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Effect of degradation on the chemical and biological properties of haptoglobin II. Cleavage of disulphide bonds
338
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
35 o15
E F F E C T OF D E G R A D A T I O N ON T H E CHEMICAL AND BIOLOGICAL P R O P E R T I E S OF H A P T O G L O B I N II. CLEAVAGE OF D I S U L P H I D E BONDS* E L W I R A LISOWSKA AND WANDA DOBRYSZYCKA
Department of Biochemistry, Institute of Immunology and Experimental Therapy and Departmenl of Scientific Investigation of the Medical Academy of Wroclaw, Wroclaw (Poland) (Received June 6th, 1966 ) (Revised manuscript received September 7th, 1966)
SUMMARY
Haptoglobin2_ 1 was separated into e and/3 chains by means of mercaptoethanol reduction with subsequent iodoacetamide alkylation, and b y S-sulphonation with sulphite in the presence of Cu 2+. The hexosamine and sialic acid contents of the separated chains were determined, as well as biological activities, namely the capacity to form with haemoglobin a complex having the properties of a "true" peroxidase and the ability to inhibit influenza virus haemagglutination. Separated chains were found not to bind with haemoglobin. /3 chain, containing all the carbohydrate part of the haptoglobin molecule, was a stronger inhibitor of haemagglutination than the native haptoglobin. After cleavage of the disulphide bonds of haptoglobin performed without separation of chains, partial recombination was obtained. Some physicochemical and biological properties of such a rearranged haptoglobin in comparison with those of the native form were examined. Under the experimental conditions used, solubility and electrophoretic mobility were changed, the sedimentation coefficient increased from 6.18 to 17. 9 and the free thiol group content increased from o.41 to 7.65 per mole. Rearranged haptoglobin was not able to bind haemoglobin but it was found to be a stronger inhibitor of haemagglutination than the native haptoglobin.
INTRODUCTION
Haptoglobin (Hp) is known to bind with haemoglobin (Hb) and the complex formed shows catalytic activity of true peroxidase type 1. As with other mucoproteins, H p was found to be an inhibitor of the haemagglutination caused by influenza virusL The object of the present study was to characterize a peptide or glycopeptide chain portion indispensable for the formation of a H b complex having the properties of a true peroxidase and also able to inhibit influenza virus haemagglutination. Abbreviations: Hp, haptoglobin; NANA, N-acetylneuraminic acid; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid). * A preliminary report of this work was presented at the Third Meeting of Federation of European Biochemical Societies, W~arsaw, April, 1966.
Biochim. Biophys. Acta, i33 (i967) 338-345
CLEAVAGE OF DISULPHIDE BONDS OF HAPTOGLOBIN
339
A study of the effects of chemical modification of a protein molecule or its degradation can yield valuable information on the relationship between structure and function if the site of modification is known. In the previous paper 2, tryptic digestion was chosen as one kind of degradation of the Hp molecule; in the present work, disulphide bonds were the site of modification. Cystine residues may form the interchain bonds between different peptide chains, or there may be intrachain bonds holding together different parts of a single chain. A molecule of HpI-1 evidently consists of four polypeptide chains held together by interchain bonds in unknown positions s. Each molecule comprises a pair of similar or identical ~ chains having an approximate mol. wt. of 9ooo and another pair of ~ chains of mol. wt. 36ooo (see ref. 4). The disulphide bonds of Hp seem to offer an opportunity for an additional approach to the problem of degradation of the molecule with consequent effects on the physicochemical and biological properties. In the present investigation Hps_ 1 was separated into s~ and ~ chains by two methods, namely 2-mercaptoethanol reduction with iodoacetamide alkylation, and S-sulphonation with sulphite in the presence of Cu 2+ ions. Moreover, attempts were made to reconstitute Hp from the reduced fragments by oxidation with air. The effects of cleavage of the disulphide bonds and of their recombination on some physicochemical properties--on the ability to activate Hb and on the inhibition of haemagglutinins--were examined. MATERIALS AND METHODS
Materials Hydrogen peroxide was obtained from AB Ferrosan, Malm6 (Sweden) ; Sephadex G-25 and Sephadex G-Ioo from Pharmacia, Uppsala (Sweden); 2-mercaptoethanol was a Koch-Light product; iodoacetamide, twice crystallized, was synthesized by one of us. The Lee strain (B) and A2-Wroclaw influenza viruses and chicken erythrocytes were kindly supplied by Professor SKURSKA and 5,5'-dithiobis-(2-nitrobenzoic acid) by Dr. M. WOLNY. Aseitic fluids used for the preparation of Hp were obtained from patients of the Third Clinic of Internal Diseases of the Medical Academy in Wroclaw. Hp was prepared from ascitic fluid by precipitation with ammonium sulphate, by chromatography on DEAL-cellulose and gel filtration on Sephadex G-2oo as described in the previous paper s. Analytical methods Hp was determined by JAYLE'S method 1, utilizing the specific peroxidase activity of the H p - H b complex formed. Protein was assayed by the absorbance at 278 m/~, taking 1.16 as the extinction coefficient for Hp2_ 1 (see ref. 5), and by the method of LOWRY et al. 6. Hexosamine was determined according to RONDLE AND MORGAN7 after hydrolysis of the material with 0.5 M HC1 for 16 h at IOO°. NANA was estimated according to SVENNERHOLM8 by the resorcinol method. For the determinations of the inhibitory activity against influenza virus haemagglutinins, the L E E strain (B) or A~-Wroclaw influenza viruses, inactivated by 3 ° min incubation at 56°, were used. Virus haemagglutinin inhibitory assays were carried out according to TAMM AND H O R S F A L L 9, using chicken erythrocytes. One Biochim. Biophys. Acta, 133 (1967) 338-345
340
E. LISOWSKA, W. DOBRYSZYCKA
