u r e 1.  SG-electropherogram of the three common h a p t o g l o b i n  phenotypes (15).  - 5 -  proposed a mechanism of polymer formation to explain the haptoglobin polymorphism.  A l l i s o n suggested the existence of  complementary binding s i t e s through which aggregation could occur.  Hp 1 subunits would have only a single combining  site  and thus only monomers would be present i n the Hp 1-1 homozygotes.  The Hp 2 subunit however, would possess two com-  plementary binding s i t e s and thus could combine with other Hp 2 subunits to form a series of polymers of increasing s i z e . 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 i n 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 i n the Hp 1-1 molecule.  Treatment of hapto-  globin with 0.01 M t h i o g l y c o l l a t e and electrophoresis instarchhgels containing 8 M urea and t h i o g l y c o l l a t e  produced  a s i m p l i f i e d haptoglobin pattern and showed a common cleavage product from a l l the haptoglobin types.  Thus disulphide  bonds are probably involved i n 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 able i n a c i d i c starch gels containing 8 M urea.  separ-  One group  of product, c a l l e d the 3 chains, are common to haptoglobins of a l l genetic types, while the other product, c a l l e d a chains d i f f e r according to genetic type. herein to be c a l l e d the a  2  The a chain from Hp 2-2,  chain, migrates behind the a chain  in Hp 1-1, which w i l l be c a l l e d the a chain. 1  are almost equal proportions of a  2  and a  1  In Hp 2-1 there  chains.  The a  polypeptide chains from Hp 1-1 are further subdivided into faster and slower migrating zones, which w i l l be c a l l e d IF IS herein Hp a and Hp a respectively, the difference i n mobility being due to substitution of a single lysine r e s i 1F IS due i n a by a glutamic acid residue i n a (23,24). It i s evident then, that only the a chains are controlled by the Hp locus, the 3 chains being determined 26).  at a separate locus (25,  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 chain contained a l l IF IS 2  the peptides found i n the Hp a  or Hp a  chains with a  r e l a t i v e l y decreased amount of the two peptides termed island C- together with an additional peptide, c a l l e d 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 p e p t i d e s suggest t h a t J p e p t i d e c o n s i s t s of a f u s i o n of C and N i n a p e p t i d e bond w i t h the l o s s of 22 r e s i d u e s a t the 2 t e r m i n a l r e g i o n s i n v o l v e d (27). The Hp a c h a i n may then be IF IS regarded as a f u s i o n of the Hp a and Hp a c h a i n s , which agrees w i t h a determined molecular weight of 17,300 ± 1,400 f o r the Hp a  2  c h a i n , almost twice t h a t of the Hp a  IF  and Hp  IS a  c h a i n m o l e c u l a r weights,  and Dixon  (23) proposed  8,860 ± 400.  Smithies, Connell  a mechanism of unequal c r o s s i n g - o v e r IF  between asymmetrically p a i r e d a l l e l e s o c c u r r i n g i n a Hp  IS /Hp  heterozygote, w i t h a r e s u l t a n t p a r t i a l gene d u p l i c a t i o n i n one chromosome and a corresponding d e l e t i o n i n the other to 2  e x p l a i n the e v o l u t i o n of the Hp F i g . 2.  gene; t h i s i s d e p i c t e d i n  F u r t h e r , i t was p r e d i c t e d t h a t by unequal but homolo-  gous c r o s s i n g - o v e r i n the o r i g i n a l Hp gene, new gene 2FF 2SS p r o d u c t s , Hp a and Hp a , c o u l d be formed. Nance and Smithies  (29) subsequently  the p r e d i c t i o n .  found these products and confirmed  A l s o by c r o s s i n g over i n an asymmetrically  p a i r e d chromosome i n the Hp 2-2 homozygote, a t r i p l i c a t i o n c o u l d r e s u l t ; t h i s appears  to be the case i n the Johnson  type h a p t o g l o b i n d i s c o v e r e d by G i b l e t t mother and her c h i l d . s i z e s of a , a 1  2  and a  (30) i n a Negro  P r e l i m i n a r y evidence of the molecular J  (Johnson) on Sephadex G-75 columns  i n d i c a t e molecular weights  of 8,000, 17,400 and 24,000 r e s -  p e c t i v e l y , thus r e p r e s e n t i n g s i n g l e , double and t r i p l e l e n g t h chains (24). .The" N-terminal amino a c i d a n a l y s i s of the a c h a i n was  - 8 -  •  -v«l-A»n-A»p-*r^ly-A»n-A«p-V«J-m-Ai^U«-Al^^ 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*  *j  C paptid*  oh.  ch.  I I  I  H  Junction poptide  1  —  eh-  Figure 2.  The amino acid sequence of the N-, C- and J -  peptide of haptoglobin a chains r e s u l t i n g from p a r t i a l duplication i n Hp a  2  chains (28).  *I  ch.  - 9shown to be valine by Smithies, Connell and Dixon  (23) and .  Smith, Edman and Owen (31) reported equal amounts of N-terminal valine and isoleucine i n haptoglobin preparations of a l l genetic types.  These results indicate that the 3 chain N-  terminal amino acid i s isoleucine and that there i s an equal number of a and 3 chains i n 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  structure of Hp 1-1  a model of the secondary  composed of each of the two a chains joined by d i s u l f i d e bonds to a 3 chain with the 3 chains also connected by d i s u l f i d e bridges.  This model of haptoglobin i s similar to  that of the immunoglobulins, which also possess two l i g h t 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:  {a $)i+, 2  (a 3)6/ 2  (a 3)e, z  ( a 3 ) I o • • • • i n contrast to ( a 3 ) 2 of the Hp 1-1 type. 2  Hp 2-1 series of bands might then be ( a 3) 2 , 1  f^a 3) 2 ( a 3 ) 4 , ( a ^ ) 2 ( a 8 ) 1  The  1  2  2  6  (a 3)2(a 3)2 / 1  2  The fact that Hp 2-2 homo-  zygotes produce a series of polymers of limited size and i n d e f i n i t e proportions, i n approximately the r a t i o of 11:35: 25:17:9:2, i s evidence that the mechanism of polymerization i s limited by some mechanism of c y c l i z a t i o n  (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 c i r c u l a r Hp 2-2 molecule.  The Hp 2-1 polymers, present i n a r a t i o of  17:29:24:19:10:1 (34) would be terminated when two a combine with 8 units (35).  chains  1  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, p a r t i a l l y 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 contained antibodies against haptoglobin i n appreciable t i t e r s . The existence of three genetically determined  haptoglobins  that d i f f e r i n physico-chemical properties might indicate that the haptoglobins would d i f f e r : i n antigenic structure. However, Beam and Franklin (18), using antisera against HbHp complexes of each gentic type, which had been absorbed with umbilical cord serum known to contain very l i t t l e globin (38), and Fine and B a t t i s t i n i  (39), employing  hapto-  anti-  sera against whole human serum of each haptoglobin type, were unable to detect any immunological Korngold  differences.  However,  (40) using antisera against p u r i f i e d Hp 2-2  - 11 preparations, demonstrated  that the antisera could d i s t i n -  guish between Hp 2-2 and Hp 1-1 when sera of these haptoglobin types reacted with the antisera.  A large number of  antigenic determinants c h a r a c t e r i s t i c of Hp 2-2 were lacking i n Hp 1-1.  Starch-gel Immunoelectrophoresis  of Hp 2-1  showed that i t was a n t i g e n i c a l l y heterogeneous;  one a n t i -  genic determinant was i d e n t i c a l with Hp 1-1, while the other was c l o s e l y 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 s i t e s of haptoglobin are involved i n some manner i n the combination of haptoglobin with i t s antibodies. With antiserum to p u r i f i e d Hp 1F-1S, Shim and Beam  (32) demon-  strated that antigenic determinants of the haptoglobin molecule reside i n 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 a n t i - a - c h a i n a n t i 2  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 i n the a chains.  - 12 Haptoglobin migrates as a single boundary with the a  2  f r a c t i o n on electrophoresis i n the T i s e l i u s apparatus and on f i l t e r - p a p e r electrophoresis (42), thus indicating that the Hp 2-1 and 2-2 polymers a l l possess i d e n t i c a l charges.  Hemo-  globin has a mobility corresponding to the 8 globulin f r a c t i o n . However, when hemoglobin i s added to serum, i t forms the HbHp complex which migrates between a 43,44).  2  and 8 i g l o b u l i n s .(38,  The haptoglobin components i n the starch gel system  migrate i n the a8 region and on addition of hemoglobin, these components are retarded i n their migration (9). The r e l a t i v e l y high carbohydrate content of haptoglobin and i t s p r e c i p i t a t i o n behaviour i n the presence of 0.6 M perchloric acid places i t i n the category of a seromucoid, a term proposed by Winzler (45) to designate a group of serum glycoproteins with very high carbohydrate content which r e main i n solution when serum i s exposed to a 0.6 M solution of perchloric acid. -Haptoglobin i s reported to consist of 74-76.5% protein, the remainder of the molecule being made up of the same content of carbohydrates i n 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 i s contained i n the 8 chain (32,48).  Gerbeck, Rafelson and Bezkarovainy (49)  provided evidence that the carbohydrate of i n t a c t 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 coe f f i c i e n t 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, essent i a l l y the same as i n the native haptoglobin, 1.9:1.5:1. Later i s o l a t i o n of the pure glycopeptides by t h i s group of investigators (50) led to the characterization of two major glycopeptides from Hp 2-1 d i f f e r i n g i n amino acid composition, but containing e s s e n t i a l l y 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 i n some units but absent in others.  On the basis of the size (molecular weights  ranging from 2000 to 3000) and the composition of the i s o l a t e d 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, J a y l e s group (47) obtained two types of carbohy1  drate chains, the smaller i s detached by trypsin i n the form of dialyzable glycopeptides and contain galactose, glucosamine and the part of s i a l i c acid not released by (51),  neuraminidase  the other fragment is. non-dialyzable and contains f i v e  kinds of carbohydrates and a l l the s i a l i c acid (70%) removed by neuraminidase Lisowska  (51).  Tryptic digestion by Dobryszycka  and  (52) also yielded a number of glycopeptides with  varying carbohydrate content, one of these glycopeptides, however, was  shown to r e t a i n the property of activating the  - 14 peroxidase a c t i v i t y of hemoglobin. The complex formed between hemoglobin and haptoglobin i s of considerable i n t e r e s t .  This combination  i s extremely  t i g h t and stable and i s highly s p e c i f i c , reminiscent of the antigen-antibody reaction, with the exception that the HbHp complex remains s o l u b l e .  That the hemoglobin-haptoglobin  reaction i s e s s e n t i a l l y 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 A s o t o p i c a l l y l a b e l l e d hemoglobin (53). Also, attempts at d i s s o c i a t i o n 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 hemoglobins with the s p e c i f i c i t y of binding residing i n the haptoglobin molecule  (44,46,55,56).  The common phenotypes of haptoglobin a l l combine s t 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 corresponds to an equimolar combination Hp 1-1 unit.  of hemoglobin with an  However, i f a haptoglobin solution i s p a r t i a l l y  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 f e t a l hemoglobin and several abnormal hemoglobins 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 i n the hemoglobin molecule for binding or to an altered configuration, which has been observed for Hb H by Perutz and Mazzarella (62) i n t h e i r 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) f i n d that haptoglobin combines with isolated a hemoglobin chains and suggests as a consequence that combination of hemoglobin with haptoglobin i s due to the presence of exposed sites peculiar to the a chains.  Studies  on the e f f e c t of altered configuration of the hemoglobin molecule on the haptoglobin binding have also been pursued with deoxyhemoglobin.  Perutz and coworkers noted that both the  deoxy- and -.oxyhemoglobin H have a similar c r y s t a l l a t t i c e to deoxyhemoglobin A (6 2) and that deoxygenation of oxyhemoglobin A results i n a markedly d i f f e r e n t quaternary structure, the contacts between the a i a  2  chains undergoing a change i n d i s -  tance and angle of contact, while the (64).  8i$2  contacts are broken  To test the importance of the oxyhemoglobin configuration  - 16 f o r b i n d i n g , Nagel et al. (65) determined  the hemoglobin  b i n d i n g c a p a c i t y of a l l types of h a p t o g l o b i n which had been exposed t o deoxyhemoglobin, by a d d i t i o n of l a b e l e d cyanomethemoglobin 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 g r e a t l y decreased  deoxyhemogJjobh was observed  and i n the presence  binding of of d i t h i o n i t e  b i n d i n g of deoxyhemoglobin t o h a p t o g l o b i n was almost  absent.  Since the 3 chains a r e r e p o r t e d t o undergo the g r e a t e s t conf o r m a t i o n a l change i n deoxygenation cule  (66), and s i n c e i t was found  unable  of the hemoglobin mole-  t h a t i s o l a t e d a chains were  t o form a complex w i t h h a p t o g l o b i n  c l u d e d t h a t 3 chains i n the conformation  (61), i t was confound  i n tetrameric  oxyhemoglobin A p a r t i c i p a t e i n complex formation  (65).  The  f a i l u r e of deoxyhemoglobin t o bind h a p t o g l o b i n was confirmed by Chiancone and c o l l a b o r a t o r s (63) by sedimentation measurements i n the u l t r a c 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 b i n d i n g of h a p t o g l o b i n to oxyhemoglobin, f o r once attached t o h a p t o g l o b i n , the hemog l o b i n c o u l d not be removed by deoxygenation.  Further  infor-  mation on the k i n e t i c mechanism of the Hb-Hp r e a c t i o n has been p r o v i d e d by the r e c e n t study of Nagel and Gibson stopped  (59) u s i n g  flow measurements of the quenching of the aromatic  amino  a c i d f l u o r e s c e n c e o f h a p t o g l o b i n upon b i n d i n g of hemoglobin. Haptoglobin was found  t o b i n d a hemoglobin chains  specifically;  once a chains have i n t e r a c t e d , r a p i d b i n d i n g of 3 c h a i n s . f o l l o w s . T h e i r p r e v i o u s i n a b i l i t y t o demonstrate b i n d i n g w i t h a chains  - 17 was a t t r i b u t e d t o d i l u t e s o l u t i o n s prepared f a c t o r y method.  The r e a c t i o n with  by a l e s s  satis-  i n t a c t hemoglobin may  proceed v i a a hemoglobin subunit s i n c e the r a t e of the r e a c t i o n does not i n c r e a s e i n a l i n e a r manner with hemoglobin c o n c e n t r a t i o n and becomes r e l a t i v e l y slower a t h i g h e r hemoglobin concentrations.  Thus i t was p o s t u l a t e d t h a t the  b i n d i n g r e a c t i o n proceeds e i t h e r by c o n s e c u t i v e b i n d i n g of a and 8 hemoglobin monomers or by attachment of a B dimers through a c h a i n s .  The l a t t e r scheme would be i n accordance  w i t h t h e i r f i n d i n g s t h a t i n 2 M s a l t s o l u t i o n s the r e a c t a n t s show r a t e s of b i n d i n g i d e n t i c a l with the r a t e i n s o l u t i o n s of low i o n i c s t r e n g t h , f o r i n 2 M s a l t hemoglobin e x i s t s l a r g e l y i n forms of a B dimers  (67). C o n s i s t e n t  with  these data would be a molecule of h a p t o g l o b i n with two i d e n t i c a l but independent b i n d i n g s i t e s , which would e x p l a i n the presence of the Hb-Hp i n t e r m e d i a t e s a t u r a t i o n of a h a p t o g l o b i n  complex observed on under-  s o l u t i o n with hemoglobin.  The  model of the h a p t o g l o b i n molecule thus bears a remarkable resemblance t o t h a t of the immunoglobulin molecule. I t i s apparent t h a t oxygen or another s i m i l a r l i g a n d i s r e q u i r e d t o induce is  some form or s t a t e of hemoglobin which  s p e c i f i c a l l y r e a c t i v e to haptoglobin  (63). Bunn (68)  proposes t h a t i t i s the reduced d i s s o c i a t i o n i n t o a8 dimers i n the deoxy-form which i s the prime f a c t o r i n i t s l a c k o f binding to haptoglobin,  a c o n c l u s i o n a r i s i n g from h i s  b i n d i n g s t u d i e s with a c h e m i c a l l y m o d i f i e d hemoglobin, BME-Hb,  which has c e r t a i n p h y s i c a l and chemical p r o p e r t i e s i n common w i t h deoxyhemoglobin.  Simon and Konigsberg  (69) r e a c t e d Hb A  with a b i f u n c t i o n a l reagent, bis(N-maleimidomethyl)ether,  (BME)  0 HC-C  C-CH BME  to y i e l d a molecule w i t h 2 moles o f BME per Hb tetramer.  One  molecule o f BME binds c o v a l e n t l y w i t h each g-93 cysteine-SH group and the second maleimide  r i n g of each BME molecule  under-  goes r e a c t i o n a t an unknown s i t e w i t h the same g c h a i n (70). BME-Hb shows no c o - o p e r a t i v e i n t e r a c t i o n , i t s oxygen  equili-  brium curve i s h y p e r b o l i c and i s independent  On the  of pH.  b a s i s of v a r i o u s p h y s i c a l and chemical c r i t e r i a X-ray a n a l y s i s  (69) and the  (70) , i t was e s t a b l i s h e d t h a t horse BME-Hb  does not change i t s conformation upon r e a c t i o n with a l i g a n d such as oxygen s i n c e i t i s a l r e a d y l o c k e d i n t o the conformat i o n of the normal horse oxyhemoglobin. the other hand, resembled  Human BME-Hb, on  n a t i v e deoxyhemoglobin i n i t s r e -  duced e x t e n t of d i s s o c i a t i o n i n t o dimers r e l a t i v e t o normal oxyhemoglobin.  When human hemoglobin was t r e a t e d w i t h s i x  d i f f e r e n t s u l f h y d r y l reagents o n l y the d e r i v a t i v e w i t h BME showed impaired b i n d i n g t o h a p t o g l o b i n and only when a l a r g e excess of BME human Hb was used d i d the b i n d i n g approach t h a t w i t h normal Hb.  Moreover, once the m o d i f i e d hemoglobin  - 19 was bound, a considerable amount of i t could be displaced by normal hemoglobin contrary to the s t a b i l i t y exhibited by haptoglobin bound with unmodified hemoglobin.  Since horse  BME-Hb c r y s t a l s are isomorphous with those of normal horse oxyhemoglobin (69), Bunn feels that i t i s not conformational a l t e r a t i o n i n the molecule which precludes formation of a stable complex but rather i t i s due to a reduced degree of reversible d i s s o c i a t i o n into symmetrical dimers i n both  BME-  and deoxyhemoglobin which i s responsible for impaired binding. This conclusion i s based on the assumption  of a haptoglobin  molecule with two i d e n t i c a l binding sites for two a$ dimers. However, although human BME-oxyhemoglobin shows limited d i s s o c i a t i o n under conditions which cause normal oxyhemoglobin to d i s s o c i a t e , 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  a pair of autosomal a l l e l e s , Hp  2  and Hp  advanced by Smithies  and Walker (12) has been shown to be e s s e n t i a l l y correct by a number of extensive pedigree studies by Galatius-Jensen (71, 72), A l l i s o n (73), Harris, Robson and S i n i s c a l c o (74), and Sutton and coworkers (7 5).  Further subdivision of the hapto-  globin phenotypes on the basis of the electrOphoretic d i f ferences i n their a polypeptide chains shows that six common  - 20 IF  haptoglobin types are determined by three a l l e l e s , Hp  }  IS  Hp  2  and Hp  (25).  In the absence of reductive cleavage these d i f -  ferences i n the a chains are indistinguishable. Aside from the three common haptoglobin phenotypes 2-1 and 2-2, a number of unusual phenotypes  1-1,  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 c a l l e d Hp Caflberg (77),  in which some bands corresponded i n mobility with those found in the Hp 2-1 pattern while other bands were c h a r a c t e r i s t i c of the 2-2 phenotype.  This pattern appears to r e s u l t from a  r e l a t i v e l y decreased production of Hp a"*" polypeptides (77). The most frequent variant Hp phenotype i s Hp 2-1M  (Fig.3),  designated thus by Connell and Smithies (81) because the rate of migration of the bands i s the same as those i n type 2-1 but the r e l a t i v e concentration of the f i r s t two bands are d i f f e r e n t and the remaining bands are either absent or scarcely d i s c e r n i b l e (82).  Hp 2-1M  occurs predominantly i n  Negro populations, the frequency of occurrence being 10% i n American Negroes (82), but this phenotype i s found at a very low frequency i n a l l other r a c i a l groups  Subtyping IF studies of Hp 2-1M show that a normal amount of Hp a or IS a i s associated with a reduced amount of the usual Hp a 2 polypeptide (77) (Fig. 3).  (83,84).  Another variant phenotype which  is much rarer than the Hp 2-1M,  i s Hp Johnson  (Hp J) (Fig. 3)  found o r i g i n a l l y i n a Negro mother and her daughter by G i b l e t t  - 21 -  Hp types  a-Polypeptides of P u r i f i e d haptoHp haptoglobin. Urea-mercapglobin. Starchsub- t o e t h a n o l s t a r c h - g e l g e l electrophore^-types e l e c t r o p h o r e s i s s i s  F i g u r e 3.  SG-electropherograms of the h a p t o g l o b i n  types and t h e i r r e s p e c t i v e subtypes (80).  pheno-  - 22 (30) but subsequently 85).  i n widely separated r a c i a l groups (23,  The o r i g i n a l phenotype appears to be a t r i p l i c a t e d a  polypeptide i n heterozygous combination  with an Hp IS chain  (23) . Many other rare Hp phenotypes have been reported. 1-P,  Hp  Hp 2-P, Hp 1-H, Hp 2-H and Hp 2-L are believed to be  heterozygous combinations of three rare a l l e l e s , Hp P  3  IS  Hp H and Hp L with one or another of the common Hp genes Hp  ,  2  IF  Hp and Hp (76). Hp 1-B and Hp 2-B are reported to be a 1 combination of a rare a l l e l e HpB with the Hp and Hp2 genes (86).  A B chain mutant i s reputedly responsible for the  phenotype, Hp 2-1 Bellevue (87).  Hp Marburg i s a variant  which has the unusual property of not being able to combine with the normal proportions of hemoglobin (88).  Hp 2-1D i s 1D  a phenotype ascribed to the expression of a new a l l e l e Hp which determines Hp a"*" which on subtyping migrates s l i g h t l y IF 0  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 i n various Caucasian populations assuming a Hardy-Weinberg d i s t r i b u t i o n 91,92,93).  (12,72,90,  In addition, Sutton's group (90) observed  f i c a n t differences between Caucasians  signi-  and Africans of Liberia  and the Ivory Coast, the frequency of the Hp gene i n the l a t t e r 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 i n  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 f o r 2  Lacandon Indians i n 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 r i s e s i n areas progresively farther r e -  moved from a p a r t i c u l a r 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  p a r t i a l 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 f r e -  9  quency of the Hp gene i n India led Ingram (96) to suggest that this gene arose i n India and since presumably i t pos7  sesses a selective advantage, i s displacing the Hp o  In support of the recent evolution of the Hp  gene.  gene i s the  finding that i n certain primates and other mammals, only a single Hp band corresponding to human Hp 1-1 i s seen on starch-gel electrophoresis (97).  Thus the multiple bands  9  c h a r a c t e r i s t i c of the Hp  gene appears to be a feature pe-  c u l i a r to humans since haptoglobin exists only i n the monomeric form  (55) i n a l l other mammals studied so f a r . 1F  G i b l e t t and Brooks (8 0) reported that whereas the Hp 2s  i s found at almost half the frequency of the Hp  gene  gene i n  Caucasians and occurs with almost equal frequency i n Negro  -  F i g u r e 4y\ World map  of Hp'  24  -  gene f r e q u e n c i e s ( 9 5 ) .  p o p u l a t i o n s , the Hp  gene i s extremely  population.  confirmed  T h i s was  extended the study and found  r a r e i n the O r i e n t a l  by Shim and B e a m  (97),  who  t h a t a l l Mongoloid p o p u l a t i o n s  and A u s t r a l i a n a b o r i g i n e s (with the e x c e p t i o n of c e r t a i n 1S  A b o r i g i n e s from Western A u s t r a l i a ) have the Hp  gene and  do  IF  not possess The  the Hp  occurrence  gene. of s e v e r a l h a p t o g l o b i n types s i d e by s i d e  i n s e v e r a l human p o p u l a t i o n s r e p r e s e n t s a t r u e polymorphism, but there i s no known reason f o r such v a r i a t i o n s , though i t 1  i s suspected  t h a t the Hp  2  and Hp  i n the past i n f l u e n c e d by one  genes are now  or have been  or more s e l e c t i v e f a c t o r s .  dence f o r t h i s hypothesis has been presented  i n a study  Evi-  (9 8)  2  i n which the Hp  gene frequency d i f f e r e n c e s between the West  A f r i c a n and the American Negro p o p u l a t i o n s c o u l d not  be  accounted  They  f o r on the b a s i s of gene m i g r a t i o n alone.  