Effect of modification on physicochemical and biological properties of haptoglobin. VI. Reaction with azlactone of p-nitrobenzoyl-valine

Biochimica et Biophysica A cta, 412 (1975) 306-316

© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37210 E F F E C T OF M O D I F I C A T I O N ON P H Y S I C O C H E M I C A L A N D B I O L O G I C A L P R O P E R T I E S OF H A P T O G L O B I N . VI. R E A C T I O N W I T H A Z L A C T O N E OF p-NITROBENZOYL-VALINE

JERZY OSADA and WANDA DOBRYSZYCKA Department of Clinical Investigation, Bio-Pharmaceutical Institute, Szewska 38/39, 50-139 Wroctaw (Poland)

(Received June 10th 1975)

SUMMARY The azlactone of p-nitrobenzoyl-valine (Nbz-Val) has been used for modification of e-amino groups of lysine in haptoglobin type 1-1, in hemoglobin, and in the haptoglobin-hemoglobin complex. By the use of this reagent 95 ~ of amino groups in haptoglobin and 9 0 ~ in hemoglobin have been blocked without any changes in peroxidase activity of the formed complexes: Nbz-Val. haptoglobin with hemoglobin, Nbz-Val. hemoglobin with haptoglobin, and Nbz-Val. (haptoglobin-hemoglobin). After reduction and reoxidation, Nbz-Val.haptoglobin was found to retain 9 0 ~ of peroxidase activity when complexed with hemoglobin, fl chains separated either from haptoglobin or Nbz-Val. haptoglobin showed 15 ~ of peroxidase activity in the complex with hemoglobin, a chains of the same origin were completely inactive. Whereas recombination of haptoglobin from a and fl chains resulted in 4 2 ~ hemoglobin-binding capacity, renaturation of Nbz-Val.haptoglobin from separated subunits was found to proceed with almost 100 ~ yield. In immunodiffusion with rabbit anti-haptoglobin or anti-Nbz-Val •haptoglobin sera, preparations of haptoglobin and Nbz-Val.haptoglobin after reduction and reoxidation or after recombination from separated subunits gave similar precipitation arcs showing the reaction of immunological identity.

INTRODUCTION Haptoglobin is an az-acid glycoprotein of serum. Haptoglobin of genetic type 1-1 is a tetramer containing 2a-chains of mol. wt 9100 each and 2fl-chains of mol. wt 40 000 each, held together by interchain disulphide bonds [1-3]. Complex of haptoglobin with hemoglobin is known to possess catalytic activity of true peroxidase type [4]. This was the reason that the method of chemical modification, very Abbreviations: Nbz-Val, p-nitrobenzoyl-valine; Nbz-Val.haptoglobin, haptoglobin with amino groups blocked by Nbz-Val; Nbz-Val. hemoglobin, hemoglobin with amino groups blocked by Nbz-Val; Nbz-Val. (haptoglobin-hemoglobin), modified complex of haptoglobin with hemoglobin ; Nbz-Val. a, Nbz-Val. fl, subunits separated from Nbz-Val •haptoglobin; pOH-Hg-BzO-, p-hydroxymercuribenzoate: N~bzSO-3, 2,4,6-trinitrobenzenesulphonic acid.

307 popular in studies on active areas of enzymes or antibodies, has been applied in investigations on the formation of an active haptoglobin, hemoglobin complex [5-7]. Modification of e-amino groups of lysine in haptoglobin by means of 2,4,6trinitrobenzenesulphonic acid (NabzSO~) was reported by Shinoda [8]. The role of these groups was then examined by Chiao and Bezkorovainy [6] and Dobryszycka and Osada [7]. The results obtained suggested that peroxidase activity of the complex with hemoglobin depended on the reagent used for the modification of amino groups. In our previous work succinyl anhydride had been applied [7]. In the present work a non-polar compound, namely the azlactone ofp-nitrobenzoyl-valine was employed. The reaction of amino groups of proteins with the azlactone ofp-nitrobenzoyl-valine proceeds as follows [9]: / Protein

