Hydrolysis of disulfide bonds in weakly alkaline media II. Bovine serum albumin dimer

BIOCHIMICAET BIOPHYSICAACTA

363

BBA 355o4 H Y D R O L Y S I S OF D I S U L F I D E BONDS IN W E A K L Y A L K A L I N E MEDIA II. BOVINE SERUM ALBUMIN D I M E R

LA RS-()I.()V AN DERS.S()N

Institute of Biochemistry and lnstztute of Physical Chemistry, Uniw'rsity of Uppsala, Uppsala (.Sweden) ( l ~ c c e i v e d A u g u s t 2 8 t h , 1969)

SUMMARY

Dimeric bovine serum albumin prepared by oxidation of mercaptalbumin went back to the monomer upon standing in weakly alkaline solutions. The kinetics of the cleavage indicated that successive reactions were involved. On addition o f - S H reagents, the rate of splitting decreased considerably. These observations were taken to indicate that the splitting proceeds by hydrolysis of an exposed disulfide bond followed by disulfide exchange reactions which finally result in cleavage of the disulfide bond that joins the two monomer units in the dimer. The implications with regard to the stability of disulfide bonds in proteins in alkaline media are discussed.

INTIIODUC'rlON

It is generally assumed that disulfide bonds in proteins are fairly stable at a weakly alkaline pH. Many studies on proteins have been performed at pH values between 9 and ii, and the possibility of disulfide bond splitting has usually been ignored. However, some studies ~ -3 on disulfide exchange reactions in low molecular weight disulfides and degraded proteins clearly show that some splitting of disulfide bonds occurs even at pH 8. It has also been shown 4,s that cleavage of disulfide bonds at a weakly alkaline pH can occur in proteins denatured in concentrated urea solutions. Studies with low molecular weight disulfides have shown63 that hydrolytic splitting occurs in weakly alkaline media by tile following mechanisnl : R S S I ( -- ( ) H

-

-

I ¢ S - -- R S O H

(~)

I¢S()1t = R S O , H .:- R S H

(2)

The equilibrium of Reaction I is far to the left, and even in o.i M NaOH the equilibrium concentration of RS is very small. However, if either of the components A b b r e v i a t i o n : P l I M B, p - h y d r o x y m e r c u r i b e n z o a t e .

Bu~chim. Bzophys, Acta, 2 o o (197 o) 3 6 3 -309

3o4

L.-O. ANDER.qSON

fiwmed in R e a c t i o n I is in some w a y r e m o v e d from the e q u i l i b r i u m , the cleavage reaction will proceed to completion. In previous studies s on the s t a b i l i t y of various serum a l b u m i n dimers, it was found t h a t dimers held t o g e t h e r b y disulfide b o n d s split when allowed to s t a n d in solutions of p H between I I and 12. It was also shown t h a t a disulfide d i m e r could be p r e p a r e d b y o x i d a t i o n of serum m e r c a p t a l b u m i n . In the present investigation this kind of d i m e r has been studied with respect to splitting and h y d r o l y s i s of disulfide b o n d s in w e a k l y alkaline media. MATERIALS AND METHODS

The bovine serum a l b u m i n used was o b t a i n e d from S t a t e n s Bakteriologiska l . a b o r a t o r i u m (SBL), Stockholm. The sample contained a b o u t 8 % dimeric serum albumin. Pure m o n o m e r was p r e p a r e d b y gel filtration on S e p h a d e x G-I5o. Disulfide d i m e r was then p r e p a r e d b y o x i d a t i o n of the m o n o m e r ~ with a 5o-fold excess of ferricyanide in o.I M Tris-o.2 M NaC1-2 mM E D T A buffer (pH 8.o). Studies on the s p l i t t i n g of the d i m e r b y reduction 8 with m e r c a p t o e t h a n o l indicated t h a t it was a b o u t 9 5 % homogeneous. The r e m a i n i n g 5 % were also split b u t at a lower rate t h a n the main part, thus i n d i c a t i n g t h a t this fraction has a n o t h e r conformation. The tool. wt. of m o n o m e r i c serum a l b u m i n was a s s m n e d to be 66 ooo. H y d r o x y m e r c u r i b e n z o a t e was p r e p a r e d from fl-toluenesulfinate according to "~VHITMORE AND ~,~,'OOD",VARI)~''.

Kinetic measurements The s t u d y of the s p l i t t i n g of the disulfide d i m e r in alkaline solution was performed in the following way. To a 1 % solution of d i m e r in o.I M N a H C O s - 2 mM E D T A buffer at 25 ° was a d d e d i M N a O H to the desired pH. W i t h the t e m p e r a t u r e m a i n t a i n e d at 25 ° , samples were w i t h d r a w n at intervals and were i m m e d i a t e l y a d j u s t e d to a b o u t p H 7, followed b y a d d i t i o n of a small a m o u n t of p - h y d r o x y m e r c u r i b e n z o a t e (PHMB) solution to s t o p disulfide exchange reactions. The comt)ositi
1)etcrmination of ..SH groups The c o n t e n t o f - S H groups was d e t e r m i n e d by s p e c t r o p h o t o m e t r i c t i t r a t i o n with P H M B in a c e t a t e buffer (pH 4.5) as described earlier a.