suggest  t h a t the Hp genes have d i f f e r e n t adaptive values i n  the two  p o p u l a t i o n s , consequently  the polymorphism may  pos-  s i b l y be u n s t a b l e i n the d i f f e r e n t r e g i o n s . A p o s s i b l e s e l e c t i v e advantage  (24) may  be i n f e r r e d  from  the s t u d i e s on a r e c e n t l y d i s c o v e r e d l i v e r enzyme, heme amethenyl oxygenase.  Nakajima and coworkers  (99,100) r e p o r t e d  the i s o l a t i o n and c h a r a c t e r i z a t i o n of t h i s enzyme which i s p r e s e n t predominantly  i n the l i v e r and kidney.  The  enzyme  c a t a l y z e s the c o n v e r s i o n of p y r i d i n e hemichromogen i n t o formylbiliverdin  ,(10 1 ) "  by o x i d a t i v e cleavage of the  p h y r i n r i n g a t the a-methenyl b r i d g e , i n the presence  por-  of NADPH,  - 26 f e r r o u s i r o n and an a c t i v a t o r .  The  substrate s p e c i f i c i t y  t h i s enzyme system i s of p a r t i c u l a r i n t e r e s t .  Data  of  indicate  t h a t the Hb-Hp complex r a t h e r than f r e e hemoglobin i s the subs t r a t e i n t h i s enzymatic  r e a c t i o n and suggest t h a t under  p h y s i o l o g i c a l c o n d i t i o n s hemoglobin i s not m e t a b o l i z e d f r e e form but r e q u i r e s combination  in i t s  with h a p t o g l o b i n f o r con-  v e r s i o n t o f o r m y l b i l i v e r d i n , which i s then h y d r o l y z e d by a second  enzyme, heme a-methenyl formylase t o b i l i v e r d i n .  Study  of the c o n v e r s i o n of complexes of methemoglobin w i t h the t h r e e g e n e t i c types of h a p t o g l o b i n showed t h a t the  initial  v e l o c i t y of c o n v e r s i o n i s g r e a t e r i n the complex w i t h Hp than i n t h a t w i t h Hp  2-1  or Hp 1-1  (99). o  t h a t the s e l e c t i v e advantage i n the Hp  Thus i t i s p o s s i b l e gene l i e s i n a more  e f f i c i e n t metabolism of the hemoglobin complex i n t o b i l e ments.  2-2  pig-  C o n s i s t e n t w i t h t h i s hypothesis i s the e s t a b l i s h e d  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 c a t a b o l i s m of the complex  (102,103).  PHYSIOLOGY AND  PATHOLOGY OF HAPTOGLOBIN  J a y l e , on o b s e r v i n g the absence of h a p t o g l o b i n i n subj e c t s w i t h h e m o l y t i c j a u n d i c e , suggested  that haptoglobin  p l a y s a r o l e i n the e l i m i n a t i o n of e x t r a c o r p u s c u l a r hemoglobin  (104).  S t u d i e s by numerous i n v e s t i g a t o r s had e s t a -  b l i s h e d t h a t when the c o n c e n t r a t i o n of hemoglobin i n the plasma i s below a t h r e s h o l d l e v e l no hemoglobin can be ted i n the u r i n e .  detec-  T h i s l e v e l , c a l l e d the r e n a l t h r e s h o l d ,  - 27 was based on the assumption that hemoglobin c i r c u l a t e s i n plasma i n the free state and passes through the glomeruli even at low plasma concentrations, only to be reabsorbed by the renal tubules.  I f the plasma concentration exceeds the  threshold l e v e l , the concentration of hemoglobin i n 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 i n d i v i d u a l , being about 135 mg/100 ml i n the majority of subjects (58).  But on repeated d a i l y injections of hemo-  globin, the threshold l e v e l i s lowered to less than half the o r i g i n a l value and i n 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 L a u r e l l and Nyman (105) and A l l i s o n and ap Rees (58) to suggest independently  a new explanation for this phenomenon.  The normal hemoglobin binding capacity of haptoglobin varies widely i n individuals 30-190 mg/100 ml (35) with an average value of about 100 mg/100 ml (73), that i s , just below the l e v e l claimed to represent the threshold l e v e l 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 i s present i n plasma i n amounts exceeding the binding capacity of haptoglobin, i t c i r c u l a t e s i n the free state and r e a d i l y passes into the glomerular f i l t r a t e .  Thus  - 28 when the i n t r a v a s c u l a r r e l e a s e of hemoglobin i s continuous, a so i n hemolytic anemias, or on repeated i n j e c t i o n s of hemog l o b i n , the r a t e of removal of the-complex may exceed the r a t e of replacement  of h a p t o g l o b i n , so t h a t the h a p t o g l o b i n  l e v e l i n the plasma may f a l l t o very low o r u n d e t e c t a b l e l e v e l s , and t h e r e f o r e show a d i m i n i s h e d t h r e s h o l d .  In v i v o  s t u d i e s o f the d i f f e r e n t i a l r e n a l t r a n s p o r t of f r e e and protein-bound  hemoglobin by Lathem and Worley  (106,107) and the  s t u d i e s of hemoglobin c l e a r a n c e w i t h l a b e l l e d i r o n by Murray and coworkers  (10 8) supported  t h r e s h o l d by h a p t o g l o b i n s .  the d e t e r m i n a t i o n o f the r e n a l  Thus one p h y s i o l o g i c a l f u n c t i o n  f o r h a p t o g l o b i n may be t o prevent the l o s s of hemoglobin from the body through  the kidneys and hence t o p l a y a r o l e  i n i r o n r e t e n t i o n and t o p r o t e c t the kidney from s i d e r o s i s produced by c h r o n i c e n t r y o f hemoglobin i n t o the kidneys. The h a p t o g l o b i n l e v e l , although  i t e x h i b i t s a wide range  i n the healthy p o p u l a t i o n , i s s t r i k i n g l y constant i n the i n dividual.  There appears t o be a g e n e t i c mechanism i n v o l v e d  i n r e g u l a t i n g the q u a n t i t y of h a p t o g l o b i n , f o r the mean v a l u e i n sera of h e a l t h y i n d i v i d u a l s of type 1-1 was 136, type 2-1, 108  and type 2-2,- 82 mg„(54).  A n a l y s i s of h a p t o g l o b i n  levels  i n monozygotic and d i z y g o t i c twins showed a l e s s e r v a r i a n c e of the mean d i f f e r e n c e between i d e n t i c a l as c o n t r a s t e d t o f r a t e r n a l twins  (109) and supports  the suggested  r o l e of gene-  t i c f a c t o r s i n the r e g u l a t i o n of h a p t o g l o b i n l e v e l of the v a r i o u s types.  Nyman  (54) suggested  t h a t hormones i n f l u e n c e  - 29 haptoglobin levels since men have a higher mean haptoglobin l e v e l than women.  I t has also been found that androgen ad-  ministration to women increases haptoglobin levels (110) and estrogen administration decreases the l e v e l (111).  However,  extensive studies by other investigators (72,112) have r e vealed no s i g n i f i c a n t difference i n levels between males and females.  There has been suggestive evidence of an increase  in haptoglobin with age i n adults-(112,113).  In newborn:  infants, a well established haptoglobin pattern i s 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 haptoglobin (114).  However, the haptoglobin l e v e l soon r i s e s  gradually during the f i r s t months of l i f e and reaches the mean l e v e l of healthy adults at 4 months (55). or  This absence  low concentration of haptoglobin was attributed to a low  rate of synthesis i n the newborn (114,115) rather than to an accelerated rate of erythrocyte destruction (116). Extensive investigations have been carried out on the v a r i a t i o n of haptoglobin levels i n pathological conditions, since the determination of haptoglobin may aid i n the d i f f e r e n t i a l diagnosis of certain diseases and a i d i n following the prognosis of the disease.  In a l l hemolytic disorders,  the plasma haptoglobin l e v e l i s either very low or absent (73).  Haptoglobin i s absent i n pernicious anemia (73,117)  but reappears within a few days of the beginning of treatment. Low levels of haptoglobin are also found i n hepatocellular  - 30 failure  (54,104); t h i s decrease was  reported  (54) to be  to an i n c r e a s e d hemoglobin c a t a b o l i s m r a t h e r than to haptoglobin. reduced  However, Owen, McKay and Got  due  decreased  (118) b e l i e v e  s y n t h e s i s of h a p t o g l o b i n to be the prime f a c t o r s i n c e  they o b t a i n e d some c o r r e l a t i o n between h a p t o g l o b i n and albumin l e v e l .  Haptoglobin  c h r o n i c inflammation, forms of s t r e s s and g l o b i n belongs  serum  l e v e l s i n c r e a s e with acute or  t i s s u e d e s t r u c t i o n or under d i f f e r e n t  i n v a r i o u s malignancies  (46,119).  Hapto-  to the serum mucoprotein f r a c t i o n which be-  haves as an acute phase r e a c t a n t , i t s serum l e v e l the a c t i v i t y of the d i s e a s e process  (120).  reflecting  Thus changes i n  serum h a p t o g l o b i n l e v e l are analogous to other n o n - s p e c i f i c changes i n serum mucoproteins i n d i s e a s e . t i o n s are t h a t although  However, i n d i c a -  there i s a p a r a l l e l i s m i n the i n -  crease of h a p t o g l o b i n and orosomucoid, there i s no correlation The  strict  (46,54,121).  c l o s e c o r r e l a t i o n between h a p t o g l o b i n , f i b r i n o g e n  and orosomucoid found  i n patients with i n f e c t i o u s diseases,  based on the s t r i k i n g c o r r e l a t i o n between d e g r a d a t i v e changes i n c o n n e c t i v e t i s s u e and high l e v e l s of c i r c u l a t i n g g l y c o proteins  (122), gave r i s e to J a y l e ' s hypothesis on the  and metabolism .of h a p t o g l o b i n  (104).  origin  Jayle postulated that  h a p t o g l o b i n i s s y n t h e s i z e d by the f i b r o c y t e s i n the connect i v e t i s s u e and  i s depolymerized  dase and r e l e a s e d .  by the a c t i o n of  T h i s hypothesis was  when Murray and C o n n e l l  (123)  neuramini-  no longer t e n a b l e  r e p o r t e d t h a t the  haptoglobin  - 31 l e v e l of the exudate withdrawn from the s i t e of subcutaneous turpentine injections which had the e f f e c t of increasing the haptoglobin l e v e l i n rabbits, was much lower than the haptoglobin l e v e l of the serum during the height of the r i s e .  It  is unlikely then that haptoglobin i s released into the blood at the s i t e of i n j e c t i o n . de  novo  The haptoglobin r i s e represents  synthesis rather than l i b e r a t i o n from a tissue pool  of the protein, since haptoglobin response to multiple i n jections i s more intense and longer l a s t i n g (121,124) and puromycin and other i n h i b i t o r s of protein synthesis prevented the elevation of plasma haptoglobin l e v e l (125).  Several  lines of evidence places the s i t e of haptoglobin biosynthesis in the l i v e r .  After perfusion of rabbit l i v e r with blood  containing l a b e l l e d amino acids (126) or radioactive leucine and galactose (127), the predominant incorporation of radioa c t i v i t y i s i n the haptoglobin f r a c t i o n .  Carbon tetrachlor-  ide-induced l i v e r damage completely prevents incorporation of l a b e l l e d amino acids into the haptoglobin f r a c t i o n and greatly decreased the s p e c i f i c r a d i o a c t i v i t y of other proteins synthesized i n the l i v e r but d i d not affect the r a d i o a c t i v i t y of y-globulin which i s known to be synthesized outside the l i v e r (128).  P a r t i a l hepatectomy also resulted i n a s t r i k i n g re-  duction i n serum haptoglobin levels produced i n response to injury (129).  The factors responsible f o r stimulation of  haptoglobin production are not known.  Increased haptoglobin  catabolism i n connection with hemolysis i s not an adequate  - 32 stimulus f o r h a p t o g l o b i n s y n t h e s i s , s i n c e the o r i g i n a l g l o b i n l e v e l i s not recovered u n t i l 5-7 m i n a t i o n of h a p t o g l o b i n  (54).  Krauss  hapto-  days a f t e r t o t a l  (130)  eli-  has shown t h a t  the plasma h a p t o g l o b i n l e v e l of adrenalectomized  rats increases  a f t e r t u r p e n t i n e i n j e c t i o n but t o a l e s s e r extent than t h a t of i n t a c t r a t s , so t h a t f a c t o r s other than s o l e l y a d r e n o c o r t i c a l s t i m u l a t i o n must be i n v o l v e d i n the s t i m u l a t i o n of  hapto-  globin synthesis. The Hb-Hp complex i s c l e a r e d as a u n i t much more r a p i d l y than f r e e h a p t o g l o b i n from the plasma  (131); the  of the former  and of the l a t t e r i s  4.5  days  i s 88-120 minutes  (133).  (132)  half-life  As long as the c o n c e n t r a t i o n of the complex  remains above 0.4  gm/1., i t i s removed from the blood  stream  a t a c o n s t a n t r a t e ; a f t e r t h i s , i t i s e l i m i n a t e d as an expon e n t i a l f u n c t i o n of time  (134).  I t i s e s t a b l i s h e d t h a t the  r e t i c u l o e n d o t h e l i a l system p l a y s a major r o l e i n the r e moval of the complex.  Both Murray, C o n n e l l and P e r t (10 2)  and Keene and J a n d l (103):. r e p o r t e d t h a t the l i v e r  accounted  f o r 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 s t u d i e s by means of s p e c i f i c  immunofluorescence  (135)  and autohistography  (134)  indicate  that the K u p f f e r c e l l s of the s i n u s o i d s of the l i v e r and  the  s p l e n i c c e l l s are the s i t e of c a t a b o l i s m of the Hb-Hp complex. S t u d i e s on the f a t e of uncomplexed h a p t o g l o b i n by and S a r c i o n e p r o t e i n had  (132)  showed t h a t 4 days a f t e r i n j e c t i o n ,  l a r g e l y degraded.  Mouray et a l . (128)  Krauss the  found t h a t  -  the h a l f - l i f e of  13 1  33 -  I-Hp was not altered by carbon t e t r a -  chloride-induced l i v e r damage.  Therefore the l i v e r also  does not appear to play a major role i n 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 s i t e of polymerization of haptoglobin polymers i s d i s t i n c t from the s i t e of hemoglobin binding, p a r t i a l l y 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.  L o c a l i z a t i o n of the hemoglobin binding s i t e  on the component haptoglobin chains i s achieved by determination of the a b i l i t y of isolated a , a 1  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 i s through the globin moiety.  Furthermore, study of the reaction of hapto-  globin with myoglobin, a protein  similar i n function and  conformation to hemoglobin, and with various vertebrate hemoglobins provide  additional information on the hemoglobin  combining s i t e .  The effects of environmental  combination  factors on the  of haptoglobin with hemoglobin has been studied  by a l t e r a t i o n of physical parameters, ionic strength and pH, and addition of a tyrosine analogue.  The involvement of  -  34 -  amino groups o f h a p t o g l o b i n i n b i n d i n g with hemoglobin has been s t u d i e d by s e l e c t i v e chemical m o d i f i c a t i o n of haptoglob i n amino groups w i t h three reagents of i n c r e a s i n g  severity  f o l l o w e d by measurement of the a b i l i t y of the 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 hemoglobin.  An approximate d e t e r -  m i n a t i o n of the area of the b i n d i n g s i t e i n the Hb-Hp comp l e x has been made by comparison o f the extent of g u a n i d i n a t i o n of the amino groups of the i n d i v i d u a l components with t h a t of the complex  and a l s o by comparison w i t h the extent  of 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 hemoglobin and h a p t o g l o b i n from the g u a n i d i n a t e d  complex.  - 35 -  1.  EXPERIMENTAL GENERAL METHODS 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 p l a s t i c  covers were inserted with either an 8 or 20 slot-former, then the cover and tray were l i g h t l y coated with Dow Corning 70 4 silicone f l u i d .  After adding 500 ml of the borate buffer to  the starch-hydrolyzed i n a 1 1. suction flask, the flask was immediately swirled and then heated with continual swirling in a water-bath u n t i l the starch suspension had passed a sudden r i s e i n v i s c o s i t y and then showed a marked decrease in viscosity.  Negative pressure was then applied to the  starch solution for a few minutes.  The degassed solution i s  immediately poured into the g e l tray, the cover c a r e f u l l y lowered into place without trapping a i r bubbles and weights placed on the cover at the perimeter of the g e l tray u n t i l the g e l i s cool.  In preparation f o r the electrophoretic run,  the cover i s l i f t e d c a r e f u l l y and the samples inserted into the slots with Pasteur pipettes.  Melted Hartz white petro-  latum i s poured into thin f i l m over the g e l surface.  After  the petrolatun had s o l i d i f i e d , the end plates of the g e l tray were removed and the g e l i s assembled i n a v e r t i c a l  - 36 position with the apparatus i l l u s t r a t e d by Smithies (136) . The outer compartments of each electrode vessel were approximately h a l f - f i l l e d with 10% NaCl solution and the inner compartments f i l l e d to a s l i g h t l y higher level than that of the s a l t solution with a bridge buffer of ten times the concentration of the gel buffer.  After e l e c t r i c a l contacts between  the two solutions i n the electrode vessel and between the bridge solution and the gel were made with several thicknesses of f i l t e r paper, the Ag/AgCl electrodes were immersed in the s a l t 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 g e l after s l i c 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 i n a solvent of composition, methanol-.distilled water-.acetic acid in r a t i o s 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 s l i c e of the gel was stained with a  benzidine reagent by mixing 1 ml of a saturated solution of benzidine i n ethanol with 1 ml of g l a c i a l acetic acid, then adding 100 ml of d i s t i l l e d water and just p r i o r 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 i s unstable and gradually fades to green and then to brown. 2.  UREA STARCH-GEL ELECTROPHORESIS Starch-gels which contained 8 M urea were prepared i n  0.05 M acetate buffer, pH 5.0 by the method of Smithies, Connell and Dixon (25).  A starch gel i n a concentration of  15 gram percent was prepared by thoroughly mixing 75 g starch-hydrolyzed with 240 gm of urea and the mixture gradua l l y added with vigorous mechanical s t i r r i n g to 300 ml of 0.05 M acetate buffer contained i n a metal beaker.  With the  beaker immersed i n a water-bath and while under continual vigorous s t i r r i n g , the mixture was heated at 70° for about 7 minutes, after which time, the r e s u l t i n g 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 s l i g h t modifications. was made of 50 gm  A solution  (10%) of acrylamide (Matheson, Coleman and  Bell) and 1 gm of N,N'-methylenebisacrylamide  (0.2%) i n 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 tetramethylethylenediamine i n 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 i s rapidly swirled and immediately poured into the gel tray.  The cover  -  38 -  was c a r e f u l l y lowered into place without trapping a i r bubbles and weights placed on the cover at the perimeter of the g e l tray.  The cover adhered to the g e l tray through a thin f i l m  of petrolatum on the upper surface of the perimeter of the g e l tray. 4.  After the g e l has set i t was ready for use.  PREPARATION OF HEMOGLOBIN (a)  The hemoglobin solutions employed  i n the electro-  phoretic runs were prepared from sedimented erythrocytes of human blood treated with ACD (sodium c i t r a t e , 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 c e l l s were  washed at least twice with a physiological saline solution and separated from the saline solution by centrifugation after each washing.  The washed p e l l e t which remained after  centrifugation was lysed with 9 times i t s volume of d i s 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 b a c t e r i a l growth.  A small portion of this stock  hemoglobin solution was stored at 4 ° f o r current use, the remaining solution was stored frozen i n several small b o t t l e s . The hemoglobin control solutions used i n 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 p u r i f i e d 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 i n the peroxidase a c t i v i t y assays or i n the Sephadex assays was i n the form of the carbonmonoxy derivative, which was prepared from t w i c e - c r y s t a l l i z e d hemoglobin according to the technique of Drabkin blood treated with ACD anti-coagulant. tion of about 8-10 mmoles Hb/1.  (139) for human  A stroma-free solu-  concentration, referred to  a molecular weight equivalent of 16,700, was obtained i n the following manner (140):  The red blood c e l l s were separated  from the plasma and washed once with a physiological saline solution, then washed three times with a mixture of saline and 0.0025 M A1C1 . 3  1.2%  The packed c e l l s after d i l u t i o n  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 r e f r i g e r a t i o n overnight  the mixture was centrifuged and the clear hemoglobin solution was siphoned o f f .  Concentration of this hemoglobin solution  was accomplished by d i a l y s i s i n a 2.8 M phosphate buffer, pH 8.0 composed of 371 gm of K HPCv3H 0 and 160 gm of KH P0 2  2  2  4  made up to 1 1., with a r a t i o of 1:3 volume within and outside the d i a l y s i s sac.  Thus, 498 ml of phosphate buffer  previously warmed to 37° was used to dialyze 166 ml of hemoglobin solution i n the cold room for a period of hours.  6-10  The dialyzate was then replaced with an equal volume  (820 ml) of fresh unwarmed 2.8 M phosphate buffer and dial y s i s continued for a further 24 hours.  At the end of t h i s  - 40 period of time, more 2.8  M phosphate buffer i s added slowly  to the dialyzate u n t i l microscopic examination of the contents of the d i a l y s i s sac revealed The material was further 24 hours.  that c r y s t a l l i z a t i o n had occurred.  then l e f t i n the cold room to dialyze a R e c r y s t a l l i z a t i o n of the hemoglobin  was  carried out i n a similar manner on a solution of the hemoglobin c r y s t a l s collected by suction f i l t r a t i o n . tained were a mixture of small and  The c r y s t a l s ob-  large sized c r y s t a l s of  i r r e g u l a r shapes and sometimes i n the t y p i c a l bipyramidal shape. The t w i c e - c r y s t a l l i z e d hemoglobin c r y s t a l s were dissolved in 55 ml of d i s t i l l e d water and dialyzed against four changes of g l a s s - d i s t i l l e d water.  The  carbonmonoxy derivative  was  prepared by saturating 230 ml of the dialyzed hemoglobin solution with carbon monoxide gas. p h i l i z e d to y i e l d 13.7  The solution was  then lyo-  g carbonmonoxyhemoglobin, which  was  stored i n the frozen state. (c) Hemoglobin solutions of various were prepared from heparinized  species of animal  blood, the concentration  of  heparin being lower than the amount which i s said to i n f l u ence the hemoglobin binding capacity or peroxidase a c t i v i t y (54), with the exception of horse blood which was in Becton-Dickdnson vacuotubules containing EDTA.  collected The  red  blood c e l l s .were sedimented by centrifugation and washed 5 times with cold 0.9% of the dog,  sodium chloride solution.  In the case  rabbit and rat hemoglobin, 5 volumes of d i s t i l l e d  - 41 water was  added to the-sedimented e r y t h r o c y t e s , i n the other  animal hemoglobins, 9 volumes of d i s t i l l e d water was  added.  A f t e r a l l o w i n g the m a t e r i a l to stand o v e r n i g h t a t 4°, the hemoglobin s o l u t i o n was fugation.  separated from the stroma by  centri-  - 42 PART I ISOLATION OF HAPTOGLOBIN AND PARTIAL SEPARATION OF HAPTOGLOBIN POLYMERS INTRODUCTION Haptoglobin was f i r s t isolated i n the form of the Hb-Hp complex by van Royen (141) on the basis of the observation by Jayle and G i l l a r d  (5) that the complex i s more soluble i n  ammonium sulfate than haptoglobin.  But a l l attempts to r e -  cover haptoglobin from i t s complex were unsuccessful. P u r i f i e d haptoglobin was f i r s t isolated-, by Jayle and Boussier (6) i n 19 54 from the urine of a nephrotic c h i l d by successive ammonium sulfate fractionation of the urinary proteins to y i e l d a Hp 1-1 preparation which was homogeneous by electrophoresis and sedimentation munoelectrophoresis  (37).  (6,7) and by Im-  In 1958, Boussier (142) succeeded  in i s o l a t i n g a haptoglobin of type 2-2 from human serum by successive ammonium sulfate fractionation, the f i n a l  hapto-  g l o b i n - r i c h f r a c t i o n being precipitated with 50% ammonium sulfate.  An additional step of p u r i f i c a t i o n 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 L a u r e l l (57) i s also based on ammonium sulfate f r a c t i o n a t i o n .  His source of haptoglobin was ascites  f l u i d obtained from cancer patients with a high l e v e l 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 in the cold.  precipitation  Further p r e c i p i t a t i o n with ethanol at low ionic  strength was used to remove impurities, notably Hb-Hp complex. A f i n a l two-step s a l t p r e c i p i t a t i o n 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 i n Laurell's method risked denaturation of the protein and that the ethanol p r e c i p i t a t i o n i s unnecessary.  By modification of Laurell's  method, Cloarec and Moretti obtained- 2-5% of 90-100% pure Hp 2-1 or Hp 2-2. P u r i f i e d haptoglobin has also been isolated by Steinbuch and Pejaudier (145) during the routine fractionation of human plasma.  