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Effect of modification on physicochemical and biological properties of haptoglobin III. Antigenic reactivity of haptoglobin with blocked tyrosine or tryptophan residues
EFFECT
OF MODIFICATION
PROPERTIES III.
ANTIGENIC
TYROSINE
ON PHYSICOCHEMICAL
AND BIOLOGICAL
OF HAPTOGLOBIN REACTIVITY
OR TRYPTOPHAN
OF HAPTOGLOBIN
WITH
BLOCKED
RESIDUES
SUMMARY
Exposed tyrosine residues of human haptoglobin (Hp) were acetylated by means of N-acetylimidazole. z-Hydroxy-5-nitrobenzyl (HNB) groups were introduced on exposed tryptophan residues. Native Hp and the two types of modified Hp were used to immunize rabbits. The obtained antisera were studied in cross-reactions with the respective antigens. From quantitative precipitin curves antibodies elicited by native Hp were found to react in a similar way with the homologous antigen as with acetylated haptoglobin (AC-HP). There was no reaction with HNB-Hp. Antibodies directed against AC-HP gave the strongest reaction with the homologous antigen, the precipitin curve with native Hp showed half the height, and a very low curve was obtained with HNB-Hp. A high degree of specificity was shown by antiserum directed against HNB-Hp which reacted only with its homologous antigen. In immunoprecipitation, native Hp and AC-HP yielded antibodies in similar amounts while HNB-Hp was a less effective immunogen. It seems likely that the changes in the Hp molecule caused by the introduction of HNB groups greatly alter the antigenic structure of Hp. The changes produced by the acetylation of tyrosine residues of Hp are less drastic. AC-HP still remains closely related to native protein. Possible reasons for the observed facts may be given: the character of the group introduced on haptoglobin, conformational changes of the protein, and modification of the hvdrophobic area on complementary sites of antigen and antibody.
Abbreviations: Hp, haptoglobin; dues acetylated by Xacetylimidazole; haptoglobin with tryptophan residues
Hb, hemoglobin; XC-HP, haptoglobm with tyrosine rcsiHXB, r-hydroxy-5.nitrobcnzyl bromide ; HXB-Hp, blocl~cd with z-hydroxy-5.nitrobenzyl groups.
MODIFICATION OF HAPTOGLOBIN
I79
INTRODUCTION
Chemical modification of proteins with concurrent observation of functional changes has been widely used to implicate particular functional groups in the activity of enzymes and other proteins (refs. 1-4). Certain activities or reactions of proteins, e.g. enzymic, antigenic or antibody, are predominantly associated with their unique native conformation. Haptoglobin (Hp), an a2-acid glycoprotein of serum, binds hemoglobin (Hb) stoichiometrically and irreversibly 5. The complex can be considered to be a "true" peroxidase. In our previous papers 6-s the effect of degradation of the Hp molecule on some physicochemical and biological properties of Hp were studied. In addition, certain aspects of the role of exposed and buried tryptophan and tyrosine residues in the formation of an active Hp-Hb complex were observed after selective modification of these residues 9. The purpose of this investigation is to present data concerning the changes in antigenic reactivity of human Hp that accompany its modification by the use of specific reagents. A previous study indicated that exposed tyrosine or tryptophan residues were essential for the formation of the H p - H b complex 9. In the present study we have been interested in the question whether antibodies to the "active site" of Hp could be produced in rabbits by injecting them with Hp modified as follows: tyrosine residues acetylated with N-acetylimidazole 1°, or chromophoric groups of 2-hydroxy5-nitrobenzyl bromide (HNB) introduced on tryptophan residues 11. An interesting problem to be studied is the specificity of antibodies formed by an antigen modified at the tyrosine or tryptophan residues. Two factors at least may play a role in these reactions: a direct result of the residue of the protein to be modified, and a conformational change induced by the respective group. MATERIALS AND METHODS
N-acetylimidazole was purchased from K and K Laboratories, Plainview, N.Y. (U.S.A.); HNB from Sigma Chem. Co. (U.S.A.); Sephadex G-25 was a Pharmacia (Sweden) product. Hb was prepared from horse erythrocytes by the method of McQUARRIE AND BENIAMSTM. Human Hp 2-1 and I - I were isolated from ascitic fluid as described elsewhere 6. Preparations that showed a purity of over 90% were lyophilised and used for modification.
Modifications Acetylation with N-acetylimidazole was performed at room temperature in o.oi M Tris buffer, pH 7.5. The reaction was allowed to proceed for I h. A 6o-fold molar excess of N-acetylimidazole over protein was employed as described by RIORDAN et al. 1°. The modified protein was separated from the excess reagent on a Sephadex G-25 column and lyophilised. The number of modified tyrosyl residues in Hp was estimated from the decrease in absorbance at 278 nm; for these calculations a molar differential extinction coefficient at 279 nm of 116o obtained on O-acetylation of N-acetyltyrosine was used. For the blockage of tryptophan residues the procedure of HORTON AND KOSHLAND11 was used. Hp dissolved in o.ooi M HC1 was treated with a 5o-fold molar Biochirn. Biophys. Acta, 243 (1971) 178-186
180
\\2 I){}I¢IC(SZV('I,L\, I. F,I.21;
excess of H N B in acetone. After 3o rain tile protein was separated from the excess reagent b y gel filtration on a Sephadex G-25 column and lyophilised. The n u m b e r of modified t r y p t o p h a n residues in H p was estimated in o.i M NaOH at 4 i o n m using a molar e x t i n c t i o n coefficient of I8 9oo. A ntisera Antibodies directed against n a t i v e Hp, Ac-Hp a n d H N B - H p were raised in rabbits, each r a b b i t being given about IO mg of the respective a n t i g e n in the foot pad, i n t r a v e n o u s l y a n d i n t r a m u s c u l a r l y . Antisera from i n d i v i d u a l animals were obt a i n e d a p p r o x i m a t e l y 4 weeks after the initial injection. Analytical methods H a p t o g l o b i n was d e t e r m i n e d b y tile peroxidase m e t h o d of JAYLE 5. Double diffusion in 1% Difco agar in 0.05 M veronal buffer at p H 8.2 was performed b y the m e t h o d of OUCHTERLONYla. Immunoelectrophoresis was carried out with 0.05 M veronal buffer, p H 8.6, for 45 rain at 7 V/era. After immunodiffusion for 48 l] at 20 °, the slides were washed in 0.9% NaC1 a n d in distilled water, dried in open air a n d stained for protein with Amido Black. Q u a n t i t a t i v e precipitin reactions were carried out as described b y ZSCHOCKE AND BEZKOROVAINY14. A n t i g e n protein solutions of a p p r o x i m a t e l y 0.05% in o.I M Tris-HC1 buffer, p H 8.0, were added to o.5-ml aliquots of antiserum. Volumes were b r o u g h t to i.o ml with the above Tris buffer, a n d the precipitates were collected b y centrifugation after an i n c u b a t i o n at 37 ° for 60 rain, a n d at 4 ° for 48 h. Precipitates were washed 3 times with o.15 M NaC1, dissolved in o.I M N a O H a n d subjected to protein d e t e r m i n a t i o n b y the t a n n i n m i c r o m e t h o d 15.
