Reactivity of sulfhydryl groups of human globin

324

BIOCHIMICAET BIOPHYSICAACTA

BBA 36046 R E A C T I V I T Y OF S U L F H Y D R Y L GROUPS OF HUMAN GLOBIN

Z. H R K A L AND Z. V O D R f t Z K A

Institute of Hematology and Blood Transfusion, Prague (Czechoslovakia) (Received A u g u s t I 3 t h , i97 I)

SUMMARY

One fast reacting and two slowly reacting sulfhydryl groups occur in the human globin aft molecule at neutral pH. From the comparison of their reactivity with that of hemoglobin it follows that the fast reacting SH group of globin is at the fl93 position. The low reactivity of sulfhydryls a l o 4 and/5112 is in agreement with the theory of the existence of al/51 type contacts in the globin molecule. Reactivity of these groups increases parallel to the unfolding and dissociation of globin dimer. The stability of globin in neutral medium decreases markedly on reaction of all its sulthydryl groups with SH reagents.

INTRODUCTION

H u m a n hemoglobin contains six cysteinyl residues two of which at t593 can react with various sulfhydryl reagents 1-3 while the remaining four, two at a l o 4 and two at flli2, are unreactive in the native tetrameric molecule. ANTONINI AND BRUNORI4 found that the reaction of the free sulihydryl group of hemoglobin with pchloromercuribenzoate (PCMB) is a pseudo first-order process with a half-life in the order of some milliseconds. Under the conditions under which liganded hemoglobin is partly dissociated into dimers even the masked SH groups react with PCMB, the half-life of their pseudo first-order reaction being about I h (ref. 5). The reactivity of sulthydryl groups of globin has not been studied in detail. JAVAHERIAN AND BEYCHOK6 found one reactive sulfhydryl group in native horse globin and two in the presence of urea. In our previous works we have shown that the conformation of human hemoglobin and globin in solution markedly differs 7,s. This paper deals with the question to what extent the different conformation of globin is reflected in the changed reactivity of its sulfhydryl groups. Abbreviations:

PCMB,

p-chloromercuribenzoate;

acid).

Biochim. Biophys. Acta, 257 (1972) 324-327

DTNB,

5,5'-dithiobis(2-nitrobenzoic

SULPHYDRYL GROUPS OF HUMAN GLOBIN

325

METHODS

H u m a n and bovine globins were prepared and renatured by the procedure of VODRA~KA et a l 2 . The reaction of sulfhydryl groups with N-ethylmaleimide was performed according to the method of GREGORYTM, that with PCMB using the method of BOYER11, in both cases in 0.05 M sodium phosphate, p H 7.0, at 20 °. The reaction with 5,5'-dithiobis(2-nitrobenzoic acid) TM (DTNB) was carried out in o.i M sodium phosphate, p H 8.0. The experiments were accomplished both in the presence and in the absence of 8 M urea. The time-courses of the reaction of globin with PCMB were monitored on a Unicam SP7oo spectrophotometer by following the changes in differential absorbance at 255 nm between o.1% globin solution (3.22.1o -5 M) to which 3 equiv of PCMB were added, and the reference solutions of globin and PCMB in separate cells. Since the initial molar concentrations of sulfhydryl groups of globin and PCMB were equal (9.66. lO -5 M) the second-order rate constants, k, were calculated using the formula kt = i / ( a -- x) -- i / a

where a is the initial concentration of SH groups and x the fraction of SH groups that reacted with PCMB in time t. The rate constants, k, were evaluated as the slope of the linear part of the curve obtained by plotting I / ( a - - x ) against time. The kinetic runs were performed in 0.05 M acetate buffer, p H 4.8, 0.05 M sodium phosphate buffers, p H 6.0 and 7.0, and in 0.025 M sodium borate, p H 9.1, at I °, 5 °, IO° and 20 °. The differential molar extinction coefficient used was 7" lO3 M-l"cm-1. RESULTS