--

CH--CH

NH 2 + O = C

I 0

I N

/CH3

CH3

~CH 3

pH 7 - 1 0

Protein --

NH

--

CO --

CH - -

, L

H--N

CH

\ CH3

L C = O

NO2

NO2

The ring of the azlactone being broken, p-nitrobenzoyl-valine (Nbz-Val) is incorporated into protein. By means of this reagent haptoglobin, hemoglobin and the haptoglobin, hemoglobin complex were modified, then peroxidase activities of different combinations were studied as well as the capacity of modified a and fl chains of haptoglobin for recombination of haptoglobin molecules. MATERIALS AND METHODS

Materials Special reagents were of the following origin: Tris, N,N,N',N'-tetramethylethylenediamine (TEMED), 1,4-bis-(5-phenyl-2-oxazolyl)-benzol (POPOP), Fluka (Switzerland); acrylamide, 2,4,6-trinitrobenzenesulphonic acid (N3bzSO3) , BDH, Poole (U.K.); N,N'-methylene-bis-acrylamide, sodium dodecyl sulphate, 2-mercaptoethanol, Koch-Light (U.K.); Ampholine pH 3.5-10, LKB (Sweden); N,N'-dicyclohexylcarbodiimide, Merck (Germany); Sephadex G-25, G-75, Pharmacia, Uppsala (Sweden); [14C]valine, Amersham (U.K.); 2,5-diphenylooxazole (PPO), Reanal (Hungary); p-hydroxymercuribenzoate (pOH-Hg-BzO-), Serva (USA). Other reagents were "Polskie Odczynniki Chemiczne" (Poland) of analytical grade. Urea was additionally purified by 3-fold crystallisation from ethanol, and guanidine hydrochloride from ether respectively. p-Nitrobenzoyl-valine and p-nitrobenzoyl-[14C]valine were obtained as follows: 2.5 g of DL-valine was dissolved in 90 ml of 1.2 N NaOH then shaken for 2 h with an ethereal solution of p-nitrobenzoic acid chloride. The ethereal layer was discarded and the rest adjusted with concentrated HCI to pH 4. The precipitate formed was washed with water and dried. The yield was 3.55 g of p-nitrobenzoyl-valine, m.p. 156-158 °C. Synthesis ofp-nitrobenzoyl-[~4C]valine was carried out in the same way,

308 adding 0.15 #Ci of [laC]valine to the reaction mixture. Respective azlactones were synthetized by the method of Baranowski et al. [10]: 3.5 g of Nbz-Val (13 mM) was dissolved in 30 ml of hot benzene with equimolar amount of N,N'-dicyclohexylcarbodiimide and left overnight. After filtration, the solution was concentrated under vacuum and the residue was extracted with hot petroleum ether. 1.5 g of crystalline product was further purified by repeated crystallization from petroleum ether. The melting point was found to be 85-86 °C, extinction coefficient of 0.1 # M at 270 n m = 1.17. Specific radioactivity of the preparation obtained with p-nitrobenzoyl-[14C]valine was 58 #Ci/M. Human haptoglobin 1-1 was prepared from ascitic fluids by the method of Smith et al. [11], horse hemoglobin according to McQuarrie and Beniams [12]. Antisera were obtained by immunization of rabbits with 14 mg of haptoglobin or 40 mg of Nbz-Val.haptoglobin with complete Freund's adjuvant in foot pads, intravenously and intramuscularly.