haemagglutinating unit is that amount of virus which causes partial agglutination of o.025 ml 5 % (v/v) chicken erythrocytes. Reductions were carried out b y two procedures. Reduction by 2-mercaptoethanol with subsequent alkylation was done according to GHIM AND BEARN3. About IOO mg of Hp2_ 1 was dissolved in 4 ml of borate buffer (pH 8.7) (0.5 M H3B0 3 and 0.2 M NaOH diluted with water, 1:2). Urea and 2-mereaptoethanol were added up to final concentrations of 8 M and 0.5 M, respectively. The mixture was allowed to stand overnight in the refrigerator, and iodoacetamide in borate buffer was added up to a final concentration of I M. Alkylation proceeded for 3 h at 4 ° in darkness. Reduction without alkylation was carried out according to SMITHIES1°. About 5 mg of Hp~_ 1 was dissolved in borate buffer (pH 8.7) (o.i M H~B03, 0.004 M EDTA and o.04 M NaOH) with 40 mM of 2-mercaptoethanol. After incubation for I h at 37 °, the samples were submitted to starch-gel electrophoresis with and without Hb, or were dialysed overnight at 4 ° against o.oi M acetate buffer (pH 4.7) and against borate buffer (pH 8.7) until the precipitate which had formed was completely dissolved. S-sulphonation was carried out according to FRAN~_K AND ZIK/~N11. About 200 mg of Hp~_ 1 was dissolved in 32 ml of ammonia buffer (pH 8.6) containing 2 ml of o.I M CuSO 4. 8 ml of the buffer containing 1.26 g Na2SO 3 was added and the mixture was allowed to stand overnight at room temperature; it was then applied to a Sephadex G-25 column equilibrated with 0.25 M ammonium carbonate (pH 7-8). Blue-coloured fractions containing copper salts were well separated from protein fractions; absorbance was measured at 280 my. Protein fractions were pooled and lyophilized; the yield of the preparation was about 19o rag. Thiol groups were determined colorimetrically b y means of DTNB as suggested b y ELLlVIAN12. The disulphide exchange between DTNB and SH groups yields reduced DTNB which has an intense yellow colour when ionized. A molar absorbance coefficient of 1.36. lO4 at 412 m/~ for the reduced DTNB was used. Since bases destroy DTNB, producing an orange-yellow coloured product, solutions of DTNB were prepared in o.I M phosphate buffer (pH 8.6). Each sample contained I mg of Hp, 15o t~moles of Tris (pH 7.8), IO t~moles of EDTA and I / , m o l e of DTNB with or without 12 mmoles of urea in a volume of 5 ml. The mixtures were incubated at 30 °. Velocity sedimentation measurements were made in the Phyw6 ultracentrifuge. The rotor speed was 400o0 rev./min (117000 × g). RESULTS
Properties of the chains of Hp separated by sulphitolysis and by reduction After sulphitolysis by means of sulphite in the presence of Cu 2+, lO5 mg of desalted and lyophilized product was applied on a Sephadex G-ioo column (2.3 cm × 30 cm) equilibrated with 0.05 M formic acid and 6 M urea. A typical elution pattern is shown in Fig. I. Three peaks were obtained in this fractionation. The yield as measured by the method of LowRY et al. Gwas as follows: 46 mg was recovered in the fl fraction, 18 mg in the a and 5 mg in the a' fraction. Two fractions which accounted for 50 % of fl fraction were pooled. The fractions comprising individual peaks were lyophilized, desalted b y gel filtration on a column of Sephadex G-25 and once more lyophilized. Biochim. Biophys. Acta, 133 (1967) 338-3¢5
341
CLEAVAGE OF DISULPHIDE BONDS OF HAPTOGLOBIN
The products of 2-mgrcaptoethanol reduction followed b y the alkylation with iodoacetamide were separated on a column of Sephadex G-Ioo equilibrated with 0.05 M formic acid containing 6 M urea. The resulting elution pattern is shown in Fig. 2. 2.0'
0 ~t 1.0
o
1.0"
10
2b
3b
4'0 Fraction
•
..50
-
r
"
lb
60
number
3b F r a c t i o n
,,:o
.~o
number
Fig. t. Gel f i l t r a t i o n d i a g r a m of S - s u l p h o n a t e d H p on S e p h a d e x G - i o o c o l u m n in 6 M u r e a a n d 0.o 5 M fo rmic acid. S - S u l p h o n a t i o n w a s c a r r i e d o u t w i t h s u l p h i t e in t h e p r e s e n c e of Cu 2+ a s d e s c r i b e d u n d e r METHODS. Hp2_ 1 w a s used. The l o a d w a s lO 5 m g of s u l p h o n a t e d produc t • Fig. 2. Gel f i l t r a t i o n d i a g r a m of r e d u c e d a n d a l k y l a t e d H p on S e p h a d e x G - i o o in 6 M u r e a a n d 0.05 M fo rmic acid. R e d u c t i o n w i t h 2 - m e r c a p t o e t h a n o l w a s c a r r i e d o u t a c c o r d i n g t o SHIM AND BEARN 3, t h e n a l k y l a t i o n w i t h i o d o a c e t a m i d e p r o c e e d e d 3 h a t 4 ° in d a r k n e s s a t a n i o d o a c e t a m i d e c o n c e n t r a t i o n of I M. Hp2_ 1 w a s used. 15o m g of r e d u c e d a n d a l k y l a t e d p r o d u c t w a s loaded•
As can be seen, the pattern obtained after fractionation of the products of H p reduction is very similar to that obtained b y sulphitolysis. One major and two minor fractions were obtained. Two minor fractions were collected together. Individual fractions were lyophilized, desalted b y gel filtration on Sephadex G-25 and finally lyophilized. Carbohydrate contents and biological properties of the fractions obtained after sulphitolysis, and those after reduction of Hp, were determined and compared with those of native Hp. The results of the determinations are assembled in Table I. The NANA contents of both the fl fractions are higher than that of the native Hp. The ~ fractions contain neither NANA nor hexosamine. Inhibitory haemagglutination activity as determined with two different strains of viruses, namely strain L E E (B) and A2-Wroclaw influenza viruses, gave identical results. The fractions devoid of carbohydrates did not show any inhibitory activity, while fl fractions prepared by reduction or b y sulphitolysis showed stronger inhibitory activity than the native Hp. Neither fraction activated Hb, as measured by the peroxidatic method of J A Y L E 1 and b y means of starch-gel electrophoresis.
Reformation of Hp after cleavage of disulphide bonds After disulphide bonds of H p had been broken, attempts were made to reconstitute the protein into its original form. Isolated ~ and fl chains of H p were dissolved in phosphate buffer of p H 7.0; 2 mg of fl chain and I mg of ~ chain were incubated for 16 h at 37 °, after which the sample did not show any peroxidase activity when H b was added. I t was then decided to reverse reduction without prior separation of the chains and without alkylation. Biochim. Biophys. Acta, 133 (I967) 3 3 8 - 3 4 5
342
E. LISOWSKA, W. DOBRYSZYCKA
TABLE I CARBOHYDRATE CONTENTS A N D /5 C H A I N S
AND BIOLOGICAL
ACTIVITIES
O]~ H A P T O G L O B I N
AND
SEPARATED
HP2 1 w a s used. Q u a n t i t a t i v e d e t e r m i n a t i o n s of N A N A a n d h e x o s a m i n e were as d e s c r i b e d u n d e r METHODS. The i n h i b i t o r y a c t i v i t y a g a i n s t influenza v i r u s L E E (B) a n d A 2-Wroc l a w v i r u s is g i v e n as t h e m i n i m a l a m o u n t (/,g) t h a t c o m p l e t e l y i n h i b i t s 4 h a e m a g g l u t i n a t i n g u n i t s of v i r u s (see MXTHODS). A c t i v e H p w a s m e a s u r e d b y t h e p e r o x i d a s e m e t h o d of JAYLE 1 a n d t h e r e s u l t c o m p a r e d w i t h t h e p r o t e i n c o n t e n t of t h e s a m p l e as d e t e r m i n e d s p e c t r o p h o t o n l e t r i c a l l y . S e p a r a t i o n of isol a t e d ~ a n d / 5 c h a i n s a f t e r r e d u c t i o n or S - s u l p h o n a t i o n w a s c a r r i e d out on S e p h a d e x G -Ioo equil i b r a t e d w i t h 0.o 5 M formic acid a n d 6 M urea.
