Reduction of disulphide bonds in human haptoglobin 2-1

Biochimica et Biophysica Acta 829 (1985) 13-18 Elsevier

13

BBA32186

Reduction of disulphide bonds in human haptoglobin 2-1

Wanda Dobryszycka and Tadeusz Guszczyflski Department of Pharmaceutical Biochemistry, Medical Academy, Szewska 38/39, 50-139 Wroclaw (Poland) (Received November 9th, 1984)

Key words: Haptoglobin; Disulfide bond reduction; Hemoglobin binding; Antibody binding; (Human)

Gradual reduction of disulphide bonds in human haptoglobin, type 2-1, was carried out either by the use of sodium borohydride or 2-mercaptoethanol, newly exposed sulphydryi groups as determined by the Eiiman's reagent and by the incorporation of [t4Clacetamide, respectively. Cleavage of disulphide bonds resulted in the formation of a number of intermediates, separated in polyacrylamide gel electrophoresis, with sulphydryl groups blocked by the radioactive label. On the basis of molecular mass determinations, subunit composition of intermediates, was postulated. The ability of haptoglobin to form an active peroxidase-like complex with hemoglobin depended to a considerable extent on the presence of intact disulphide bonds. On the contrary, throughout the course of reduction of inter- and intrachain disulphides, antigenic reactivity was found to remain unchanged.

Introduction Human haptoglobin, an a2-acid glycoprotein of serum, exists in three main genetic types: 1-1, 2-2 and 2-1. Haptoglobin 1-1 is a tetramer composed of two light subunits ct1 (9.1 kDa) and two heavy subunits fl (40 kDa), the structure being maintained by the disulphide bonds joining subunits ct1 to fl, and al to a 1. However, haptoglobin types 2-2 and 2-1 show electrophoretic polymorphism as a result of the presence of Ot2 subunit, which is almost a duplicate of cd subunit (17.6 kDa). The haptoglobin 2-2 polymeric series (ct2fl)n is distinctly different from that of haptoglobin 2-1 (polymeric series contains subunits ~1, c~2, and fl), (for review see Ref. 1). No haptoglobin type contains free sulphydryl groups. Abbreviations: ~t1, Ol2, j~, subunits of haptoglobin type 2-1; SDS, sodium dodecyl sulphate; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid), Ellman's reagent; POPOP, 1,4-bis(5-phenyloxazol-2-yl)benzene; PPO, 2,5-diphenyloxazole.

Haptoglobin forms with hemoglobin a very stable complex showing the catalytic properties of a ' true' peroxidase, although neither haptoglobin nor hemoglobin is an enzyme. This unique property has been useful for quantitative determination of haptoglobin [2]. Reduction of haptoglobin 1-1 yielded a number of intermediates followed by the complete dissociation into a I and fl subunits [3]. Thereafter we have been interested in the role of interchain disulphide bonds in the formation of quaternary structure of haptoglobin and its biological activities (hemoglobin- and antibody-binding). In this paper we describe sulphydryl groups of the intermediates appearing in the process of successive reduction of haptoglobin 2-1, as determined directly or by incorporation of [14C]acetamide into polyacrylamide gel electrophoretic fractions. On the ground of molecular mass measurements of the reduction products, their subunit composition could be proposed.

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14 Materials and Methods

Materials. Human haptoglobin 2-1 was isolated from ascitic fluid [4]. The preparations used were of 100% purity as checked spectrophotometrically using extinction coefficient for haptoglobin 2-1 of 1.16 and comparison with the peroxidase method [5] as well as by polyacrylamide gel electrophoresis. Preparation of /3 subunit was carried out according to Bernini and Borri-Voltattorni [6]. The following reagents were used. DEAE-52 cellulose, POPOP, PPO, Triton X-100 from Serva, Heidelberg (F.R.G.); Coomassie Brilliant Blue R250, BDH, Poole (U.K.); SDS, International Enzymes Ltd., Windsor (U.K.); 2-mercaptoethanol, guanidine hydrochloride, iodoacetamide, Fluka (Switzerland); iodo[a4C]acetamide, Amersham (U.K.); sodium borohydride, DTNB (Ellman's reagent), Sigma (U.S.A.); sodium arsenite, Riedel de HaEn, Hannover (F.R.G.). For the determinations of molecular mass, the following markers were used: bovine albumin (67 kDa), and chymotrypsinogen (25 kDa) from Serva, Heidelberg (F.R.G.); haptoglobin 1-1 (100 kDa), fl subunit (40 kDa), a I subunit (8.9 kDa), and a 2 subunit (17.6 kDa) were prepared in our laboratory. Other reagents were analytical grade from P.O.Ch., Gliwice (Poland). Sulphydryl group assays following reduction. Titration of sulphydryl groups using sodium borohydride as reducing agent was carried out by the method of Habeeb [7]: to 1 mg of protein in Tris-glycine buffer (pH 8.0) containing 8 M guanidine and EDTA, a drop of antifoam and 2% solution of sodium borohydride were added. After a 30 min incubation at 40°C, the excess sodium borohydride was destroyed by adding 1 M HCI and acetone, followed by the addition of DTNB. The absorbance was read at 412 nm and the sulphydryl content was calculated using a molar extinction coefficient of 1.36.104 . Titration of sulphydryl groups following 2mercaptoethanol reduction was carried out according to Krawczyk and Dobryszycka [3]. At the defined time intervals, 50 /~1 samples (50 btg of protein) were withdrawn and 50 /~1 of 0.06 M iodoacetamide, containing 13 /~Ci iodo[laC]acet amide in 0.01 M phosphate buffer, (pH 7.0) was added. The aliquots were boiled and subjected to