Analytical methods Protein concentration was estimated according to Lowry et al. [13] or spectrophotometrically using an extinction coefficient for haptoglobin 1-1 of F 0.1% = 1.15 ~278 nm (see ref. 14). Haptoglobin was assayed by the peroxidase method of Jayle [4] and amino groups were determined with N3bzSO 3 by the method of Habeeb [15]. Incorporation of Nbz-Val on amino groups of protein was performed according to Siemieniewski and Baranowski [16] as follows: to 100 mg of a protein dissolved in 50 ml of carbonate buffer pH 10.0, 5 ml of ethanolic solution of the azlactone of Nbz-Val (5.8 mg/ml) was added; final concentrations of ethanol and the azlactone were 1 0 ~ and 3/~M/ml respectively. The mixture was gently stirred for 3 h at 4 °C. The reaction was stopped by adding 5 ml of 10 ~ solution of hydroxylamine hydrochloride. After centrifugation, excess of reagents was dialyzed against distilled water and the modified protein was lyophilized. Reduction and reoxidation of native haptoglobin and Nbz-Val.haptoglobin as well as preparation of a and fl chains were carried out according to Bernini and Borri-Voltattorni [17]: the unfolding and cleavage of haptoglobin and Nbz-Val.haptoglobin into a and fl chains were achieved by reductive sulphitolysis in 6 M guanidine HCI followed by blocking of SH groups with pOH-Hg-BzO-. Reduction/reoxidation procedure without separation of subunits was carried out by dissolving 100 mg of native haptoglobin or Nbz-Val. haptoglobin in 20 ml of 0.5 M Tris/HC1 buffer pH 8.5 containing 8 M urea, 0.5 M 2-mercaptoethanol and 7 mM EDTA. Following a 4-h reduction in an atmosphere of N2 the proteins were dialyzed against a buffer pH 8.15 containing 33 mM Tris, 16 mM NaHzPO4, 2.7 mM EDTA, and 3 mM 2-mercaptoethanol, then were allowed to reoxidize for 24 h in an open vessel at room temperature with slow mixing. Reconstitution of haptoglobin or Nbz-Val.haptoglobin from separated chains was achieved as follows: to 22 mg of fl chain of haptoglobin or Nbz-Val. haptoglobin in 5 ml of the same buffer (Tris/phosphate/EDTA/mercaptoethanol) as in the previous experiment, 5 mg of respective a chain in 3 ml of the buffer were added. After 17 h incubation at room temperature the preparations were checked by a peroxidase test, polyacrylamide gel electrophoresis and immunodiffusion. Disc electrophoresis in 7.5 ~ polyacrylamide gel at pH 8.3 was carried out as

309 described by Davis [18] and at pH 2.1 in propionic acid by Maurer [19] respectively. Thin-layer gel electrofocusing was performed on 4.2~o polyacrylamide gel with 2 ~ Ampholine, pH range 3-10 according to Awdeh et al. [20]. Double immunodiffusion in 1 ~ agar gel was done by the method of Ouchterlony [21]. Velocity sedimentation measurements were done in an MOM 3170 (Hungary) ultracentrifuge. The rotor speed was 60 000 rev./min (260 000 × g), 20 °C. Absorbance spectra were recorded on a Specord UV VIS, C. Zeiss (G.D.R.) spectrophotometer. Radioactivity was measured in USB-2 (Poland) apparatus using scintillation cocktail: 4 g PPO, 0.2 g POPOP, 20 ml of ethylene glycol, 100 ml methanol, 60 g naphtalene, made up to 1000 ml with dioxane [22]. RESULTS

Ultraviolet spectra of Nbz- Val. haptoglobin and separated subunits The mercury derivatives of a and fl subunits were separated on a column of Sephadex G-75 equilibrated with 8 M urea. The resulting elution pattern is shown in Fig. 1. In spite of the same amount of particular proteins taken for this experiment, absorbance values of a and/3 chains obtained from Nbz-Val- haptoglobin were found to be higher than those from unmodified haptoglobin. Fractions containing particular

i

4.0 O2.0'

c

"~ 1,0. <

O.5.