All three sulthydryl groups of the human globin a f t dimer react in 8 M urea with PCMB and N-ethylmaleimide at p H 7.0 and with D T N B at p H 8.0. In the absence of urea one SH group reacts rapidly, while the reaction of the remaining two is very slow and followed by precipitation of globin. The reaction of human globin with PCMB was studied in more detail by examining the p H and temperature dependence of the reaction kinetics. On addition of I equiv of PCMB to o.1% globin solution at p H 4.8, 6.0, 7.0 and 9.1, one SH group reacts rapidly, its kinetics being too fast to be measurable under the present experimental conditions (tl/2 < I min). These PCMB-treated globins are equally stable as the starting material. On addition of 3 equiv PCMB to the globin solution at p H 6.0, 7.0 and 9.1, apart from the first fast reacting sulthydryl group, also the remaining two react. However, their reactivity is much lower; the reaction proceeds with a half-life of several hours and its kinetics are, except at the beginning, in agreement with the time-course of the second-order reaction (Fig. I). The initial non-linear part of the second-order plot is probably the result of the overlapping kinetics of the fast reacting SH group. The reactivity of the masked sulfhydryl groups is lowest at neutral p H ; on increasing or decreasing the p H the rate of reaction increases (Table I). The kinetics are temperature dependent, the reaction is 50% faster at 5 ° than at I °. At higher temperature the reaction cannot be followed spectrophotometrically due to the gradual precipitation of globin on reaction with PCMB. The activation energy of the Biochim. Biophys. Acta, 257 (1972) 324-327

326

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F i g . I. The second-order rate plot of the reaction of s u l f h y d r y l groups of h u m a n globin w i t h P C M B a t I ° in o.o 5 M s o d i u m p h o s p h a t e , p H 7.o ( 0 ) , o.o25 M s o d i u m borate, p H 9.1 ( O ) , a n d o.o 5 M s o d i u m p h o s p h a t e , p H 6.0 (~)). The globin c o n c e n t r a t i o n was 3 . 2 2 ' lO .5 M, i.e. 9.66" lO .5 M in S H groups; c o n c e n t r a t i o n o f P C M B , 9.66" IO-5 M.

TABLE

I

THE SECOND-ORDER RATE CONSTANTS, k, HALF-LIVES,

tO,

AND THE ACTIVATION ENERGIES, I~a, OF

THE REACTION OF MASKED SULFHYDRYL GROUPS OF HOMAN GLOBIN WITH P C M B

pH

6.o 7.o 9.1

k (l.mole-X.sec

1)

tt* ( r a i n )

E a ( h c a l . m o l e -1)

i °

5°

i °

5°

3.6o 1.29 3-63

-1.96 5.23

71 200 71

-132 49

-15.9 13.9

* The values were calculated for a globin c o n c e n t r a t i o n o f 3 . 2 2 ' IO -~ M, i.e. for an initial c o n c e n t r a t i o n of m a s k e d S H groups o f 6 . 4 4 . lO -5 M.

reaction calculated from the rate constants is approx. 15 kcal/mole. On reaction of 3 equiv PCMB with human globin in acetate buffer, pH 4.8, two SH groups react in less than i rain and the reaction of the third is completed in 5 rain. Under these conditions globin with three SH groups blocked by PCMB is stable at room temperature for several days. Human globin with one sulihydryl group blocked by reaction with I equiv PCMB yields, on reconstitution with CO-heine, carbonylhemoglobin in which no reactive SH group can be found. It appears therefore that the fast reacting SH group of human globin is identical with the reactive sulflaydryl of hemoglobin at the fl93 position. This suggestion is further supported by the finding that the single sulfhydryl Biochim. Biophys. Acta,

257 (1972) 3 2 4 - 3 2 7

SULFHYDRYL GROUPS OF HUMAN GLOBIN

327

group occurring in bovine globin which is located in a position analogous to fl93 (see ref. 13) is also fast reacting with PCMB at neutral pH. DISCUSSION