Conn's f r a c t i o n IV, the only f r a c t i o n contain-  ing haptoglobin i n s i g n i f i c a n t quantities, was obtained at pH 5.8 by p r e c i p i t a t i o n with ethanol.  Successive additions  of 0.5% r i v a n o l (a c a t i o n i c detergent) to this f r a c t i o n IV removed the ceruloplasmin, and albumin was removed by p r e c i p i t a t i o n with 3.5% ethanol of the supernatant at pH 5.9, followed by r i v a n o l fractionation at pH 8.5.  Haptoglobin  could then be separated from t r a n s f e r r i n by alcoholic prec i p i t a t i o n at pH 4.4-4.6.  A preferable method of removing  the t r a n s f e r r i n contaminant i s by chromatography on DEAEc e l l u l o s e i n 0.03 M acetate buffer, pH 5.0. At this pH hapto-  - 44 globin i s adsorbed whereas t r a n s f e r r i n i s not.  Elution i s  effected with 0.5 M acetate buffer at the same pH and a haptoglobin preparation of about 80% purity i s obtained Steinbuch and Quentin  (146).  (147) subsequently used DEAE-cellulose  chromatography i n a rapid method of i s o l a t i o n of haptoglobin from whole plasma.  After d i l u t i o n of the plasma and adjusting  the pH to 5.0 with 0.3 M acetic acid, i t i s chromatographed on DEAE-cellulose i n 0.0 3 M acetate buffer, pH 5.0.  Adsorp-  t i o n of haptoglobin at t h i s pH i s less selective than at lower pH values but by development with 0.1 M acetate buffer, pH 5.0, haptoglobin i s eluted whereas ceruloplasmin and other proteins are retained.  By this rapid technique 20% of Hp  in 90-100% purity and 8-30% be obtained (144).  1-1  of Hp 2-1 i n 90-95% purity could  This method i s 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 anion-exchange chromatographic of haptoglobin.  technique for the i s o l a t i o n  In this elegant method, serum was dialyzed  a g a i n s t a pH 4.2 buffer comprised 0.04 4.2.  (81) introduced the f i r s t  of 0.2 M acetic acid and  M NaOH and then chromatographed on Dowex 2-X10  at pH  At t h i s pH, haptoglobin whose i s o e l e c t r i c point i s  about 4.2  (46) i s the main serum protein bearing a net nega-  t i v e charge while most other serum proteins have a net positive charge.  Thus i t i s observed that haptoglobin of pro-  gressively greater purity i s 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 i n 48% y i e l d i s obtained.  However, under conditions of such an acidic  pH,  haptoglobin i n whole serum i s denatured rapidly (148) . Connell and Shaw (148) improved the preparation of haptoglobin and made the method more convenient on a large scale by using DEAE-cellulose.  DEAE-cellulose i s employed as the  adsorbent because i t s capacity for protein i s several hundredf o l d greater than Dowex 2.  A pH of 4.6 was chosen for ad-  sorption since below pH 4.4 haptoglobin i s rapidly  denatured  as indicated by loss of a b i l i t y to form a complex, while above pH 4.8 the s e l e c t i v i t y of adsorption decreases and less pure preparations are obtained.  After the serum i s adjusted  to pH 4.6 with 1.0 M acetic acid, i t i s desalted on a Sephadex G-25  column.  The desalted serum i s 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 t i t r a t e d to pH 4.6 with 1 N HC1.  The  DEAE-cellulose i s then packed i n a small column and eluted with 0.2 M NaCl.  The eluates which are considered s u f f i -  c i e n t l y r i c h i n haptoglobin as determined  by measuring the  hemoglobin binding capacity (81) are diluted with water to give an o p t i c a l density of 10.0 at 280 my.  Haptoglobin i s  then precipitated by treatment with ammonium sulfate at 55% saturation and a solution of the p r e c i p i t a t e i s dialyzed and lyophilized.  Haptoglobin of approximately  94% purity i s  - 46 obtained i n 32% y i e l d . Smith, Edman and Owen (31) have modified the method of Connell and Shaw.  Pooled human plasma i s 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 i s washed with 0.01 M acetate buffer containing 0.01  M NaCl, after which the haptoglobin i s eluted with ace-  tate buffer containing 0.04 M NaCl adjusted to pH 4.7. The effluent i s brought to pH 7.0 and the volume reduced on a rotary evaporator.  The solution i s dialyzed against water  and then l y o p h i l i z e d .  A further fractionation with ammonium  sulfate i s necessary to purify the protein preparation.  Hp  2-1 and 2-2 preparations appeared pure on starch gel electrophoresis; however, Hp 1-1 preparations contained traces of impurities.  With a s l i g h t modification i n the elution tech-  nique, Cloarec and Moretti (144) reported obtaining pure Hp 1-1 i n excellent y i e l d s , 75%, but the Hp 2-1 was p a r t i a l l y denatured. I t i s apparent that the method of i s o l a t i o n of haptoglobin by the methods of Jayle or L a u r e l l are too laborious and the yields of protein e n t i r e l y too low.  Also certain  of these methods (31,57,81) may y i e l d p a r t i a l l y products.  denatured  Connell and Shaw's method appeared to be the  method of choice since a f a i r l y good y i e l d of protein of high purity i s obtained.  Therefore at the s t a r t 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 e a s i l y denatured during preparation by this technique and when native haptoglobin was obtained, the y i e l d 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-  t e i n was obtained from the pooled plasma. developed  Thus a method was  i n which ascites f l u i d was used as the source of  the protein, a method which could be e a s i l y adapted to preparation of large amounts of genetically homogeneous haptoglobin i n high purity.  Ascites f l u i d , which was  f i r s t used  by L a u r e l l (57), proved to be an excellent source of haptoglobin since large quantities (up to 25 1.) can be obtained from a single patient, thus ensuring genetic homogeneity. It has been reported that i n certain malignancies the haptoglobin l e v e l i s elevated (54,104).  It was observed during  this study that ascites f l u i d associated with abdominal carcinoma generally had a high l e v e l of haptoglobin when examined by starch-gel electrophoresis and thus this source of ascites f l u i d was used for i s o l a t i o n of haptoglobins. PURIFICATION OF HAPTOGLOBIN AND 1.  PARTIAL SEPARATION OF POLYMERS  PREPARATION OF HAPTOGLOBIN BY CONNELL AND  SHAW METHOD  Plasma was aspirated from human ACD-treated  blood i n  which the c e l l s had settled after standing i n the cold room. A sample of the plasma was g e n t i c a l l y typed once on a 20 s l o t  - 48 starch gel to enable a large number of samples to be examined and once on an 8 s l o t g e l to confirm the typing. each donor was  Plasma from  stored i n the frozen state either i n d i v i d u a l l y  or pooled according to phenotype and allowed to thaw p r i o r to a preparation.  C l o t t i n g of the plasma was promoted by the  addition of an amount of 3.0 M C a C l equivalent to the amount 2  of c i t r a t e present.  After standing overnight at 4° the c l o t  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 s t i r r i n g , 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 i n water and t i t r a t e d to pH 4.6 with N HC1.  1.0  The concentration was then adjusted to 2 5 mg/ml.  desalted serum was  The  treated with 6 successive 1 g.v lots of  DEAE-cellulose suspension. each l o t of adsorbent was  After s t i r r i n g for 15 minutes, 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 i n water  was poured into a column and elution carried out with 0.2 M NaCl.  A f a i n t green band moves with the sodium chloride front  and haptoglobin was  eluted p r a c t i c a l l y at the s a l t front.  A  starch-gel electropherogram of the 6 eluate fractions indicated  - 49 that the f i r s t three eluates had the highest content.  Combination of these haptoglobin-rich eluates gave  an O.D. of 18 at 280 my. O.D.  The solutions were diluted to an  of 10 followed by p r e c i p i t a t i o n of the haptoglobin i  with ammonium sulfate to 55% saturation. was  haptoglobin  c o l l e c t e d by suction f i l t r a t i o n .  The precipitates-  I t was dissolved i n  4.5 ml of water and the solution dialyzed exhaustively against d i s t i l l e d water and then l y o p h i l i z e d . globin was 132.6  mg.  Y i e l d of hapto-  Electrophoresis on starch gel indicated  that the protein combined readily with hemoglobin to give the t y p i c a l Hb-Hp complex and that there were only traces of impurities. Although this p a r t i c u l a r preparation of haptoglobin yielded native product, often the preparation was found to be p a r t i a l l y or wholly denatured as demonstrated by the lack or the very low binding of hemoglobin.  Also the y i e l d 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 i n order to overcome these 2.  disadvantages.  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 a c e t i c acid buffer, ad-  - 50 justed to pH 4.7 with NaOH referred to as 0.01 M sodium acetate buffer, pH 4.7 hereafter. times.  The dialyzate was  changed several  During d i a l y s i s an insoluble precipitate formed i n the  d i a l y s i s sac, this was removed by centritfugation at the end of the d i a l y s i s . 4.8;  t h i s was  serum was  The pH of the serum after the d i a l y s i s  was  adjusted to pH 4.7 with 1.0 M acetic acid.  The  then applied to a DEAE-cellulose column (5 cm x  7.5 cm) equilibrated i n 0.01  M sodium acetate buffer, pH  The column was washed with 14 0 ml of the same buffer.  4.7.  A  narrow pale green band could be seen at the uppermost part of the column.  Elution was  carried out with a l i n e a r s a l t  gradient made with 100 ml of 0.01  M NaCl i n 0.01  M sodium  acetate buffer, pH 4.7 and 100 ml of 0.1 M NaCl i n 0.01 sodium acetate buffer, pH 4.7.  M  The f i n a l concentration of  NaCl proved to be i n s u f f i c i e n t to elute the proteins, therefore the column was  eluted with an additional 50 ml of buffered  :0..I MlNaCl followed by 0.2 M NaCl i n the acetate buffer u n t i l the elution was complete.  The protein-rich effluents were  pooled into three fractions and the pH adjusted to approximately 7.0 for frozen storage since i n this p a r t i c u l a r 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 r i c h i n haptoglobin, but albumin and post-albumin  impurities were present.  Further  - 51 p u r i f i c a t i o n of the protein was carried out by saturation with 55% ammonium s u l f a t e .  However, t h i s procedure  caused  considerable denaturation of the protein as demonstrated by the considerable amount of insoluble material after d i a l y 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 p r e c i p i t a t e after lyophilization. bin  The starch gel of the undenatured haptoglo-  showed that most impurities had been removed, but a f a s t -  migrating contaminant was  s t i l l present.  Therefore, an  additional p u r i f i c a t i o n step was carried out on an a n a l y t i c a l 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 i n 0.005 M acetate buffer.  The haptoglobin fractions were combined,  dialyzed and l y o p h i l i z e d . This method of preparation of haptoglobin i s unsuitable for a number of reasons.  Besides the low y i e l d , the method  i s tedious and the ammonium sulfate p u r i f i c a t i o n step causes p a r t i a l denaturation of the product.  Also, the impurities  are not completely removed even after the second p u r i f i c a t i o n 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 y i e l d of protein by choosing ascites f l u i d from cancer patients with a high haptoglobin l e v e l .  - 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, decanted from c l o t s and  frozen for storage and  was  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  s o l i d 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  s a l t concentration of 55% saturation.  Each haptoglobin type  was  After 1 hour of  subsequently treated i n p a r a l l e l .  s t i r r i n g i n an ice-bath, centrifugation. 0.01  the p r e c i p i t a t e was  A 100 ml solution of the p r e c i p i t a t e i n  M acetic acid, pH 4.7  (with NaOH), was  the same buffer in the cold room. 3-4  times.  collected by  dialyzed  against  The dialyzate was  changed  At the end of d i a l y s i s ,  some precipitated  proven to not contain haptoglobin by starch-gel s i s , was  removed by centrifugation.  was  applied to a preparative  cm)  which was  pH 4.7.  electrophore-  supernatant solution  DEAE-cellulose column (5 x  equilibrated with 0.01  The column was  The  protein,  9.5  M sodium acetate buffer,  washed with the same buffer u n t i l  washings showed an absorbance at 280 my  lower than 0.02  the O.D.  units.. A f a i n t green band near the top of the column could be observed.  Haptoglobin was  consisting of 300 ml of 0.01  eluted with a linear M NaCl i n 0.01  gradient  M sodium acetate  - 53 buffer, pH 4.7 and 300 ml.of 0.3 M NaCl i n 0.01 M sodium acetate buffer, pH 4.7.  The absorption at 280 my, the pH and the con-  d u c t i v i t y 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. 5),  In the DEAE-chromatography of Hp 1-1 (Fig.  fractions from the f i r s t half and those from the l a s t half  of the o p t i c a l density peak were combined i n 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 l y o p h i l 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 p r o f i l e of the preparative DEAE column of Hp 1-1 ascites f l u i d , peak i s observed.  (Fig. 5), a single 280 my absorbing  The SG electropherogram of the DEAE column  effluent fractions indicated that the haptoglobin-rich f r a c tion corresponded to the o p t i c a l density peak, as revealed by the benzidine-stained half of the g e l .  The amido black-  stained section of the g e l showed that at t h i s stage of i t s preparation, haptoglobin i s contaminated with mainly albumin, some pre- and post-albumins, t r a n s f e r r i n and minute amounts of a slow-migrating impurity (Fig. 7). In the DEAE chromatography of the ascites, f l u i d of type  -  Figure 5.  54  -  DEAE-cellulose chromatography of crude Hp 1-1 on a  5.0 x 9.5 cm column i n 0 . 0 1 M sodium acetate buffer, pH 4 . 7 .  - 55 -  Effluent  Figure 6.  Volume , ml  DEAE-celiluiese chromatography of crude Hp 2-1 on  a 5.0 x 9.5 cm column i n 0.01 M sodium acetate buffer, pH 4.7.  - 56 -  F i g u r e 7.  SG-electropherogram of the D E A E - c e l l u l o s e 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 a t 250 ml through t o 440 ml.  ( S l o t s are numbered  l e f t to r i g h t f o r each 10 ml. f r a c t i o n ) .  commencing consecutively  F i g u r e 7.  SG-electropherogram of the D E A E - c e l l u l o s e 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 at 250 ml through t o 440 ml.  commencing  ( S l o t s are numbered c o n s e c u t i v e l y  l e f t to r i g h t f o r each 10 ml f r a c t i o n ) .  - 57 2-1, the e l u t i o n diagram (Fig. 6) shows 2 major peaks, a larger front-running peak merging with a heterogeneous slowrunning peak.  The SG electropherogram  of these effluent  fractions shows that the foremost o p t i c a l 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 postalbumin impurities. Further p u r i f i c a t i o n of the haptoglobin was achieved by gel  f i l t r a t i o n on Sephadex G-200.  crude Hp 1-1,  Approximately  half the  f r a c t i o n A Hp 2-1, or f r a c t i o n B Hp 2-1 were  each treated i n the following manner: dissolved i n 5 ml of 0.05  the haptoglobin  was  M ammonium acetate buffer, pH  8.5  and chromatographed on a Sephadex G-200 column (2.5 cm x 186 cm)  i n 0.05  M ammonium acetate, pH 8.5 buffer.  effluents were monitored  Column  at 280 my and analyzed for hapto-  globin content by starch-gel electrophoresis and the Hp fractions combined and l y o p h i l i z e d .  1-1  In the case of the Hp  2-1 chromatography, the eluates were i n d i v i d u a l l y l y o p h i l i z e d to preserve the p a r t i a l resolution of haptoglobin polymers obtained on g e l f i l t r a t i o n .  F i n a l y i e l d of p u r i f i e d 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 The Sephadex G-200 p r o f i l e  fluid.  (Fig. 8) of Hp 1-1 shows a  small peak running ahead of the main protein f r a c t i o n ,  F i g u r e 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 a c e t a t e , pH 8.5.  - 59 f o l l o w e d by some s m a l l molecular  weight components.  be seen t h a t good r e s o l u t i o n of h a p t o g l o b i n i n a n t s can be achieved  I t may  from i t s contam-  by g e l f i l t r a t i o n and by o m i t t i n g the  o v e r l a p p i n g p r o t e i n f r a c t i o n s when i s o l a t i n g the h a p t o g l o b i n peak, a h i g h l y p u r i f i e d h a p t o g l o b i n A SG-electropherogram  preparation i s obtained.  ( F i g . 9) of r e p r e s e n t a t i v e f r a c t i o n s o f  the Sephadex G-20 0 column shows t h a t the main peak i s very r i c h i n h a p t o g l o b i n w h i l e the s m a l l peak ahead o f i t i s a high molecular  weight impurity and the slow e l u t i n g p r o t e i n s  are albumin and post-albumins. f i n a l Hp 1-1 product  A SG-electropherogram of the  a f t e r l y o p h i l i z a t i o n of combined  t i o n s o f the main peak shows t h a t i t combines with  frac-  hemoglobin  to g i v e a sharp and i n t e n s e Hb-Hp band w i t h only a minute amount of a slow-migrating dase and Sephadex assays  i m p u r i t y being p r e s e n t .  Peroxi-  (to be d e s c r i b e d i n a subsequent  s e c t i o n ) i n d i c a t e t h a t the h a p t o g l o b i n  combines i n an a p p r o x i -  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 a n a l y s i s o f another Hp 1-1 prepared  by the same method con-  f i r m s the e s s e n t i a l homogeneity o f the product, only a t r a c e of i m p u r i t y .  Thus the h a p t o g l o b i n  high degree of p u r i t y and i s f u l l y The  there  being  i s o f a very  native.  Sephadex G-200 e l u t i o n diagram of the p r o t e i n s o f  both peaks A and B f o l l o w i n g D E A E - c e l l u l o s e  chromatography  of the Hp 2-1 p r e p a r a t i o n may be seen i n F i g u r e 10.  The  i n i t i a l peaks i n both cases  A starch  gel  c o n s i s t of h a p t o g l o b i n .  (Figure 11) of c o n s e c u t i v e  f r a c t i o n s throughout the hapto-  - 60 -  +  1 1 F i g u r e 9.  2  3  4  5  6  7  8  SG-electropherogram of f r a c t i o n s f o l l o w i n g  dex G-200 chromatography t i o n s a t the f o l l o w i n g ml,  (3) 450 ml,  ml,  (8) hemoglobin  ( F i g . 8) of Hp 1-1.  e l u t i o n volumes: il)  (4) 490 ml, control.  (5) 530 ml,  Sepha-  Protein  frac-  280 ml,  (2) 420  (6) 560 ml,  (7) 590  - 60 -  +  0  I 1 F i g u r e 9.  2  3  4  5 6  7  8  SG-electropherogram of f r a c t i o n s f o l l o w i n g  dex G-200 chromatography t i o n s a t the f o l l o w i n g ml,  (3) 450 ml,  ml,  (8) hemoglobin  ( F i g . 8) of Hp 1-1.  Protein  e l u t i o n volumes: $1) 280 ml,  (4) 490 ml, control.  (5) 530 ml,  Sepha-  (6) 560 ml,  frac-  (2) 420 (7) 590  - 61 -  T  1  —r  1  1  1  1  r-\  Fraction Number  Figure 10.  P u r i f i c a t i o n 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) i n 0.05 M ammonium acetate, pH 8.5.  F i g u r e 11.  SG-electropherogram of Hp 2-1 polymer  from Sephadex G-200 chromatography  fractions  commencing a t f r a c t i o n  number 28 c o n s e c u t i v e l y through t o f r a c t i o n number 46. ( S l o t s are numbered l e f t t o r i g h t ) .  - 63 g l o b i n peaksshows a p a r t i a l r e s o l u t i o n from predominantly  of polymers ranging  s l o w l y m i g r a t i n g ones to  r a p i d l y m i g r a t i n g ones.  predominantly  There are no d i s c e r n i b l e  i n the SG electropherogram  impurities  of the polymer f r a c t i o n s .  Sepha-  dex assays of the polymer f r a c t i o n s g i v e s an average v a l u e of 1.05  moles hemoglobin bound per 9 5,00 0 gm  This indicates 100%  that  the h a p t o g l o b i n p r e p a r a t i o n i s c l o s e  to  purity. A SG electropherogram  2-1  haptoglobin.  of the i n i t i a l  p r e p a r a t i o n i s shown i n F i g u r e 12.  stages of the  The  from the extreme l e f t c o n t a i n the f o l l o w i n g  Hp  s l o t s numbered samples to each  of which hemoglobin had been added and the r e s u l t s are as follows: (1) the u n t r e a t e d Hp haptoglobin  2-1  a s c i t e s f l u i d which shows a h i g h  level,  (2) the i n s o l u b l e  p r e c i p i t a t e formed d u r i n g d i a l y s i s of  ammonium s u l f a t e p r e c i p i t a t e  55%  i n which no h a p t o g l o b i n i s  discernible, (3) the 5,5% ammonium s u l f a t e p r e c i p i t a t e , r i c h i n h a p t o g l o b i n , (4) the s o l u t i o n a f t e r D E A E - c e l l u l o s e a d s o r p t i o n , d e p l e t e d of haptoglobin, (5) the D E A E - c e l l u l o s e a t the h e i g h t of peak A  ( F i g . 6), the  f r a c t i o n r i c h e s t i n h a p t o g l o b i n which has bound a l l the added hemoglobin and  i s contaminated  with some  impurities,  (6) the D E A E - c e l l u l o s e f r a c t i o n midway on the d e c l i n i n g of peak A  slope  ( F i g . 6), the h a p t o g l o b i n l e v e l i s d e c r e a s i n g  -  64  -  WW  1 2  Figure 1 2 .  3  6  SG-electropherogram of Hp 2 - 1  stages of i t s p r e p a r a t i o n to t e x t ,  4 5  p. 57).  7 8  d u r i n g the  initial  ( f o r i d e n t i f i c a t i o n of s l o t s  refer  Figure 12.  SG-electropherogram of Hp 2-1 d u r i n g the  stages of i t s p r e p a r a t i o n to t e x t ,  p. 5 7 ) .  initial  ( f o r i d e n t i f i c a t i o n of s l o t s  refer  - 65 as judged by a small amount of excess hemoglobin, (7) the foremost part of peak B, DEAE-cellulose column (Fig. 6), haptoglobin l e v e l i s decreasing and amount of imp u r i t i e s increasing, (8) the l a t t e r part of peak B, DEAE-cellulose column (Fig. 6),  a large excess of hemoglobin and a lower l e v e l of  haptoglobin present. A p u r i f i c a t i o n 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 i s shown i n 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 l a t e r studies suggested a molecular weight of 100,000 (149).  The most recent value reported by this group  (150) i s 98,770 ± 2270.  Since both the molecular weight  determination by Mr. Choy Hew  and the value obtained with  succinylated Hp 1-1 i n these studies (see p. 151)  suggest a  molecular weight of approximately 95,000, the calculations here are based on this value. i s used for hemoglobin.  A molecular weight of 66,800  I t can be seen that ammonium s u l -  fate f r a c t i o n a t i o n 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 f r a c t i o n C representing the t a i l end of the peak, so that fractions A and B containing the bulk of the haptoglobin, showed approximately 5-fold enrichment.  After Sephadex p u r i f i c a t i o n , the  main haptoglobin f r a c t i o n B more than doubled i n s p e c i f i c  TABLE I P u r i f i c a t i o n o f H a p t o g l o b i n 1-1 TOTAL TOTAL PROTEIN TOTAL PROTEIN TOTAL 4 07 UNITS TOTAL VOLUME OD VOL. x OD OD 40 7 x VOL. Hb BOUND Hp (MG) TOTAL 40 7 (ML) 280 my 280 my x DIL'N TOTAL 28 0 PURIF. YIELD (MG)  STEP 1.  Crude ascites fluid  2.  55% ammonium s u l 242. 5 fate precipitate  3.  DEAE-cellulose 185 Peak: A  1.73  320 . 1  518.6  B  170  3.94  669.8  1013.0  C  201. 5  0.52  104.8  63. 25  A  22. 7  1. 32  29.96  9.43  B  89.7  4.44  398. 3  C  92.5  2. 69  248.8  21.4  1.94  4.  15.8  9480  3039  445  586.6  0.320  1  12. 2  2959  2683  392.8  517.6  0.907  2.83  1.62  5.06  1307.8  1.51  4.73  }  0. 60  1 . 89  5  0.315  0.98  }  233.6  100  88.3  } 52. 5  Sephadex G-200  5. ' A & C re-run 6.  600  B re-run  41. 52  1392  258.1  361. 7 154.7 204.4  116.5  2.68  312.2  1241  •340.1  1  ( •269.3  3.49 1.45 3.73  10.92  158.0  4. 53 I 11.66  1  145.9 3.98  12.42  - 67 activity.  F r a c t i o n s A and C a r e the f r o n t and back o v e r l a p  s e c t i o n s c o n t a i n i n g c o n s i d e r a b l e i m p u r i t i e s and t h e r e f o r e these f r a c t i o n s were r e p u f i f i e d .  F r a c t i o n B was a l s o r e -  p u r i f i e d t o remove a small amount of a high molecular i m p u r i t y d e t e c t e d by the Sephadex G-200 assay.  weight  The f i n a l  h a p t o g l o b i n p r e p a r a t i o n from r e p u r i f i e d f r a c t i o n s A, B and C a r e o f a high degree of p u r i t y s i n c e the Sephadex assay i n d i c a t e d t h a t 1 mole of h a p t o g l o b i n has bound w i t h almost 1 mole of hemoglobin.  A final purificatfon  of about 1 2 - f o l d  was achieved and a y i e l d of 45.9% of pure h a p t o g l o b i n was obtained.  The d i f f e r e n c e i n the p u r i f i c a t i o n r a t i o  obtained  i n this method and the 2 8 - f o l d p u r i f i c a t i o n i n the C o n n e l l and Shaw method  (148) i s due t o a s c i t e s f l u i d having a lower  t o t a l p r o t e i n c o n c e n t r a t i o n than plasma and to h a p t o g l o b i n forming a f a r g r e a t e r p r o p o r t i o n , of the a s c i t e s p r o t e i n than the plasma p r o t e i n s .  