Material
NA NA (%)
Hexosamine (%)
A etive Hp (per cent of the protein content)
Viral inhibitory activity (/,g per 4 haemagglutinating units)
Haptoglobin S-sulphonation : /5 c h a i n chain
3.9
4.7
IOO
o. i
4 .6 o
5.5 o
o o
4.6
5.3
IOO
6.2 o
5.9 o
o o
Haptoglobin Reduction : fl c h a i n c¢ c h a i n
o.o25 3.2 o.2 0.05 6.25
TABLE II THIOL GROUPS AND BIOLOGICAL ACTIVITIES OF REFORMED HAPTOGLOBIN AND REFORMED /5 CHAIN Hp2_ 1 w a s used. H p or s u l p h o n a t e d / 3 c h a i n were r e d u c e d w i t h 40 m m o l e s of 2 - m e r c a p t o e t h a n o l a t 37 ° for i h in b o r a t e buffer (pH 8.7). The s a m p l e s w e re d i a l y s e d o v e r n i g h t a g a i n s t o.oi M a c e t a t e buffer (pH 4-7) t h e n a g a i n s t b o r a t e buffer (p H 8.7) u n t i l c o m p l e t e d i s s o l u t i o n of t h e p r e c i p i t a t e f o r m e d occurred. The p r o t e i n is called " r e f o r m e d " . The c o m p o s i t i o n of t h e s a m p l e s for S H - g r o u p d e t e r m i n a t i o n s w a s as follows : I m g of H p or/3 chain, 15o/*moles of Tris (pH 7.8), i o / , m o l e s E D T A , I /,mole of D T N B , 12 m m o l e s of u r e a in a v o l u m e of 5 ml. The m i x t u r e s were i n c u b a t e d a t 3 °0 for 45 min. D e t e r m i n a t i o n s of b i o l o g i c a l a c t i v i t i e s as in T a b l e I.
Material
SH groups (moles~mole
Viral inhibitory activity (#g per 4 haemagglutinating units)
Active Hp (per cent of the protein content of the sample)
o.41 7.65
o.i 0.05
ioo o
o.22 3-75
o.oi 0.007
of lip*)
Haptoglobin Reformed Hp fl-chain o b t a i n e d by S-sulphonation R e f o r m e d fl-chain
o o
* C a l c u l a t e d for mol. wt. of 85 ooo.
5 mg of HP2-1 was reduced with 4 ° mM of 2-mercaptoethanol at pH 8. 7 and 37 ° for I h. After this time the sample was monitored by starch-gel electrophoresis and it was shown that reduced Hp did not bind any Hb. The sample was submitted to dialysis overnight at 4 ° against o.oi M acetate buffer (pH 4.7) followed by dialysis against borate buffer (pH 8.7). During this last step the precipitate formed in acidic medium was completely dissolved. The product obtained was examined for Hb binding Eiochim. Biophys. Acta, 133 (1967) 333-345
CLEAVAGE OF DISULPHIDE BONDS OF HAPTOGLOBIN
343
and haemagglutinins inhibition. Moreover, it was submitted to ultracentrifugation and to paper electrophoresis. Amounts of SH groups, in comparison with the native Hp, were determined. The same procedure was applied to the isolated /~ fraction obtained by sulphitolysis of Hp. Some properties of H p and ]3 chain material modifieated in this way are shown in Table II. The molar amounts of SH groups, as determined in Hp2-1 used in this experiment, were found to be o and 0.41 when measured with urea, while after reduction and recombination the amount increased to 7.65 per mole of Hp (calculated for the molecular weight of 85 ooo). A similar increase was observed when sulphonated ]~ chain was reduced and then partially oxidized. 3.75 moles of SH groups were found in comparison with 0.22 mole obtained with isolated /3 chain. These reconstituted compounds lost the capacity to bind Hb as measured by the peroxidase method as well as by starch-gel electrophoresis, but inhibition of influenza virus haemagglutinins was stronger, both in reconstituted H p and in reconstituted/~ chain. It should be pointed out that the native Hp is soluble over a broad pH range, while the reconstituted protein appears to be completely insoluble in o.oi M acetate buffer (pH 4.7). Ultracentrifugation of a 1% solution of Hp2_ 1 showed a single boundary with a sedimentation coefficient of 6.18, while reformed Hp was found to have a sedimentation coefficient of 17. 9 S. On electrophoresis in o.I M phosphate buffer (pH 6.6), Hp migrated as a single band while reformed Hp in the same conditions showed a diffused pattern and only 5 % of the protein applied on paper migrated with an electrophoretic mobility similar to that of the native Hp. DISCUSSION
The elution patterns reported above for Hp treated with 2-mercaptoethanol, as well as those obtained after sulphitolysis are very similar. One peak comprising twice as much protein as two other peaks was obtained. The large peak is evidently the ~ chain, but it was surprising to find two different peaks for the a chains. SHIM AND BEARNa isolated ~ and/3 chains of HpI_ 1 and found only two peaks. The present experiments described above were carried out by the same method, but with Hp2_ 1. It is known 4 that while Hpl_ 1 and Hp2_ 2 are composed of equal amounts of ~ and /3 chains, Hp2_ 1 represents another kind of polymerisation of the same chains; artificial mixture of types i - i and 2-2 does not result in 2-1 type. It seems likely that in the molecule of Hp2-1 there exist two types of e chains differing slightly in molecular weight. Gel filtration on Sephadex G-ioo makes possible their separation. Isolated chains did not bind any Hb. This fact was not due to denaturation caused by urea because the chains prepared without urea were also unable to combine with Hb. If the disulphide bonds are broken and then reformed, one would expect with a high probability that the protein would be reconstituted into its original form, especially when all the disulphide bonds concerned are purely intrachain ones~3,~. ROHOLT, RADZIMSKIAND P1RESSMAN15 demonstrated that mixing of H and L chains from a pool of antibodies from several rabbits can give a large recovery of effective sites, and the chains from different rabbits combine without forming such sites. These facts signify that the combinations of chains which give effective sites must be preferred over other combinations. Biochim. Biophys. Acta, 133 (1967) 338-345
344
E. LISOWSKA, W. DOBR¥SZYCKA
Incubation of alkylated ~ and fi chains of H p did not result in recombination to the original form, and for this reason, the experimental conditions for reversal of reduction or sulphitolysis were chosen. The evidence presented indicates that reconstituted H p was formed with completely changed physicochemical and biological properties. This has been found for solubility, electrophoretic mobility, sedimentation coefficient, for the amount of free SH groups, the capacity of H b binding and for the inhibition of virus haemagglutinins. CLOAREC16 found for H p l _ 1 15 SH groups, WAKS AND ALFSEN17 for H p i - 1 and for Hp2_ 2 found 16 SH groups. This accounts for 7-8 disulphide bonds and at least three of them occupy interchain positions. It seems evident that there is no free SH group in Hp, the DTNB-reduced content being about 0.4 mole per molecule and 0.22 mole per/3 chain. This amount increased to 7.65 when, after reduction and reoxidation by exposure to air, the protein seemed to be reformed. Nearly half of the thiol groups formed under the conditions described were found in fl chain. The fact that only 7.65 out of 16 SH groups was found is due to the conditions of the experiment. Partial oxidation caused reformation of about 4 disulphide bonds. Results of ultracentrifugation, when the change of sedimentation coefficient from 6.18 to 17. 