RESULTS Characteristics of the o b t a i n e d modifications of H p are given in Table I. The ability of H p to form a stable complex with H b was used to estimate the a m o u n t of f u n c t i o n a l protein recovered with the respective modifications, assuming TABLE I EFFECT
OF T H E M O D I F I C A T I O N
OF E X P O S E D
TYROSINE
OR T R Y P T O P H A N
RESIDUES
OF H p
ON T H E
A C T I V A T I O N OF H b
Mean values obtained for at least three preparations are given. Peroxidase activity was measured as described by JAYLE5. The number of acetylated tyrosine residues was calculated using a molar differential extinction coefficient at 279 nm of 116o (ref. io); the number of modified tryptophan residues was estimated in o. i M NaOH at 4to nm, using a molar extinction coefficient of I8 900 (ref. II). Preparation
Residues modified per mole of Hp*
Peroxidase activity of the complex with Hb (%)
Native Hp Ac-Hp HNB-Hp
13 9
lOO i6.i o
*
Calculated for a mol.wt, of 85 ooo. Human Hp 2-i was used.
Biochim. Biophys. dcta, 243 (1971) 178-186
MODIFICATION OF HAPTOGLOBIN
181
Fig. I. I m m u n o r e a c t i v i t y of native Hp, Ac-Hp, and H N B - H p . a. I n the center well, anti-native H p 2-1 s e r u m ; in the outer wells: I, H p I - I ; 2, H N B - H p ; 3, Ac-Hp; 4, H p 2-1; each in a concentration of o.o5%, b. Ill the center well, antinative H p serum; in the outer wells, a 2°,~, solution of H N B - H p . c. I n the center well, a n t i s e r u m to Ac-Hp; in the outer wells: i, H N B - H p ; 2, H p ; 3, A c - H p ; 4, A c - H p ; 5, H p ; each in o.O5~o solution, d. In the center well, a n t i s e r u m to Ac-Hp; in the outer wells, a I % solution of Hp. e. I n the center well, a n t i s e r u m to H N B - H p ; in the o u t e r wells: i, H N B - H p ; 2, A c - H p ; 3, H N B - H p ; 4, H p ; 5, H N B - H p ; each in o.o5% solution.
Biochim. Biophys. Acta, 243 (1971) 178 186
182
W. I)OBRVSZYCKA, I. BK("
'ii~ii~i!!iiiiiiiiiiiii!iiii
Fig. 2. hmnunoelectrophoresis of native Hp, Ac-Hp, and HNB-Hp. a. Antiserum to native Hp in the trough; upper well, Hp; lower well, Ac-Hp. b. Antiserum to Ac-Hp in the trough; upper well, Hp; lower well, Ac-Hp. c. Antiserum to HNB-Hp in the trough; in the wells, HNB-Hp.
the p e r o x i d a s e a c t i v i t y of the complex of n a t i v e H p with H b to be lOO%. After t h e blockage of nine exposed t r y p t o p h a n residues with H N B , a complete loss of a c t i v i t y was observed, while after a c e t y l a t i o n of 13 exposed tyrosine residues 16.1% of t h e original p e r o x i d a s e a c t i v i t y was found. R a b b i t s were i m m u n i z e d with n a t i v e Hp, A c - H p a n d H N B - H p . Six r a b b i t s i m m u n i z e d with n a t i v e H p r e s p o n d e d at a regular time, in comparison with five out of six r a b b i t s i m m u n i z e d with A c - H p , and only two out of six i m m u n i z e d with H N B - H p , respectively. Biochim. Biophys. Acta, 243 (1971) i78-186
183
MODIFICATION OF HAPTOGLOBIN
The effect of the modifications on the formation of antibodies and on the precipitin reaction was studied with the obtained antisera to Hp, Ac-Hp and HNBHp. The results of double gel diffusion experiments are shown in Fig. I. As can be seen in Fig. Ia, antiserum to H p 2-1 (the center well) gave distinct precipitin lines with Hp 2-1, H p i - I , Ac-Hp and a very faint line with HNB-Hp. In order to make this latter combination visible, H N B - H p was applied at a concentration of 2 g/Ioo nil, i.e. 4 ° times higher than the concentrations of Hp and Ac-Hp used in this experiment (Fig. ib). When the center well contained antiserum to Ac-Hp (Fig. IC), definite precipitation arcs developed with Ac-Hp and a very faint one with native Hp; no line was formed with HNB-Hp. Precipitin arcs of anti-Ac-Hp serum with native Hp were made visible when applying Hp at a higher concentration (Fig. Id). Antiserum to H N B - H p reacted only with its homologous antigen, i.e. H N B - H p (Fig. Ie). Results of immunoelectrophoresis are shown in Yig. 2. In Fig. 2a, antiserum to Hp 2-1 was put in the trough. The arc obtained with Ac-Hp is slightly diffused and displaced toward the anode as compared with the parallel arc formed with native Hp. A similar pattern developed when antiserum to Ac-Hp was placed in the trough, against Ac-Hp and native Hp (Fig. 2b). In Fig. 2c the arcs obtained in the reaction of antiserum to H N B - H p with its homologous antigen, i.e. HNB-Hp, are shown.