Gel filtration experiments Gel filtration e x p e r i m e n t s on S e p h a d e x (;-x5o were performed on both a prep a r a t i v e a n d an a n a l y t i c a l scale. The p r e p a r a t i v e column was 2oo cm long a n d had l l t o c h i m . Iliophys. Acta. . o o (lcUo) 3o3 3,5~

I)I.qUI.FII.)E BOND HYDROLYSIS

305

a bed volume of I6OO ml. The analytical column was 7 0 cm long and had a bed w)lume of 2oo ml. The buffer used was o.I bl Tris-o.2 M Na('l (pH 7.3).

Sedimentation measurements All centrifugations were carried out in a Svedberg type oil-turbine ultracentrifuge at a spee(t of IOOO rev./sec.

('oncentration measurements The concentrations of serum albunfin were determined by measuring either the ultraviolet absorption at a78 nm or the difference in refractive index between the albumin solution and the corresponding buffer, or by' weighing the dry sample. RESULTS

Kinetics of the splitting of the disulfide dimer The splitting of disulfide dimer to monomer was studied in carbonate buffer at 25 °. Some experinaents were also performed in phosphate buffer. The experimental procedure is described above. Fig. I illustrates the kinetics of the splitting at pH lO.65. The curve has a lag period in the beginning which indicates that consecutive reactions are inw)lved in the splitting. The pH dependence of the splitting is shown in Fig. 2. pH

,oo

I1.0'

>_

10.0 "~ 50

'

5

110

ti me (h)

,o 2o 3'o ,'o Half-life of the dlmer (h)

Fig. I. T i m e - c o u r s e of s p l i t t i n g of disulfide d i m e r in a l k a l i n e s o l u t i o n a t 25". ,o m g / m l d i m e r s o l u t i o n in o.i M c a r b o n a t e - 2 m.M F.DTA buffer (pH 1o.6I). Fig. 2. R a t e of s p l i t t i n g of disulfide d i m e r in a l k a l i n e s o l u t i o n s of different pH values. The r a t e of s p l i t t i n g is e x p r e s s e d as the h a l f q i f e of t h e d i m e r a n d is p l o t t e d a g a i n s t t h e p H a t w hi c h t h e r e a c t i o n w a s run. The t e m p e r a t u r e was 25 ° and o. I M c a r b o n a t e e mM E D T A buffers of v a r i o u s p H v a l u e s were used.

Tile half-life of the dimer is plotted against the pH at which the reaction was run. The shapes of tile curves were similar in the pH interval Io.4 II.2. Below pH Io.I the initial parts of the curves were nearly linear. An interesting feature is the break in the pH dependency curve at pH lO.3-1o.4. This is probably related to the change in omformation that is known to occur with serum albumin in this pH intervaP °. The sedimentation coefficient of the dimer decreased when the pH of the solution was raised above io.3, indicating that the molecule begins to assume a more extended conformation at this pH. The buffer solutions used contained EDTA. The reaction rate increased about 25°:o in the absence of EDTA, which probably reflects the presence of catalvtic Biochim. Biophys. ,4eta, 20o (i97 o) 303-369

366

I . . - o . AN I ) E R S S O N

a m o u n t s of metal ions in the solutions. E D T A p r e s u n m b l y chelates the metal ions so t h a t t h e y no longer influence and c o m p l i c a t e the reactions. The l)ossibility t h a t dissolved o x y g e n might be involved in the splitting of the d i m e r was i n v e s t i g a t e d by performing an e x p e r i m e n t wherein a d i m e r solution was d i v i d e d into two parts, one of which was d e o x y g e n a t e d , while the o t h e r was not. The relnoval of o x y g e n was effected b y b u b b l i n g nitrogen through the solution for Io rain. No differences were observed between the two solutions with regard to the rate of disulfide cleavage. The effect o f SH reagents on the s p l i t t i n g was also investigated. The presence of o.5 mM P H M B in a disulfide d i m e r solution of p H lO.8 7 decreased the rate ~t" splitting a b o u t IO fold. The kinetics of the reaction was also c h a n g e d ; instead of an Ss h a p e d eurw.~, a first-order curve was o b t a i n e d . A d d i t i o n of AgNO a to disulfide dimer solutions also decreased the rate of s p l i t t i n g trot also induced aggregation to t r i m e r and t e t r a m e r . No aggregation occurred in the solutions containing PHM B.