9 was found, indicate that an aggregation occurred which is quite different from polymerisation of c~ and fl chains to a structure characteristic for the genetic type of Hp2_ 1. After cleavage of the disulphide bonds of Hp, both isolated chains and the reformed protein lost the property of H b binding. The evidence presented seems to indicate that a part of the H p molecule indispensable for the formation of an active complex with H b might contain interchain bonds. SHIM, LEE AND KANG4 suggested that H p is bound with H b in the fl chain and that two Hb-binding sites for the (~ fi) 2 subunits of H p exist, each for one-half of the Hb molecule. Since, in the previous work 2, only one out of four glycopeptides obtained after tryptic digestion of H p was found to have Hb-binding capacity, it seems likely that this glycopeptide might have in its molecule this "core" containing interchain bonds indispensable for H b binding and formation of an active complex. The data presented here give further support for the role of NANA residues in inhibitory haemagglutinins properties and this is in accord with present general opinion. In the previous paper ~, NANA-free glycopeptide, the product of tryptic digestion of Hp, was found to be an inhibitor of haemagglutination. It has been suggested that other sugars could replace NANA in the combination of influenza virus with H p and with the particulate cellular receptors at the surface of the red blood cell. However, the glycopeptide devoid of NANA was found to contain about 4 % of carbohydrate in its molecule. In the present paper, ~ chains of H p which do not contain any NANA are not found to be inhibitors of haemagglutination, while fl chains containing the carbohydrate part of the H p molecule are stronger inhibitors than the native Hp. Reformed H p was found to exhibit higher inhibitory activity than native Hp. It is known is that neuraminidase removes only 7 ° % of NANA from the Hp molecule while the remaining 30 % sited inside the molecule is not accessible to the action of the enzyme. A possible conclusion is that the cleavage of disulphide bonds of H p results in unfolding of the chains so that the whole carbohydrate part becomes accessible; the additional, newly accessible, NANA probably causes an increase of inhibition of haemagglutinins of the reformed in contrast with the original Hp. On the other hand, an aggregation of H p and increase in the size of the molecule Biochim. Biophys. Acta, 133 (1967) 338-345
CLEAVAGE OF DISULPHIDE BONDS OF HAPTOGLOBIN
345
could cause an increase in inhibitory properties, on the same principle that polymerised orosomucoid has higher inhibitory activity than the native form, as described b y MORAWIECKI AND LISOWSKA19. ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k P r o f e s s o r Dr. T. BARANOWSKI f o r his i n t e r e s t a n d e n c o u r a g e m e n t . T h e y e x p r e s s t h e i r g r a t i t u d e t o Dr. A. MORAWIECKI f o r m a k i n g a v a i l a b l e t h e u l t r a c e n t r i f u g e , t o P r o f e s s o r Dr. Z. SKURSKA for s u p p l y i n g v i r u s e s a n d t o Dr. M. WOL~Y for t h e g e n e r o u s g i f t of D T N B . T h e e x c e l l e n t t e c h n i c a l a s s i s t a n c e of Miss 1V[. DUK is g r a t e f u l l y a c k n o w l e d g e d .
REFERENCES M. F. JAYLE, Bull. Soc. Chim. Biol., 33 (1951) 876. W. DOBRYSZYCKAAND E. LISOWSKA, Biochim. Biophys. Acta, i2I (1966) 42. B. S. SHIM AND A. G. BEARN, J. Exptl. Med., 12o (1964) 611. B. S. SHIM, T. H. LEE ANn Y. S, KANG, Nature, 207 (1965) 1264. L. CLOAREC, Contribution ~ l't~tude Physico-Chimique des Haptoglobines Humaines, R. Foulon, Paris, 1964, p. 6. 6 0 . H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265. 7 C. J. M. RONDLE AND W. T. J. MORGAN, Biochem. J., 61 (1955) 586. 8 L. SVENNERHOLM, Biochim. Biophys. Acta, 24 (1957) 604. 9 I. TAMM AND F. L. HORSFALL, J. Exptl. Med., 95 (1952) 71. IO O. SMITHIES, Science, 15o (1965) 1595. I i F. FRAN~K AND J. ZIKf~N, Collection Czech. Chem. Commun., 29 (1964) 14Ol. 12 G. L. ELLMAN,Arch. Biochem. Biophys., 82 (1959) 7O. 13 G. H. DIXON AND A. C. WARDLAW,Nature, 188 (196o) 721. 14 F. H. WHITE, JR., ./. Biol. Chem., 236 (1961) 1353. 15 O. A. ROHOLT, G. RADZlMSKI AND D. PRESSMAN,Science, 147 (1965) 613. 16 L. CLOAREC, Compt. Rend., 257 (1963) 983 . 17 M. WAKS AND A. ALFSEN, Arch. Biochem. Biophys., 113 (1966) 304 . 18 L. CLOAREC, Contribution ~ l't2tude Physico-Chimique des Haptoglobines Humaines, R. Foulon, Paris, 1964, p. 5 o. 19 A. MORAWlECKI AND E. LISOWSKA, Biochem. Biophys. Res. Commun., 18 (1965) 6o6. i 2 3 4 5
Biochim. Biophys. Acta, 133 (1967) 338-345
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
35 o15
E F F E C T OF D E G R A D A T I O N ON T H E CHEMICAL AND BIOLOGICAL P R O P E R T I E S OF H A P T O G L O B I N II. CLEAVAGE OF D I S U L P H I D E BONDS* E L W I R A LISOWSKA AND WANDA DOBRYSZYCKA
Department of Biochemistry, Institute of Immunology and Experimental Therapy and Departmenl of Scientific Investigation of the Medical Academy of Wroclaw, Wroclaw (Poland) (Received June 6th, 1966 ) (Revised manuscript received September 7th, 1966)
SUMMARY
Haptoglobin2_ 1 was separated into e and/3 chains by means of mercaptoethanol reduction with subsequent iodoacetamide alkylation, and b y S-sulphonation with sulphite in the presence of Cu 2+. The hexosamine and sialic acid contents of the separated chains were determined, as well as biological activities, namely the capacity to form with haemoglobin a complex having the properties of a "true" peroxidase and the ability to inhibit influenza virus haemagglutination. Separated chains were found not to bind with haemoglobin. /3 chain, containing all the carbohydrate part of the haptoglobin molecule, was a stronger inhibitor of haemagglutination than the native haptoglobin. After cleavage of the disulphide bonds of haptoglobin performed without separation of chains, partial recombination was obtained. Some physicochemical and biological properties of such a rearranged haptoglobin in comparison with those of the native form were examined. Under the experimental conditions used, solubility and electrophoretic mobility were changed, the sedimentation coefficient increased from 6.18 to 17. 9 and the free thiol group content increased from o.41 to 7.65 per mole. Rearranged haptoglobin was not able to bind haemoglobin but it was found to be a stronger inhibitor of haemagglutination than the native haptoglobin.