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Fig. 3. Q u a n t i t a t i v e precipitin curves of native Hp, Ac-Hp, and H N B - H p with the respective antisera raised in rabbits. A. A n t i s e r u m to native Hp. B. Antiserum to Ac-Hp. C. A n t i s e r u m to H N B - H p . The following antigens were used: native H p ( A - - A ) , Ac-Hp ( 0 . . . . . O), and I t N B - H p (O - - - O).
Fig. 3 represents quantitative precipitin curves involving native Hp, Ac-Hp, H N B - H p and specific antisera. In Fig. 3A, antiserum to Hp 2-1 was used against Hp 2-1, Ac-Hp and HNB-Hp. Antibodies directed against Hp 2-1 reacted similarly with native Hp and Ac-Hp, precipitating m a x i m u m antibody protein at the same antigen concentration of o.15/~g. However, at the peak, native H p precipitated 0.26/~g of antibodies while Ac-Hp precipitated 0.2o #g. The curve obtained with H N B - H p was almost flat with a very weak m a x i m u m at o.I/~g of the antigen, precipitating o.o3 ~g of antibody protein. Fig. 3 B represents precipitin curves obtained with antiserum to Ac-Hp. This antiserum showed the highest precipitating efficiency with its homologous antigen, i.e. with Ac-Hp, at a concentration of o.23/~g. The peak obtained with native Hp was half that obtained with Ac-Hp. With this antiserum H N B - H p showed a slight m a x i m u m at o.o5-o.1/~g, precipitating o.o6/~g of antibody protein. Biochim. Biophys. Acla, 243 (1971) 178-186
18 4
w. I)OBRYSZYCKA, I. BEC
Antiserum to H N B - H p showed a high degree of specificity, giving a distinct precipitation curve only with H N B - H p (Fig. 3C). The peak was attained at a concentration of o.z #g of antigen, precipitating o.~5 #g of antibodv protein. Native Hp and Ac-Hp did not react with antiserum to HNB-Hp. DISCUSSION
The approach of chemical modification for probing the nature of antigenic sites in proteins is extremely valuable, especially if the specificity of the reagent is well established and the derivative is purified and characterized. N-acetylimidazole has been shown by RIORDAN el el. 1° to be a relatively selective acylating reagent which preferentially acylates exposed tyrosyl groups, although amino groups and cysteine sulthydryl groups can also react. The extent of the reaction of N-acetylimidazole with e-amino groups has been found to be different for various proteins and usually much lower than in the case of other acylating reagents. For example, acetic anhydride blocked 68% of the amino groups of carboxypeptidase A, 57% of Hb, and 9o% of ovomucoid, respectively; however, N-acetylimidazole failed to acetylate the groups of carboxypeptidase A, acetylated only 3% of the amino groups of Hb, and H % of ovomucoid 1°. The possibility of acetylation of eamino groups of Hp was taken into consideration in one of our previous papersK However, the number of eight exposed tyrosyl residues as determined by the use of N-acetylimidazole was very similar to that of seven found by spectrophotometric titration; also the number of 28 total tyrosyl residues determined by N-acetylimidazole was very similar to that found by spectrophotometric titration, i.e. 28 29, and to that of 27-28 moles/mole of Hp obtained by amino acid analysis. Hp does not contain free cysteine groupsT; hence the possibility of the reaction of N-acetylimidazole with free SH groups would be rather improbable. It has been shown that H N B reacts specifically with tryptophan residues in proteins, especially when the latter do not contain any free SH groups u, as is the case with Hp. In comparison, the determination of tryptophan residues in canine Hp showed 12 moles/mole of Hp when using HNB, ZL 5 spectrophotometrically, and 9Io by amino acid analysis, respectively 9. When complexed with Hb, Hp behaves as an enzyme and we took advantage of this property in order to define the actual amount of a functional protein with the native conformation remaining after a modification. The preparation of canine Hp modified at exposed tryptophan or tyrosine residues ° or the analogous human Hp derivatives of the present work, indicated that such a procedure resulted in a significant depression of the ability to activate Hb in the case of acetylation, or in a complete loss of this ability when using HNB. In the present paper it has been shown that tyrosine or tryptophan residues modified in the above manner differ in their reactivity toward respective antibodies as well as in cross-reactions with antibodies produced to native or modified protein. It is well known that certain modifications m a y profoundly affect the function of a protein without greatly altering its antigenic structure. For example, there was no correlation between the enzymatic activity and immunoreactivity of maleylphosphofructokinasO 6, and even greater antigenic reactivity than in native albumin was found in conformationally altered albumin aT. In contrast, certain immunological Iliochin~. Biophys. dcla, 243 (I97 x) I 7 8 - 1 8 6
MODIFICATION OF HAPrOGLOBIN
18 5