20

40

6tD

80

Fraction number

Fig. 1. Gel filtration pattern of the mercury derivatives of a and fl chains of haptoglobin type 1-1 (0--0) and Nbz-Val-haptoglobin 1-1 (O--O) on Sephadex G-75 in 8 M urea. Reduction and sulphitolysis according to Bernini and Borri-Voltatorni [17]. subunits were assembled (30-50 and 55-70). Following the removal o f p O H - H g - B z O groups by 2-mercaptoethanol in high concentration, they were dialysed and concentrated or lyophilized. Near-ultraviolet spectra of haptoglobin, Nbz-Val. haptoglobin and isolated subunits are shown in Fig. 2.

310 A280m i 1,21.0, 0.8 0.6

2

0.4 0.2 0 240

260 280 300 Wavelength / nrn

320

Fig. 2. Absorbance spectra of native haptoglobin (1), a chain (2), and fl chain of haptoglobin (3), Nbz-Val" haptoglobin (4), Nbz-Val- a chain (5), Nbz-Val. fl chain (6), p-nitrobenzoyl-valine obtained by hydrolysis of the azlactone (7). The samples containing 500/~g of protein/ml in 0.01 M Tris/HCl buffer, pH 7.0; the concentration of p-nitrobenzoyl-valine was 0.1 #M/ml of the same buffer.

As can be seen in Fig. 2 the absorption spectra of Nbz-Val. haptoglobin and of the subunits obtained from the modified protein in the near-ultraviolet region show higher specific extinction and are slightly shifted towards a shorter wavelength i.e. 253 ~+ 249, 280 -+ 272 nm as compared with those of the native haptoglobin and respective subunits. As absorbance of 0.1 # M / m l of p-nitrobenzoyl-valine obtained by hydrolysis of the azlactone was 1.17, the higher absorption intensities of the polypeptide chains derived from Nbz-Val. haptoglobin (Fig. 1) demonstrated the incorporation of Nbz-Val residues into haptoglobin.

Determination of amino groups in Nbz- Val" haptoglobin Exposed amino groups in particular preparations were determined according to Habeeb [15] and estimations of the incorporated Nbz-Val residues either by differential absorbance value at 270 nm or by measurements of radioactivity of included Nbz-[14C]valine were carried out. The results of these determinations are summarized in Table I. 53 exposed amino groups in native haptoglobin were determined, in haptoglobin submitted to the action of 8 M urea the number amounted to 61. In separated native a and fl chains 8 and 22 amino groups were found respectively. About 57 amino groups in haptoglobin were available to the modifier. When a and/3 chains were obtained from Nbz-Val. haptoglobin, 5 amino groups in a chain and 19 in fl chain were blocked. Modification of hemoglobin with Nbz-Val resulted in 90 % blockage of amino groups. Peroxidase activities of complexes of native hemoglobin or Nbz-Val. hemoglobin with native haptoglobin, Nbz-Val" haptoglobin or separated subunits of haptoglobin The results of the measurements of peroxidase activity of complexes of native hemoglobin and Nbz-Val.hemoglobin with native haptoglobin, Nbz-Val.haptoglobin, and particular s bunits of haptoglobin are summarized in Table II.

311 TABLEI REACTION OF THE AZLACTONE OF p-NITROBENZOYL-VALINE WITH AMINO GROUPS OF HAPTOGLOBIN, SEPARATED SUBUNITS OF HAPTOGLOBIN AND WITH HEMOGLOBIN Amino groups were determined with N3bzSOg by the method of Habeeb [15], residues incorporated into protein were estimated spectrophotometricaUy [16] or by the measurements of radioactivity of Nbz-[14C]valine(results indicated*). In parentheses, calculated number of NH2 groups is given, taking into account the molecular weights of haptoglobin, 100 000; a chain, 9100 (ref. 1); fl chain, 40 000 (ref. 2); hemoglobin, 66 000, respectively. Compounds

Exposed NH2 groups (mol/mol)

Nbz-Val residues (mol/mol)

Native haptoglobin a chain of haptoglobin fl chain of haptoglobin Nbz-Val. haptoglobin Nbz-Val- a chain of haptoglobin Nbz-Val. fl chain of haptoglobin Haptoglobin in 8 M urea Nbz-Val- hemoglobin