SDS-polyacrylamide gel electrophoresis. Protein bands were visualized by staining with 0.05% Coomassie brilliant blue R-250. The gels were cut into pieces according to separated protein bands. Each piece was dissolved by a 30 min boiling in 1 ml of hydrogen peroxide followed by a transfer into 15 ml of the scintillation mixture (POPOP and PPO in toluene). Radioactivity was measured in a B-counter of Kontron (Switzerland). The specific activity ratio of labelled protein to radioactive iodoacetamide is equal to the molar ratio of -SH groups to protein and therefore represents the number of -SH groups per molecule. Controls of non-labelled preparations were included in each experiment.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was carried out in 7.5% gels by the procedure of Weber and Osborn [8]. Relative content of the separated electrophoretic bands was determined in a Carl Zeiss, Jena (G.D.R.) densitometer. From the electrophoretic mobility of the protein bands and appropriate standard markers, molecular mass of intermediates appearing during reduction was measured. Protein and haptoglobin assays. The protein content was determined by the method of Lowry et al. [9], and haptoglobin according to Jayle [2], respectively. Immunodiffusion. Double immunodiffusion in agar gel was performed according to Ouchterlony [10]. Anti-haptoglobin 2-1 antiserum was raised in rabbits as previously described [11]. Results

Sodium borohydride reduction Throughout the course of the reduction of haptoglobin 2-1 and fl subunit by sodium borohydride, the liberated -SH groups were determined by DTNB (Table I). Five disulphides were reduced almost immediately, next two were cleaved in the 1st minute, one in the 3rd and two in the 5th minute, respectively. This corresponded to 20 -SH groups, which was in good agreement with the theoretical number of -SH groups present in a completely reduced molecule of haptoglobin 2-1 (Fig. 1). Reduction of /3 subunit resulted in the rapid appearance of three -SH groups followed by

15 TABLE I SULPHYDRYL GROUPS APPEARING DURING SODIUM BOROHYDRIDE REDUCTION OF H A P T O G L O B I N 2-1 -SH groups were determined by DTNB according to Habeeb [7], and expressed in m o l e s / m o l e of protein. The results are mean values from at least five determinations. Time (rain)

Haptoglobin 2-1 -SH groups

0 0.5 1 3 5 15

0 10.2 13.8 15.8 20.4 20.4

Subunit fl a -SH groups 2.8 4.5

a Subunit fl was isolated according to Bernini and Borii-Voltattorni [61.

further one to two groups as early as in the 1st minute of the reaction.

2-Mercaptoethanol reduction The speed of the reaction with sodium borohydride made difficult the proper detection of

intermediates; thus, in succeeding experiments 2mercaptoethanol was used for gradual reduction of the disulphide bonds in haptoglobin 2-1. In the course of a 90 min reduction of haptoglobin 2-1 with the use of 2-mercaptoethanol, the samples were alkylated with the labelled iodo[14C]acetamide and subjected to SDS-polyacrylamide gel electrophoresis. The relative content of the separated protein bands was measured densitometrically. On the basis of the radioactivities of particular protein bands (Fig. 2), the number of -SH groups liberated in the given times was calculated (Table II). The mobilities of the intermediates separated electrophoretically were used for molecular mass determinations. These in turn served for propositions regarding subunit composition of the intermediates (Table III). The reduction with sodium borohydride proceeded much more quickly than that with 2mercaptoethanol; nevertheless, in both cases the quantitative dissociation of disulphide bonds was found to be comparable, amounting to 5-2-1-2 borohydride-reduced disulphides, and 4-3-1-2 mercaptoethanol-reduced, respectively.