techniques can distinguish between proteins differing by a single amino acid is. ATASSI AND CARUSO19 found that apomyoglobin modified with H N B at tryptophan- 7 did not show any alterations in antigenic reactivity, and when modified also at t r y p t o p h a n - i 4, showed a decrease in antigenic reactivity, most likely caused by unfolding or a marked deviation from the spherical shape. Native Hp and 8 M urea-treated Hp with unfolded polypeptide chains were immunochemically very similar, although they differed significantly in the peroxidase activity of the complex with Hb (our unpublished data). This fact demonstrates that the native conformation of the Hp molecule, which is of great importance in the formation of the active complex with Hb, does not play an essential role in antigenic reactivity. Acetylation of exposed tyrosine residues of Hp resulted in a decrease to onesixth of tile peroxidase activity of the complex with Hb. Antibodies elicited by Ac-Hp were directed mainly against the homologous antigen. The reaction with native Hp was much weaker, and with H N B - H p almost absent. It is very interesting that the quantitative precipitin curves of anti-native Hp serum with native Hp and with Ac-Hp are quite similar. This immunological relationship between native Hp and Ac-Hp suggests that either tyrosine residues are not an essential part of the antigenic site of Hp, or that the shape of tile acetyl group on the exposed tyrosine residue does not represent a steric hindrance for the antigen antibody reaction. This problem will be studied further in this laboratory by the introduction of different groups on tyrosine residues of Hp. A rather high degree of immunological specificity was shown by the Hp derivative modified by the use of HNB at exposed tryptophan residues. This Hp derivative is devoid of the ability to activate Hb. Antibodies directed against H N B - H p reacted only with their homologous antigen. In immunoprecipitation, native Hp and Ac-Hp yielded antibodies in similar amounts, while in this regard H N B - H p was less effective. HABEEB2° pointed out that the nitrotyrosyl residues of a protein are more immunogenic than tyrosyl residues, and nitroguanyl residues more than guanyl residues. In our experiments, the nitro group of HNB did not enhance the immunogenicity of modified Hp. In contrast, native Hp and Ae-Hp were far better immunogens than HNB-Hp. It has been suggested by BENJAMINIe~ al. 2~ that a hydrophobic area enhances a possible hydrophobic interaction between the specific sterically complementary areas on the antigen and antibody sites, respectively. This implies that the antibody site contains hydrophobic areas which interact with the hydrophobic area(s), or in their proximity, of the antigenic determinant. The apparent affinity of HNB for hydrophobic residues in proteins is well known. In proteins the HNB group yields indolenine adducts suffering a number of fates depending on the juxtaposition of such nucleophiles as serine or tyrosine hydroxyls, lysine amino groups, etc. 22. Are the differences in antigenic reactivity of native Hp, Ac-Hp and H N B - H p observed in this work due solely to changes in the hydrophobic nature of areas on the antigen and antibody sites ? Such an explanation would be highly improbable. The shape of both groups introduced on Hp, and conformational changes of the protein should be taken into consideration. It seems likely that the changes in the Hp molecule caused by the introduction of HNB groups greatly alter the antigenic structure of Hp. New antigenic determiBiochim. Biophys. Acta,
243 (1971) 178 t86
i86
W. DOBRYSZYCKA, I. BEC
n a n t s could be f o r m e d w h i c h would h a r d l y be d e t e c t e d by an a n t i s e r u m p r e p a r e d against n a t i v e H p or Ac-Hp. The changes p r o d u c e d by the a c e t v l a t i o n of tvrosine r e s i d u e s are less d r a s t i c : t h e y d o n o t c h a n g e t h e a b i l i t i e s o f t h e a n i m a l s t o r e c o g n i z e a n a r e a as a n t i g e n i c or t o s y n t h e s i z e a n t i b o d y a r e a s c o m p l e m e n t a r y t o t h e a n t i g e n i c area. M o d i f i e d in t h i s w a y , t h e H p m o l e c u l e still r e m a i n s closely r e l a t e d t o t h e n a t i v e p r o t e i n . H o w e v e r , it is n o t p o s s i b l e a t t h i s s t a g e t o d e t e r m i n e w h e t h e r a n t i g e n i c s p e c i f i c i t y o f t h e H N B d e r i v a t i v e o f H p is c a u s e d b y a c h a n g e in t h e h y d r o p h o b i c a r e a , b y a c o n f o r n l a t i o n a l c h a n g e or w h e t h e r it h a p p e n s as a d i r e c t r e s u l t o f m o d i f y ing the respective groups. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 I8 19 20 21 22
K. YAMAGAMIAND K. SCHMID, J. Biol. Chem,, 242 (1967) 4176. Y. NAKAGAWAAND M. L. BENDER, Biochemistry, 9 (197o) 259. L. L. HOUSTON AND K. A. WALSH, Biochemistry, 9 (197o) 156. R. B. FREEDMAN AND G. K. RADDA, Biochem. J., 114 (1969) 611. M. F. JAYLE, Bull. Soc. Chim. Biol., 33 (1951 ) 876W. DOBRYSZYCKA AND E. LISOWSKA, Biochim. Biophys. Acta, 12i (1966) 42. E. LISOWSKA AND ~¢V. I)OBRYSZYCKA, Biochim. Biophys. Acta, 133 (1967) 338. W. DOBRYSZYCKA AND J. C. KUKRAL, Arch. Immunol. Ther. Exp., 18 (197o) 527 . W. DOBRYSZYCKA, A. PUSZTAI AND J. C. KUKRAL, Biochim. Biophys. Acta, 175 (1969) 271. J. F. RIORDAN, W. E. C. WACKER AND 13. L. VALLEE, Biochemistry, 4 (1965) 1758. H. R. HORTON AND D. t5. KOSHLAND JR., J. Am. Chem. Sue., 87 (1965) 1126. E. B. McQUARRIE AND H. N. BENIAMS, Proc. Soc. Expt. Biol. Med., 86 (1954) 627 . O. OUCHTERLONY, Acta Pathol. Microbiol. Scan&, 26 (1949) 507 • R. H. ZSCHOCKE AND A. BEZKOROVAINY, Biochim. Biophys. Acta, 200 (197o) 241. ~V. MEJBAUM-KATZENELLENBOGEN, Acta Biochim. Pol., 2 (1955) 279. K. [~YEDA, Biochemistry, 8 (1969) 2366. J. H. PETERS AND E. GOETZL, J. Biol. Chem., 244 (1969) 2068. G. T. COCKS AND A. C. W*ILSON, Science, 164 (1969) 188. M. Z. ATASSI AND D. R. CARUSO, Biochemistry, 7 (1968) 699. A. F. S. A. HABEEB, J. lmmunol., 99 (1967) 1264. E. BENJAMINI, M. SHIMIZU, J. D. YOUNG AND C. Y. LEUNG, Biochemistry, 8 (1969) 2242. G. M. LOUDON AND D. E. KOSttLAND JR., J. Biol. Chem., 245 (197 o) 2247.
Biochim. Biophys. Acta, 243 (1971) 178-186
OF MODIFICATION
PROPERTIES III.