53.20 (62) 8.27 (8) 22.00 (23) 5.89 3.10 3.93 61.37 (62) -

57.80, 56.7" 5.46 18.70 53.10" (60)

Peroxidase activities of particular h a p t o g l o b i n , h e m o g l o b i n complexes were u n c h a n g e d when N b z - V a l - h a p t o g l o b i n or N b z - V a l . h e m o g l o b i n were used, the same was observed with fl chains as a chains were inactive in the complex f o r m a t i o n . P r e i n c u b a t i o n of the examined c o m p o u n d s in Tris buffer p H 8.15 c o n t a i n i n g E D T A a n d 2 - m e r c a p t o e t h a n o l resulted in significant elevation o f peroxidase activity as measured by Jayle's [4] method. P r e i n c u b a t e d native h a p t o g l o b i n formed the complex with native h e m o g l o b i n d e m o n s t r a t i n g an enzymatic activity 62 ~ higher t h a n w i t h o u t this a d d i t i o n a l procedure, p r e i n c u b a t e d N b z - V a l . h a p t o g l o b i n formed with native h e m o g l o b i n a complex with peroxidase activity 51 ~ higher etc. Preformed haptoTABLE II PEROXIDASE ACTIVITY OF THE COMPLEX OF Nbz-Val-HAPTOGLOBIN AND ITS SUBUNITS WITH HEMOGLOBIN AND Nbz-Val.HEMOGLOBIN Peroxidase activity was measured by the method of Jayle [4]. In parentheses are given values obtained by preincubation of particular compounds in 33 mM Tris/16 mM NaH2PO4 buffer pH 8.15, containing 2.7 mM EDTA and 3 mM 2-mercaptoethanol. Compounds

Native haptoglobin a chain of haptoglobin fl chain of haptoglobin Nbz-Val.haptoglobin a chain of Nbz-Val. haptoglobin fl chain of Nbz-Val.haptogiobin

Peroxidase activity (%) with Native hemoglobin

Nbz-Val hemoglobin

100 (162) 0 (0) 14 (15) 100 (151) 0 (0) 16 (19)

106 (126) 0 (0) 14 (11) 102 (147) 0 (0) 15 (14)

312

Fig. 3. Schlieren images obtained during a simultaneous sedimentation experiment of native haptoglobin (1) and Nbz-Val.haptoglobin (2). Protein concentrations were 10 mg/ml; exposures were made at 30 min (a) and 48 min (b) after the rotor attained 60 000 rev./min. Sedimentation is from left to right. globin-hemoglobin complex submitted to the action of the azlactone did not change peroxidase activity as compared with the native complex under the conditions of the experiment.

Ultracen trifugation Ultracentrifugation of 1 ~ solution of haptoglobin 1-1 or Nbz-Val. haptoglobin showed a single boundary with a sedimentation coefficient of 4.19 S (Fig. 3).

Electrophoresis, isoelectric focusing The electrophoretic pattern of Nbz-Val. haptoglobin in polyacrylamide gel, pH 8.3 was very similar to that of native haptoglobin (see Fig. 4, gels no. 1 and 2), but at pH 2.1 the difference between them was clearly visible (Fig. 5). When the same preparations were submitted to thin-layer gel electrofocusing on polyacrylamide gel with Ampholine, pH range 3-10, isoelectric point of native haptoglobin was found to be 4.2-4.3 but only a small part of NbZ-Val-haptoglobin entered the gel, demonstrating isoelectric point of 5.3.