INTRODUCTION
Haptoglobin (Hp) is known to bind with haemoglobin (Hb) and the complex formed shows catalytic activity of true peroxidase type 1. As with other mucoproteins, H p was found to be an inhibitor of the haemagglutination caused by influenza virusL The object of the present study was to characterize a peptide or glycopeptide chain portion indispensable for the formation of a H b complex having the properties of a true peroxidase and also able to inhibit influenza virus haemagglutination. Abbreviations: Hp, haptoglobin; NANA, N-acetylneuraminic acid; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid). * A preliminary report of this work was presented at the Third Meeting of Federation of European Biochemical Societies, W~arsaw, April, 1966.
Biochim. Biophys. Acta, i33 (i967) 338-345
CLEAVAGE OF DISULPHIDE BONDS OF HAPTOGLOBIN
339
A study of the effects of chemical modification of a protein molecule or its degradation can yield valuable information on the relationship between structure and function if the site of modification is known. In the previous paper 2, tryptic digestion was chosen as one kind of degradation of the Hp molecule; in the present work, disulphide bonds were the site of modification. Cystine residues may form the interchain bonds between different peptide chains, or there may be intrachain bonds holding together different parts of a single chain. A molecule of HpI-1 evidently consists of four polypeptide chains held together by interchain bonds in unknown positions s. Each molecule comprises a pair of similar or identical ~ chains having an approximate mol. wt. of 9ooo and another pair of ~ chains of mol. wt. 36ooo (see ref. 4). The disulphide bonds of Hp seem to offer an opportunity for an additional approach to the problem of degradation of the molecule with consequent effects on the physicochemical and biological properties. In the present investigation Hps_ 1 was separated into s~ and ~ chains by two methods, namely 2-mercaptoethanol reduction with iodoacetamide alkylation, and S-sulphonation with sulphite in the presence of Cu 2+ ions. Moreover, attempts were made to reconstitute Hp from the reduced fragments by oxidation with air. The effects of cleavage of the disulphide bonds and of their recombination on some physicochemical properties--on the ability to activate Hb and on the inhibition of haemagglutinins--were examined. MATERIALS AND METHODS
Materials Hydrogen peroxide was obtained from AB Ferrosan, Malm6 (Sweden) ; Sephadex G-25 and Sephadex G-Ioo from Pharmacia, Uppsala (Sweden); 2-mercaptoethanol was a Koch-Light product; iodoacetamide, twice crystallized, was synthesized by one of us. The Lee strain (B) and A2-Wroclaw influenza viruses and chicken erythrocytes were kindly supplied by Professor SKURSKA and 5,5'-dithiobis-(2-nitrobenzoic acid) by Dr. M. WOLNY. Aseitic fluids used for the preparation of Hp were obtained from patients of the Third Clinic of Internal Diseases of the Medical Academy in Wroclaw. Hp was prepared from ascitic fluid by precipitation with ammonium sulphate, by chromatography on DEAL-cellulose and gel filtration on Sephadex G-2oo as described in the previous paper s. Analytical methods Hp was determined by JAYLE'S method 1, utilizing the specific peroxidase activity of the H p - H b complex formed. Protein was assayed by the absorbance at 278 m/~, taking 1.16 as the extinction coefficient for Hp2_ 1 (see ref. 5), and by the method of LOWRY et al. 6. Hexosamine was determined according to RONDLE AND MORGAN7 after hydrolysis of the material with 0.5 M HC1 for 16 h at IOO°. NANA was estimated according to SVENNERHOLM8 by the resorcinol method. For the determinations of the inhibitory activity against influenza virus haemagglutinins, the L E E strain (B) or A~-Wroclaw influenza viruses, inactivated by 3 ° min incubation at 56°, were used. Virus haemagglutinin inhibitory assays were carried out according to TAMM AND H O R S F A L L 9, using chicken erythrocytes. One Biochim. Biophys. Acta, 133 (1967) 338-345
340
E. LISOWSKA, W. DOBRYSZYCKA
haemagglutinating unit is that amount of virus which causes partial agglutination of o.025 ml 5 % (v/v) chicken erythrocytes. Reductions were carried out b y two procedures. Reduction by 2-mercaptoethanol with subsequent alkylation was done according to GHIM AND BEARN3. About IOO mg of Hp2_ 1 was dissolved in 4 ml of borate buffer (pH 8.7) (0.5 M H3B0 3 and 0.2 M NaOH diluted with water, 1:2). Urea and 2-mereaptoethanol were added up to final concentrations of 8 M and 0.5 M, respectively. The mixture was allowed to stand overnight in the refrigerator, and iodoacetamide in borate buffer was added up to a final concentration of I M. Alkylation proceeded for 3 h at 4 ° in darkness. Reduction without alkylation was carried out according to SMITHIES1°. About 5 mg of Hp~_ 1 was dissolved in borate buffer (pH 8.7) (o.i M H~B03, 0.004 M EDTA and o.04 M NaOH) with 40 mM of 2-mercaptoethanol. After incubation for I h at 37 °, the samples were submitted to starch-gel electrophoresis with and without Hb, or were dialysed overnight at 4 ° against o.oi M acetate buffer (pH 4.7) and against borate buffer (pH 8.7) until the precipitate which had formed was completely dissolved. S-sulphonation was carried out according to FRAN~_K AND ZIK/~N11. About 200 mg of Hp~_ 1 was dissolved in 32 ml of ammonia buffer (pH 8.6) containing 2 ml of o.I M CuSO 4. 8 ml of the buffer containing 1.26 g Na2SO 3 was added and the mixture was allowed to stand overnight at room temperature; it was then applied to a Sephadex G-25 column equilibrated with 0.25 M ammonium carbonate (pH 7-8). Blue-coloured fractions containing copper salts were well separated from protein fractions; absorbance was measured at 280 my. Protein fractions were pooled and lyophilized; the yield of the preparation was about 19o rag. Thiol groups were determined colorimetrically b y means of DTNB as suggested b y ELLlVIAN12. The disulphide exchange between DTNB and SH groups yields reduced DTNB which has an intense yellow colour when ionized. A molar absorbance coefficient of 1.36. lO4 at 412 m/~ for the reduced DTNB was used. Since bases destroy DTNB, producing an orange-yellow coloured product, solutions of DTNB were prepared in o.I M phosphate buffer (pH 8.6). Each sample contained I mg of Hp, 15o t~moles of Tris (pH 7.8), IO t~moles of EDTA and I / , m o l e of DTNB with or without 12 mmoles of urea in a volume of 5 ml. The mixtures were incubated at 30 °. Velocity sedimentation measurements were made in the Phyw6 ultracentrifuge. The rotor speed was 400o0 rev./min (117000 × g). RESULTS