ANTIGENIC
TYROSINE
ON PHYSICOCHEMICAL
AND BIOLOGICAL
OF HAPTOGLOBIN REACTIVITY
OR TRYPTOPHAN
OF HAPTOGLOBIN
WITH
BLOCKED
RESIDUES
SUMMARY
Exposed tyrosine residues of human haptoglobin (Hp) were acetylated by means of N-acetylimidazole. z-Hydroxy-5-nitrobenzyl (HNB) groups were introduced on exposed tryptophan residues. Native Hp and the two types of modified Hp were used to immunize rabbits. The obtained antisera were studied in cross-reactions with the respective antigens. From quantitative precipitin curves antibodies elicited by native Hp were found to react in a similar way with the homologous antigen as with acetylated haptoglobin (AC-HP). There was no reaction with HNB-Hp. Antibodies directed against AC-HP gave the strongest reaction with the homologous antigen, the precipitin curve with native Hp showed half the height, and a very low curve was obtained with HNB-Hp. A high degree of specificity was shown by antiserum directed against HNB-Hp which reacted only with its homologous antigen. In immunoprecipitation, native Hp and AC-HP yielded antibodies in similar amounts while HNB-Hp was a less effective immunogen. It seems likely that the changes in the Hp molecule caused by the introduction of HNB groups greatly alter the antigenic structure of Hp. The changes produced by the acetylation of tyrosine residues of Hp are less drastic. AC-HP still remains closely related to native protein. Possible reasons for the observed facts may be given: the character of the group introduced on haptoglobin, conformational changes of the protein, and modification of the hvdrophobic area on complementary sites of antigen and antibody.
Abbreviations: Hp, haptoglobin; dues acetylated by Xacetylimidazole; haptoglobin with tryptophan residues
Hb, hemoglobin; XC-HP, haptoglobm with tyrosine rcsiHXB, r-hydroxy-5.nitrobcnzyl bromide ; HXB-Hp, blocl~cd with z-hydroxy-5.nitrobenzyl groups.
MODIFICATION OF HAPTOGLOBIN
I79
INTRODUCTION
Chemical modification of proteins with concurrent observation of functional changes has been widely used to implicate particular functional groups in the activity of enzymes and other proteins (refs. 1-4). Certain activities or reactions of proteins, e.g. enzymic, antigenic or antibody, are predominantly associated with their unique native conformation. Haptoglobin (Hp), an a2-acid glycoprotein of serum, binds hemoglobin (Hb) stoichiometrically and irreversibly 5. The complex can be considered to be a "true" peroxidase. In our previous papers 6-s the effect of degradation of the Hp molecule on some physicochemical and biological properties of Hp were studied. In addition, certain aspects of the role of exposed and buried tryptophan and tyrosine residues in the formation of an active Hp-Hb complex were observed after selective modification of these residues 9. The purpose of this investigation is to present data concerning the changes in antigenic reactivity of human Hp that accompany its modification by the use of specific reagents. A previous study indicated that exposed tyrosine or tryptophan residues were essential for the formation of the H p - H b complex 9. In the present study we have been interested in the question whether antibodies to the "active site" of Hp could be produced in rabbits by injecting them with Hp modified as follows: tyrosine residues acetylated with N-acetylimidazole 1°, or chromophoric groups of 2-hydroxy5-nitrobenzyl bromide (HNB) introduced on tryptophan residues 11. An interesting problem to be studied is the specificity of antibodies formed by an antigen modified at the tyrosine or tryptophan residues. Two factors at least may play a role in these reactions: a direct result of the residue of the protein to be modified, and a conformational change induced by the respective group. MATERIALS AND METHODS
N-acetylimidazole was purchased from K and K Laboratories, Plainview, N.Y. (U.S.A.); HNB from Sigma Chem. Co. (U.S.A.); Sephadex G-25 was a Pharmacia (Sweden) product. Hb was prepared from horse erythrocytes by the method of McQUARRIE AND BENIAMSTM. Human Hp 2-1 and I - I were isolated from ascitic fluid as described elsewhere 6. Preparations that showed a purity of over 90% were lyophilised and used for modification.
Modifications Acetylation with N-acetylimidazole was performed at room temperature in o.oi M Tris buffer, pH 7.5. The reaction was allowed to proceed for I h. A 6o-fold molar excess of N-acetylimidazole over protein was employed as described by RIORDAN et al. 1°. The modified protein was separated from the excess reagent on a Sephadex G-25 column and lyophilised. The number of modified tyrosyl residues in Hp was estimated from the decrease in absorbance at 278 nm; for these calculations a molar differential extinction coefficient at 279 nm of 116o obtained on O-acetylation of N-acetyltyrosine was used. For the blockage of tryptophan residues the procedure of HORTON AND KOSHLAND11 was used. Hp dissolved in o.ooi M HC1 was treated with a 5o-fold molar Biochirn. Biophys. Acta, 243 (1971) 178-186
180
\\2 I){}I¢IC(SZV('I,L\, I. F,I.21;
excess of H N B in acetone. After 3o rain tile protein was separated from the excess reagent b y gel filtration on a Sephadex G-25 column and lyophilised. The n u m b e r of modified t r y p t o p h a n residues in H p was estimated in o.i M NaOH at 4 i o n m using a molar e x t i n c t i o n coefficient of I8 9oo. A ntisera Antibodies directed against n a t i v e Hp, Ac-Hp a n d H N B - H p were raised in rabbits, each r a b b i t being given about IO mg of the respective a n t i g e n in the foot pad, i n t r a v e n o u s l y a n d i n t r a m u s c u l a r l y . Antisera from i n d i v i d u a l animals were obt a i n e d a p p r o x i m a t e l y 4 weeks after the initial injection. Analytical methods H a p t o g l o b i n was d e t e r m i n e d b y tile peroxidase m e t h o d of JAYLE 5. Double diffusion in 1% Difco agar in 0.05 M veronal buffer at p H 8.2 was performed b y the m e t h o d of OUCHTERLONYla. Immunoelectrophoresis was carried out with 0.05 M veronal buffer, p H 8.6, for 45 rain at 7 V/era. After immunodiffusion for 48 l] at 20 °, the slides were washed in 0.9% NaC1 a n d in distilled water, dried in open air a n d stained for protein with Amido Black. Q u a n t i t a t i v e precipitin reactions were carried out as described b y ZSCHOCKE AND BEZKOROVAINY14. A n t i g e n protein solutions of a p p r o x i m a t e l y 0.05% in o.I M Tris-HC1 buffer, p H 8.0, were added to o.5-ml aliquots of antiserum. Volumes were b r o u g h t to i.o ml with the above Tris buffer, a n d the precipitates were collected b y centrifugation after an i n c u b a t i o n at 37 ° for 60 rain, a n d at 4 ° for 48 h. Precipitates were washed 3 times with o.15 M NaC1, dissolved in o.I M N a O H a n d subjected to protein d e t e r m i n a t i o n b y the t a n n i n m i c r o m e t h o d 15.