Reduction and reoxidation of Nbz- Val" haptoglobin. Reconstitution of subunits Peroxidase activity of hemoglobin complex with reduced/reoxidized haptoglobin was found to be 92 ~ ; with Nbz-Val-haptoglobin submitted to the same procedure, 90 ~ . Peroxidase activity of hemoglobin bound to reconstituted haptoglobin obtained from a and/3 chains amounted to 42 ~ as compared with the activity of the native haptoglobin-hemoglobin complex assumed to be 100K. On the contrary, Nbz-Val. haptoglobin renatured from respective subunits retained 98 ~o of peroxidase activity when complexed with hemoglobin. Electrophoretic patterns of obtained preparations are shown in Fig. 4. It is very interesting that reduced/reoxidized haptoglobin, reconstituted haptoglobin and/3 chain originated from native haptoglobin (Fig. 4 gels no. 7, 11, 13) show a fraction which does not enter the gel, a chain-band (Fig. 4) is doubled, while

313

Fig. 4. Electrophoresis in 7.5 ~ polyacrylamide gel, pH 8.3. 1, Native haptoglobin type 1-1 ; 2, NbzVal.haptoglobin; 3, 4, a chain from native haptoglobin; 5, 6, a chain from Nbz-Val.haptoglobin; 7, 8, fl chain from native haptoglobin; 9, 10, fl chain from Nbz-Val.haptoglobin; 11, Reduced and reoxidized haptoglobin; 12, Reduced and reoxidized Nbz-Val.haptoglobin; 13, Haptoglobin reconstituted from a and fl subunits; 14, Nbz-Val.haptoglobin reconstituted from Nbz-Val.a and Nbz-Val.fl subunits. To samples 4, 6, 8, 10 containing 250pg of protein in 1 ml of 2 0 ~ sacharose was added 0.1 ml of 10~ sodium dodecyl sulphate and 0.05 ml 2-mercaptoethanol. Then they were incubated at room temperature for 15 min. The gels were stained with amido black 10 B in 7 ~ acetic acid. a n a l o g o u s p r e p a r a t i o n s o r i g i n a t e d f r o m N b z - V a l . h a p t o g l o b i n consist o f one welldefined b a n d . H o w e v e r , when these native h a p t o g l o b i n p r e p a r a t i o n s h a d been subm i t t e d before electrophoresis to reducing a n d dissociating agents, mobilities o f p a r t i c u l a r p r e p a r a t i o n s originating either f r o m native h a p t o g l o b i n o r N b z - V a l . h a p t o g l o b i n were f o u n d to be identical. It seems likely t h a t the b l o c k a g e o f a m i n o g r o u p s m a y prevent a n a g g r e g a t i o n occurring t h r o u g h o u t the course o f the f o r m a t i o n o f the p r o t e i n molecule o u t o f subunits.

Fig. 5. Patterns of native haptoglobin 1-1 (a) and Nbz-Val.haptoglobin (b) in 7.5 ~ polyacrylamide gel electrophoresis in 1 M propionic acid, pH 2.1 according to Maurer [19].

314

4

5

/

3 A

I1

--B

5"

q

C

Fig. 6. Agar gel immunodiffusion patterns of haptoglobin and Nbz-Val.haptoglobin. (A) Center well, anti-haptoglobin serum; 1, Nbz-Val. haptoglobin; 2, reduced-reoxidized haptoglobin; 3, reduced-reoxidized Nbz-Val.haptoglobin; 4, haptoglobin recombined from isolated subunits; 5, Nbz-Val-haptoglobin recombined from isolated subunits; 6, native haptoglobin. (B) Center well, anti-haptoglobin serum; 1, Nbz-Val./3 chain; 2, fl chain; 3, a chain; 4, Nbz-Val.a chain. (C) Center well, anti-Nbz-Val, haptoglobin serum; 1, fl chain; 2, native haptoglobin; 3, Nbz-Val-haptoglobin; 4, Nbz-Val./3 chain; 5, Nbz-Val. a chain; 6, a chain. The antigens were of 0.1 ~ concentrations except fl chain which was 0.2 ~, and a chain 2 700respectively.