Properties of the chains of Hp separated by sulphitolysis and by reduction After sulphitolysis by means of sulphite in the presence of Cu 2+, lO5 mg of desalted and lyophilized product was applied on a Sephadex G-ioo column (2.3 cm × 30 cm) equilibrated with 0.05 M formic acid and 6 M urea. A typical elution pattern is shown in Fig. I. Three peaks were obtained in this fractionation. The yield as measured by the method of LowRY et al. Gwas as follows: 46 mg was recovered in the fl fraction, 18 mg in the a and 5 mg in the a' fraction. Two fractions which accounted for 50 % of fl fraction were pooled. The fractions comprising individual peaks were lyophilized, desalted b y gel filtration on a column of Sephadex G-25 and once more lyophilized. Biochim. Biophys. Acta, 133 (1967) 338-3¢5
341
CLEAVAGE OF DISULPHIDE BONDS OF HAPTOGLOBIN
The products of 2-mgrcaptoethanol reduction followed b y the alkylation with iodoacetamide were separated on a column of Sephadex G-Ioo equilibrated with 0.05 M formic acid containing 6 M urea. The resulting elution pattern is shown in Fig. 2. 2.0'
0 ~t 1.0
o
1.0"
10
2b
3b
4'0 Fraction
•
..50
-
r
"
lb
60
number
3b F r a c t i o n
,,:o
.~o
number
Fig. t. Gel f i l t r a t i o n d i a g r a m of S - s u l p h o n a t e d H p on S e p h a d e x G - i o o c o l u m n in 6 M u r e a a n d 0.o 5 M fo rmic acid. S - S u l p h o n a t i o n w a s c a r r i e d o u t w i t h s u l p h i t e in t h e p r e s e n c e of Cu 2+ a s d e s c r i b e d u n d e r METHODS. Hp2_ 1 w a s used. The l o a d w a s lO 5 m g of s u l p h o n a t e d produc t • Fig. 2. Gel f i l t r a t i o n d i a g r a m of r e d u c e d a n d a l k y l a t e d H p on S e p h a d e x G - i o o in 6 M u r e a a n d 0.05 M fo rmic acid. R e d u c t i o n w i t h 2 - m e r c a p t o e t h a n o l w a s c a r r i e d o u t a c c o r d i n g t o SHIM AND BEARN 3, t h e n a l k y l a t i o n w i t h i o d o a c e t a m i d e p r o c e e d e d 3 h a t 4 ° in d a r k n e s s a t a n i o d o a c e t a m i d e c o n c e n t r a t i o n of I M. Hp2_ 1 w a s used. 15o m g of r e d u c e d a n d a l k y l a t e d p r o d u c t w a s loaded•
As can be seen, the pattern obtained after fractionation of the products of H p reduction is very similar to that obtained b y sulphitolysis. One major and two minor fractions were obtained. Two minor fractions were collected together. Individual fractions were lyophilized, desalted b y gel filtration on Sephadex G-25 and finally lyophilized. Carbohydrate contents and biological properties of the fractions obtained after sulphitolysis, and those after reduction of Hp, were determined and compared with those of native Hp. The results of the determinations are assembled in Table I. The NANA contents of both the fl fractions are higher than that of the native Hp. The ~ fractions contain neither NANA nor hexosamine. Inhibitory haemagglutination activity as determined with two different strains of viruses, namely strain L E E (B) and A2-Wroclaw influenza viruses, gave identical results. The fractions devoid of carbohydrates did not show any inhibitory activity, while fl fractions prepared by reduction or b y sulphitolysis showed stronger inhibitory activity than the native Hp. Neither fraction activated Hb, as measured by the peroxidatic method of J A Y L E 1 and b y means of starch-gel electrophoresis.
Reformation of Hp after cleavage of disulphide bonds After disulphide bonds of H p had been broken, attempts were made to reconstitute the protein into its original form. Isolated ~ and fl chains of H p were dissolved in phosphate buffer of p H 7.0; 2 mg of fl chain and I mg of ~ chain were incubated for 16 h at 37 °, after which the sample did not show any peroxidase activity when H b was added. I t was then decided to reverse reduction without prior separation of the chains and without alkylation. Biochim. Biophys. Acta, 133 (I967) 3 3 8 - 3 4 5
342
E. LISOWSKA, W. DOBRYSZYCKA
TABLE I CARBOHYDRATE CONTENTS A N D /5 C H A I N S
AND BIOLOGICAL
ACTIVITIES
O]~ H A P T O G L O B I N
AND
SEPARATED
HP2 1 w a s used. Q u a n t i t a t i v e d e t e r m i n a t i o n s of N A N A a n d h e x o s a m i n e were as d e s c r i b e d u n d e r METHODS. The i n h i b i t o r y a c t i v i t y a g a i n s t influenza v i r u s L E E (B) a n d A 2-Wroc l a w v i r u s is g i v e n as t h e m i n i m a l a m o u n t (/,g) t h a t c o m p l e t e l y i n h i b i t s 4 h a e m a g g l u t i n a t i n g u n i t s of v i r u s (see MXTHODS). A c t i v e H p w a s m e a s u r e d b y t h e p e r o x i d a s e m e t h o d of JAYLE 1 a n d t h e r e s u l t c o m p a r e d w i t h t h e p r o t e i n c o n t e n t of t h e s a m p l e as d e t e r m i n e d s p e c t r o p h o t o n l e t r i c a l l y . S e p a r a t i o n of isol a t e d ~ a n d / 5 c h a i n s a f t e r r e d u c t i o n or S - s u l p h o n a t i o n w a s c a r r i e d out on S e p h a d e x G -Ioo equil i b r a t e d w i t h 0.o 5 M formic acid a n d 6 M urea.