fluid  The lower amount, 269.3  mg from 600 ml o f t h i s a s c i t e s f l u i d compared t o 988.9 mg from 4 70 ml i n the previous p r e p a r a t i o n o f h a p t o g l o b i n from a d i f f e r e n t sample of a s c i t e s f l u i d ,  i s due t o the c o n s i d e r -  able v a r i a t i o n i n h a p t o g l o b i n l e v e l s i n d i f f e r e n t p a t i e n t s and a t d i f f e r e n t stages o f the d i s e a s e i n a s i n g l e p a t i e n t . I t i s e v i d e n t t h a t t h i s simple method of p r e p a r a t i o n of h a p t o g l o b i n from a s c i t e s f l u i d those p r e v i o u s l y used. and  the product  i s a f a r s u p e r i o r method t o  The technique  i s rapidly  i s pure and not denatured.  advantage t o t h i s procedure  executed,  A significant  i s that large q u a n t i t i e s of  -  68  -  haptoglobin can be readily obtained from a single patient and thus i n a genetically homogeneous state.  Haptoglobin of  a single phenotype proved invaluable i n amino acid sequence studies of the a , a 1  2  and 3 chains, which are  uncomplicated  by heterogeneity due to the presence of other possible haptoglobins which might vary at unrecognized  loci.  As might be expected, i f Hp 2-2 consists of a heterogeneous populations of polymeric molecules as postulated by Smithies and Connell (14) and by A l l i s o n  (21), resolution of  Hp 2-2 and Hp 2-1 should be possible on Sephadex G-200. has been observed by Killander (151) and by Javid (152) confirmed here,, (for further discussion see p. 82).  This and  - 69 PART II SEPHADEX G-200 ASSAY INTRODUCTION Quantitation of the haptoglobin l e v e l of a b i o l o g i c a l f l u i d or of a p u r i f i e d haptoglobin solution i s based on the a b i 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 i n d i r e c t method i n which the peroxidase a c t i v i t y of the Hb-Hp complex i s measured by iodometry (153) or spectrophotometry  (81,154,155).  On the other hand, the  amount of hemoglobin bound has been estimated by t i t r a t i o n of a haptoglobin solution with a series of hemoglobin solutions of increasing concentration.  The resulting complexes  were then separated from excess hemoglobin by paper electrophoresis and the end point determined as the f i r s t appearance of excess hemoglobin (105,118).  A l t e r n a t i v e l y , the determin-  ation of the amount of hemoglobin present i n the complex can be achieved by densitometry turbidometry  (156), photometry (10 6) or tannin  (157), and excess hemoglobin can be separated  from the complex by means of starch electrophoresis (131), agar electrophoresis (158,159), cellulose-acetate e l e c t r o phoresis (160,161), g e l f i l t r a t i o n phoresis (164).  (162,163), Immunoelectro-  The determination of serum haptoglobin has  also been automated (165,166).  The multitude of methods avai-  lable f o r the quantitation of haptoglobin i s a r e f l e c t i o n of the c l i n i c a l significance of this serum protein, which has  - 70 n e c e s s i t a t e d the development of r a p i d and p r e c i s e means o f d e t e r m i n a t i o n of h a p t o g l o b i n content of b i o l o g i c a l 1.  fluids.  PEROXIDASE ASSAY I t had been known f o r some time t h a t blood c o u l d c a t a l y z e  o x i d a t i o n of aromatic chromogens i n the presence peroxide, Wu  of hydrogen  (167) showed t h a t t h i s a c t i v i t y i s a l l account-  ed f o r by the hemoglobin content and t h a t the peroxidase a c t i v i t y o f hemoglobin and i t s d e r i v a t i v e s i s l i n k e d t o the presence  of the heme component.  Although hemoglobin can  behave l i k e a peroxidase i n c e r t a i n systems, i t does not s a t i s f y ' a l l the c r i t e r i a r e c o g n i z e d f o r c l a s s i f i c a t i o n as a " t r u e p e r o x i d a s e " , t h e r e f o r e i t i s termed a  "pseudo-peroxi-  dase" (54). P o l o n o v s k i and J a y l e (2) f i r s t  observed  t h a t the peroxidase  a c t i v i t y o f hemoglobin i s markedly enhanced by the a d d i t i o n of serum or plasma; furthermore, the pH optimum of the r e a c t i o n was d i s p l a c e d from 5.6 t o 4.4 ( 3 ) . Subsequently,  these  i n v e s t i g a t o r s showed (153^8) t h a t i t was the s t o i c h i o m e t r i c combination  o f hemoglobin t o the h a p t o g l o b i n i n the serum or  plasma which markedly i n c r e a s e d the peroxidase a c t i v i t y of f r e e hemoglobin and transformed (168).  I t i s not understood  i t i n t o a t r u e peroxidase  how h a p t o g l o b i n when bound t o  hemoglobin i n f l u e n c e s the peroxidase a c t i v i t y of the heme groups.  Bajic  (169) has suggested  the hemoglobin molecule;  that haptoglobin s t a b i l i z e s  t h i s may be through p r o t e c t i o n of  the hemoglobin from the d e n a t u r i n g e f f e c t of a c i d  solutions,  - 71 since Connell and Smithies (81) reported that as the pH increases the peroxidase a c t i v i t y of hemoglobin i s progress i v e l y 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 r e s u l t of his i n i t i a l observation, Jayle (4) developed a method of determining the haptoglobin index i n serum by measuring the p a r t i c i p a t i o n of the Hb-Hp complex i n an oxidation reaction system i n which ethyl hydrogen peroxide was the oxidizing substrate and iodide the electron donor. The stoichiometry of this system i s as follows: C H OOH + 2I~ + 2H 2  5  +  >• C H OH + I 2  5  2  + H0 2  The extent of the reaction may be followed by t i t r a t i o n with thiosulfate of the iodine liberated i n 5 minutes at pH 4.4 and 32°.  The v e l o c i t y 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 a c t i v i t y in the system i s proportional to the amount of serum added u n t i l a saturation point i s reached.  The haptoglobin content  of the sample i s then equal to the smallest volume of serum for which maximum a c t i v i t y can be obtained; this value i s expressed as the Haptoglobin Index (H.I.), the concentration of haptoglobin i n a serum which saturates a M/20,000 hemoglobin solution. per 100 ml.  One H.I. corresponds to 105.4 mg haptoglobin  -  72  -  I t i s generally recognized values but  t h a t t h i s method g i v e s p r e c i s e  i t i n v o l v e s a t i t r a t i o n c a r r i e d out w i t h very  f u l t i m i n g and uses e t h y l hydrogen peroxide, e x p l o s i v e reagent.  care-  a potentially  For r o u t i n e c l i n i c a l work, J a y l e  (4)  has  proposed a more r a p i d method, the " a c t i v a t i o n method", i n which hemoglobin i n excess of t h a t r e q u i r e d f o r b i n d i n g haptoglobin  i s added to the serum and  to  a f t e r the excess hemo-  g l o b i n has been p a r t i a l l y i n a c t i v a t e d by the a d d i t i o n of i o d i n e , the peroxidase  a c t i v i t y of the complex i s measured by the same  r e a c t i o n system.  T h i s a c t i v a t i o n method g i v e s r e s u l t s  cient for routine analysis C o n n e l l and peroxidase  Smithies  suffi-  (168). (81) confirmed  the p r i n c i p l e of  the  method i n t h e i r thorough study of the r e a c t i o n con-  d i t i o n s , t h a t i s , the pH of the r e a c t i o n , c o n c e n t r a t i o n of hydrogen peroxide the peroxidase  and  c o n c e n t r a t i o n of g u a i a c o l , under which  a c t i v i t y of the f r e e hemoglobin i s e s s e n t i a l l y  zero w h i l e t h a t of i t s complex i s maximal. as the reducing  G u a i a c o l was  chosen  s u b s t r a t e s i n c e as the c o n c e n t r a t i o n used i n  the assay, i t proved to be a powerful  i n h i b i t o r of the p e r o x i -  dase a c t i v i t y of f r e e hemoglobin w h i l e the complexes were i n h i b i t e d to a much l e s s e r extent. more s t a b l e d u r i n g s t o r a g e ,  Methemoglobin, which i s  i s the standard.  a pH of 4.0,is c a r r i e d out i n a c u v e t t e and  The  r e a c t i o n at  the formation  t e t r a g u a i a c o l d u r i n g the r e a c t i o n i s f o l l o w e d at 470 my spectrophotometer thermostatted lag  phase of a few  at 30°.  .'.ignoring the  of  in a initial  seconds, the slope of the l i n e a r p o r t i o n  - 73 of the plot of the observed extinction against time i s used as a measure of the peroxidase a c t i v i t y .  The haptoglobin  l e v e l of the sample i s obtained from this a c t i v i t y by r e f e r ence to a c a l i b r a t i o n curve prepared with standard haptoglobin solutions of increasing d i l u t i o n .  This quantitative method  has been found to be sensitive and r e l i a b l e (168). quirement  of a spectrophotometer  c a l l y controlled chamber may  The re-  f i t t e d with a thermostati-  present an obstacle, and  skill  i s required i n this technique since i t requires the taking of several readings at short intervals unless the spectrophotometer i s f i t t e d with a recording unit. Owen and coworkers (154) proposed a s i m p l i f i e d 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 o p t i c a l density at 470 my reaches a maximum before 10 minutes after which the color fades.  Subtraction of a serum blank corrects for any  peroxidase a c t i v i t y of the serum i t s e l f , which i s 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 d i f f e r e n t p r i n c i p l e underlies the electrophoretic  methods of quantitation of haptoglobin which u t i l i z e s the difference i n migration between the Hb-Hp complex and free hemoglobin.  L a u r e l l and Nyman (105) developed a technique  which measures the maximum amount of hemoglobin that can be bound by the haptoglobin i n the b i o l o g i c a l f l u i d before any free hemoglobin i s demonstrable by paper electrophoresis at pH 7.0.  Near the i s o e l e c t r i c point of hemoglobin, a good  separation between the complex and free hemoglobin i s achieved, the complex migrates to the anode and the hemoglobin which is close to i t s i s o e l e c t r i c point i s carried towards the cathode due to endosmotic flow.  Increasing amounts of hemo-  globin are added to a standard amount of serum with the d i 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 i s 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 a 2 - g l o b u l i n s , as the complex does, i s present i n sera, i t s a f f i n i t y for hemoglobin i s much lower than that of haptoglobin, even when haptoglobin i s f u l l y saturated with hemoglobin, and therefore, this component does not s i g n i f i c a n t l y a f f e c t the r e s u l t s .  Although an  error of ± 10 mg/100. ml i s 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 r e s u l t s , i s time-consuming, necessitating several electrophoreses for a single haptoglobin determination. The three p r i n c i p l e methods of haptoglobin quantitation, Jayle's saturation method, Connell and Smithies' spectrophotometric assay and L a u r e l l 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 i n perfect agreement (168)..  For c l i n i c a l  analysis, Jayle's a c t i v a t i o n method and Owen's modification of Connell and Smithies' technique are less laborious and exacting.  As for the many other s i m p l i f i e d electrophoretic  methods, their precision i s believed to be less s a t i s f a c t o r y (168). 3.  SEPHADEX ASSAY It i s apparent that the above procedures depend either  d i r e c t l y , as i n Jayle's or Connell and Smithies' peroxidase assay, or i n d i r e c t l y , as i n the electrophoretic method, on the peroxidase a c t i v i t y of the heme-containing proteins. I t i s known that many substances  i n serum influence: the oxida-  tion of benzidine and similar substrates.  Nyman (54) reported  that the peroxidase a c t i v i t y appears to depend not only on the haptoglobin concentration i n the sample, but also on the composition of the serum, since she found evidence for the presence of serum components other than haptoglobin, capable  - 76 of activating the peroxidase a c t i v i t y of hemoglobin.  Also,  there were indications of factors i n h i b i t i n g the same reaction. Thus errors may  arise i n assessing the l e v e l by the peroxidase  a c t i v i t y of the serum.  Although, a catalase i s present i n  variable quantitites i n serum which i s sensitive to hydrogen peroxide but not to ethyl hydroperoxide and serum can exhibit a peroxidase due to the presence of leucocytes, these are presumably taken care of by a serum blank.  Heparin i s reported  not only to decrease the a f f i n i t y of haptoglobin for hemoglobin but also to i n h i b i t the peroxidase a c t i v i t y of the complex, although the amount of heparin used as an anticoagulent has no influence (54).  Evidently, there i s need for a d i r e c t  assay of the haptoglobin content which would preclude the many influences on the peroxidase reaction.  Another factor leading  to the development of the d i r e c t Sephadex assay was  the re-  peated observation that the peroxidase assay on haptoglobin polymer solutions which had been frozen and thawed several times gave r i s e to e r r a t i c results and a steady i n a c t i v a t i o n of the hemoglobin binding, indicating that haptoglobin i s rather e a s i l y denatured by this procedure, contrary to the observation reported by Nyman (54). SEPHADEX ASSAY The assay method for the binding of hemoglobin to haptoglobin used i n this study takes advantage of the difference i n molecular weight between free hemoglobin, molecular weight 66,800, and the Hb-Hp complex, which i s at least molecular  - 77 weight 161,800 f o r the Hp 1-1 complex and higher f o r the polymeric species i n Hp 2-1 and 2-2.  An amount of hemoglobin  in excess of that required to f u l l y saturate the binding s i t e s on haptoglobin, i s added to a sample and the mixture subjected to g e l f i l t r a t i o n on Sephadex G-200.  Since the rate of elu-  tion of proteins on Sephadex i s dependent on molecular size (172), the Hb-Hp complex i s separated from the uncomplexed free hemoglobin.  The stoichiometry of binding i s obtained  by measuring the r a t i o 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 i s used for the assay, excepting i n a few preliminary runs and i n those cases where a d i f f e r e n t buffer i s indicated, f o r example, i n the study of the  e f f e c t of pH upon the binding.  In the control Hp 1-1  assay shown i n Figure 13, 0.5 ml of 1.2 mg hemoglobin solution i n 0.02 M Tris-HCl buffer, pH 7.42 was added to 1 mg Hp 1-1 i n 0.5 ml of the same buffer. and  The solutions are mixed  then chromatographed on the Sephadex G-200 column.  Eluates of approximately 1 ml volume are collected and the absorption of each f r a c t i o n 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 r a t i o of the absorbance at 40 7 my due to the heme to  -  Figure  13.  (frame A) in  0.1  A s s a y o f Hp and Hp  2-1  M phosphate  1-1  78  -  i n 0.02  polymersoh  buffer,  pH  7.0  M Tris-HCl  Sephadex G-200 ( f r a m e s B,CC  buffer,  pH  7.42  (1 cm  x 50  cm)  and  D).  - 79 the absorbance at 2 80 my due to the protein portions i s approxr imately 3.9 i n free hemoglobin, while i n the complex with pure Hp 1-1 the r a t i o f a l l s to 2.0.  This l a t t e r value corresponds  c l o s e l y to that calculated for a 1:1 complex.  Based on the  extinction c o e f f i c i e n t s 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 Hp 1-1 respectively,  and  the t h e o r e t i c a l r a t i o of the absorban-  cies at 407 my to 280 my for stoichiometric binding i s 1.99. This method of assay of haptoglobin thus gives a d i r e c t measure of the amount of hemoglobin bound by the haptoglobin molecule and i s therefore not subject to the influence of known and unknown factors on the peroxidase reaction.  In  addition, the elution p r o f i l e r e s u l t i n g from this assay gives an immediate  i n d i c a t i o n of any change i n the conformation of  the protein molecule since besides molecular size, the shape of the molecule also a f f e c t s 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 e l u t i o n volume for the native protein. This was observed by Habeeb (173) who u t i l i z e d the s e n s i t i v i t y of the elution volume of a protein molecule on a column of Sephadex G-200 to i t s Stokes radius to evaluate the conformat i o n a l changes associated with chemical modification of bovine serum albumin.  Also, the presence of any impurities which  - 80 -  d i f f e r i n molecular weight from the complex or the free hemoglobin i s immediately detected.  This assay method besides  being applicable to pure haptoglobin solutions i s also s u i t able for assay of serum, ascites or other b i o l o g i c a l f l u i d s , although additional calculations are required for these assays, since haptoglobins are the only class of serum proteins shown conclusively to combine s i g n i f i c a n t l y with hemoglobin.  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 i s f u l l y saturated and there i s an excess of free hemoglobin, thus they do not influence the haptoglobin assay  (174).  In addition, hemoly-  s i s i n the serum samples does not v i t i a t e the analysis. many as four assays have been performed the technique i s simple.and quick.  As  simultaneously and  I f , on the other hand,  only one assay i s required to be carried out, this i s e a s i l y done without necessitating fresh reagents to be made up and t i t r a t e d each time an assay i s performed,  as would be the  case ihi the-peroxidase assays. It was subsequently noted that a somewhat similar g e l f i l t r a t i o n method had been performed by R a t c l i f f e and Hardwicke (162) and by L i o n e t t i , V a l e r i , Bond and F o r t i e r  (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).  R a t c l i f f e and Hardwicke computed the hemo-  globin binding capacity of the sample by pooling the component fractions of the f i r s t peak, the Hb-Hp peak, and by measuring the 415 my absorption.  The hemoglobin concentration  in t h i s peak i s read o f f a c a l i b r a t i o n chart and extrapolated to the amount of hemoglobin present i n 100 ml of the serum. L i o n e t t i and coworkers (163) i n 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 r e l a t i v e to the t o t a l 418 my absorption of the effluent.  Since these methods rely  on the quantitative recovery of the f r a c t i o n s , the Sephadex assay proposed i n this study i s more accurate.  R a t c l i f f e and  Hardwicke preferred to use G-100, since G-200 p a r t i a l l y resolved the haptoglobin complexes and produced a broader band of elution of bound hemoglobin.  P a r t i a l resolution of hapto-  globin polymer complexes has also been observed i n the assays reported here, however, since as w i l l be shown l a t e r , the polymers bind the same amount of hemoglobin regardless of the size of the polymers, t h i s p a r t i a l resolution of the complex does not invalidate the assay with Sephadex G-200.  Also,  there was only a s l i g h t broadening of the peaks observed with the complexes with haptoglobin polymers, much less than that observed by R a t c l i f f e and Hardwicke, and the peak was well separated from the free uncomplexed hemoglobin peak.  These  authors report reproducible results which correlated well  - 82 -  with Rowe s agar g e l method 1  (162) and with the peroxidase and  e l e c t r o p h o r e t i c methods (163).  Immediately a f t e r the Sephadex  G-200 assay method had been developed, Gordon and B e a m (175) presented  the r e s u l t s on the b i n d i n g o f the component a and  3 chains o f h a p t o g l o b i n w i t h hemoglobin i n which these  experi-  ments were conducted on Sephadex G-100 and the amount of b i n ding determined by measurement of the absorbancies and  a t 280 my o f each f r a c t i o n .  a t 418 my  These measurements were used  q u a l i t a t i v e l y t o determine the extent of b i n d i n g and the absorbancies  were not used q u a n t i t a t i v e l y as has been done  i n the Sephadex G-200 assay here. Cloarec and M o r e t t i  A l s o a t the same time,  (176) r e p o r t e d on the p r e p a r a t i o n of a  Hb-Hp 2-1 complex and subsequent f r a c t i o n a t i o n of t h i s comp l e x on a Sephadex G-200 column.  They a l s o found t h a t the  r a t i o of the 406 my t o the 278 my absorbancies e f f l u e n t f r a c t i o n i s a constant  o f each  f i g u r e , t h a t i s , i n the  v i c i n i t y o f 2.0. ASSAY OF Hp 2-1 POLYMERS Recently,  Javid  (152,177) concluded  t h a t the hemoglobin  b i n d i n g of p a r t i a l l y r e s o l v e d polymers o f Hp 2-1 and 2-2 v a r i e s i n v e r s e l y as the s i z e of the polymer.  After g e l  f i l t r a t i o n on Sephadex G-200 of Hp 2-1 and 2-2, J a v i d obt a i n e d a s e r i e s of f r a c t i o n s r e p r e s e n t i n g d e c r e a s i n g polymer s i z e .  average  The p r o t e i n c o n c e n t r a t i o n was determined on  e q u a l l y spaced f r a c t i o n s throughout the h a p t o g l o b i n  peak by  the b i u r e t v a l u e and--the hemoglobin b i n d i n g c a p a c i t y was  - 83 -  measured by Smith and Owen's peroxidase assay  (178).  From  these v a l u e s the amount of hemoglobin bound t o each m i l l i g r a m of h a p t o g l o b i n was c a l c u l a t e d .  The r e s u l t s showed t h a t the  l a r g e r the polymer, the s m a l l e r the amount of hemoglobin bound. The c o n c l u s i o n reached was t h a t the s i t e through which p o l y m e r i z a t i o n occurs must a l s o be i n v o l v e d i n the hemoglobin binding.  However, J a y l e (4 6) had r e p o r t e d t h a t 1 mg o f hemo-  g l o b i n combines with 1.3 mg of h a p t o g l o b i n of a l l g e n e t i c types.  I t i s thus necessary t o c l a r i f y the b i n d i n g between  hemoglobin and the v a r i o u s h a p t o g l o b i n polymers.  For t h i s  reason, the p a r t i a l r e s o l u t i o n of Hp 2-1 polymers o b t a i n e d d u r i n g i t s p u r i f i c a t i o n on Sephadex G-200 was p r e s e r v e d and polymeric s p e c i e s r e p r e s e n t i n g the l a r g e polymers,  the medium  polymers and the s m a l l polymers were t e s t e d f o r t h e i r b i n d i n g c a p a c i t y by the Sephadex assay. d e f i n i t e disagreement  The r e s u l t s obtained were i n  with those of J a v i d  (177); however,  s i n c e J a v i d used the peroxidase assay f o r hemoglobin b i n d i n g to h a p t o g l o b i n , i t was p o s s i b l e t h a t t h i s method c o u l d be measuring a d i f f e r e n t a s p e c t of the b i n d i n g from the d i r e c t o b s e r v a t i o n of. b i n d i n g w i t h the Sephadex assay.  Therefore,  peroxidase assays u s i n g the method of C o n n e l l and Smithies (81) were a l s o c a r r i e d out on r e p r e s e n t a t i v e polymeric  frac-  tions. For the assay, an e a r l y f r a c t i o n from the Sephadex G-200 column o f Hp 2-1, f r a c t i o n A, ( F i g . 10), tube 29, was taken to r e p r e s e n t the l a r g e polymers,  tube 37, a t the h e i g h t of  - 84 the h a p t o g l o b i n polymers, and to r e p r e s e n t these  peak was  considered  tube 4 3, a slower e l u t i n g the s m a l l polymers.  f r a c t i o n s ( F i g . 11)  The  Tube 37 has  f r a c t i o n , was  taken  SG electropherogram  of  shows t h a t tube 29 c o n s i s t s o f  s e v e r a l s p e c i e s o f high molecular polymers.  r e p r e s e n t a t i v e o f medium  weight s l o w l y ^ n i g r a t i n g  3 p r i n c i p l e components along with a  minor component^all m i g r a t i n g w i t h 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 s i z e s i m i l a r to Hp  1-1.  In each case,  M  the Sephadex assay was  phosphate b u f f e r , pH was mg  added to 1 ml  7.0.  c a r r i e d out i n 0.1  An excess of hemoglobin, 1.2  mg,  s o l u t i o n of phosphate b u f f e r c o n t a i n i n g 1  of the polymer and  the mixture s u b j e c t e d to the Sephadex  G-200 assay. The mers and  e l u t i o n diagrams of the l a r g e polymers, medium p o l y s m a l l polymers are shown i n frames B, C and  F i g u r e 13 r e s p e c t i v e l y . 407 my  to 2 80 my  The  are 1.81  medium polymers and  2.10  r a t i o s of the absorbancies  f o r the l a r g e polymers, 2.0 f o r s m a l l polymers  These values are e q u i v a l e n t to 0.85,  1.04  hemoglobin bound per mole of h a p t o g l o b i n  s p e c i e s of the same h a p t o g l o b i n d i c t s the f i n d i n g s of J a v i d , who 2-1  type.  at for  (Table I I ) .  and  1.26  moles of  (Table I I ) .  there i s l i t t l e d i f f e r e n c e i n b i n d i n g between the  Hp  D of  Thus,  polymeric  This d i r e c t l y  contra-  r e p o r t e d t h a t the l a r g e s t  polymers, t h a t i s , the l e a s t r e t a r d e d on Sephadex  G-200, possessed  only 8.7%  c i t y of the non-retarded Peroxidase  of the hemoglobin b i n d i n g capa-  or s m a l l e s t polymers.  assays were performed on the same  f r a c t i o n s a c c o r d i n g to the method of C o n n e l l and  polymeric Smithies  - 85 TABLE II Hemoglobin Binding By Haptoglobin Polymers  Preparation  Fraction  Hp 1-1  whole  Hp 2-1  whole large, f r a c t i o n 29  OP 407 my OP 2 80 my  Mole Hb 95,000 g Hp G-200 assay  1.92  0.99  small,  1.81  0.85: 0.67  2.00  1.04  0.98  2.10  1.26  1.00  fraction  43 Hp 2-2  0.94 0.94  large, f r a c t i o n 30 medium, f r a c t i o n 37  Mole Hb 95,000 g Hp Peroxidase assay  whole large, f r a c t i o n 41+42+43 large, f r a c t i o n 42+43  0.54 1.21  0.44 .  0.46  large, f r a c t i o n 44 medium, f r a c t i o n 49  0.53 1.47  0.60  medium, f r a c t i o n 50  0.61  medium, f r a c t i o n 51  0.68  small, f r a c t i o n 65+66 small, f r a c t i o n 67  1.50  0.63  0.53 0.46  - 86 (81).  