RESULTS Characteristics of the o b t a i n e d modifications of H p are given in Table I. The ability of H p to form a stable complex with H b was used to estimate the a m o u n t of f u n c t i o n a l protein recovered with the respective modifications, assuming TABLE I EFFECT
OF T H E M O D I F I C A T I O N
OF E X P O S E D
TYROSINE
OR T R Y P T O P H A N
RESIDUES
OF H p
ON T H E
A C T I V A T I O N OF H b
Mean values obtained for at least three preparations are given. Peroxidase activity was measured as described by JAYLE5. The number of acetylated tyrosine residues was calculated using a molar differential extinction coefficient at 279 nm of 116o (ref. io); the number of modified tryptophan residues was estimated in o. i M NaOH at 4to nm, using a molar extinction coefficient of I8 900 (ref. II). Preparation
Residues modified per mole of Hp*
Peroxidase activity of the complex with Hb (%)
Native Hp Ac-Hp HNB-Hp
13 9
lOO i6.i o
*
Calculated for a mol.wt, of 85 ooo. Human Hp 2-i was used.
Biochim. Biophys. dcta, 243 (1971) 178-186
MODIFICATION OF HAPTOGLOBIN
181
Fig. I. I m m u n o r e a c t i v i t y of native Hp, Ac-Hp, and H N B - H p . a. I n the center well, anti-native H p 2-1 s e r u m ; in the outer wells: I, H p I - I ; 2, H N B - H p ; 3, Ac-Hp; 4, H p 2-1; each in a concentration of o.o5%, b. Ill the center well, antinative H p serum; in the outer wells, a 2°,~, solution of H N B - H p . c. I n the center well, a n t i s e r u m to Ac-Hp; in the outer wells: i, H N B - H p ; 2, H p ; 3, A c - H p ; 4, A c - H p ; 5, H p ; each in o.O5~o solution, d. In the center well, a n t i s e r u m to Ac-Hp; in the outer wells, a I % solution of Hp. e. I n the center well, a n t i s e r u m to H N B - H p ; in the o u t e r wells: i, H N B - H p ; 2, A c - H p ; 3, H N B - H p ; 4, H p ; 5, H N B - H p ; each in o.o5% solution.
Biochim. Biophys. Acta, 243 (1971) 178 186
182
W. I)OBRVSZYCKA, I. BK("
'ii~ii~i!!iiiiiiiiiiiii!iiii
Fig. 2. hmnunoelectrophoresis of native Hp, Ac-Hp, and HNB-Hp. a. Antiserum to native Hp in the trough; upper well, Hp; lower well, Ac-Hp. b. Antiserum to Ac-Hp in the trough; upper well, Hp; lower well, Ac-Hp. c. Antiserum to HNB-Hp in the trough; in the wells, HNB-Hp.
the p e r o x i d a s e a c t i v i t y of the complex of n a t i v e H p with H b to be lOO%. After t h e blockage of nine exposed t r y p t o p h a n residues with H N B , a complete loss of a c t i v i t y was observed, while after a c e t y l a t i o n of 13 exposed tyrosine residues 16.1% of t h e original p e r o x i d a s e a c t i v i t y was found. R a b b i t s were i m m u n i z e d with n a t i v e Hp, A c - H p a n d H N B - H p . Six r a b b i t s i m m u n i z e d with n a t i v e H p r e s p o n d e d at a regular time, in comparison with five out of six r a b b i t s i m m u n i z e d with A c - H p , and only two out of six i m m u n i z e d with H N B - H p , respectively. Biochim. Biophys. Acta, 243 (1971) i78-186
183
MODIFICATION OF HAPTOGLOBIN
The effect of the modifications on the formation of antibodies and on the precipitin reaction was studied with the obtained antisera to Hp, Ac-Hp and HNBHp. The results of double gel diffusion experiments are shown in Fig. I. As can be seen in Fig. Ia, antiserum to H p 2-1 (the center well) gave distinct precipitin lines with Hp 2-1, H p i - I , Ac-Hp and a very faint line with HNB-Hp. In order to make this latter combination visible, H N B - H p was applied at a concentration of 2 g/Ioo nil, i.e. 4 ° times higher than the concentrations of Hp and Ac-Hp used in this experiment (Fig. ib). When the center well contained antiserum to Ac-Hp (Fig. IC), definite precipitation arcs developed with Ac-Hp and a very faint one with native Hp; no line was formed with HNB-Hp. Precipitin arcs of anti-Ac-Hp serum with native Hp were made visible when applying Hp at a higher concentration (Fig. Id). Antiserum to H N B - H p reacted only with its homologous antigen, i.e. H N B - H p (Fig. Ie). Results of immunoelectrophoresis are shown in Yig. 2. In Fig. 2a, antiserum to Hp 2-1 was put in the trough. The arc obtained with Ac-Hp is slightly diffused and displaced toward the anode as compared with the parallel arc formed with native Hp. A similar pattern developed when antiserum to Ac-Hp was placed in the trough, against Ac-Hp and native Hp (Fig. 2b). In Fig. 2c the arcs obtained in the reaction of antiserum to H N B - H p with its homologous antigen, i.e. HNB-Hp, are shown.
g
R ~qNTI 14 SERUN P
Et g.:
O2
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/ o,,.....o,, , .i ~ _ ~ " ' ~
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,'7
I 0.l
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C RNTI H N B - H ~ 5ERUP'I
RNTI Rc-Hp £ERUI'I
0.3
O. I
.... o.I
i 0.2
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Fig. 3. Q u a n t i t a t i v e precipitin curves of native Hp, Ac-Hp, and H N B - H p with the respective antisera raised in rabbits. A. A n t i s e r u m to native Hp. B. Antiserum to Ac-Hp. C. A n t i s e r u m to H N B - H p . The following antigens were used: native H p ( A - - A ) , Ac-Hp ( 0 . . . . . O), and I t N B - H p (O - - - O).