Material
NA NA (%)
Hexosamine (%)
A etive Hp (per cent of the protein content)
Viral inhibitory activity (/,g per 4 haemagglutinating units)
Haptoglobin S-sulphonation : /5 c h a i n chain
3.9
4.7
IOO
o. i
4 .6 o
5.5 o
o o
4.6
5.3
IOO
6.2 o
5.9 o
o o
Haptoglobin Reduction : fl c h a i n c¢ c h a i n
o.o25 3.2 o.2 0.05 6.25
TABLE II THIOL GROUPS AND BIOLOGICAL ACTIVITIES OF REFORMED HAPTOGLOBIN AND REFORMED /5 CHAIN Hp2_ 1 w a s used. H p or s u l p h o n a t e d / 3 c h a i n were r e d u c e d w i t h 40 m m o l e s of 2 - m e r c a p t o e t h a n o l a t 37 ° for i h in b o r a t e buffer (pH 8.7). The s a m p l e s w e re d i a l y s e d o v e r n i g h t a g a i n s t o.oi M a c e t a t e buffer (pH 4-7) t h e n a g a i n s t b o r a t e buffer (p H 8.7) u n t i l c o m p l e t e d i s s o l u t i o n of t h e p r e c i p i t a t e f o r m e d occurred. The p r o t e i n is called " r e f o r m e d " . The c o m p o s i t i o n of t h e s a m p l e s for S H - g r o u p d e t e r m i n a t i o n s w a s as follows : I m g of H p or/3 chain, 15o/*moles of Tris (pH 7.8), i o / , m o l e s E D T A , I /,mole of D T N B , 12 m m o l e s of u r e a in a v o l u m e of 5 ml. The m i x t u r e s were i n c u b a t e d a t 3 °0 for 45 min. D e t e r m i n a t i o n s of b i o l o g i c a l a c t i v i t i e s as in T a b l e I.
Material
SH groups (moles~mole
Viral inhibitory activity (#g per 4 haemagglutinating units)
Active Hp (per cent of the protein content of the sample)
o.41 7.65
o.i 0.05
ioo o
o.22 3-75
o.oi 0.007
of lip*)
Haptoglobin Reformed Hp fl-chain o b t a i n e d by S-sulphonation R e f o r m e d fl-chain
o o
* C a l c u l a t e d for mol. wt. of 85 ooo.
5 mg of HP2-1 was reduced with 4 ° mM of 2-mercaptoethanol at pH 8. 7 and 37 ° for I h. After this time the sample was monitored by starch-gel electrophoresis and it was shown that reduced Hp did not bind any Hb. The sample was submitted to dialysis overnight at 4 ° against o.oi M acetate buffer (pH 4.7) followed by dialysis against borate buffer (pH 8.7). During this last step the precipitate formed in acidic medium was completely dissolved. The product obtained was examined for Hb binding Eiochim. Biophys. Acta, 133 (1967) 333-345
CLEAVAGE OF DISULPHIDE BONDS OF HAPTOGLOBIN
343
and haemagglutinins inhibition. Moreover, it was submitted to ultracentrifugation and to paper electrophoresis. Amounts of SH groups, in comparison with the native Hp, were determined. The same procedure was applied to the isolated /~ fraction obtained by sulphitolysis of Hp. Some properties of H p and ]3 chain material modifieated in this way are shown in Table II. The molar amounts of SH groups, as determined in Hp2-1 used in this experiment, were found to be o and 0.41 when measured with urea, while after reduction and recombination the amount increased to 7.65 per mole of Hp (calculated for the molecular weight of 85 ooo). A similar increase was observed when sulphonated ]~ chain was reduced and then partially oxidized. 3.75 moles of SH groups were found in comparison with 0.22 mole obtained with isolated /3 chain. These reconstituted compounds lost the capacity to bind Hb as measured by the peroxidase method as well as by starch-gel electrophoresis, but inhibition of influenza virus haemagglutinins was stronger, both in reconstituted H p and in reconstituted/~ chain. It should be pointed out that the native Hp is soluble over a broad pH range, while the reconstituted protein appears to be completely insoluble in o.oi M acetate buffer (pH 4.7). Ultracentrifugation of a 1% solution of Hp2_ 1 showed a single boundary with a sedimentation coefficient of 6.18, while reformed Hp was found to have a sedimentation coefficient of 17. 9 S. On electrophoresis in o.I M phosphate buffer (pH 6.6), Hp migrated as a single band while reformed Hp in the same conditions showed a diffused pattern and only 5 % of the protein applied on paper migrated with an electrophoretic mobility similar to that of the native Hp. DISCUSSION
The elution patterns reported above for Hp treated with 2-mercaptoethanol, as well as those obtained after sulphitolysis are very similar. One peak comprising twice as much protein as two other peaks was obtained. The large peak is evidently the ~ chain, but it was surprising to find two different peaks for the a chains. SHIM AND BEARNa isolated ~ and/3 chains of HpI_ 1 and found only two peaks. The present experiments described above were carried out by the same method, but with Hp2_ 1. It is known 4 that while Hpl_ 1 and Hp2_ 2 are composed of equal amounts of ~ and /3 chains, Hp2_ 1 represents another kind of polymerisation of the same chains; artificial mixture of types i - i and 2-2 does not result in 2-1 type. It seems likely that in the molecule of Hp2-1 there exist two types of e chains differing slightly in molecular weight. Gel filtration on Sephadex G-ioo makes possible their separation. Isolated chains did not bind any Hb. This fact was not due to denaturation caused by urea because the chains prepared without urea were also unable to combine with Hb. If the disulphide bonds are broken and then reformed, one would expect with a high probability that the protein would be reconstituted into its original form, especially when all the disulphide bonds concerned are purely intrachain ones~3,~. ROHOLT, RADZIMSKIAND P1RESSMAN15 demonstrated that mixing of H and L chains from a pool of antibodies from several rabbits can give a large recovery of effective sites, and the chains from different rabbits combine without forming such sites. These facts signify that the combinations of chains which give effective sites must be preferred over other combinations. Biochim. Biophys. Acta, 133 (1967) 338-345
344
E. LISOWSKA, W. DOBR¥SZYCKA