The reagents were prepared as f o l l o w s :  0.600 M H 0 2  2  s o l u t i o n i s prepared immediately b e f o r e use b y d i l u t i o n w i t h c o l d d i s t i l l e d water of an approximately 30% w/v s o l u t i o n , which i s s t a n d a r d i z e d (179) about once a week by t i t r a t i o n with 0.100 N KMn0 . 4  The 0.600 M H 0 2  reagent i s kept i n the  2  r e f r i g e r a t o r i n a t i g h t l y capped b o t t l e d u r i n g the assay and a f r e s h a l i q u o t used f o r the assay approximately every 15-20 minutes.  A 0.03 M b u f f e r e d s o l u t i o n of g u a i a c o l i s made up  by d i s s o l v i n g 1.86 g;a o f g u a i a c o l i n 50 ml o f 1.0 M a c e t i c a c i d , a d j u s t i n g the volume t o 400 ml w i t h water,  then  titra-  t i n g t o pH 4.0 w i t h aqueous NaOH and a f i n a l a d j u s t i n g o f the volume t o 50 0 ml w i t h water.  The methemoglobin reagent  i s prepared a c c o r d i n g t o the procedure of Smith and Owen (178) i n which 10 ml o f potassium f e r r i c y a n i d e i s added t o a s o l u t i o n of 250 mg of hemoglobin oxidize  the hemoglobin  i n 25 ml of d i s t i l l e d water to  t o methemoglobin.  After  the volume was made up t o 450 ml w i t h water.  10 minutes,  P r i o r to the  assay the methemoglobin s o l u t i o n was d i l u t e d t o a c o n c e n t r a t i o n of 18.6 mg Hb/100 ml, a c c o r d i n g t o a m i l l i m o l a r t i o n of 38.2 a t 500 my  (58).  extinc-  C a l i b r a t i o n curves were con-  s t r u c t e d w i t h pure Hp 1-1, 2-1 and 2-2.  A s o l u t i o n of 3 mg  of h a p t o g l o b i n i n 3 ml of 0.15 M sodium c h l o r i d e was made, from which 22 t o 25 s o l u t i o n s The H 0 2  2  of i n c r e a s i n g  d i l u t i o n s were made.  reagent, g u a i a c o l reagent and methemoglobin  solutions  were each p l a c e d i n a water bath thermostatted a t 30°. of the d i l u t e d h a p t o g l o b i n s o l u t i o n s  Each  and the stock h a p t o g l o b i n  solution was mixed with an equal volume of methemoglobin reagent and placed i n the water bath.  In a cuvette, 0.20 ml  of the mixture i s placed and 2.75 ml of the guaiacol reagent i s added.  Immediately after the addition of 0.05 ml of H 0 2  2  reagent to the mixture, the cuvette i s capped with a groundglass stopper, quickly inverted 2 to 3 times, then inserted along with a blank i n a recording Beckman DB-spectrophotometer which i s thermostatted at 30° and zeroed at 470 my with blanks Simultaneously, the automatic recorder i s started.  On com-  p l e t i o n of the assay f o r the series of haptoglobin solutions, the slope i n extinction units x 1 0 / s e c , 3  that i s , the peroxi-  dase a c t i v i t y f o r each solution, i s calculated from the progress curve of the reaction ignoring the i n i t i a l time lag of a few seconds.  A plot of the peroxidase a c t i v i t y of each  solution versus the haptoglobin concentration yields the c a l i b r a t i o n 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 i n the above manner.  Due., to i n s u f f i c i e n t material, f r a c t i o n 30 was  used i n place of f r a c t i o n 29 to represent the large polymers. A concentration range of 0.168 mg/ml to 0.331 mg/ml haptoglobin polymers was used i n the peroxidase assay and the peroxidase a c t i v i t y was obtained by reference to the c a l i b r a tion curve of unfractionated Hp 2-1. The results i n Table II are expressed as the f r a c t i o n of a mole of hemoglobin (molecular weight 66,800) bound to 95,000  -  g haptoglobin  88  -  and are averages of f o u r d e t e r m i n a t i o n s .  The  b i n d i n g data by the two methods show a f a i r l y good agreement and  the peroxidase  assay confirms  the f a c t t h a t there i s no  e s s e n t i a l d i f f e r e n c e i n b i n d i n g of hemoglobin by the l a r g e s m a l l polymeric  species.  Although the l a r g e polymers appear  to show s l i g h t l y lower b i n d i n g than the medium and c u l a r weight polymers, i t i s i n no way r e p o r t e d by J a v i d .  and  the l a r g e decrease  However, i n t h i s case, r e s o l u t i o n was evidence  2-2,  Dixon, f u r t h e r s u b s t a n t i a t e s  The hemoglobin b i n d i n g of Hp  there was  peroxidase  s m a l l polymer f r a c t i o n s of Hp  which were i s o l a t e d by Dr. G.H.  low.  s m a l l mole-  A s i m i l a r s e r i e s of Sephadex and  assays on heavy, medium and  this finding.  and  of a h i g h molecular  2-2  is slightly  not as  weight non-hapto-  g l o b i n contaminant i n the l a r g e polymer f r a c t i o n . f o r t h i s m a t e r i a l , i t i s c l e a r t h a t the l a r g e Hp mers a l s o show strong hemoglobin b i n d i n g .  effective  Correcting 2-2  poly-  I t has been r e -  p e a t e d l y observed d u r i n g a very e x t e n s i v e s e r i e s of assays by the peroxidase of h a p t o g l o b i n and a steady  method t h a t repeated  f r e e z i n g and  thawing  s o l u t i o n s g i v e r i s e to e r r a t i c assay r e s u l t s  i n a c t i v a t i o n of hemoglobin b i n d i n g ,  that haptoglobin  indicating  i s r a t h e r e a s i l y denatured by t h i s procedure.  I t i s p o s s i b l e t h a t the l a r g e r polymers are more s u s c e p t i b l e to t h i s treatment and would t h e r e f o r e become p r e f e r e n t i a l l y i n a c t i v a t e d , thus accounting i n J a v i d ' s work.  f o r the l a c k of b i n d i n g observed  Moreover, C l o a r e c and M o r e t t i  (176), i n  agreement with our r e s u l t s , were a l s o unable to c o n f i r m  - 89 Javid's findings.  These i n v e s t i g a t o r s r e p o r t e d t h a t t h e i r  Hb-Hp 2-1 f r a c t i o n s from a G-200 column gave r a t i o s of the absorbancies  of 407 my t o 280 my of about 2.0 throughout the  peak and thus the same amount of hemoglobin i s bound t o h a p t o g l o b i n r e g a r d l e s s of the s i z e of the polymers. ASSAY GF a , a 1  The  2  AND 8 HAPTOGLOBIN CHAINS  i n t e r a c t i o n between hemoglobin and h a p t o g l o b i n to form  the Hb-Hp complex i s very r a p i d as i n d i c a t e d by the k i n e t i c measurements of Nagel and Gibson by the stopped (59).  flow  technique  A l s o the r e a c t i o n between the two p r o t e i n s i s an ex-  tremely s p e c i f i c one which can be l i k e n e d t o the s p e c i f i c i t y of  an a n t i g e n f o r i t s s p e c i f i c antibody.  In order t o g a i n  more i n f o r m a t i o n on the molecular a r c h i t e c t u r e of the b i n d i n g s i t e so as t o c o r r e l a t e i t s s t r u c t u r e w i t h i t s f u n c t i o n , i t was of i n t e r e s t to i n v e s t i g a t e the b i n d i n g c a p a c i t i e s o f the component chains of the h a p t o g l o b i n molecule. s i t e on the h a p t o g l o b i n molecule  The b i n d i n g  may comprise amino a c i d  r e s i d u e s from both the a and the 8 chains of the h a p t o g l o b i n molecule  or s o l e l y from the a or the 8 c h a i n .  These amino  a c i d r e s i d u e s may be l o c a t e d on both chains or on only one type of c h a i n s ; i n any event,  they a r e probably remote from  each other when c o n s i d e r e d as a l i n e a r sequence, but a r e i n c l o s e juxt-a p o s i t i o n i n space. of  enzyme molecules  Studies on the a c t i v e  sites  have i n d i c a t e d t h a t the c a t a l y t i c ac-  t i v i t y i s a f u n c t i o n of the d e l i c a t e o r i e n t a t i o n of c r i t i c a l groups.  - 90 -  An i n h e r e n t d i f f i c u l t y  i n s t u d y i n g the hemoglobin  c a p a c i t y of the i n d i v i d u a l c h a i n s i s t h a t c o n d i t i o n s  binding  neces-  sary t o s e p a r a t e the c o n s t i t u e n t c h a i n s u s u a l l y a l s o decrease the  b i n d i n g a b i l i t y of the molecule as a whole.  T h i s pro-  blem has a l s o been encountered i n the many attempts t o determine the involvement o f i s o l a t e d heavy and l i g h t chains of  the antibody molecule i n the f o r m a t i o n of the combining  site.  Whatever technique i s used t o d i s s o c i a t e the p e p t i d e  c h a i n s , c o n s i d e r a b l e l o s s of a f f i n i t y (180).  Thus, Gordon and B e a m  f o r antigen follows  (175) found t h a t c o n d i t i o n s  s i m i l a r t o those used i n d i s s o c i a t i o n of immunoglobulins causes c o n s i d e r a b l e l o s s of b i n d i n g c a p a c i t y i n the haptog l o b i n molecule.  Hp 2-2 was reduced w i t h 0.2 M mercapto-  e t h a n o l f o r 1 hour a t room temperature, a l k y l a t e d with 0.24 M i d d o a c e t i c a c i d f o r 30 minutes a t 4° f o r 15 hours.  Gel  f i l t r a t i o n through Sephadex G-100 i n 0.01 N p r o p i o n i c  acid,  pH 3.5, y i e l d e d a f r o n t - r u n n i n g peak comprised of both 3 and a c h a i n s , a middle peak of v i r t u a l l y pure 3 chains and a 2  s m a l l s l o w - e l u t i n g peak of a  2  c h a i n s which were probably  contaminated w i t h s m a l l amounts of 3 c h a i n s . experiments w i t h Sephadex G-100 chromatography  In t h e i r  binding  and measure-  ment of the 418 my and 280 my a b s o r b a n c i e s , i t was observed t h a t the 3 c h a i n s had bound a s u b s t a n t i a l amount of hemoglobin, in. s p i t e of the harsh c o n d i t i o n s used i n d i s s o c i a t i o n of the chains.  The i s o l a t e d a c h a i n s showed a t r a c e of b i n d i n g 2  a b i l i t y which was probably due.to contamination by the s m a l l  - 91 amount of 8 c h a i n s .  The mixture of u n d i s s o c i a t e d 3 and a  2  chains i n the f r o n t - r u n n i n g peak of the column had lower b i n d i n g c a p a c i t y than pure 3 c h a i n .  These r e s u l t s  w i t h the f i n d i n g s by Shim, Lee and Kang  agree  (33) t h a t a n t i - 3 -  c h a i n antiserum r e a c t e d o n l y w i t h f r e e h a p t o g l o b i n but not w i t h the Hb-Hp complex while i n c o n t r a s t , a n t i - a - c h a i n 2  antiserum r e a c t e d w i t h both f r e e and bound h a p t o g l o b i n . appears  It  then t h a t h a p t o g l o b i n binds with hemoglobin a t the  3 c h a i n , w h i l e i n the complex the a n t i g e n i c determinants"of the 3 c h a i n a r e covered by the hemoglobin molecule t h e r e f o r e u n a v a i l a b l e t o the antibody. to  7  and a r e  I t was f e l t a d v i s a b l e  c o n f i r m the f i n d i n g of Gordon and B e a m s i n c e i n t h e i r  experiments  the chains were i n c o m p l e t e l y d i s s o c i a t e d .  Hp a , a 1  2  and 3 chains were i s o l a t e d from  purified  Hp 2-1 by Dr. J.A. Black f o r amino a c i d sequence a n a l y s i s a f t e r i t was reduced with 0.5 M mercaptoethanol  i n 8 M urea  and the f r e e t h i o l s a l k y l a t e d w i t h 1.0 M iodoacetamide (26). The p o l y p e p t i d e c h a i n s were separated by Sephadex G-7 5 chromatography . i n  1.0 M a c e t i c a c i d .  The h a p t o g l o b i n chains  i s o l a t e d under these c o n d i t i o n s were probably p a r t i a l l y denatured s i n c e the 8 and the a bility  2  chains showed l i m i t e d  i n 0.1 M phosphate b u f f e r , pH 7.0.  observed  solu-  I t had been  by Mr. Choy Hew t h a t the 3 c h a i n c o u l d be brought  i n t o s o l u t i o n by f i r s t  d i s s o l v i n g i t i n 0.2 M.NH^OH and  then lowering the pH w i t h a c e t i c a c i d .  Since under the  proper c o n d i t i o n s 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  chains  1  could be car ried'out. The Sephadex assay was carried out on 2 mg of the 8 chain which was dissolved i n 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 i n 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 i n 0.25 ml of the ammonium acetate buffer and the mixture assayed on Sephadex. The e a r l i e r elution of the 8 chain  (Figure 14, frame A),  that i s , i n the void volume instead of i n the v i c i n i t y of 20 ml, indicates possible change i n the shape of the molecule such as an unfolding phenomenon.  This i s l i k e l y since the  8 chain appeared p a r t i a l l y denatured.  Another p o s s i b i l i t y  i s that the 8 chain i s aggregated, as has been observed by Mr. C. Hew during ultracentrifuge studies of 8 chain. spite of possible changes,in conformational  In  state or aggre-  gation i n the molecule, the 8 chain binds a s i g n i f i c a n t amount of hemoglobin, the 407 my/280 my r a t i o being 0.268, approximately one-eighth of the normal binding. The isolated a  1  chain was soluble i n the 0.1 M phos-  phate, pH 7.0 buffer usually used i n the Sephadex G-200 assay.  A solution of 2 mg of the a  1  chain i n 1 ml of the  0.1 M phosphate buffer was added to 5.5 mg of hemoglobin i n 0.25  ml of the same buffer.  The mixture was then assayed  0.9  0  0.6  0.3  6  2 8 0 rr.u  O <•  05 O  CO  >-  u  < O  »DL  O  0  EFFLUENT F i g u r e 14.  Assay o f 3 , a  G-200 (1 cm x 50 cm). pH 7.0 ( a c e t i c a c i d ) , pH 7.0, (C) a  1  and  1  10  20  30  VOLUME, mi 2 a  haptoglobin  chains on Sephadex  (A) g c h a i n i n 0.2 M ammonium (B)  2 a  40  acetate,  c h a i n i n 0.1 M phosphate b u f f e r ,  c h a i n i n 0.1 M phosphate b u f f e r , pH 7.0.  - 94 on Sephadex G-200. The elution p r o f i l e (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. uncombined a  1  The  chain emerges just s l i g h t l y behind the hemo-  globin peak as i s evidenced  by a s l i g h t t a i l i n g of the hemo-  globin peak. The a  2  chain was  buffer, pH 7.0,  p a r t i a l l y soluble i n 0.10  M phosphate  therefore a 0.9 5 ml supernatant  solution i n  this buffer with an absorption at 280 my of 1.55 The a 0.25  2  chain solution was  was  used.  combined with 5.5 mg hemoglobin i n  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 i s observed i n the e l u t i o n pattern (Fig. 14, frame B) which i s eluted i n 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 a  2  i s eluted just behind the hemoglobin peak.  The  chain i s l i k e l y contaminated.with some 3 chain since i n  the i s o l a t i o n of the chains by gel f i l t r a t i o n , the a is eluted immediately after the 3 chain.  2  chain  This would also be  i n agreement with the observation that the a  1  chain shows no  hemoglobin binding capacity. In spite of the severe conditions used i n dissociation of the polypeptide chains, the 8 chain bound an appreciable amount of hemoglobin, as has been observed by Gordon and Beam.  - 95 The  f i n d i n g t h a t the a  1  c h a i n shows no a b i l i t y t o b i n d hemo-  g l o b i n i s more c o n c l u s i v e evidence Gordon and B e a m  here confirms  the a  2  2  c h a i n was contaminated by  c h a i n used here.  Thus the evidence  the f i n d i n g t h a t the b i n d i n g s i t e f o r hemoglobin  i s l o c a t e d on the h a p t o g l o b i n and B e a m  by  t h a t the a c h a i n i s not d i r e c t l y i n v o l v e d i n  the b i n d i n g s i t e , s i n c e t h e i r a 8 c h a i n , as wa<s:  than t h a t presented  8 chains as suggested by Gordon  and by Shim, Lee and Kang.  The q u e s t i o n as t o  whether the a c h a i n p l a y s any r o l e i n the b i n d i n g , such as a s e m i s p e c i f i c one i n m a i n t a i n i n g the molecule and thereby  the c o r r e c t conformation  of  enhancing the b i n d i n g c a p a c i t y of  the 3 c h a i n s , as has been suggested f o r the heavy and l i g h t c h a i n s o f the antibody molecule  (1801  remains t o be answered.  BINDING OF GLOBIN AND MYOGLOBIN TO HAPTOGLOBIN 1.  GLOBIN Turning  t o the hemoglobin moiety o f the complex, i t  appears t h a t r e a c t i o n s i n v o l v i n g the i r o n atom or n e i g h b o r i n g atoms do not i n t e r f e r e w i t h complex formation with  haptoglobin  s i n c e carbonmonoxy-, cyano-, and methemoglobin as w e l l as the H S-derivative 2  oxyhemoglobin  are bound by h a p t o g l o b i n (54,168).  t o the same extent as  I t has been reasoned t h a t s i n c e the  l i g a n d on the heme does not i n f l u e n c e complex formation the heme group probably  does not p a r t i c i p a t e i n the i n t e r a c t i o n  between hemoglobin and h a p t o g l o b i n .  That t h i s i s / l i k e l y , - e o m e s  from paper e l e c t r o p h o r e t i c experiments of Nyman (174) and of Neale, Aber and Northam (181), i n which i t was shown t h a t  h a p t o g l o b i n d i d not i n t e r a c t w i t h p y r i d i n e hemichrome or hematin.  In the e a r l y s t u d i e s on h a p t o g l o b i n , i t was r e -  p o r t e d by J a y l e (18 2) and by van Royen (141) t h a t n a t i v e g l o b i n binds h a p t o g l o b i n ; however, no p u b l i s h e d evidence was presented.  Competition  experiments by Nyman (54) with hemo-  g l o b i n and g l o b i n d i d show t h a t g l o b i n can b l o c k the hemog l o b i n b i n d i n g groups of the h a p t o g l o b i n . Soothill  (183), u s i n g antiserum  A l s o Rowe and  a g a i n s t g l o b i n and mixtures  of g l o b i n and serum as a n t i g e n , demonstrated an a r c of prec i p i t a t e of i d e n t i c a l :'m-obility t o the p r e c i p i t i n l i n e of h a p t o g l o b i n bound hemoglobin and suggested a l s o binds g l o b i n .  that haptoglobin  E l e c t r o p h o r e t i c i n v e s t i g a t i o n s (55) a l s o  showed f o r m a t i o n of a globin-Hp  complex.  Nevertheless, i t  was  c o n s i d e r e d of i n t e r e s t t o determine by the d i r e c t Sepha-  dex  assay method t h a t g l o b i n binds with G l o b i n was prepared  treatment  o f hemoglobin  haptoglobin.  i n the u s u a l manner by a c i d (18,4) .  acetone  A s o l u t i o n 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 t o 20 ml of 20% v/v cone. HC1 i n acetone, p r e v i o u s l y c h i l l e d i n a d r y i c e - a c e t o n e mixture  (-30°).  After  the f l a s k r a p i d l y , the p r e c i p i t a t e was immediately  shaking collected  by s u c t i o n f i l t r a t i o n and washed s e v e r a l times w i t h  cold  acetone  The p r e -  (-30°) and f i n a l l y washed with c o l d e t h e r .  c i p i t a t e was d r i e d under a n i t r o g e n atmosphere. pared  Globin pre-  i n t h i s manner i s e s s e n t i a l l y f r e e of heme, s i n c e the  a b s o r p t i o n a t 407 my was l e s s than 0.03% of the normal ab-  -  sorption.  The  preparation  buffer,  7.0  and  pH  However,  by  tion  of- t h e  this  case,  applying 8  titration  pH  of  is  globin  in  solution to  0.2 of  in  0.2  M  with an  Gel  acetic  any M  N H 4 O H  and  the  M  at  ammonium  filtration  of  acetate  was  globin  pH  in  0.2  If with  M  titrating to  the  in  control  soluIn  NH^OH,  before  a  instead, a  0.2  could  Thus, of  for  solution.  mixture  prepared  globin  used  9.2.  2 mg  subjected  mg  phosphate  precipitates  the  to  after  buffer  1.5  of  M  aggregated. was  i s combined  added  mixture,  a  of  globin  pH  0.1  into  precipitation. was  acid,  of  solution  the  as  brought  approximately  N H 4 O H  acetic  was  in  probably  technique  solution  sign  0.2  insoluble  acid,  haptoglobin,  1 ml  1.0  globin  NH4OH  M  was  same  after  reached,  9 without  bin  in  with  -  therefore,  the  chains,  however,  on  9  was,  97  1.5  NHi*OH  M be  mg  the  lowered  of  glo-  haptoglobin  in  to  9.0  a  pH  of  Sephadex  05  assay  the  same  manner.  was  also  carried  out. The ture  elution  (Fig.  volume  15)  diagram  shows  hemoglobin  edge  the  The  of  main  quantities  ment were excess,  of  of  chosen  since  particular point  that  corresponding  between  407  peak  main  and  absorption  the  A  would not  formation  complex,  eluted  found  very  is  haptoglobin  is  for  hump  be  a  at  small  haptoglobin  globin  the  a  and  peak  usually  indicates  Thus, of  globin  haptoglobin.  that  my  the  the  that  globin  assay.  elution  to  and  so  of  at  instead  a  complex  the  tailing  excess  of  used  in  this  just  slightly  applicable of  one of  main  two  mix-  in  globin. experiin  this  peak  large  at  peaks  the  ml  - 98 -  EFFLUENT  F i g u r e 15.  VOLUME  (ML)  B i n d i n g of g l o b i n to h a p t o g l o b i n on  Sephadex G-200 (1 cm x 50 cm) i n 0.2 M ammonium hydroxide, pH 9.0 ( a c e t i c  acid).  - 99 of uncomplexed h a p t o g l o b i n f o l l o w e d by uncombined g l o b i n , i n d i c a t e s complex f o r m a t i o n . by the g l o b i n c o n t r o l p r o f i l e .  This i s further substantiated Another l i n e o f evidence i s  t h a t a s o l u t i o n of g l o b i n i n the presence  o f h a p t o g l o b i n does  not p r e c i p i t a t e as r e a d i l y d u r i n g lowering of the pH o f the s o l u t i o n as a g l o b i n s o l u t i o n r . i n the absence of h a p t o g l o b i n . T h i s may be taken t o mean t h a t a complex has formed and t h a t h a p t o g l o b i n on combining w i t h g l o b i n has s t a b i l i z e d the g l o b i n molecule  i n some way.  confirms  the previous suggestions t h a t h a p t o g l o b i n binds t o  hemoglobin through 2.  Thus the r e s u l t s o f the g l o b i n assay  the g l o b i n moiety.  MYOGLOBIN Myoglobin has a s i m i l a r f u n c t i o n t o hemoglobin i n t h a t  it  i s the oxygen c a r r y i n g pigment found  b r a t e s and c e r t a i n i n v e r t e b r a t e s (185).  i n muscles of v e r t e While both hemo-  g l o b i n and myoglobin c o n t a i n an i d e n t i c a l p r o s t h e t i c group, the heme, myoglobin i s a r e l a t i v e l y simple s t r u c t u r e , composed of a s i n g l e p o l y p e p t i d e c h a i n c o n t a i n i n g 153 amino a c i d s and a s i n g l e heme moiety, i n c o n t r a s t t o the f o u r pept i d e chains of 574 amino a c i d s and f o u r heme molecules  i n the  hemoglobin molecule.  struc-  A comparison of the known primary  t u r e of sperm whale myoglobin and of human hemoglobin (186) shows t h a t although there a r e many d i f f e r e n c e s i n amino a c i d c o n s t i t u t i o n i n the a and 8 chains of hemoglobin and the myoglobin p o l y p e p t i d e c h a i n , many s i m i l a r i t i e s a r e a l s o apparent between the three types of p e p t i d e chains  (186,187),  -  there being  100  -  68 amino a c i d r e s i d u e s i n i d e n t i c a l p o s i t i o n s i n  the a and 3 chains  (186), and about 37 i d e n t i c a l  between myoglobin and the a and-8 chains  (188).  residues A compari-  son of a l l t h r e e chains shows t h a t they a r e c e r t a i n l y  still  homologous but there are only 21 amino a c i d s i n i d e n t i c a l positions. Perutz  Moreover, the X-ray c r y s t a l l o g r a p h i c s t u d i e s by  (188) and by Kendrew and coworkers  (189) r e v e a l e d  t h a t sperm whale myoglobin and the a and 8 hemoglobin chains were s t r i k i n g l y s i m i l a r i n t h e i r t e r t i a r y s t r u c t u r e .  Each  p o l y p e p t i d e c h a i n i s f o l d e d i n a t e t r a h e d r a l arrangement with the heme group l y i n g on the s u r f a c e of the molecule i n pockets formed by the f o l d s i n the p o l y p e p t i d e c h a i n .  This  polypep-  t i d e c h a i n - f o l d , f i r s t d i s c o v e r e d i n sperm whale myoglobin, has s i n c e been found  a l s o i n s e a l myoglobin and i t s appearance i n  horse hemoglobin suggests of  t h a t a l l hemoglobins and myoglobins  v e r t a b r a t e s f o l l o w the same p a t t e r n  C188,190,191).  E x t e n s i v e i n v e s t i g a t i o n of v e r t e b r a t e hemoglobin shows a b a s i c s i m i l a r i t y i n chemical  s t r u c t u r e , the molecule i n  each case i s composed of 4 p o l y p e p t i d e chains of approximately 17,000 molecular  weight w i t h one heme group each, except i n  the case o f the hemoglobin of lamprey, Lampetra g e n e r a l l y accepted  fluviatilis  }  as the most p r i m i t i v e l i v i n g v e r t e b r a t e ,  which i s c h a r a c t e r i z e d by a s i n g l e p o l y p e p t i d e c h a i n of molecular weight 17,500 and the presence of a s i n g l e heme group.  Hagfishl (Myxine  glutinosa)  hemoglobin may be s i m i l a r  or p o s s i b l y a dimer of 34,000 molecular  weight  (192).  - 101 Since myoglobin appears to be c l o s e l y r e l a t e d t o hemog l o b i n , i t i s o f c o n s i d e r a b l e i n t e r e s t to determine whether h a p t o g l o b i n w i l l b i n d to t h i s oxygen-carrying p r o t e i n . F i s c h e r and Spaet  Javid,  (193) r e p o r t e d no b i n d i n g of myoglobin to  h a p t o g l o b i n and i n f a c t c o u l d not demonstrate b i n d i n g of myog l o b i n by any serum p r o t e i n i n t h e i r s t a r c h g e l e l e c t r o p h o r e sis.  Myoglobin when added i n v a r y i n g c o n c e n t r a t i o n s to serum  always moved as a s i n g l e band w i t h the same m o b i l i t y of myog l o b i n alone and caused no change  in. m o b i l i t y of h a p t o g l o b i n  bands i n c o n t r a s t t o the r e s u l t s shown by the a d d i t i o n of hemoglobin to serum.  