Fig. 3 represents quantitative precipitin curves involving native Hp, Ac-Hp, H N B - H p and specific antisera. In Fig. 3A, antiserum to Hp 2-1 was used against Hp 2-1, Ac-Hp and HNB-Hp. Antibodies directed against Hp 2-1 reacted similarly with native Hp and Ac-Hp, precipitating m a x i m u m antibody protein at the same antigen concentration of o.15/~g. However, at the peak, native H p precipitated 0.26/~g of antibodies while Ac-Hp precipitated 0.2o #g. The curve obtained with H N B - H p was almost flat with a very weak m a x i m u m at o.I/~g of the antigen, precipitating o.o3 ~g of antibody protein. Fig. 3 B represents precipitin curves obtained with antiserum to Ac-Hp. This antiserum showed the highest precipitating efficiency with its homologous antigen, i.e. with Ac-Hp, at a concentration of o.23/~g. The peak obtained with native Hp was half that obtained with Ac-Hp. With this antiserum H N B - H p showed a slight m a x i m u m at o.o5-o.1/~g, precipitating o.o6/~g of antibody protein. Biochim. Biophys. Acla, 243 (1971) 178-186
18 4
w. I)OBRYSZYCKA, I. BEC
Antiserum to H N B - H p showed a high degree of specificity, giving a distinct precipitation curve only with H N B - H p (Fig. 3C). The peak was attained at a concentration of o.z #g of antigen, precipitating o.~5 #g of antibodv protein. Native Hp and Ac-Hp did not react with antiserum to HNB-Hp. DISCUSSION
The approach of chemical modification for probing the nature of antigenic sites in proteins is extremely valuable, especially if the specificity of the reagent is well established and the derivative is purified and characterized. N-acetylimidazole has been shown by RIORDAN el el. 1° to be a relatively selective acylating reagent which preferentially acylates exposed tyrosyl groups, although amino groups and cysteine sulthydryl groups can also react. The extent of the reaction of N-acetylimidazole with e-amino groups has been found to be different for various proteins and usually much lower than in the case of other acylating reagents. For example, acetic anhydride blocked 68% of the amino groups of carboxypeptidase A, 57% of Hb, and 9o% of ovomucoid, respectively; however, N-acetylimidazole failed to acetylate the groups of carboxypeptidase A, acetylated only 3% of the amino groups of Hb, and H % of ovomucoid 1°. The possibility of acetylation of eamino groups of Hp was taken into consideration in one of our previous papersK However, the number of eight exposed tyrosyl residues as determined by the use of N-acetylimidazole was very similar to that of seven found by spectrophotometric titration; also the number of 28 total tyrosyl residues determined by N-acetylimidazole was very similar to that found by spectrophotometric titration, i.e. 28 29, and to that of 27-28 moles/mole of Hp obtained by amino acid analysis. Hp does not contain free cysteine groupsT; hence the possibility of the reaction of N-acetylimidazole with free SH groups would be rather improbable. It has been shown that H N B reacts specifically with tryptophan residues in proteins, especially when the latter do not contain any free SH groups u, as is the case with Hp. In comparison, the determination of tryptophan residues in canine Hp showed 12 moles/mole of Hp when using HNB, ZL 5 spectrophotometrically, and 9Io by amino acid analysis, respectively 9. When complexed with Hb, Hp behaves as an enzyme and we took advantage of this property in order to define the actual amount of a functional protein with the native conformation remaining after a modification. The preparation of canine Hp modified at exposed tryptophan or tyrosine residues ° or the analogous human Hp derivatives of the present work, indicated that such a procedure resulted in a significant depression of the ability to activate Hb in the case of acetylation, or in a complete loss of this ability when using HNB. In the present paper it has been shown that tyrosine or tryptophan residues modified in the above manner differ in their reactivity toward respective antibodies as well as in cross-reactions with antibodies produced to native or modified protein. It is well known that certain modifications m a y profoundly affect the function of a protein without greatly altering its antigenic structure. For example, there was no correlation between the enzymatic activity and immunoreactivity of maleylphosphofructokinasO 6, and even greater antigenic reactivity than in native albumin was found in conformationally altered albumin aT. In contrast, certain immunological Iliochin~. Biophys. dcla, 243 (I97 x) I 7 8 - 1 8 6
MODIFICATION OF HAPrOGLOBIN
18 5
techniques can distinguish between proteins differing by a single amino acid is. ATASSI AND CARUSO19 found that apomyoglobin modified with H N B at tryptophan- 7 did not show any alterations in antigenic reactivity, and when modified also at t r y p t o p h a n - i 4, showed a decrease in antigenic reactivity, most likely caused by unfolding or a marked deviation from the spherical shape. Native Hp and 8 M urea-treated Hp with unfolded polypeptide chains were immunochemically very similar, although they differed significantly in the peroxidase activity of the complex with Hb (our unpublished data). This fact demonstrates that the native conformation of the Hp molecule, which is of great importance in the formation of the active complex with Hb, does not play an essential role in antigenic reactivity. Acetylation of exposed tyrosine