Incubation of alkylated ~ and fi chains of H p did not result in recombination to the original form, and for this reason, the experimental conditions for reversal of reduction or sulphitolysis were chosen. The evidence presented indicates that reconstituted H p was formed with completely changed physicochemical and biological properties. This has been found for solubility, electrophoretic mobility, sedimentation coefficient, for the amount of free SH groups, the capacity of H b binding and for the inhibition of virus haemagglutinins. CLOAREC16 found for H p l _ 1 15 SH groups, WAKS AND ALFSEN17 for H p i - 1 and for Hp2_ 2 found 16 SH groups. This accounts for 7-8 disulphide bonds and at least three of them occupy interchain positions. It seems evident that there is no free SH group in Hp, the DTNB-reduced content being about 0.4 mole per molecule and 0.22 mole per/3 chain. This amount increased to 7.65 when, after reduction and reoxidation by exposure to air, the protein seemed to be reformed. Nearly half of the thiol groups formed under the conditions described were found in fl chain. The fact that only 7.65 out of 16 SH groups was found is due to the conditions of the experiment. Partial oxidation caused reformation of about 4 disulphide bonds. Results of ultracentrifugation, when the change of sedimentation coefficient from 6.18 to 17. 9 was found, indicate that an aggregation occurred which is quite different from polymerisation of c~ and fl chains to a structure characteristic for the genetic type of Hp2_ 1. After cleavage of the disulphide bonds of Hp, both isolated chains and the reformed protein lost the property of H b binding. The evidence presented seems to indicate that a part of the H p molecule indispensable for the formation of an active complex with H b might contain interchain bonds. SHIM, LEE AND KANG4 suggested that H p is bound with H b in the fl chain and that two Hb-binding sites for the (~ fi) 2 subunits of H p exist, each for one-half of the Hb molecule. Since, in the previous work 2, only one out of four glycopeptides obtained after tryptic digestion of H p was found to have Hb-binding capacity, it seems likely that this glycopeptide might have in its molecule this "core" containing interchain bonds indispensable for H b binding and formation of an active complex. The data presented here give further support for the role of NANA residues in inhibitory haemagglutinins properties and this is in accord with present general opinion. In the previous paper ~, NANA-free glycopeptide, the product of tryptic digestion of Hp, was found to be an inhibitor of haemagglutination. It has been suggested that other sugars could replace NANA in the combination of influenza virus with H p and with the particulate cellular receptors at the surface of the red blood cell. However, the glycopeptide devoid of NANA was found to contain about 4 % of carbohydrate in its molecule. In the present paper, ~ chains of H p which do not contain any NANA are not found to be inhibitors of haemagglutination, while fl chains containing the carbohydrate part of the H p molecule are stronger inhibitors than the native Hp. Reformed H p was found to exhibit higher inhibitory activity than native Hp. It is known is that neuraminidase removes only 7 ° % of NANA from the Hp molecule while the remaining 30 % sited inside the molecule is not accessible to the action of the enzyme. A possible conclusion is that the cleavage of disulphide bonds of H p results in unfolding of the chains so that the whole carbohydrate part becomes accessible; the additional, newly accessible, NANA probably causes an increase of inhibition of haemagglutinins of the reformed in contrast with the original Hp. On the other hand, an aggregation of H p and increase in the size of the molecule Biochim. Biophys. Acta, 133 (1967) 338-345
CLEAVAGE OF DISULPHIDE BONDS OF HAPTOGLOBIN
345
could cause an increase in inhibitory properties, on the same principle that polymerised orosomucoid has higher inhibitory activity than the native form, as described b y MORAWIECKI AND LISOWSKA19. ACKNOWLEDGEMENTS T h e a u t h o r s t h a n k P r o f e s s o r Dr. T. BARANOWSKI f o r his i n t e r e s t a n d e n c o u r a g e m e n t . T h e y e x p r e s s t h e i r g r a t i t u d e t o Dr. A. MORAWIECKI f o r m a k i n g a v a i l a b l e t h e u l t r a c e n t r i f u g e , t o P r o f e s s o r Dr. Z. SKURSKA for s u p p l y i n g v i r u s e s a n d t o Dr. M. WOL~Y for t h e g e n e r o u s g i f t of D T N B . T h e e x c e l l e n t t e c h n i c a l a s s i s t a n c e of Miss 1V[. DUK is g r a t e f u l l y a c k n o w l e d g e d .
REFERENCES M. F. JAYLE, Bull. Soc. Chim. Biol., 33 (1951) 876. W. DOBRYSZYCKAAND E. LISOWSKA, Biochim. Biophys. Acta, i2I (1966) 42. B. S. SHIM AND A. G. BEARN, J. Exptl. Med., 12o (1964) 611. B. S. SHIM, T. H. LEE ANn Y. S, KANG, Nature, 207 (1965) 1264. L. CLOAREC, Contribution ~ l't~tude Physico-Chimique des Haptoglobines Humaines, R. Foulon, Paris, 1964, p. 6. 6 0 . H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL,J. Biol. Chem., 193 (1951) 265. 7 C. J. M. RONDLE AND W. T. J. MORGAN, Biochem. J., 61 (1955) 586. 8 L. SVENNERHOLM, Biochim. Biophys. Acta, 24 (1957) 604. 9 I. TAMM AND F. L. HORSFALL, J. Exptl. Med., 95 (1952) 71. IO O. SMITHIES, Science, 15o (1965) 1595. I i F. FRAN~K AND J. ZIKf~N, Collection Czech. Chem. Commun., 29 (1964) 14Ol. 12 G. L. ELLMAN,Arch. Biochem. Biophys., 82 (1959) 7O. 13 G. H. DIXON AND A. C. WARDLAW,Nature, 188 (196o) 721. 14 F. H. WHITE, JR., ./. Biol. Chem., 236 (1961) 1353. 15 O. A. ROHOLT, G. RADZlMSKI AND D. PRESSMAN,Science, 147 (1965) 613. 16 L. CLOAREC, Compt. Rend., 257 (1963) 983 . 17 M. WAKS AND A. ALFSEN, Arch. Biochem. Biophys., 113 (1966) 304 . 18 L. CLOAREC, Contribution ~ l't2tude Physico-Chimique des Haptoglobines Humaines, R. Foulon, Paris, 1964, p. 5 o. 19 A. MORAWlECKI AND E. LISOWSKA, Biochem. Biophys. Res. Commun., 18 (1965) 6o6. i 2 3 4 5
Biochim. Biophys. Acta, 133 (1967) 338-345