The nature of the monomer formed The m o n o m e r fi)rmed in the alkaline s p l i t t i n g of the d i m e r could be c o m p l e t e l y s e p a r a t e d from the r e m a i n i n g d i m e r b y gel filtration on S e p h a d e x (;-15o. l:ig. 3 shows the gel filtration d i a g r a m of a disulfide d i m e r solution t h a t had been k e p t at p H lO.77 for 9 h. No o t h e r p r o d u c t s were formed in the reaction except at the highest pH studied (pH 11.2) where small a m o u n t s of t r i m e r and t e t r a m e r were flJrmed. There were no differences between the regenerated n l o n o n l e r a n d native m o n o m e r with respect to gel filtration and s e d i m e n t a t i o n I)ehavior. D e t e r m i n a t i o n s of the - S H c o n t e n t s of the m o n o m e r s by spectrot)hotometric t i t r a t i o n gave values m the range of o.48 o.57 SH/mole. W h e n the t i t r a t i o n s were performed i m m e d i a t e l y after the s e p a r a t i o n of the monomer, the - S I t c o n t e n t s o b t a i n e d were s o m e w h a t higher. The values given are the stable ones, o b t a i n e d after s t a n d i n g 3 or 4 dab's at p t t 7.3. The residual d i m e r was also found to contain o. I-o.2 - S H / m o l e . This - S H slowly d i s a p p e a r e d . The amino acid composition of the m o n o m e r was d e t e r m i n e d with special a t t e n t i o n to the possible presence of l y s i n o - a l a n i n e and cysteic or cysteine sulfinic acid. Prior to acid hydrolysis, the sample was t r e a t e d with excess iodoacetic acid for 2 h at pH 8.4 in order to t r a n s f o r m the cysteine present in the sample to 5-carboxb'm e t h y l c y s t e i n e . The a m i n o acid c o m p o s i t i o n of the carboxymethb, lated regenerated m o n o m e r was the same as t h a t of i o d o a c e t a t e - t r e a t e d n a t i v e bovine serum a l b u m i n m o n o m e r with the exception t h a t o.4-o.5 e q u i v a l e n t of cvsteic or cysteine sulfinic acid was present and the cystine c o n t e n t was 2 or 3'I,, lower. No l v s i n o - a l a n i n e was detected.

Hydrolysis of disulfide bonds in dimeric and monomcric serum albumin The reaction of P H M B with - S H groups formed in the alkaline t r e a t m e n t of the disulfide d i m e r could be followed d i r e c t l y by m e a s u r e m e n t s of the changes in u l t r a v i o l e t a b s o r p t i o n at 25o nm. W h e n P H M B reacts with - S H groups, its a b s o r p t i o n at this w a v e l e n g t h increases considerably, l:ig. 4 illustrates the t i m e - c o u r s e of the reaction at p H I I.O8 and at p H lO.82. The e x p e r i m e n t a l conditions were the same as those described above. According to R e a c t i o n 1 calculation of the n u m b e r of disulfide b o n d s split, resulting in the f o r m a t i o n of i P H M B - m e r c a p t i d e per disulfide bond split, 13.wktm. Biophvs..4cta, 2 0 o (xO7 o) ~b 3 3tu)

367

DISULFIDE BOND HYDROLYS[.~

°21~

A::t l 0.!

0.10. AA2NI 0.05'

0.6 0.7 0.8 09 1.0 1.1 1.2 Elution volume (l)

3'o 6'0 9~ 1~o 1~o 1~o Time (rain)

Fig. 3- Elution diagram of gel filtration on Sephadex G i5o of a io mg/ml disulfide dimer solution which had been kept at p H zo.77 for 9 h in o.i M c a r b o n a t e - 2 mM EI)TA buffer at 25 ° . The buffer used in the gel filtration was o.i M Tris-o.2 M NaCI (pH 7.3). Fig. 4. Time-course of hydrolysis of disulfide lxmds in disulfide dimer in the presence of PHMB. The increase in absorption at 250 n m caused by the formation of P H M B - m e r e a p t i d e s is plotted against time. The conditions were: 0.8 mg/ml disulfide dimer in o.i M c a r b o n a t e - e mM F D T A 5-1o -6 M P H M B solution at 25 ~. @, p H 1I.O8; O, p t t io.82.

shows that at pH 11.o8, o. 5 disulfide bond per serum albumin dimer is split after 45 min and I.O after about I h and 4o rain. Probably several disulfide bonds split but at different rates. This is in accordance with the observed kinetics. Comparison of bovine serum albumin dimer with oxidized glutathione 7 shows that the rate of hydrolysis of disulfide bonds is much lower in the protein. Even if we assume that only one disulfide bond is split in the bovine serum albumin, the rate of splitting is higher in the glutathione. Accessibility and strain on the disulfide bonds are probably the main factors which determine the rate of hydrolysis in different compounds. Iodoacetate-treated native serum albumin showed about the same PHMB reaction curve as the disulfide dimer, but the rate of the reaction was somewhat lower. DISCUSSION