residues of Hp resulted in a decrease to onesixth of tile peroxidase activity of the complex with Hb. Antibodies elicited by Ac-Hp were directed mainly against the homologous antigen. The reaction with native Hp was much weaker, and with H N B - H p almost absent. It is very interesting that the quantitative precipitin curves of anti-native Hp serum with native Hp and with Ac-Hp are quite similar. This immunological relationship between native Hp and Ac-Hp suggests that either tyrosine residues are not an essential part of the antigenic site of Hp, or that the shape of tile acetyl group on the exposed tyrosine residue does not represent a steric hindrance for the antigen antibody reaction. This problem will be studied further in this laboratory by the introduction of different groups on tyrosine residues of Hp. A rather high degree of immunological specificity was shown by the Hp derivative modified by the use of HNB at exposed tryptophan residues. This Hp derivative is devoid of the ability to activate Hb. Antibodies directed against H N B - H p reacted only with their homologous antigen. In immunoprecipitation, native Hp and Ac-Hp yielded antibodies in similar amounts, while in this regard H N B - H p was less effective. HABEEB2° pointed out that the nitrotyrosyl residues of a protein are more immunogenic than tyrosyl residues, and nitroguanyl residues more than guanyl residues. In our experiments, the nitro group of HNB did not enhance the immunogenicity of modified Hp. In contrast, native Hp and Ae-Hp were far better immunogens than HNB-Hp. It has been suggested by BENJAMINIe~ al. 2~ that a hydrophobic area enhances a possible hydrophobic interaction between the specific sterically complementary areas on the antigen and antibody sites, respectively. This implies that the antibody site contains hydrophobic areas which interact with the hydrophobic area(s), or in their proximity, of the antigenic determinant. The apparent affinity of HNB for hydrophobic residues in proteins is well known. In proteins the HNB group yields indolenine adducts suffering a number of fates depending on the juxtaposition of such nucleophiles as serine or tyrosine hydroxyls, lysine amino groups, etc. 22. Are the differences in antigenic reactivity of native Hp, Ac-Hp and H N B - H p observed in this work due solely to changes in the hydrophobic nature of areas on the antigen and antibody sites ? Such an explanation would be highly improbable. The shape of both groups introduced on Hp, and conformational changes of the protein should be taken into consideration. It seems likely that the changes in the Hp molecule caused by the introduction of HNB groups greatly alter the antigenic structure of Hp. New antigenic determiBiochim. Biophys. Acta,
243 (1971) 178 t86
i86
W. DOBRYSZYCKA, I. BEC
n a n t s could be f o r m e d w h i c h would h a r d l y be d e t e c t e d by an a n t i s e r u m p r e p a r e d against n a t i v e H p or Ac-Hp. The changes p r o d u c e d by the a c e t v l a t i o n of tvrosine r e s i d u e s are less d r a s t i c : t h e y d o n o t c h a n g e t h e a b i l i t i e s o f t h e a n i m a l s t o r e c o g n i z e a n a r e a as a n t i g e n i c or t o s y n t h e s i z e a n t i b o d y a r e a s c o m p l e m e n t a r y t o t h e a n t i g e n i c area. M o d i f i e d in t h i s w a y , t h e H p m o l e c u l e still r e m a i n s closely r e l a t e d t o t h e n a t i v e p r o t e i n . H o w e v e r , it is n o t p o s s i b l e a t t h i s s t a g e t o d e t e r m i n e w h e t h e r a n t i g e n i c s p e c i f i c i t y o f t h e H N B d e r i v a t i v e o f H p is c a u s e d b y a c h a n g e in t h e h y d r o p h o b i c a r e a , b y a c o n f o r n l a t i o n a l c h a n g e or w h e t h e r it h a p p e n s as a d i r e c t r e s u l t o f m o d i f y ing the respective groups. REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13 14 15 16 17 I8 19 20 21 22
K. YAMAGAMIAND K. SCHMID, J. Biol. Chem,, 242 (1967) 4176. Y. NAKAGAWAAND M. L. BENDER, Biochemistry, 9 (197o) 259. L. L. HOUSTON AND K. A. WALSH, Biochemistry, 9 (197o) 156. R. B. FREEDMAN AND G. K. RADDA, Biochem. J., 114 (1969) 611. M. F. JAYLE, Bull. Soc. Chim. Biol., 33 (1951 ) 876W. DOBRYSZYCKA AND E. LISOWSKA, Biochim. Biophys. Acta, 12i (1966) 42. E. LISOWSKA AND ~¢V. I)OBRYSZYCKA, Biochim. Biophys. Acta, 133 (1967) 338. W. DOBRYSZYCKA AND J. C. KUKRAL, Arch. Immunol. Ther. Exp., 18 (197o) 527 . W. DOBRYSZYCKA, A. PUSZTAI AND J. C. KUKRAL, Biochim. Biophys. Acta, 175 (1969) 271. J. F. RIORDAN, W. E. C. WACKER AND 13. L. VALLEE, Biochemistry, 4 (1965) 1758. H. R. HORTON AND D. t5. KOSHLAND JR., J. Am. Chem. Sue., 87 (1965) 1126. E. B. McQUARRIE AND H. N. BENIAMS, Proc. Soc. Expt. Biol. Med., 86 (1954) 627 . O. OUCHTERLONY, Acta Pathol. Microbiol. Scan&, 26 (1949) 507 • R. H. ZSCHOCKE AND A. BEZKOROVAINY, Biochim. Biophys. Acta, 200 (197o) 241. ~V. MEJBAUM-KATZENELLENBOGEN, Acta Biochim. Pol., 2 (1955) 279. K. [~YEDA, Biochemistry, 8 (1969) 2366. J. H. PETERS AND E. GOETZL, J. Biol. Chem., 244 (1969) 2068. G. T. COCKS AND A. C. W*ILSON, Science, 164 (1969) 188. M. Z. ATASSI AND D. R. CARUSO, Biochemistry, 7 (1968) 699. A. F. S. A. HABEEB, J. lmmunol., 99 (1967) 1264. E. BENJAMINI, M. SHIMIZU, J. D. YOUNG AND C. Y. LEUNG, Biochemistry, 8 (1969) 2242. G. M. LOUDON AND D. E. KOSttLAND JR., J. Biol. Chem., 245 (197 o) 2247.
Biochim. Biophys. Acta, 243 (1971) 178-186