However, Lathem  (194) l a t e r p r e s e n t e d  evidence of p r o t e i n b i n d i n g of myoglobin to form.a  complex  which has e l e c t r o p h o r e t i c 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 w i t h what he terms "protein-bound hemoglobin" which appears t o be the Hb-Hp compex. B a r r e t t and Crosby  Subsequently, Wheby,  (195) used :;.ahaptoglobinemic serum  and  demonstrated t h a t 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 w i t h the non-haptoglobin protein-heme complexes serum on s t a r c h g e l e l e c t r o p h o r e s i s .  seen w i t h normal  The p r o t e i n b e l i e v e d to  be r e s p o n s i b l e f o r t h i s b i n d i n g i s what i s termed the heme b i n d i n g 8 - g l o b u l i n by Nyman (54).  S i n c e the above evidence  i s not c l e a r - c u t , i t appeared a d v i s a b l e to show more d i r e c t l y whether Two mg  or not myoglobin i s bound by h a p t o g l o b i n . of sperm whale myoglobin  t o r i e s ) was d i s s o l v e d i n 0.25  (Mann Research Labora-  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 i s eluted free, not combined with any myoglobin, since the front-running peak shows no 407 my absorption due to the heme group of the myoglobin. bin  A l l the myoglo-  i s observed as a single peak eluting late i n the g e l  f i l t r a t i o n i n accordance with i t s smaller molecular weight. This r e s u l t then confirms the observations of Wheby and associates that the myoglobin binding noted by Lathem i s not due to haptoglobin.  I t i s evident that the single poly-  peptide chain i n myoglobin does not carry a binding s i t e for haptoglobin i n contrast to the results observed with i s o lated a hemoglobin chains C59). This r e s u l t further i l l u s trates the great s p e c i f i c i t y of binding of haptoglobin with hemoglobin and may be inherent i n what i s described as the b i o l o g i c a l role of haptoglobin, that of combining s p e c i f i c a l l y with extracorpuscular (160) hemoglobin to conserve  iron.  ASSAY OF HAPTOGLOBIN WITH HEMOGLOBIN FROM OTHER SPECIES Tryptic hydrolyzates of hemoglobin of various animals give f i n g e r p r i n t patterns i n which most of the peptide fragments are very s i m i l a r .  The patterns from f i s h hemo-  globin were most d i f f e r e n t from mammals, consistent with their separation i n the phylogenetic scale (196).  The ob-  servation that hybridization can be achieved between hemoglobins from d i s t a n t l y related animals under a variety of  - 103 -  Figure  16.  Binding  of sperm whale myoglobin t o hapto-  g l o b i n on Sephadex G-200 (1 cm x 50 cm) phate b u f f e r , pH  7.0.  i n 0.1 M phos-  experimental gross  (197),  architecture of subunits  However, of  conditions  104 -  i ti s readily  f u r t h e r suggests i s similar  apparent  evidence  tween a g i v e n  that the primary  suggests  polypeptide  chain  ( e g . a o r 8) f o u n d  i s roughly  animals  as e s t a b l i s h e d by s t a n d a r d  classification. more d i s t a n t Since  which  myoglobin,  i s related  species  oxygen  t h e number o f d i f f e r e n c e s t h e  a molecule  with  similar  the capability  of binding with  t o a s c e r t a i n whether  of animals,  lar  way.  for  Jayle's peroxidase  All  haptoglobins  also bind  assay  from i t has  and a d a p t i o n  to the  to haptoglobin binds  (44,46,56).  t o be d e t e r m i n e d  i n a  horse  simi-  hemoglobin  hemoglobin  (153).  have been r e p o r t e d t o  hemoglobins,  r a t and mousey  hemoglobin appears  haptoglobin,  although  employs horse  f a r examined  numerous a n i m a l  scale,  retained the basic function of  I t i s known t h a t h a p t o g l o b i n  thus  molecule  hemoglobins  whose h e m o g l o b i n ,  has n e v e r t h e l e s s  monkey, r a b b i t ,  f u n c t i o n and  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  t r a n s p o r t , would  combine w i t h  these  methods o f p h y l o g e n e t i c  independent n a t u r a l s e l e c t i o n  environment  i n different  c o n f i g u r a t i o n t o the hemoglobin  i s of interest  undergone  another.  the relationship.  does n o t possess  other  to  proportional to the relatedness of  The g r e a t e r  three-dimensional  not  structure  t h a t t h e number o f d i f f e r e n c e s b e -  animals  it  i n a l l hemoglobins.  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  Present  and  that the  horse,  cow, d o g ,  The b i n d i n g o f  by t h e h a p t o g l o b i n  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  and  (44,56).  - 105  -  However, the peroxidase a c t i v i t y of a complex with hemoglobin i s twice as g r e a t as the corresponding  horse complex  w i t h human hemoglobin, although the b i n d i n g c a p a c i t y of human h a p t o g l o b i n i s the same f o r these two g l o b i n as determined (54).  The  by.the  paper e l e c t r o p h o r e t i c method  complex w i t h dog hemoglobin has  dase a c t i v i t y and  types of hemo-  l i t t l e peroxi-  t h a t w i t h r a t hemoglobin has none (46).  The peroxidase a c t i v i t y i s v a r i a b l e depending on the  origin  of the hemoglobin and therefore.the a b i l i t y of h a p t o g l o b i n to enhance the peroxidase, a c t i v i t y .is c l e a r l y d i s s o c i a t e d i t s a b i l i t y to combine w i t h hemoglobin.  The peroxidase  from  assay  i s i n e f f e c t measuring a d i f f e r e n t p r o p e r t y .of ..the. system and is,  t h e r e f o r e , more e q u i v o c a l as a measure of. b i n d i n g than  the d i r e c t ' Sephadex assay.  No q u a n t i t a t i v e , s t u d i e s of the  b i n d i n g of d i f f e r e n t animal hemoglobins by pure h a p t o g l o b i n have been r e p o r t e d p r e v i o u s l y . c a r r i e d out with serum or plasma qualitative;  I n v e s t i g a t i o n s have been (44,56) but these have been  Since h a p t o g l o b i n binds to the g l o b i n 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  s t r u c t u r e 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 w i t h h a p t o g l o b i n may  p r o v i d e some  i n f o r m a t i o n on the nature of the b i n d i n g between hemoglobin and h a p t o g l o b i n .  To t h i s end, a s e r i e s of Sephadex assays  were c a r r i e d out w i t h hemoglobin from a s e r i e s of v e r t e b r a t e s ranging from the c l o s e l y r e l a t e d domestic  mammals t o the  more d i s t a n t l y r e l a t e d .bird, amphibia  fishes.  and  - 106 Hemoglobin solutions were prepared from heparinized blood of the following animals:  cow, sheep, pig, dog,  rabbit, cat, r a t , mouse, chicken, frog and rainbow trout. The amount of heparin used to prevent coagulation of the blood i s an amount which i s less than that which i s said to i n h i b i t binding of haptoglobin to hemoglobin, than 40 mg heparin per 100 ml plasma (54).  that i s , less  Horse blood c o l -  lected i n 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 pH 7.0.  An amount of hemoglobin  buffer,  i n excess of that required  for equimolar haptoglobin binding was added to 1.5 mg or 2.0 mg of pure haptoglobin dissolved i n 0.5 ml of 0.1 M phosphate buffer.  The mixture was then separated by the Sephadex G-200  method.  With the hemoglobins  of r a t , rabbit, dog, trout and  frog, controls of the hemoglobin  solution alone were also  performed. The results of these assays are summarized i n Table I I I . In every case a Hb-Hp complex has formed between human haptoglobin and the vertebrate hemoglobin, tions by previous investigators.  confirming the observa-  These complexes have approxi-  mately the same molecular weights as the human Hb-Hp complex, since the point of elution on the column i s the same as that in the l a t t e r case.  The observed r a t i o of the absorbancies  at 407 my to 280 my i s , i n the horse, cow,  sheep, pig and cat  TABLE I I I Haptoglobin Binding with Animal H i g h m o l . w t . peak Assay  407 my/280 my  Hemoglobins  Hb-Hp 407 my/280 myr-obs.  peak  Free  407 my/280 my c o r r e c t e d " " 1  Hb  407 my/280 my  1.  R a t 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.  C a t 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.  Pig  -  2.00  2.05  3.71  0.81  1.84  2.00  3.33  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.  H o r s e Hb + Hp  -  2.16  2.01  4.49  Hb + Hp  8. . Mouse Hb + Hp 9.  f  Values corrected for  a l t e r e d 407/280 r a t i o s i n t h e  hemoglobin.  - 108 hemoglobins, very similar to that found with human hemoglobin. In hemoglobins of the dog, rabbit, chicken, r a t and mouse, the observed r a t i o i s s l i g h t l y lower and i s due to a lower 407/280 r a t i o i n the native hemoglobin.  Correcting for this  deviation from the observed value of 3.9 for human haptoglobin, the 407/280 r a t i o of a l l the complexes value of 2.0 except i n two cases.  i s very close to the  Fish and trout, the two  more d i s t a n t l y related species, show a marked v a r i a t i o n i n the 407/280 r a t i o , the corrected values being 0.68 and respectively.  0.76  The stoichiometry of binding appears to be one  molecule of haptoglobin to one-quarter molecule of hemoglobin. If the lower absorbancy r a t i o s found with the more d i s tantly related vertebrate hemoglobins are a r e f l e c t i o n 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 e q u i l i brium i n the complex.  To test this p o s s i b i l i t y , 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 i n experiment 1 (Table IV) were combined and re-run on the Sephadex G-200 assay column for experiment 6.  The results  summarized i n Table IV shows that the 407/280 absorbancy r a t i o remains e s s e n t i a l l y constant regardless of the quantity of hemoglobin, whether i t i s just s l i g h t l y over the s t o i c h i o metric amount required for binding as i n experiment 1 or  - 109 -  TABLE IV Haptoglobin Binding-with Varying Concentrations of Trout Hemoglobin Experiment  Trout Hb mg/ml  Human Hp mg/ml  Hb/Hp  OD 407 OD 28 0 my  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  -  no  -  whether a large excess of hemoglobin i s present as i n experiment 5.  The s l i g h t l y higher r a t i o i n experiment 1 compared  to the other values i n the remaining experiments i s due to a larger scale separation to provide s u f f i c i e n t Hb-Hp complex for a re-run i n experiment 6 and as a consequence, there i s a s l i g h t overlap of the free hemoglobin peak.  The re-run of  the trout Hb-Hp complex 2 days later i n experiment 6, shows that the complex i s not r e a d i l y reversible for no free hemoglobin i s observed, although there i s a very s l i g h t skewing of the peak at the t a i l end.  The conclusion arrived at from  these experiments i s that the binding between f i s h hemoglobin and human haptoglobin i s not r e a d i l y reversible and the lowered r a t i o observed combination  i n the binding i s not due to a loose  between the two proteins.  The lower r a t i o i n the complexes with frog and f i s h 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 p a r t i c u l a r hemoglobin i n the mixture of components.  Another more l i k e l y  p o s s i b i l i t y i s that the heme i s bound more loosely i n f i s h and frog hemoglobins, p a r t i c u l a r l y when these hemoglobins are bound by haptoglobin.  There i s evidence of an altered  conformation i n the mammalian Hb-Hp complex (201) and the heme i n 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 f i s h  - Ill hemoglobin i s added to human serum, a s t r o n g heme-albumin band appears  on a SG-electropherogram.  Since t h e r e i s very  l i t t l e heme-albumin complex formed w i t h s i m i l a r amounts of human hemoglobin, t h i s suggests t h a t f i s h hemoglobin i s i n e q u i l i b r i u m w i t h a p p r e c i a b l e c o n c e n t r a t i o n of f r e e heme and thus the complex of heme with g l o b i n i s much weaker than i n human hemoglobin.  Reaction of f i s h o r f r o g hemoglobin with  human h a p t o g l o b i n may cause.some l o s s of the heme analogous  t o the r e a c t i o n of a n t i - g l o b i n  n a t i v e myoglobin observed by S e l a (202)'.  groups,  (myoglobin) w i t h Myoglobin  w i t h the antibody d i r e c t e d a g a i n s t the g l o b i n moiety  reacts o f the  myoglobin t o produce a c o l o r l e s s p r e c i p i t a t e ; t h i s i m p l i e s t h a t a c o n f o r m a t i o n a l change i n the myoglobin molecule has r e s u l t e d i n e x p u l s i o n of the heme group. I t was a l s o observed  from the e l u t i o n p r o f i l e s i n assays  of c e r t a i n mammalian hemoglobins t h a t a heme-containing was o f t e n e l u t e d a t the v o i d volume (Table I I I ) .  peak  T h i s peak  when present i n the Hb-Hp assay was a l s o present i n c o n t r o l samples o f the hemoglobin a l o n e .  In most cases, i t was a  minor component of the s o l u t i o n ; however, when dog hemoglob i n was allowed t o stand f o r a p e r i o d o f 5 days a t 4°, the h i g h molecular weight pigment i n c r e a s e d c o n s i d e r a b l y and on f u r t h e r s t o r a g e , a p r e c i p i t a t e appeared.  I n o n l y one case  out of twelve s p e c i e s of hemoglobin s t u d i e d , namely r a t , t h i s 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 p r o t e i n s i n the b l o o d .  In mouse t h i s value  - 112 -  was 3.8%.  Hemoglobin from the p a r t i c u l a r s t r a i n of mouse  used, DBA/2J (Roscoe B. Jackson Memorial Laboratories, Bar Harbor, Maine), has been shown to possess i n addition to i t s normal tetrameric hemoglobin, a polymer with a sedimentation c o e f f i c i e n t of 6.32, formed by d i s u l f i d e interaction between chains of 2 hemoglobin molecules (203).  Polymerization of  hemoglobin molecules has also.been observed i n other strains of mice (204) termed "diffuse" (205,206), i n which the approximately 7S component increases upon storage, as contrasted to the strains of mice termed "single" i n which the hemoglobin never exhibits polymerization either fresh or i n storage (206). This phenomenon has been explained as being due to a single mutation (207).which f a c i l 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) i n r e p t i l e s and amphibian hemoglobin hemolyzates, and studied i n the frog (209) and t u r t l e (210) by other investigators.  The appearance of the  larger molecular weight component i n dog hemoglobin upon storage, which has not so f a r been reported, is similar to the phenomenon found with frog, mice and t u r t l e hemoglobin.  It  is also interesting that storage of an abnormal human hemoglobin, Hb Porte Alegre, also results i n dimerization (211). The above series of Sephadex assays on haptoglobin polymers, isolated a , a 1  2  and 8 haptoglobin chains, globin,  myoglobin and with animal hemoglobins demonstrate the usefulness of this method of quantitation.  The procedure i s  -  113 -  r a p i d and simple and s e v e r a l assays may be c a r r i e d out s i m u l taneously.  A d i r e c t measure o f the amount o f hemoglobin  bound t o the component under i n v e s t i g a t i o n i s g i v e n by the heme a b s o r p t i o n a t 407 my i n r e l a t i o n t o the a b s o r p t i o n due to p r o t e i n i n the Hb-Hp complex.  A l s o , the assay appears not  to be i n f l u e n c e d by any unknown f a c t o r s as has been observed by Nyman (54) i n the peroxidase assay and the e f f e c t of s p e c i e s v a r i a t i o n o f hemoglobin Furthermore,  on the peroxidase r e a c t i o n i s avoided.  the e l u t i o n p r o f i l e s p r o v i d e s an immediate p i c -  t u r e of the p r o c e s s , thus i n the assay of a p u r i f i e d  hapto-  g l o b i n p r e p a r a t i o n i m p u r i t i e s p o s s e s s i n g m o l e c u l a r weights d i f f e r e n t from those of the complex or f r e e hemoglobin be immediately  may  d e t e c t e d and any c o n f o r m a t i o n a l change or  p o l y m e r i z a t i o n i n e i t h e r hemoglobin  or h a p t o g l o b i n i s e v i d e n t  from a change i n the p o i n t of e l u t i o n o f the molecule.  - 114 PART I I I 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 l i m i t s without affecting the assay.  This provides consid-  erable advantage over gel electrophoresis or ion-exhange chromatography which are possible only within very narrow l i m i t s of s a l t concentration and pH.  In the peroxidase  reaction, the pH of the reaction i s a c r i t i c a l factor and cannot be varied to any great extent without  adversely  affecting the reaction (81), s i m i l a r l y , i n paper e l e c t r o phoresis the pH cannot be varied greatly without  affecting  the separation between the Hb-Hp complex and excess hemoglobin. It has been observed repeatedly  (53,54,63,153) that the  binding between hemoglobin and haptoglobin i s tight and essentially irreversible.  For instance, there i s almost no  exchange between labeled components i n the complex with nonradioactive molecules i n the system (53,131) and although haptoglobin does not bind deoxyhemoglobin, once i t i s bound to oxyhemoglobin, deoxygenation does not remove the haptoglobin (63).  The great s t a b i l i t y of binding between hemo-  - 115 globin and haptoglobin i s not due to the formation of a d i s u l f i d e bond for haptoglobin contains no free sulfhydryl groups (212,213) and treatment of hemoglobin with H g , Ag , ++  Cu , p-mercuribenzoate +  (212) or iodoacetamide,  maleimide, cystine and cystamine globin binding.  +  N-ethyl-  (68) did not i n h i b i t hapto-  Thus blocking of the reactive sulfhydryl  groups of hemoglobin has no e f f e c t 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 galacturonic acid and glucosamine i n h i b i t the reaction between hemoglobin and haptoglobin (214).  These observations led to  the suggestion that the interaction i s of an e l e c t r o s t a t i c nature with the intervention of the carbohydrate moiety of haptoglobin i n the combination with hemoglobin (214).  Sub-  sequently, however, neuraminidase action on haptoglobin and on the Hb-Hp complex (19,51) was shown to a f f e c t neither complex formation nor the peroxidase a c t i v i t y of the complex, therefore the carbohydrates be essential i n binding.  themselves appear not to  Bajic (169) claims that the s t a b i -  l i t y of the Hb-Hp complex i s ascribed to e l e c t r o s t a t i c forces and the action of van der Waal's forces between surfaces with pronounced complementarity,  since the formation of the complex  can be i n h i b i t e d equally by e l e c t r i c a l l y p o s i t i v e , negative and neutral polymeric substances  (protamine, heparin and  sodium alginate). The s t a b i l i t y of the complex i s very high and i t has  - 116 r e s i s t e d various attempts at d i s s o c i a t i o n (104).  In v i t r o ,  the complex i s 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 i r r e v e r s i b l y destroyed (54). The i n a b i l i t y of haptoglobin i n acid media to bind hemoglobin was confirmed by Pavlicek and Kalous  (215) whose t i t r a t i o n  curves, d i f f e r e n t i a l absorption spectra and o p t i c a l rotation measurements indicated that i n the region below pH 5.0 the haptoglobin molecule undergoes far-reaching s t r u c t u r a l changes. In the work described here, the e f f e c t of increasing s a l t concentrations to produce very high ionic strength and the e f f e c t of varying the pH over a wide range on the formation of the complex was studied i n order to c l a r i f y the nature of the linkage between hemoglobin and haptoglobin. single experiment was conducted  Also a  to test the importance of an  analogue of tyrosyl-residues i n complex formation. EFFECT OF IONIC STRENGTH AND pH UPON HEMOGLOBIN-HAPTOGLOBIN BINDING A series of Sephadex G-200 assays  was performed i n 0.2  M Tris-HCl buffer, pH 7.5 containing 0, 0.5, 1.0 and 2.0 M (NHOaSOi*.  In each case, 1 mg haptoglobin was dissolved i n  0.5 ml of the buffer and 1.2 mg hemoglobin was dissolved i n 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 s a l t concentrations are summarized i n Table V.  Even at 1 M ammonium  - 117 -  TABLE V E f f e c t of S a l t C o n c e n t r a t i o n on Hb/Hp Combination (Sephadex G-200 Method) 0.0 2 M T r i s - H C l 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 t o p r e c i p i t a t e  - 118 sulphate and probably above, combination occurs at the usual stoichiometry.  This r e s u l t was  observed both i n the presence  of the divalent anion, sulfate as well as i n the monovalent anion, chloride. was  At the highest s a l t concentration, there  evidence of p r e c i p i t a t i o n and the e l u t i o n p r o f i l e  somewhat e r r a t i c , which may  was  account for the s l i g h t l y lower  binding observed. In another series of binding experiments, buffers ranging from pH 3.0-pH 11.0 were used.  of composition  l i s t e d i n Table VI,  A solution of 1.2 mg of hemoglobin i n 0.5 ml of  the buffer was  added to 1.0 mg of haptoglobin i n 0.5 ml of  the same buffer.  The mixture was  then assayed for hemo-  globin binding capacity on Sephadex. The r e s u l t s of the experiments i n which the pH varied are summarized i n Table VI.  was  I t i s evident that  stoichiometric complex formation occurred between pH and pH 11.0.  4.0  The apparent stoichiometry appears to change  above pH 9.0 but this was  found due to s p e c i f i c absorption  changes at the higher pH s  i n which the absorption spectra  1  of hemoglobin at higher pH s 1  show a decrease i n 407  absorption and an increase i n 280 my absorption.  my  When  corrected for these changes the 407/280 r a t i o from pH to 11.0  i s close to the value of 2.0.  a c i d i c pH s, 1  pH 3.0 and 3.5,  4.0  At the extreme  an instant darkening of the  hemoglobin solution indicated an i r r e v e r s i b l e denaturation of the molecule, which has been reported by F i e l d and  - 119 -  TABLE VI E f f e c t of pH on Hb/Hp Combination (Sephadex G-200 Method) Buffer (0 .1 M)  OD 407 my/OD 280  3.0  citric-citrate  (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**  bicarbonate-carbonate (Na)  1.95**  11.0  * 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 i n the haptoglobin molecule (215).  Therefore, an altered stoichiometry of bind-  ing i s to be expected and a s i g n i f i c a n t lower amount of hemoglobin i s observed to be bound to the haptoglobin. Thus, both the results of the experiments with increasing s a l t concentration and. varying pH indicate that e l e c t r o s t a t i c interactions cannot be the sole intermolecular forces involved i n binding, since masking the e l e c t r i c a l l y charged groups by a high concentration of ions does not a f f e c t the interaction nor does wide v a r i a t i o n i n the ionic state of charged groups on the two proteins produce any s i g n i f i c a n t e f f e c t on the binding.  These results minimize the importance.of electro-  s t a t i c binding i n complex formation, contrary to the observations of Robert, Bajic and Jayle (214).  Although electro-  s t a t i c forces may not be the sole factor involved i n the binding, there i s s t i l l the p o s s i b i l i t y that e l e c t r o s t a t i c forces together with other types of interactions acting i n a co-operative manner may be responsible for the s t a b i l i t y of the Hb-Hp complex. Anfinsen  (217) has found that the t e r t i a r y folding of  reduced RNase i s severely i n h i b i t e d by analogu'es of tyrosine.  In a single experiment to test the e f f e c t of a tyro-  syl-analogue, phenol, i n the binding, solutions of 1.2 mg hemoglobin and 1 mg haptoglobin each i n 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 d i a l y s i s to remove the aromatic phenol 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 i n 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 i n this assay was considerably r e duced to render the method unpractical for further studies, the presence of the aromatic compound tending to a f f e c t the porosity of the dextran g e l .  However, the phenol experi-  ment tends to question the involvement of tyrosine i n the Hb-Hp complex as postulated by Kalous and P a v l i e e k (218) . ;  A further aspect of the Hb-Hp binding i s revealed by the experiments  i n which s a l t concentration and pH are varied.  Several investigators have shown that hemoglobin dissociates r e a d i l y into half molecules at high s a l t concentrations and at both acid and a l k a l i n e pH s (219). 1  The osmotic pressure  data by Gutfreund (220), the sedimentation and d i f f u s i o n studies by Benbamou and coworkers (221) and the l i g h t scattering and sedimentation studies by R o s s i - F a n e l l i , Antonihi and Caputo (222), a l l indicate that a reversible d i s s o c i a t i o n of hemoglobin into subunits occurs above 0.5 M NaCl (219), there being  - 122 -  F i g u r e 17. x 50 cm)  Assay of Hp 1-1 on Sephadex G-200 (1 cm  i n 0.1 M phosphate b u f f e r , pH 7.0  0.1 M phenol.  containing  - 123 similar effects with other s a l t s (222).  At the highest s a l t  concentration studied by Rossi-Fanelli and associates, 3 M NaCl, the weight average molecular weight indicated that the great majority of the molecules exist i n a dissociated form. S i m i l a r l y at the extreme pH's reversible d i s s o c i a t i o n occurs, from pH 6.0 to 3.5 a d i s s o c i a t i o n 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 d i s s o c i a t i o n i s no longer reversible (219).  