Hemoglobin- and antibody-binding N

1

lO5 r s - s 7 rs-sq C ~; 148 179190 219245 /

15 34 687493 12ff C N1 SH LS-SJsH LS-SJ131142 N ! SlH rS-S77263c 15 34 68~

~2chai n~ form SH a 1choinJ polymers

The fundamental criterion for the native structure of oligomeric proteins is their biological activity. Peroxidase activity of the complex with hemoglobin was detected during a 90 min reduction of haptoglobin 2-1 with 2-mercaptoethanol (Fig. 3). Almost immediately after the addition of the re-

!

N

I

SI

FS_S.7 rS_S7 245C

105 148 1'79190 219 SH

SH

T A B L E II INCORPORATION OF [14C]ACETAMIDE INTO SULPHYDRYL GROUPS DURING 2-MERCAPTOE T H A N O L R E D U C T I O N O F H A P T O G L O B I N 2-1

@_s_s_@ (=1 /3)2. (=2/J,)n where n=0,1,2,. .... Fig. 1. Structural representation and scheme of the subunit arrangements in haptoglobin 2-1 according to Bowman and Kurosky [1]. -SH groups at Cys-15 and Cys-74 are postulated to be involved in polymer formation.

Time

Radioactivity

(min)

dpm a

-SH groups

-S-S- bonds

0 8.1 13.2 16.7 21.0

4 7 8 10

% of recovery

0 0.5 5 50 90

0 1922 3180 3 985 4998

38.4 63.0 79.7 100.0

a Sum of the radioactivity incorporated into electrophoretic protein bands in the gels shown in Fig. 2.

16

Postuloted subunit composition of intermediates . . . .

216

--520

- 51~

347 356

--508 --477

-284 -37C

340

_1371

2121

532 131

194

A

/~. ~'1. ~1'2. ['J'

G2. G2

.....

/.-3.

.....

/,9 . G2

.....

~ • G1

~2

339 297

110

C

B

D

E

Fig. 2. Polyacrylamide gel electrophoresis [8] of haptoglobin 2-1 without SDS (sample A), and at successive stages of the reduction by 2-mercaptoethanol with SDS (samples B E). (A) Haptoglobin 2-1; (B) 0.5 min reduction; (C) 5 min reduction; (D) 50 min reduction; (E) 90 min reduction. The protein bands were stained with Coomassie brilliant blue R-250. The numbers indicate radioactivities (dpm) of [14C]acetamide incorporated into particular bands. This was used for the calculations of the -SH groups content (Tables II and 11I). The postulated subunit composition of the electrophoretic bands is also given.

TABLE lII S U L P H Y D R Y L G R O U P S A N D M O L E C U L A R MASS MERCAPTOETHANOL R E D U C T I O N OF HAPTOGLOBIN 2-1

OF

INTERMEDIATES

APPEARING

DURING

2-

Sulphydryl groups were determined by the ratio of labelled electrophoretic protein bands to incorporated [~4C]acetamide (Fig. 2). The molecular mass of intermediates was measured in SDS-polyacrylamide gel electrophoresis [8]. Composition of intermediates was calculated taking into account molecular mass of haptoglobin 2-1 subunits: fl, 40 kDa, 0/2, 17.6 kDa, and c0, 8.9 kDa [1]. Mean values of five determinations are given. Reduction time (min):

Total

Sulphydryl groups 0.5

5

Molecular mass (kDa)

Postulated subunit

determined

theoretical

composition of intermediates /3.0/1. a2./3 /3.0/lfl ft. a 2- a 2 /3. a 2 /3. a 1 /3

50

90

2 2 1 7

10

110.0 89.0 69.2 57.6 49.0 40.0

106.5 88.9 75.2 57.6 48.9 40.0

1 2 1 2 1 1

2 1 6

-

2

3

7

17.5

17.6

0/2

-

1

2

4

9.0

8.9

aI

8

14

17

21

2

17

As shown in Fig. 4, all the samples exhibited full immunological identity. The isolated /3 subunit was found to be deficient in antigenic determinants as related to haptoglobin 2-1 and to reduction products.

'I I

o •

'1

I

~ ~oc

~0

..........

0

[3. u'l ~ 0 50 0"0 L'~ ~'3D_ I

I

I

I

I

I

I

I

I

10

20

30

40

50

60

70

80

90

Time {rain)

Fig. 3.Peroxidase reaction [2] of the products of haptoglobin 2-1 reduction by 2-mercaptoethanol. The peroxidase activity of native haptoglobin 2-1 complexed with hemoglobin was taken as 100%. Percentage of -SH groups reduced is also shown,

ducing agent and the appearance of free -SH groups, peroxidase activity was diminished by 50%, followed by a further decrease up to approx. 10% in the 90th minute. Reoxidation with atmospheric oxygen resulted in the renaturation of 80% of peroxidase activity in the complex with hemoglobin, independently of the time of the previous reduction. The same samples were used for immunodiffusion with anti-haptoglobin 2-1 rabbit antiserum.