Similarly to hemoglobin under dissociation conditions s one fast reacting and two slowly reacting sulihydryl groups occur in the human globin a/5 molecule. This similarity in reactivity of the SH groups suggests that the fast reacting sulfllydryl group of globin is identical with the reactive cysteinyl residue of hemoglobin at the /393 position. This idea is supported by the finding that no reactive SH group can be detected in recombined carbonylhemoglobin in which the fast reacting SH group has been blocked with PCMB prior to recombination. The slowly reacting SH groups of globin should therefore be the remaining two sulihydryls alo 4 and/~II2. Since the low reactivity of sulfhydryls alo 4 and fllI2 in the hemoglobin dimer is caused by their participation in the alfll subunit contacts* it seems probable that the equally low reactivity of these groups in the globin aft molecule has the same origin. If the globin molecule were a dimer of the alfl~ type, the alflX contacts would be interrupted and SH groups alo 4 and/3112 would be free to react. In this latter case we would expect an approximately uniform reactivity of all three SH groups. As this is not the case and two types of sulfhydryl groups can be clearly distinguished in globin at pH values close to neutral we conclude that the molecule in neutral medium is represented by a dimer of the alfll type. Under conditions promoting the unfolding and dissociation of the globin molecule (low pHT,s, presence of ureamS,1~) masked sulihydryls become accessible and their reactivity increases. Blocking of all three SH groups of the globin molecule at neutral pH probably promotes its dissociation in the a andfl chains due to the inclusion of the bulky PCMB molecules in the alfll subunit contacts. Since the free a and fl globin chains at pH values close to neutral are practically insoluble 17, precipitation of the protein occurs as the reaction proceeds. Blocking of sulihydryl fl93 only, which does not participate in the contact regions, has no influence on the stability of globin. REFERENCES i 2 3 4 5

6 7 8 9 IO ii 12 13 14 15 16 17

G. GUIDOTTI AND W. KONINGSBERG, J. Biol. Chem., 239 (1964) 1474. A. RIGGS, J. Biol. Chem., 236 (1961) 19¢8. R. BENESCH AND R. E. BENESCH, J . Biol. Chem., 236 (1961) 405 . E. ANTONINI AND M. BRUNORI, dr. Biol. Chem., 244 (1969) 3909. E. CHIANCONIt, D. L. CURREL, P. VECCHINI, E. ANTONINI AND J. WYMAN, J. Biol. Chem., 245 (197 ° ) 41o5 • K. JAVAHERIAN AND S. BEYCHOK, J. Mol. Biol., 37 (1968) I. Z. HRKAL AND Z. VODRf~.KA, Biochim. Biophys. Acta, 133 (1967) 527 . Z. HRKAL AND Z. VODRXZKA, Biochim. Biophys. Acta, 16o (I968) 269. Z. VODRfi~ZKA, H. HOLEYSOVSK~. AND H. ~iPALOVX, Collect. Czech. Chem. Commun., 29 (1964) 1287 . J. D. GREGORY, J. Am. Chem. Soc., 77 (1955) 3922. P. D. BOYER, J. Am. Chem. Soc., 76 (1954) 4331. G. L. ELLMAN, Arch. Biochem. Biophys., 82 (1959) 7 O. M. O. DAYHOFF, Atlas of Protein Sequence and Structure, Vol. 4, N a t i o n a l B i o m e d i c a l R e s e a r c h F o u n d a t i o n , Silver Spring, Ohio, 1969, D-6I. M. F. PERUTZ, H. MUIRHEAD, J. 3/1. COX AND L. C. G. GOAMAN, Nature, 219 (I968) I 3 I . A. I. CHERNOFF AND N. M. PETTIT, Blood, 24 (1964) 75 ° . T. ASAKURA, S. MINAKAMI, Y. YONEYAMA AND H. YOSHIKAWA, J. Bioehem. Tokyo, 56 (1964) 594. H. FOi~TOVk-~fPALOVk AND Z. VOOR~ZKA, Collect. Czech. Chem. Commun., 35 (197 o) 1261.

• W e p r e s u m e t h a t c y s t e i n y l fii 12 in t h e h u m a n h e m o g l o b i n m o l e c u l e is s i m i l a r l y s i t u a t e d as l e u c y l fli 12 in h or se h e l n o g l o b i n for w h i c h X - r a y c r y s t a l l o g r a p h y d a t a a re a v a i l a b l e 14.

Biochim. Biophys. Acta, 257 (1972) 324-327