In the  alkaline d i r e c t i o n , at a pH just beyond 10 d i s s o c i a t i o n begins u n t i l at pH 11.0 the molecule i s completely dissociated into 2 subunits (223).  At pH 11.6, the changes i n the hemoglobin  molecule are no longer reversible (223).  Thus, at the higher  s a l t concentrations used i n the present study, as well as at the  extreme pH s, hemoglobin would c e r t a i n l y exist largely 1  as half molecules and since binding by Sephadex G-200 appears unchanged, i t would seem l i k e l y that the binding s i t e on haptoglobin i s able to combine with these half molecules. This i s a point of considerable i n t e r e s t , since comparison of haptoglobin with the 7S immunoglobulins  shows a simi-  l a r i t y i n basic structure i n that both molecules possess two pairs of d i s s i m i l a r (two l i g h t and two heavy) chains' connected by disulphide bonds.  Furthermore, the combination of hapto-  globin with hemoglobin i s similar to the antigen-antibody reaction i n that the combination i s extremely t i g h t and very  specific.  One major difference, however, i s that the complex  between hemoglobin and haptoglobin remains soluble.  An i n t e r -  esting question i s whether haptoglobin, l i k e the IS antibody, i s bivalent, but since the stoichiometry of binding i s one hemoglobin to one haptoglobin, there are two p o s s i b i l i t i e s i n the binding, either there i s one s i t e per haptoglobin molecule which could bind the whole hemoglobin or there are two sites per haptoglobin molecule each of which bind one-half molecule of hemoglobin.  L a u r e l l (57) and A l l i s o n and ap Rees  (58) by paper electrophoresis observed the formation of two kinds of Hb-Hp complexes.  On starch gel electrophoresis  L a u r e l l (55) and Shim, Lee and Kang (33) provided evidence that when haptoglobin i s i n excess over hemoglobin, an i n t e r mediate complex i s formed which i s l i k e l y to have the s t o i chiometry of one-half molecule of hemoglobin to one haptoglobin, since the intermediate complex migrates i n 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 k i n e t i c studies by Nagel and Gibson  (59) i n  which a reaction between aB hemoglobin dimers with two simil a r but independent binding s i t e s on the haptoglobin molecule i s 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 p u r i f i c a t i o n on DEAE-Sephadex chromatography. From the molecular weight and the heme content of the i n t e r mediate complex, Hamaguchi (225) concluded that i t consists of one molecule of haptoglobin and one-half a molecule of hemoglobin.  - 125 -  PART IV CHEMICAL MODIFICATION OF AMINO GROUPS IN HAPTOGLOBIN INTRODUCTION Chemical modification of proteins with s p e c i f i c group reagents plays an important r o l e i n elucidating the groups involved i n the active s i t e of a b i o l o g i c a l l y active protein. Ideally the reagent under suitable conditions should a f f e c t only one type of group or one p a r t i c u l a r 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 e f f e c t of the modification on the b i o l o g i c a l a c t i v i t y must also be available so that changes i n the b i o l o g i c a l a c t i v i t y can be interpreted i n r e l a t i o n to the modification.  I t i s to be  expected that random reaction of a p a r t i c u l a r chemical group would produce a family of proteins varying i n number of subs t i t u t e d groups as well as d i s t r i b u t i o n of these groups throughout the molecule.  Correlation of the change i n b i o l o g i c a l  a c t i v i t y with the actual modification i n the fractionated material as d i s t i n c t from the o v e r a l l modification i n the heterogeneous protein population would provide more meaningf u l interpretations.  Furthermore, i t i s necessary to d i s -  tinguish between loss of b i o l o g i c a l a c t i v i t y due to chemical modification on a s p e c i f i c group i n the active s i t e from an unfolding of the t e r t i a r y structure of the molecule from a modification of a group located outside the active s i t e but which i s involved i n maintaining  the molecule i n the native  - 126 conformation by p a r t i c i p a t i o n in t e r t i a r y interactions  (226).  If loss of b i o l o g i c a l a c t i v i t y can be shown to occur on modif i c a t i o n of a s p e c i f i c group without conformational changes in the protein, then i t may be concluded that the amino acid modified i s an important component of the active s i t e .  On  the other hand, i f changes i n 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 modification 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 i s complicated and i t s usefulness as a s p e c i f i c 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 r e a c t i v i t y and further condense with amide, guanidyl, phenolic and heterocyclic groups to y i e l d cross-linking methylene bridges (227). Shinoda  (228) labeled the amino groups by their conver-  sion into trinitrophenyl-residues (TNP-residues) with 2,4,6trinitrobenzene-l-sulfonic acid (TNBS).  This reagent i s  reported to react almost quantitatively with amino groups i n s l i g h t l y a l k a l i n e solution without detectable substitution of amino groups or hydroxyl groups (229).  I t was found that  with 15.5,-16.4 and 11.8 moles of TNP-residues  i n Hp 1-1,  2-1  - 127 and  2-2 molecules r e s p e c t i v e l y , the hemoglobin b i n d i n g  c i t y was 50% o f the i n i t i a l c a p a c i t y and a  semilogarithmic  p l o t showed a l i n e a r r e l a t i o n s h i p between i n c r e a s e s residues city.  capa-  of TNP-  i n the p r o t e i n and l o s s o f hemoglobin b i n d i n g  capae  I t was concluded from these r e s u l t s t h a t some of the  amino groups comprise one of the e s s e n t i a l c o n s t i t u e n t s o f the a c t i v e s i t e of h a p t o g l o b i n .  However, the TNP group i s  a r a t h e r bulky moiety and there was no evidence t h a t the l o s s of b i n d i n g c a p a c i t y c o u l d not have been due to a nonspecific  i n a c t i v a t i o n such as an a l t e r e d  conformational  state. Thus, the chemical m o d i f i c a t i o n experiments so f a r ted a r e i n c o n c l u s i v e . accurate  Furthermore, i n order  repor-  t o o b t a i n an  p i c t u r e of the changes t h a t may be t a k i n g p l a c e i n  the molecular s t r u c t u r e when a p r o t e i n i s subjected t o chemical m o d i f i c a t i o n , i t i s necessary t o f o l l o w the e f f e c t s of m o d i f i c a t i o n by employing as many methods as p o s s i b l e . the work to be d e s c r i b e d  here m o d i f i c a t i o n s with three  In  dif-  f e r e n t reagents of i n c r e a s i n g s e v e r i t y have been examined t o study the e s s e n t i a l i t y of the amino groups. n a t i o n with l-guanyl-3,5-dimethyl p y r a z o l e converts due  First,  guanidi-  n i t r a t e (GDMP)  l y s i n e t o homoarginine, a c h e m i c a l l y  different resi-  but one which remains p o s i t i v e l y charged.  The extent  of m o d i f i c a t i o n i s e a s i l y determined by measuring the homoa r g i n i n e content f o l l o w i n g a c i d h y d r o l y s i s of the guanidinated p r o t e i n ; homoarginine appears on the amino a c i d  analyzer  - 128 c l e a r l y separated after arginine (Fig. 18).  The second type  of modification of amino groups, acetylation with acetic anhydride, i n the presence of sodium acetate, i s somewhat more drastic than guanidination, since p o s i t i v e l y charged e - l y s y l side chains are converted to neutral, c-N-acetyl side chains. Lastly, the most drastic modification i s succinylation of amino groups with succinic anhydride which converts the positive amino group to the negatively charged N-succinyl compounds.  In the l a s t 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 r a d i c a l change i n the nature of the chemical group, the basic amino group of lysine i s replaced by the more basic guanidino group. of the reaction to chymotrypsinogen  Application  by Chervenka and Wilcox  (231) and to ribonuclease by Klee and Richards (.232) resulted in modification of lysine residues without causing major changes i n secondary structure.  Guanidination i s also essen-  t i a l l y s p e c i f i c for the e-amino groups of lysine (233) and i s therefore a good method for investigating the essential nature of these residues for b i o l o g i c a l a c t i v i t y . Guanidination was c a r r i e d out with GDMP instead of with the c l a s s i c reagent, o-methylisourea, because of i t s lower optimum pH, 9.5 as compared with 10.5-11 for the l a t t e r reagent (234).  In the present studies, i t was found that reaction  - 129 -  A  1  Figure  18.  2 3  Separation  (1) l y s i n e (2) h i s t i d i n e (3) ammonia (4)  arginine  (5) homoarginine  5  o f known b a s i c amino a c i d s on the  Beckman/Spinco 120 C amino a c i d Peaks  4  analyzer.  -  130 -  at a pH of 9.0 for a period of 3 days with 0.5 M GDMP reagent produced s u f f i c i e n t l y extensive modification so that the lower pH was used throughout. EXPERIMENTAL Guanidination  of haptoglobin,  Hb-Hp and hemoglobin was  carried out by a procedure s i m i l a r to the procedure of Habeeb (235) with GDMP.  P u r i f i e d Hb-Hp complex was prepared for the  reaction by addition of 118 mg hemoglobin dissolved i n 2.5 ml of 0.05 M  NH4HCO3,  pH 8.0 buffer to 100 mg Hp 1-1 i n 2.5 ml  of the same buffer and separation of the complex on a Sephadex G-200 reverse flow column, 2.8 cm x 97 cm equilibrated i n 0.05  M  NH4HCO3,  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-  t i o n into the bottom of the column.  I t had been observed pre-  viously that i n 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 it.  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 l y o p h i l i z e d . guanidination  For the  reaction, a 0.5 M GDMP solution was made up  - 131 by adding  2.515 go. GDMP t o 7 ml of d i s t i l l e d water and the  pH a d j u s t e d t o 9.0 w i t h 1.0 N NaOH and the volume then made up t o 25 ml.  Separate  25 mg p o r t i o n s o f h a p t o g l o b i n were  each d i s s o l v e d i n 2.5 ml of 0.1 M GDMP, 0.2 M GDMP and 0.5 M GDMP s o l u t i o n s a t pH 9.0 together w i t h a c o n t r o l i n 0.1 M phosphate b u f f e r , pH 7.0. In a d d i t i o n , 25 mg o f Hb-Hp complex and 25 mg of hemoglobin were each d i s s o l v e d i n 2.5 ml 0.2 M GDMP.  Each o f the above s o l u t i o n s were a d j u s t e d  to pH 9.0 w i t h NaOH and allowed t o r e a c t a t 4° f o r a p p r o x i mately  72 hours.  A t the end o f the s p e c i f i e d p e r i o d , each  s o l u t i o n was d e s a l t e d on a Sephadex G-25, 2.5 x 30 cm column, to remove excess reagent and t o stop f u r t h e r reaction.  The  g u a n i d i n a t e d hemoglobin mixture had a g r e a t d e a l of p r e c i p i t a t e which was removed b e f o r e d e s a l t i n g . were then l y o p h i l i z e d .  The s o l u t i o n s  Sephadex GfSOO assays were  performed  on the h a p t o g l o b i n s m o d i f i e d w i t h 0.1 M, 0.2 M and 0.5 M reagent and on the c o n t r o l .  In each case, 2 mg hemoglobin  i n 0.2 5 ml o f 0.1 M phosphate b u f f e r , pH 7.0 was added t o 2.5 mg o f the m o d i f i e d and c o n t r o l h a p t o g l o b i n i n 0.25 ml o f phosphate b u f f e r . Sephadex method.  The mixtures were then assayed by the To determine  the extent o f m o d i f i c a t i o n ,  5 mg o f each o f the m o d i f i e d p r o t e i n s and the c o n t r o l were s e p a r a t e l y d i s s o l v e d i n 1 ml 6 N HC1 and the mixture l y z e d i n s e a l e d ampoules a t 105° f o r 18 hours. of the h y d r o l y s i s p e r i o d , the b l a c k p r e c i p i t a t e  hydro-  A t the end (humin) i n  the h y d r o l y z a t e s was removed by c e n t r i f u g a t i o n and the super-  - 132 natant dried i n vacuo over NaOH p e l l e t s .  The residue was  taken up i n 1 ml of pH 2.2 c i t r a t e buffer and aliquots of t h i s solution analyzed i n 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 i n 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, i n which 26.3%  and 52.9%  of the e-amino groups were guanidinated,  there i s no e f f e c t on the binding capacity, as may be seen by the 407 my/280 my r a t i o .  From the e l u t i o n p r o f i l e s i n  Figure 19, frames B and C representing these two modified proteins respectively, the guanidinated haptoglobins combined with hemoglobin to give a symmetrical peak eluting i n the same volume for the Hb-Hp complex as that seen i n the control (Fig. 20, frame A).  However, at the greatest extent  of modification with 0.5 M GDMP reagent, there i s evidence that a new phenomenon i s occurring (Fig. 19, frame D).  A  portion of the highly substituted haptoglobin i s eluted e a r l i e r from the Sephadex G-200 column, indicating that a conformational change has occurred.  Following this change,  the binding i s very much decreased, the r a t i o having dropped to 0.73.  However, the greater part of the protein, which i s  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 II CH -C  C-CH 3 I N  3  NH 3 ^ (CH K  NH IC=NH  W  2  N C=NH  +  2  I  I©  P  H 9  >  (CH ) 2  l-guanyl-3,5dimethyl pyrazole n i t r a t e (GDMP)  4  I  G>  NK2 NO3  -NH-CH-CO... l y s y l residues  NH  ..NH-CH-CO.. homoarginine  OD 407 Reaction Conditions  % lysine  Hp 1-1, pH 9.0, 0°, 72 hrs 100 Control 73.7 0.1 M GDMP 47.1 0.2 M GDMP 0.5 M EDMP peak A 17.8 peak B  % homoarginine OD 28 0  26.3 52.9  82.2  2.19 2.23 2.12 0.73* 2.07  Hb, pH 9.0,0°, 7 2 hrs 0.2 M GDMP  41. 7  Hb-Hp, pH 9.0, 0°, 72 hrs 0.2 M GDMP  4 7.8  58.3 52.2  * Lower elution volume on Sephadex G-200 indicates mational change  confor-  ~r  i  r~~  - 134 1 ™r  EFFLUENT  j——i  2—™i  V O L U M E , ml  F i g u r e 19. Assay o f Hp 1-1 g u a n i d i n a t e d w i t h 0.1 M GDMP (B), 0.2 M GDMP (C), 0.5 M GDMP (D) compared with a c o n t r o l Sephadex G-200 i n 0.1 Mv-phbsphate b u f f e r , pH 7.0.  (AXcon  - 135 s t i l l binds e s s e n t i a l l y the same amount of hemoglobin, the 407 my/280 my r a t i o being 2.07.  It appears then that binding  remains e s s e n t i a l l y constant u n t i l a very large percentage of the amino groups have been converted to guanidino groups when there i s an attendant conformational change i n the molecule and the binding i s greatly reduced. An attempt to ascertain the extent of the area involved in the binding s i t e 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 d i s t r i b u t i o n of the lysine residues over the surface of the haptoglobin molecule since lysine i s a frequently occurring amino acid residue i n proteins and, being highly polar, usually occurs at the surface where i t is able to interact with solvent.  Table VII shows that  hemoglobin i s modified to a s l i g h t l y 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 i n haptoglobin (236). 52.2%  Guanidination of the complex leads to  of the combined lysine content of hemoglobin and hapto-  globin being converted to homoarginine. compared with a value of 55.2%  This r e s u l t can be  calculated from the homoarginine  content of hemoglobin and haptoglobin when guanidinated separately under the same conditions.  The difference between the  observed and t h e o r e t i c a l values gives an indication of the extent of the area of the binding s i t e .  The small difference  - 136  of 3% indicates that the extent of involvement of lysine residues i n the active s i t e i s very small.  However, another  p o s s i b i l i t y exists that on binding of hemoglobin by haptoglobin there are alterations i n the conformation of the molecule(s) such".that more lysine residues become available for the modification reaction. such conformational  There are indications that  changes i n the hemoglobin molecule do  occur on combination of hemoglobin with haptoglobin and a f f e c t the heme to globin linkage.  The fact that the Hb-  Hp complex has a high a f f i n i t y f o r oxygen and lacks a Bohr e f f e c t may r e f l e c t conformational molecule that make impossible  changes i n the hemoglobin  the s l i g h t a l t e r a t i o n s i n  quaternary interactions underlying heme-heme i n t e r a c t i o n and the Bohr e f f e c t (237).  A l t e r a t i o n of the peroxidase  pH optimum (3) and spectral changes i n the spectrum due to heme also suggest a change i n the heme environment i n the complex (201).  Nevertheless,  i n those globular proteins  whose detailed three-dimensional structures have been solved, l y s y l - s i d e chains are always exposed on the surface of the molecule so that i t would be u n l i k e l y that change could expose other  "buried" l y s i n e s .  conformational Iii the case  of chymotrypsinogen, Chervenka and Wilcox (231) have shown that a l l the l y s y l residues can be substituted by guanidination and the protein remains activatable.  The conclusion,  therefore, remains that the area of contact between hemoglobin and haptoglobin  i n the complex involves only a small  - 137 area of the surface of the haptoglobin molecule or else the area 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 chains. ACETYLATION Acetylation of amino groups with acetic anhydride converts the p o s i t i v e l y charged ammonium ion to a neutral Nacetyl group and the chemical change i s therefore more r a d i c a l i n nature than guanidination.  Acetylation of amino groups  i s 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 r e a d i l y accessible to react i o n with one of the s p e c i f i c acetylation reagents under mild conditions and the extent of the modification i s assessed by r e l a t i v e l y simple a n a l y t i c a l means.  The reagent of choice  for the substitution of acetyl groups on amino groups i s acetic anhydride, which under certain conditions i s a highly s p e c i f i c reagent for amino groups (238,239).  The reaction  is most often c a r r i e d out i n half-saturated sodium acetate, which functions as a buffer of the reaction mixture and also appears to serve as a c a t a l y s t (240). high concentrations  In addition, the  of acetate ions catalyzes the hydrolysis  of O-acetyltyrosyl residues and thereby increases the specif 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 i n 5 ml of h a l f -  - 138 saturated sodium acetate and cooled i n an ice-bath during the reaction.  A 0.1 M acetic anhydride solution i n ace-  t o n i t r i l e was made up and also kept i n the ice-bath.  Cold  acetic anhydride solution was added to each flask during an i n t e r v a l of 1 hour so that the f i n a l molar r a t i o s of acetic anhydride to haptoglobin i s 10:1, 40:1, -80:1 and 400:1  i n flasks designated A, B, C, and D respectively.  Flask C corresponds to an approximately 1:1 r a t i o for acetic anhydride to lysine on the basis of 64 lysine r e s i dues i n Hp 1-1. Addition of reagent was as follows:  a  t o t a l 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 y l 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 t o t a l of 200 y l of reagent.  Flask C contained a f i n a l 400 y l 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. added i n i t i a l l y ,  To Flask D, 200 y l was  then 150 y l every 5 minutes, which results  in a t o t a l 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 l y o p h i l i z e d .  The  binding capacity of each of the modified proteins was determined 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  -  139  -  in 0 . 1 M phosphate buffer, pH 7 . 0 to 1 . 5 mg of hemoglobin i n 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 i n the native conformation, these two grossly d i f f e r e n t species of acetylated haptoglobins were p a r t i a l l y resolved on a Sephadex  G-200,  in 0 . 1 M phosphate buffer, pH 7 . 0 .  1  cm x  50  cm column  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 i n 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  Solutions of  (230).  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 d i s solved i n 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 solutions representing p a r t i a l l y separated species of haptoglobin acetylated with a r a t i o 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 F i g u r e 20. hydride.  V O L U M E , m?  Assay of Hp 1-1 a f t e r a c e t y l a t i o n w i t h a c e t i c anR a t i o s of reagent t o h a p t o g l o b i n 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 b u f f e r , pH 7.0  - 141  -  i — —  EFFLUENT Figure  21.  P a r t i a l separation  a c e t y l a t e d Hp 400:1  VOLUME,  1-1  of two  r  ml species  of  f o l l o w i n g r e a c t i o n with a r a t i o  of  a c e t i c anhydride to p r o t e i n on Sephadex G-200  (1 cm x 50 cm)  i n 0.1  M phosphate b u f f e r , pH  7.0.  - 142 at 40° for 2 hours i n the dark. 1.0 N HC1 was  At the end of this period,  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 i n Table VIII.  I t can be seen  that binding of hemoglobin by the modified protein i s l i t t l e affected by nearly 50% acylation of the lysine groups.  At  these levels of acetylation, i n which there i s no great excess of reagent, the elution p r o f i l e s  (Fig. 20, frames A,  B and C) shows that the bulk of the haptoglobin i s i n the native conformation,  although a s l i g h t skewing of the peaks  suggests the beginning of a change i n a very small tion of the proteins.  propor-  At the highest degree of acetylation,  a conformational change i n the haptoglobin i s c l e a r l y apparentand a new unfolded species appear (Fig. 20, frame D). peak appearing  The  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 i n the normal elution volume shows almost 40% of the normal binding (Table VIII).  This i s a further i l l u s t r a t i o n  of the profound e f f e c t 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 i n the  - 143 TABLE V I I I A c e t y l a t i o n of Haptoglobin CH  3  \:=o I  NH © 3  CH N-terminal and e-lysylresidues  1-1  /  0 | C=0  C H  Na a c e t a t e pH 8.2-> ( h a l f saturated)  3  I  C=0 | NH I  3  acetic anhydride  Reaction Conditions  e-N-acetyl-lysyl-  % Acetylation*  Control  OD 407 OD 28 0 2.25  10:1 a c e t i c anhydride^ to Hp 1-1  23.4  2.01  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 t r i n i t r o b e n z e n e s u l f o n i c  acid  ' ** Lower e l u t i o n volume on G-200 i n d i c a t e s  reaction conformational  change t  Dissolved  i n a c e t o n i t r i l e and added i n approximately  equal a l i q u o t s over 60" a t 0°.' -  - 144 chemical nature of the molecule i n which the p o s i t i v e l y charged amino group i s replaced by a negatively charged Nsuccinyl group.  Succinic anhydride has been reported  attack the l y s y l amino groups s p e c i f i c a l l y (243)  to  and being  a r e l a t i v e l y stable, non-volatile substance i s 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 hydrolyzed under the reaction conditions employed here. EXPERIMENTAL Succinylated haptoglobins were prepared according the procedure of Habeeb, Cassidy and Singer  to  (245) with a  r a t i o of succinic anhydride to lysine of approximately 15:1  (246).  of 0.01  To a solution of 25 mg of Hp 1-1  M Tris-HCl buffer, pH 8.0,  cooled i n an  throughout the entire reaction, 31.5 anhydride was  8.0  mg of s o l i d succinic stirred.  haptoglobin solution served as a  The pH of. the reaction mixture was  by addition of 1.0  ml  ice-bath  added and the mixture continuously  Another 2.5 ml of a 1.0% control.  i n 2.5  maintained near  N NaOH from a syringe.  of 10 minutes, 30 minutes, 1 hour and  At i n t e r v a l s  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 column.  to chromatography on a Sephadex G-25,  0.9 x 30  cm  The protein column effluents were used for binding  studies and analysis on starch gel electrophoresis.  Deter-  - 145 mination of the sedimentation  c o e f f i c i e n t s and the molecular  weights by the Archibald sedimentation  equilibrium method  was carried out on 10 minutes and 2 hours succinylated haptoglobins prepared  i n a subsequent experiment.  Ultracentri-  fugation of the 10 minutessuccinylhaptoglobin  was i n 0.1 M  phosphate buffer, pH 7.0 containing 0.1 M NaCl and the 2 hours succiny.l-haptoglobin was i n 0.01 M phosphate buffer, pH 7.0 with 0.05 M KC1.  Various attempts at f r a c t i o n a t i n g  the 10 minutes and 2 hours succinylated haptoglobins by DEAE-cellulose  chromatography with d i f f e r e n t gradient systems  proved unsuccessful. RESULTS In Figure 22, frames A, B, C and D are the e l u t i o n diagrams from the binding assay of the succinylhaptoglobins prepared by 10 minutes, 30 minutes, 1 hour and 2 hours of reaction respectively.  I t i s apparent that at the shortest  reaction time, 10 minutes, a conformational change i n the molecule i s occurring and at the same time the binding capac i t y has decreased  by 65% (Table i x ) .  As the reaction time  i s allowed to increase there i s a progressive decrease i n binding a b i l i t y ; by 1 hour and 2 hours, succinylated haptoglobins are e s s e n t i a l l y devoid of hemoglobin binding capac i t y (Table IX).  The Sephadex assays of the 10 minutes and  2 hours haptoglobin controls show .no decrease i n hemoglobin binding and therefore precludes any influence on the hemoglobin binding by the shearing action of the magnetic  i  1  i  1  T —  EFFLUENT F i g u r e 22.  