4

Fig. 4. Cross-reactions of the products of haptoglobin 2-1 reduction by 2-mercaptoethanol. In the center well is rabbit antiserum directed against human haptoglobin 2-1. In the outer wells are antigens, 50 /xg each: (1,4), haptoglobin 2-1; (3) subunit; (2) products of a 15 min reduction; (5) products of a 50 min reduction; (6) products of a 90 rain reduction. Double immunodiffusion in agar gel was carried out according to Ouchterlony [10].

Discussion

In the molecule of haptoglobin 2-1 there are interchain disulphide bonds linking a t to fl, a 2 to fl, and c~1 to a 2. Moreover, two intrachain disulphides are in the fl chain, two in the a 2 and one in the a 1 (Fig. 1). Four classes of disulphide bond may be distinguished in the structure of haptoglobin 2-1 on the basis of their relative sensitivity to reduction agents. The number of disulphides in particular classes would be approximately 4, 3, 1, 2. However, it would have been an oversimplification to ascribe the experimentally determined four classes to four kinds of disulphide existing in the structure of haptoglobin 2-1. The electrophoretic patterns of intermediates appearing during gradual reduction of haptoglobin 2-1 were found to be rather complex. Taking into consideration all the information, experimental and theoretical, it seems that one of disulphides linking cd with a 2 may be dissociated very quickly, almost simultaneously with the cleavage of some bonds l i n k i n g ot 2 with ft. Sulphydryl groups formed at this initial stage were probably reshuffled through disulphide interchange, which resulted in the fast appearance of the following intermediates: ft. a t • O~2" /3; /3" OtI • /3; /3" 0/2. O~2; /3" O/2; /3" otl; /3. In the next step two compounds disappeared (/3. a ] • a 2/3 and /3. a 2. a2). Instead, there were dissociated a 2 and a ] subunits, though the intermediates/3, a 2 a n d / 3 , a 1-/3 remained. Obviously, some linkages /3. a I and /3-a 2 are more accessible to the solvent than identical linkages probably buried in the interior of the haptoglobin 2-1 molecule. At a 50 min determination, the same electrophoretic intermediates were found as at 5 rain; thus, the two newly reduced disulphides had to be intrachain ones. Within 50-90 min, last interchain disulphide bonds releasing the separated subunits, as well as the most resistant intrachain disulphide in 13 subunit, were cleaved.

18

Evidence has been presented that the interchain disulphide bonds are much more susceptible to reduction than the intrachain ones [12]. Moreover, disulphides that link two symmetrical subunits can be dissociated more easily than the bonds between two kinds of subunit [13]. Therefore, the bond a 1. a 2 (-SH group No. 15 in a I linked with -SH group no. 74 in a 2) and that linking -SH group No. 15 in a 2 with further part of the molecule (Fig. 1) should have been the most susceptible to reduction. However, the folding of the polypeptide chains causes the asymmetry and consequently, different accessibility of -S-S- bonds to the reagents. Such an asymmetry of the haptoglobin 1-1 molecule was observed in our laboratory by spin and fluorescence labelling [14]. As a rule, biological properties of proteins depend on their uniquely folded conformation, but the effect of the reduction of disulphide bonds on biological activities may vary. A loss or significant decrease of biological properties could occur [15,16]; sometimes stimulation was observed [12] or a native set of disulphide bridges was found to be inessential for activities [13,17]. Gradual reduction of disulphide bonds in haptoglobin 2-1 resulted in the rapid decrease of hemoglobin-binding capacity by 50%, even in the sample which contained the tetramer a I • ft. 13- a 2 (Figs. 2, 3). As an isolated fl subunit exhibits approx. 15% of the peroxidase activity of the complex of native haptoglobin with hemoglobin, and a subunits are devoid of this activity [18], it seems that residual hemoglobin-binding of the sample completely reduced may be ascribed to the relative high content of the/3 subunit. On the other hand, 30-20% activities of the samples inbetween would be related to the presence of trimers and dimers besides /3 subunits. It can be concluded that disulphide linkages in haptoglobin 2-1 are essential for the maintenance of such a conformation of the molecule, which enables the formation of an active complex with hemoglobin. Thus, renaturation of almost entire peroxidase activity following oxidative reassociation of the reduced samples might

suggest that a native set of disulphide bonds was formed. This was demonstrated for haptoglobin 1-1 [19]. On the other hand, immunological reactivity was found to be independent of the reduction of disulphide bonds.

Acknowledgement This work was supported by the grant No. I1.1.2.3 from the Polish Academy of Sciences.

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