Antigenic properties of Nbz- Val. haptoglobin Anti-haptoglobin 1-1 serum was obtained after 4 weeks by immunization of the rabbit with 14 mg of the protein. Only one out of three rabbits which were given Nbz-Val.haptoglobin generated antibodies after 12 weeks, being immunized with 40 mg of the antigen. Obtained antisera were tested in immunodiffusion against Nbz-Val.haptoglobin and isolated subunits (Fig. 6). Nbz-Val.haptoglobin behaved in immunoprecipitation against anti-haptoglobin serum exactly in the same way as native haptoglobin even after reductionreoxidation procedures or after recombination from isolated subunits. The same was observed with the use of anti-Nbz-Val, haptoglobin serum, showing precipitin arcs characteristic for immunological identity, a chains originated from native haptoglobin or Nbz-Val.haptoglobin were inactive in immunoprecipitation against both the antisera used. DISCUSSION

The present study is an evaluation of the reactivity of the amino groups in haptoglobin with respect to complex formation with hemoglobin or with antibody, and their role in stabilization of the quaternary structure of the protein. Modification of amino groups in haptoglobin was carried out by the means of the azlactone of p-nitrobenzoyl-valine (Nbz-Val), a reagent which attacks specifically amino groups in protein molecules. It was introduced by Kochman and Baranowski [9] who reported modification of aldolase by Nbz-Val with partial loss of enzymatic activity. In our previous paper it was shown that modification of tyrosine residues in haptoglobin independently of the reagent used (N-acetylimidazole, tetranitromethane, iodine) resulted in rather similar changes in interaction of haptoglobin with hemoglobin and in antigenic reactivity [23]; tryptophan residues modified either with 2-hydroxy-5-nitrobenzyl bromide [5] or with N-bromosuccinimide (unpublished) were found to play an essential part in both the activities examined. The problem of the role of amino groups in haptoglobin has not been so explicit when modifications were

315 carried out with reagents of different chemical character as for instance with N3bzSOg [8], which prevents complex formation with hemoglobin, or with succinyl anhydride (peroxidase activity of the complex with hemoglobin decreased by 33 ~ [7]. These findings could suggest that an effect of amino group modification in haptoglobin on haptoglobin-hemoglobin interaction might depend rather on polarity of the reagent used than on any kind of spheric hindrance. Results obtained in the present paper with the use of a non-polar compound, namely the azlactone of p-nitrobenzoyl-valine, as an amino group-modifying agent seem to maintain this hypothesis. Within the range of experimental uncertainty the measured properties of the modified haptoglobin are indistinguishable from those of native protein. Haptoglobin with 95 ~ of amino groups blocked with Nbz-Val behaved in the same way as the native protein in complex formation with hemoglobin, in pH 8.3 polyacrylamide gel electrophoresis, in immunoprecipitation with antihaptoglobin or anti-Nbz-Val, haptoglobin sera. The same was observed when amino groups of hemoglobin were modified or the preformed haptoglobin.hemoglobin complex was submitted to the action of the azlactone of Nbz-Val; in each case peroxidase activity was unchanged as compared with the respective reaction of native proteins. Equally satisfactory was a 98 70 recovery of peroxidase activity of the hemoglobin complex with the molecules of Nbz-Val.haptoglobin formed by combining separated chains, while haptoglobin activity restored from isolated chains amounted only to 42 70. Moreover, blockage of amino groups with Nbz-Val prevented aggregation when Nbz-Val.haptoglobin underwent reduction-reoxidation procedure or reconstitution from isolated ~ and/3 subunits (Fig. 4, gels no. 5, 9, 12, 14). The native haptoglobin under the same conditions demonstrated a tendency to aggregation (Fig. 4, gels no. 3, 7, 11, 13). Reappearance of the full examined properties in NbzVal-haptoglobin signifies that modification has abolished the aggregation of the chains and restored the residues in question to an exposed position as occurs in native subunits. The behaviour of haptoglobin and Nbz-Val. haptoglobin in electrophoresis at pH 2.1 and in electrofocusing should be pointed out. In spite of the replacement of amino groups by non-polar residues of N-benzoyl-valine, the modified protein migrated towards the anode faster than the native one and demonstrated an isoelectric point of 5.3 compared to 4.2 for untreated haptoglobin. On the other hand, because of a few bands in the electrophoretic patterns at pH 2.1 of both preparations (see Fig. 5) and the fact that only an inadequate part of Nbz-Val.halogptobin entered the gel in electrofocusing (an artefact?), a question arises about the effects of acid medium on Nbz-Val.haptoglobin which demonstrated in pH 8.3 electrophoresis exactly the same mobility as the native haptoglobin. However, although Nbz-Val.haptoglobin gave a reaction of immunological identity with the native haptoglobin in double immunodiffusion test, it should be emphasized that the only property of Nbz-Val. haptoglobin which does not agree with the corresponding one in the native haptoglobin is that the modified protein was found to be a less effective antigen than native haptoglobin. Only one out of three rabbits responded to immunization with a much higher dose of Nbz-Val. haptoglobin than with haptoglobin which has been a very good antigen. Replacement of e-amino groups of lysine by N-benzoyl-valine residues did not enhance the immunogenicity even with the presence of such a good immunogen as a nitro-benzoyl group. It seems