i  i——T  VOLUME,  1——T——-r~  ml  Assay o f Hp 1-1 s u c c i n y l a t e d f o r v a r i o u s times on  Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate R e a c t i o n times a r e :  CA) 10 minutes,  b u f f e r , pH 7.0.  CB) 30 minutes,  (C) 1 hour,  (D) 2 hours, w i t h a f i f t e e n - f o l d excess of s u c c i n i c anhydride. NB - Frame C - due t o m a l f u n c t i o n o f the f r a c t i o n c o l l e c t o r not all  f r a c t i o n s were c o l l e c t e d .  -  147 -  TABLE I X Succinylation  CO— I CH •I  NH  2  CO— succinic  N-succinyl-  e-lysyl  anhydride  derivative  Reaction  OP 4 0 7 OP 2 8 0  Conditions  1 - 1 , 0.01 M T r i s - H C l ,  10  2  I. NH  a-NH o r 2  Hp  1-1  <3 CO-CH -CH -COO 2  0  2  CH  of Haptoglobin  %  Succinylation  p H 8.0  1  control  2. 55  120'  control  2.20  t 1260:1 s u c c i n i c protein  anhydride  to  10 '  0.76**  30  0.68**  1  0.24**  60'  0.11**  120 ' 10' 120'  t  added to  as a s o l i d  8 by a d d i t i o n  ** l o w e r  a t 0°  'with  0.41**  85.6  0.07**  96.9  stirring  a n d pH m a i n t a i n e d  close  o f NaOH  e l u t i o n v o l u m e o n G-200 i n d i c a t e s  conformational  change  - 148 s t i r r i n g 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 hemoglobin binding capacity while the slower migrating modified component having l o s t 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 l a t t e r i s scarcely discernible i n the 30 minutes preparation (slot 6).  At the greatest extent of modifica-  t i o n , 1 hour and 2 hours reactions, only one slow migrating band i s 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 i n starch gel or polyacrylamide electrophoresis.  In 8.0 M urea starch gels i n 0.05 M acetate  buffer, pH 5 however, the control haptoglobin remained close to the o r i g i n as would be expected from i t s i s o e l e c t r i c point of 4.2  (46), and the succinylhaptoglobins migrated at a  149 -  free  0  ^--^  CD  CZD  C  ^  )  Hp  __ '  c  ,-- Hp-Hb  .eakage  A  8  7  F i g u r e 23.  5  4  Hp 1-1  and t h e i r c o n t r o l s .  1  The samples a r e as (2) 10  (3) 10 minutes c o n t r o l Hp with Hb  (4) 10 minutes c o n t r o l Hp,  added,  (5) 30 minutes s u c c i n y l - H p w i t h  (6) 30 minutes s u c c i n y l - H p ,  Hb added,  2  (1) 10 minutes s u c c i n y l - H p w i t h Hb added,  minutes s u c c i n y l - H p ,  added,  3  SG-electropherogram of 10 minutes and 30 minutes  succinylated follows:  6  o  (8) 30 minutes c o n t r o l  Hb  (7) 30 minutes c o n t r o l Hp  Hp.  with  - 149 -  8  7  F i g u r e 23.  5  4  1  The samples are as  (1) 10 minutes s u c c i n y l - H p w i t h Hb added,  (2) 10  (3) 10 minutes c o n t r o l Hp with Hb  (4) 10 minutes c o n t r o l Hp,  added,  (5) 30 minutes s u c c i n y l - H p with  (6) 30 minutes s u c c i n y l - H p ,  Hb added,  2  Hp 1-1 and t h e i r c o n t r o l s .  minutes s u c c i n y l - H p ,  added,  3  SG-electropherogram of 10 minutes and 30 minutes  succinylated follows:  6  (8) 30 minutes c o n t r o l  (7) 30 minutes c o n t r o l  Hp.  Hb Hp.with  - 150 -  8 F i g u r e 24.  7  6  5  4  3  2  1  SG-electropherogram of 1 hour and 2 hours s u c c i n y -  l a t e d Hp 1-1 and t h e i r c o n t r o l s . s u c c i n y l - H p w i t h Hb added, c o n t r o l Hp with Hb added, s u c c i n y l - H p with Hb added, c o n t r o l Hp with Hb added,  The s o l u t i o n s a r e : (1) 1 hour  (2) 1 hour s u c c i n y l - H p , (4) 1 hour c o n t r o l Hp,  (5) 2 hours  (6) 2 hours s u c c i n y l - H p , (8) 2 hours c o n t r o l  Hp.  (3) 1 hour  (7) 2 hours  - 150 -  +  0  8 F i g u r e 24.  7  6  5  4  3  2  1  SG-electropherogram of 1 hour and 2 hours s u c c i n y -  l a t e d Hp 1-1 and t h e i r c o n t r o l s . succinyl-Hp with Hb added, c o n t r o l Hp with Hb added, s u c c i n y l - H p w i t h Hb added, c o n t r o l Hp with Hb added,  The s o l u t i o n s a r e : (1) 1 hour  (2) 1 hour s u c c i n y l - H p , (4) 1 hour c o n t r o l Hp,  (5) 2 hours  (6) 2 hours s u c c i n y l - H p , (8) 2 hours c o n t r o l  Hp.  (3) 1 hour  (7) 2 hours  -  considerably  151  -  greater rate towards the anode.  The slower mi-  gration of the succinylated components than the unmodified haptoglobins i n borate starch gel could not be attributed to an aggregation phenomenon as a molecular weight of (103,475;  97,485;  99,200  was determined for the 2 hours  96,792)  succinylated haptoglobin by the Archibald approach to e q u i l i brium technique (Fig. 2 5 ) , and assuming quantitative reaction a correction factor of  6500  for the 64 l y s y l residues and the  N-terminal amino group y i e l d s Hp 1 - 1 unit.  A sample c a l c u l a t i o n based on measurements of  the u l t r a c e n t r i f u g a l patterns c  m  o  _  c  e s s e n t i a l l y a single  92,700,  ~ ~ — 2 - ~w~  1  x  n n'' 2z  n =0  i n F i g . 25 i s the following =  dx m  0.60  o  C  m  = 0.67  Mm =  1 3  =  4  RT /-, w  3  4  '  -y-^  (63.8404)  (dc/dx) \ T  nc-i r = 4.951FX A  m  (l-vp)(jo^  m  The  -  -  JIL  =  0.052  ir, *. 10 1  Xm°m  ~p—or  0.60 n nan  5.86  x  0.052  97,485  slower migration  of the succinylhaptoglobins  i n starch  gel can be correlated to the unfolding phenomenon observed on the Sephadex  G-200  columns.  The unfolded nature of these  molecules would hinder t h e i r migration gel  through the starch  so that the f r i c t i o n a l retardation outweighs t h e i r i n -  crease i n 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 unfolded by the denaturing e f f e c t of urea and the charge  Time bar  2.7 angle -  speed  =  hr  3.2  70  12,000 r p m  Figure R  p  1  the  _  1  hr  25. ±  n  Q  ^  3.7 Ultracentrifuge  Q 5  Archibald  M  K C 1  ^  o.01  approach  hr  patterns  M phosphate  4.3 of  2 hours  buffer,  to sedimentation  pH  hr  4.8  succinylated 7.0  equilibrium.  during  hr  - 153 differences become apparent. In subsequent succinylation reactions, haptoglobin was modified to a s l i g h t l y greater extent, that i s , the 10 minutes and 2 hours succinylated haptoglobin  show r a t i o s of  0.41 and 0.07 respectively i n the binding assay (Table IX). The v a r i a t i o n i n the degree of modification during the same time i n t e r v a l s may be explained by the influences of the pH fluctuations and the effectiveness of the s t i r r i n g on the reaction since succinic anhydride i s only slowly soluble i n the aqueous medium.  Coincident with the considerable de-  crease, almost 80%, i n binding a b i l i t y of the 10 minutes succinylated haptoglobin 85.6%  i n the second preparation, i s an  reaction of the l y s y l groups and binding i s completely  l o s t i n the quantitatively substituted 2 hours modified protein. In t h i s kind of chemical modification, the conversion of the p o s i t i v e l y 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 i s the major influence on the binding a c t i v i t y . 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 c o e f f i c i e n t s f o r these modified proteins.  These highly  charged succinylated proteins exhibit marked e l e c t r o s t a t i c effects on sedimentation which causes a decrease i n S2 0/ w.  - 154 The  -  10 minutes s u c c i n y l h a p t o g l o b i n showed a heterogeneous  p a t t e r n i n a s y n t h e t i c boundary c e l l , a 3.70  S and a minor peak w i t h 3.57  a major component with  S ( F i g . 26).  The  2 hours  m o d i f i e d h a p t o g l o b i n has a sedimentation constant of 3.18 (Fig.  27)  c o n s i d e r a b l y lower than the 4.4  haptoglobin  (46).  S of  S  unmodified  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 r e -  l a t e d to volume and  shape of the sedimenting u n i t  expanded and unfolded molecule  (247) , an  i s subject to greater  fric-  t i o n a l f o r c e 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 s u c c i n y l a t e d haptoglobins i s not due t o a d i s s o c i a t i o n i n t o s u c c i n y l a t e d subunits due  to e l e c t r o s t a t i c  r e p u l s i o n between the n e g a t i v e l y charged c a r b o x y l a t e groups as has , been r e p o r t e d f o r other p r o t e i n s (248,249), i s e v i d e n t 1  from the m o l e c u l a r weight of 99,200 f o r the 2 hours m o d i f i e d protein.  These data i n d i c a t e then t h a t the observed  decreased  hemoglobin b i n d i n g by the s u c c i n y l a t e d haptoglobins i s mainly a r e f l e c t i o n of the g r e a t c o n f o r m a t i o n a l change i n the molec u l e which p r o f o u n d l y a l t e r s the conformation s i t e of the  of the a c t i v e  molecule.  Chemical m o d i f i c a t i o n by s u c c i n y l a t i o n was i n Hp for  2-1  and Hp  2-2.  10 minutes and  same procedure  studied further  In each case the p r o t e i n s were r e a c t e d  2 hours and c o n t r o l s were prepared by  as t h a t f o r the Hp  1-1.  the  Each p r e p a r a t i o n was  assayed f o r b i n d i n g a b i l i t y by the Sephadex G-200 method and analyzed by s t a r c h g e l e l e c t r o p h o r e s i s .  Time  0  Bar Angle  25°  Speed = 56,000 rpm Temp. = 25.17° D i r e c t i o n of Sedimentation  4'  8'  12'  16  50°  50°  50°  50"  F i g u r e 26.  Sedimentation p a t t e r n s of 10 minutes  g l o b i n i n 0.1 M NaCl, 0.1 M phosphate boundary  cell.  succinylated  b u f f e r , pH 7.0  hapto-  i n a synthetic  Time  0  4'  8'  12'  16'  30°  70°  50°  50°  50°  B ci 3T  Angle  Speed = 56,000 rpm  Figure  Temp. = 21.95° D i r e c t i o n of  j _ .05 M KC1, -. cell.  Sedimentation  n 0  27.  Sedimentation patterns  o f 2 hours s u c c i n y l a t e d  0.01 M phosphate b u f f e r , pH 7.0  haptoglobin  in a synthetic  boundary  - 157 A reduced hemoglobin binding i s also observed i n both 10 minutes succinylated Hp 2-1 and 2-2  (Table X).  On  longer  treatment of the proteins, 2 hours, a further reduction i n binding i s seen.  Again the decreased binding i s 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 p a r t i a l resolution of the polymeric complexes on Sephadex G-200.  The succinylated protein  complexes, on the other hand (Fig. 28, frames A and  C,  F i g . 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 i s s l i g h t l y retarded although the sharpness and r e l a t i v e positions of the bands are maintained (Fig. 30).  The succinylated Hp 2-2 polymers are s i m i l a r l y  retarded i n their migration rate and s t i l l maintain t h e i r c h a r a c t e r i s t i c series of bands. t i o n of haptoglobin t u r a l changes.  This means that succinyla-  does not produce non-specific struc-  Rather i t appears that the subunits  of  each polymer molecule on succinylation must unfold i n a highly s p e c i f i c manner with the r e s u l t that each unfolded polymer molecule i s retarded to a similar extent on the starch gels, thereby maintaining  the c h a r a c t e r i s t i c 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  1260:1 succinic anhydride protein  t  to  10'  0.83**  120'  0.32**  Hp 2-2, 0.01 M Tris-HCl, pH 8.0 10' control 120' control 1260:1 succinic anhydride^ to protein  1.69 1.73  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' 120'  •  _  2.15 0.15  + added as a s o l i d at 0°f with s t i r r i n g and pH maintained close to 8 by addition of NaOH ** lower elution volume on G-2 00 indicates conformational change  -159 -i  0  10  1  1  20  30  1  40  1  j  1  50  0  10  EFFLUENT F i g u r e 28.  VOLUME,  ~r-  1  i  r  20  30  40  50  ml  Assay of 10 minutes and 2 hours s u c c i n y l a t e d Hp 2-1  and t h e i r c o n t r o l s on Sephadex G-200 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0. (B) 10 minutes c o n t r o l , (D) 2 hours c o n t r o l .  (A) 10 minutes s u c c i n y l a t e d Hp 2-1,  (C) 2 hours s u c c i n y l a t e d Hp 2-1,  - 160 T  _ _  1  1  [———i  EFFLUENT F i g u r e 29.  r — — i — — i  —  1  i  T  V O L U M E , mi  Assay of 10 minutes and 2 hours s u c c i n y l a t e d Hp  and t h e i r c o n t r o l s on Sephadex G-200 (1 cm x 50 cm) phosphate b u f f e r , pH 7.0. (B) 10 minutes  (A) 10 minutes  (D) 2 hours c o n t r o l .  i n 0.1 M  s u c c i n y l a t e d Hp  c o n t r o l , (C) 2 hours s u c c i n y l a t e d Hp  2-2  2-2,  2-2,  - 161 +  o  8 F i g u r e 30. succinylated  7  6  5  4  2  1  SG-electropherogram of 10 minutes and 2 hours Hp 2-1 and t h e i r c o n t r o l s .  s u c c i n y l - H p 2-1 with Hb,  (1) 10 minutes  (2) 10 minutes s u c c i n y l - H p  (3) 10 minutes c o n t r o l Hp 2-1 with Hb, Hp 2-1,  3  2-1,  (4) 10 minutes  (5) 2 hours s u c c i n y l - H p 2-1 with Hb,  control  (6) 2 hours  s u c c i n y l - H p 2-1,  (7) 2 hours c o n t r o l Hp 2-1 with Hb,  hours c o n t r o l Hp  2-1.  (8) 2  - 162 changes i n the haptoglobin molecule, the p o s s i b i l i t y exists that such great changes i n conformation may r e s u l t i n a d i s s o c i a t i o n of the very stable Hb-Hp complex.  I t was, there-  fore, of i n t e r e s t to determine the e f f e c t 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 i n 2 ml  of 0.01 M Tris-HCl buffer, pH 8.0, cooled i n an ice-bath, 25.2 mg of s o l i d 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 t o t a l of 2 hours, after which time the mixture was desalted on Sephadex G-25. The complex s t i r r e d i n 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 e l u t i o n p r o f i l e s of the succinylated complexes (Fig. 31, frames A and C), shows a highly i n t e r e s t i n g r e s u l t of the chemical modification.  The protein modified for 10  0  10  20  30  40  50  EFFLUENT F i g u r e 31.  60  0  VOLUME,  10  20  30  40  ml  Assay of 10 minutes and 2 hours s u c c i n y l a t e d Hb-Hp  complexes and t h e i r c o n t r o l s on Sephadex G-20 0 (1 cm x 50 cm) i n 0.1 M phosphate b u f f e r , pH 7.0.  (A) 10 minutes  Hb-Hp, (B) 10 minutes c o n t r o l Hb-Hp, (C) 2 hours Hb-Hp, (D) 2 hours c o n t r o l Hb-Hp.  succinylated succinylated  - 164 minutes shows a main peak of complex which has e s s e n t i a l l y retained the same amount of bound hemoglobin as may from the absorbancy r a t i o of 2.15  (Table X).  be seen  However, there  i s evidence of the beginning of a d i s s o c i a t i o n of the complex since a small peak corresponding to free hemoglobin may seen along with two other minor components.  be  These disso-  ciated proteins do not appear i n 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 s t i r r i n g of the reaction mixture. complex, the protein i s now  In ithe 2 hours succinylated  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 d i s s o c i a t i o n phenomenon i s of considerable  interest  since a l l previous attempts at s p l i t t i n g the very stable Hp complex had proved unsuccessful studies, i t was  (55).  Hb-  In the present  found that neither 4.0 M urea i n 0.2  M acetic  acid, pH 5.0,nor 8.0 M urea i n 0.1 M Tris-HCl buffer, pH  8.0,  were able to separate the complex into i t s components although strong urea i s usually able to dissociate non-covalently subunits.  The observed d i s s o c i a t i o n of the succinylated com-  plex i s analogous to that observed by Klotz and Nagy (250)  bound  Keresztes-  that e l e c t r o s t a t i c repulsions introduced by suc-  c i n y l a t i o n resulted i n complete d i s s o c i a t i o n 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 d e t e r g e n t .  That the Hb-Hp complex i s d i s s o c i a t e d by s u c c i n y l a t i o n confirms that covalent  bonds a r e not i n v o l v e d  i n complex f o r -  mation. Further studies  on the nature of the hemoglobin  s i t e were c a r r i e d out by the use of t h i s d i s s o c i a t i o n nique on the complex. dinated  tech-  The Hb^-Hp complex, which was guani-  with 0.2 M GDMP reagent d e s c r i b e d  i n a previous  s e c t i o n , and i n which 47.8% l y s i n e r e s i d u e s ted, was s u c c i n y l a t e d  binding  remained unreac-  f o r 2 hours by r e a c t i o n of 50.4 mg of  s u c c i n i c anhydride w i t h 10 mg of g u a n i d i n a t e d Hb-Hp complex i n 1 ml o f 0.01 M T r i s - H C l b u f f e r , pH 8.0.  After  desalting  on G-25 and l y o p h i l i z a t i o n , an a l i q u o t of the doubly-modif i e d complex was a p p l i e d t o a Sephadex G-200 column (1 x 50 cm) column i n 0.1 M phosphate b u f f e r , pH 7.0, t o d e t e r mine the e x t e n t 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 s u c c i n y l a t e d time w i t h 10.49 mg of s o l i d reagent f o r 2 hours.  a second A f t e r de-  s a l t i n g and l y o p h i l i z a t i o n , the d i s s o c i a t e d 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 b u f f e r , pH 7.0. A  Fractions  comprising peak  ( d i s s o c i a t e d haptoglobin) and those comprising peak B  ( d i s s o c i a t e d hemoglobin)  ( F i g . 32, frame C) were  combined and d i a l y z e d e x h a u s t i v e l y then l y o p h i l i z e d .  against  separately  distilled  water,  The s p l i t complex, peak A and peak B were  h y d r o l y z e d by a d d i t i o n of 0.4 ml of 6.0 N HC1 t o 2 mg of each  - 166 T  EFFLUENT F i g u r e 32.  ~1  1  ~T~  T  V O L U M E , ml  M o d i f i e d Hb-Hp on Sephadex G-200 i n 0.1 M phosphate-  b u f f e r , pH 7.0.  (A) Hb-Hp g u a n i d i n a t e d  with 0.2 M GDMP, 72 h r . ,  0°, pH 9.0 chromatographed on Sephadex G-200 (1 x 50 cm column). (B) Guanidinated  Hb-Hp complex s u c c i n y l a t e d 2 h r . , pH 8.0 on an  1 cm x 50 cm column, (C) S u c c i n y l a t e d and guanidinated Hb-Hp complex s u c c i n y l a t e d a f u r t h e r 2 h r . , pH 8.0, on an 1 cm x 87 cm column.  - 167 protein f r a c t i o n and heated at 105° for 18 hours i n evacuated ampoules.  After centrifuging to remove the black p r e c i p i -  tate, the hydrolyzates were dried under vacuum and then taken up i n 0.45 ml of pH 2.2 c i t r a t e buffer.  Aliquots of the hy-  drolyzates were analyzed i n 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 i s occurring in the molecule since a faster eluting peak i s separating from the complex i n the native conformation which has retained the bound hemoglobin.  However, modification with this rea-  gent has not caused any d i s s o c i a t i o n of the complex.  On  introduction of negative succinyl groups into this guanidinated Hb-Hp complex, considerable d i s s o c i a t i o n of the hemoglobin and haptoglobin components occurs (Fig. 32, frame B). Succinylation a second time carries this d i s s o c i a t i o n almost to completion  (Fig. 32, frame C).  Thus the r e l a t i v e l y mild  guanidination reaction has produced a conformational change i n the Hb-Hp complex molecule but d i s s o c i a t i o n i s not achieved u n t i l a high density of negative charges are i n t r o duced i n place of the p o s i t i v e charge. Determination of the homoarginine content of the components of the dissociated complex should give a further i n d i cation of the area of the binding s i t e or any conformational change of the molecules upon binding of hemoglobin by haptoglobin, again assuming a s t a t i s t i c a l d i s t r i b u t i o n of l y s y l residues i n the molecule.  I t i s expected that i n the complex  - 168  -  the l y s i n e r e s i d u e s i n the b i n d i n g s i t e would be and prevented  covered  from r e a c t i o n w i t h the GDMP reagent,  the  ex-  t e n t of p r o t e c t i o n being a measure of the area of c o n t a c t . Thus the decrease  i n homoarginine of the h a p t o g l o b i n  and  hemog T.orbin s p l i t from the complex as compared to the h a p t o g l o b i n and  the hemoglobin g u a n i d i n a t e d s e p a r a t e l y  should g i v e an i n d i c a t i o n of the approximate area of the b i n d i n g 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 f r e e p r o t e i n s and the bound p r o t e i n .  In Table  peak A, c o n t a i n i n g m o d i f i e d h a p t o g l o b i n d i s s o c i a t e d the complex, has of 52.9%  52.6%  XI,  from  homoarginine as compared to the amount  i n the f r e e g u a n i d i n a t e d h a p t o g l o b i n .  Comparison  of the amount of homoarginine i n the m o d i f i e d hemoglobin d e r i v e d from the complex, peak B, p o s s e s s i n g 51.0%, to the v a l u e of 58.3%, i n a g u a n i d i n a t e d hemoglobin w i t h the b i n d i n g s i t e exposed, shows a d i f f e r e n c e of about 7%. i n an average decrease  of 3.5%  This r e s u l t s  i n homoarginine content i n  the g u a n i d i n a t e d complex and agrees with the r e s u l t s  obtained  by comparison of the extent of g u a n i d i n a t i o n of the Hb-Hp complex w i t h a t h e o r e t i c a l l y c a l c u l a t e d v a l u e based on d i n a t i o n of f r e e hemoglobin and  free haptoglobin.  guani-  These  r e s u l t s can b e s t be e x p l a i n e d by the area of c o n t a c t between the p r o t e i n s being q u i t e l i m i t e d . DISCUSSIONS OF CHEMICAL MODIFICATION Three reagents  of i n c r e a s i n g s e v e r i t y have been used  f o r the chemical m o d i f i c a t i o n of h a p t o g l o b i n 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 GDMP, pH 9.0, 72 hrs, 0°  47.1  52.9  41.7  58.3  47.4 49.0  52.6 51.0  Hb guanidination with 0.2 M 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)_ peak B (Hb)  - 170  -  study the r o l e of amino groups i n the p r o t e i n . tageous  I t i s advan-  i n a study of chemical m o d i f i c a t i o n to use  several  reagents both as a check on the s p e c i f i c i t y of the a c t i o n toward  the d e s i r e d f u n c t i o n a l group and t o minimize  non-  s p e c i f i c s t r u c t u r a l changes or d e n a t u r a t i o n . The molecular conformation of the h a p t o g l o b i n molecule i s important i n m a i n t a i n i n g the proper o r i e n t a t i o n of the hemoglobin b i n d i n g s i t e .  Chemical m o d i f i c a t i o n of the pro-  t e i n by i n t r o d u c t i o n of new modify  c o v a l e n t l y bound groups may  so  the i n t r a m o l e c u l a r f o r c e s as t o cause r e o r g a n i z a t i o n  depending  upon the groups i n t r o d u c e d .  Habeeb (173)  the s e n s i t i v i t y of the e l u t i o n volume of a p r o t e i n  used molecule  on a Sephadex G-20 0 column to i t s Stokes r a d i u s t o e v a l u a t e c o n f o r m a t i o n a l changes a s s o c i a t e d with chemical m o d i f i c a t i o n of bovine serum albumin.  I t was  found t h a t  significant  changes i n asymmetry of the p r o t e i n molecule due to s u c c i n y l a t i o n and a c e t y l a t i o n o c c u r r e d , whereas the shape changes a s s o c i a t e d w i t h g u a n i d i n a t i o n were s m a l l . In the p r e s e n t s t u d i e s , Sephadex G-200 assays of the c h e m i c a l l y m o d i f i e d h a p t o g l o b i n s f o r hemoglobin b i n d i n g capac i t y a l s o served t o d e t e c t any u n f o l d i n g of the molecules which might a f f e c t the b i n d i n g a b i l i t y  non-specifically.  In agreement w i t h Habeeb s f i n d i n g s and as expected from the 1  m i l d chemical nature of the s u b s t i t u t i o n , even a t an o v e r a l l c o n v e r s i o n of about groups  82% of the amino groups to guanidino  (Table V I I ) , o n l y a s m a l l p o r t i o n ,  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 a l t e r a t i o n i n the hemoglobin binding capacity (Table VII). In the acetylation reaction, i n which there i s a r a t i o of a c e t i c anhydride to haptoglobin of 80:1, corresponding to just over an equimolar r a t i o of reagent to lysine residues, the reaction would be expected to be s p e c i f i c a l l y directed towards the amino groups (238) .  In this case, there i s no  s i g n i f i c a n t conformational change when almost 50% of the posit i v e charges have been replaced with neutral groups and the hemoglobin binding a b i l i t y i s reduced only s l i g h t l y VIII).  (Table  With a large excess of acetic anhydride reagent, a  greater portion of the molecule unfolds (Fig. 20, form D) and i n this form, the haptoglobin shows much reduced binding. The remainder of the molecules with normal e l u t i o n volume are 70% modified o v e r a l l and now show less than 50% of the binding (Table VIII).  The reduced binding observed at the high-  est l e v e l of acetylation may be due to more extensive reaction with groups other than the amino groups or undetected physicochemical  changes i n the molecule which a f f e c t the  hemoglobin binding property.  I t i s u n l i k e l y that this reduced  binding i s due to the loss of a f u n c t i o n a l l y important  lysine  residue since an extensive guanidination of the lysines d i d not influence the binding.  I t could not be the loss of  - 172. p o s i t i v e l y charged groups at the higher l e v e l of acetylation that d i r e c t l y influenced the binding capacity since binding experiments i n strong s a l t solutions showed that the e l e c t r o s t a t i c interactions are not of prime importance. In the succinylation reaction, s t r u c t u r a l changes i n the haptoglobin molecule are immediately evident both from the gel chromatography p r o f i l e s (Fig. 22) and the pherograms (Fig. 23 and 24) .  SG-electro-  Tire:, l a t t e r show that a f t e r 1  ten minutes succinylation two components migrating more slowly than control haptoglobin  are formed.  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