316 then likely that the modification alters greatly the h y d r o p h o b i c character of the haptoglobin molecule causing inability of the rabbits to synthetize the antibodies or a certain delay in their production. ACKNOWLEDGMENTS The authors are greatly indebted to Professer Ignacy Siemion for rendering accessible his u n p u b l i s h e d m e t h o d for the synthesis o f p - n i t r o b e n z o y l - v a l i n e . REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Black, J. A. and Dixon, G. H. (1968) Nature 218, 736-741 Gordon, S., Cleve, H. and Bearn, A. G. (1968) Proc. Soc. Exp. Biol. Med. 127, 52-59 Malchy, B., Rorstad, O. and Dixon, G. H. (1973) Can. J. Biochem. 51,265-273 Jayle, M. F. (1951) Bull. Soc. Chim. Biol. 33, 876-880 Dobryszycka, W. and Bec, I. (1971) Biochim. Biophys. Acta 243, 178-186 Chiao, M. T. and Bezkorovainy, A. (1972) Biochim. Biophys. Acta 263, 60-69 Dobryszycka, W. and Osada, J. (1973) Acta Biochim. Polon. 20, 145-151 Shinoda, T. (1965) J. Biochem. Tokyo 57, 100-102 Kochman, M. and Baranowski, T. (1967) Acta Biochim. Polon. 14, 221-233 Baranowski, T., Kochman, M., Nowak, K. and Siemion, I. (1963) Bull. Acad. Polon. Sci. Ser. Biol. 11, 107-111 Smith, H., Edman, P. and Owen, J. A. (1962) Nature 193, 286 McQuarrie, M. B. and Beniams, H. N. (1954) Proc. Soc. Exp. Biol. Med. 86, 627-632 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265275 Cloarec, L. (1964) Contribution h l'Etude Physico-chimique des Haptoglobines Humaines (Foulon, R., ed.), p. 6, Paris Habeeb, A. F. S. A. (1966) Anal. Biochem. 14, 328-336 Siemieniewski, H. and Baranowski, T. (1969) Acta Biochim. Polon. 16, 243-251 Bernini, L. F. and Borri-Voltattorni, C. (1970) Biochim. Biophys. Acta 200, 203-219 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427 Maurer, H. R. (1968) Disk-Elektrophorese, p. 44, Walter de Gruyter and Co., Berlin Awdeh, Z. L., Williamson, A. R. and Askonas, B. A. (1968) Nature 219, 66-67 Ouchterlony, O. (1949) Acta Pathol. Microbiol. Scand. 26, 507-515 Bray, G. A. (1960) Anal. Biochem. 1,279-285 Dobryszycka, W. and Katnik, I. (1975) Acta Biochim. Polon. 22, 143-153