The time-course of the splitting of disulfide dimer indicates that successive reactions are involved. One might, therefore, suspect that disulfide exchange reactions occur. Support for this is obtained from tile fact that the disulfide bond that joins the 2 monomer units is rather shielded from direct attack by an hydroxyl ion. Definite proof is afforded by the experiments wherein PHMB was added to the dimer solution. PHMB inhibits disulfide exchange reactions by reacting with the - S H groups, whereas it definitely promotes the hydrolytic cleavage reaction itself 7. The results obtained thus clearly show that about 9o% of the splitting of dimer is mediated through disulfide exchange reactions. The remaining lO% is probably a direct reaction between O H - and the intramolecular disulfide bond, as indicated by the first-order kinetics. The sequence of reactions involved in the splitting of the dimer might thus be schematized as follows: Biochim. t3iophys. Acta, 20o (i97 o) 363-309

368

I..-O. ANI)t-RSSON

IA'b°~i° I

I Aii~uml'~

I

S

I

I

S

I

OH-

+

S--S--S

,~ "

HOS i

S-

AIl~ami~

S

,. J

~OS I

S--S

AIl:xamln

+

S-

(3)

',

r h i s reaction scheme accords with all the experimental observations. First, the scheme involves a sequence of reactions which can acccmnt for the S-shaped t i m e course of the overall dimer , nionomer reaction. Second, it involves disulfide exchange reactions that are inhibited by the presence of PHMB. Third, the - S H content of the m o n o m e r formed w, mld be o. 5 -%t-l/mole in good agreement with the observed values of o.48-o.57 -SHimole. It is, of course, possible to write reaction schemes involving 3 or more steps with 3 or more disulfide bonds participating in the reaction instead cff 2, but the net effect would still be the sauie. The reaction sequence proposed is similar to that suggested for the splitting of disulfide dimer by reduction ~ with mereaptoethanol. "rhe driving force for the hydrolysis of the disulfide bond probably arises from electrostatic repulsion between different chain segments, which should tend to force the equilibrium (I) toward the right, i.e. toward dissociation. In the reaction scheme suggested, it has been assumed t h a t hydrolytic splitting of the disulfide bond occurs. It has been shown, however, that in strongly alkaline media another type of splitting occurs, the [1 splitting which is shown in Reaction 4. i

()1t

('I-t-('I-t2--S

i

S ('H2-('It--+

{ . ' - . ( ' t l 2 -- % - . 5 . - ( ' 1 4 2 - ( ' I I

: It.,()

(I)

The fl splitting has been shown to occur in several proteins n but only at pH values above I2. This reaction is often followed by a second step in which the dehydroalanine residue formed in the first step reacts with a lvsine residue and forms lvsino-alanine. However, no lysino-alanine was found in hydrolysates of alkali-cleavaged disulfide dimer. Studies: on the splitting of oxidized glutathione in weakly alkaline media revealed no fl splitting below p H I I. Apparently, the fl splitting reaction does not occur under the conditions specitied here for the splitting of the disulfide dimer. The unstable sulfenic acid group fl)rmed in the hydrolysis is probably oxidized further to sulfinic or sulfonic acid by dissolved oxygen. The detection of cvsteinc sulfinic or sulfonic acid in the hvdrolvsates of alkali-split dimer supports this. Dismutation according to Reaction e might occur but is not very likely as there are steric factorsS; oxidation of bovine serum albumine cmly gives dimer when the protein is denatured, which probably stops the - ,%OH groups from coming sufficiently close to each other. "l'his is also the reason whv the second step in the reaction scheme suggested is written as irreversible. Serum albumin assume.s a more extended conformation at pH io.2 io. 4. This conformational transition greatly affects the rate of splitting of the disulfide dimer. Probably the equilibrium in the hydrolysis step is pulled to the right its a consequence of the disulfide bond being under strain in the changed conformaticm. It is also possible that the rates of disulfide exchange reactions are increased as a consequence ¢ff the conformation being more flexible in the extended form. The results of this investigation show that disulfide braids hydrolyze rather rapidly even in weakly alkaline niedia when the .<,H f(~rmed is solnehow withdrawn tiu~chtm tiiofgiys..'tcla.

>~,:J ficJ7o ) 3ft~

i~

I)ISI;I.FIDF. B O N D H Y D R O L Y S I S

309

from the hydrolysis equilibrium. The withdrawal o f - S H can be effected either by reacti(m with - S H reagents or by disulfide exchange reactions. These facts should be kept in mind when for some reason it is necessary to expose a disulfide-containing protein to weakly alkaline media. If an --SH reagent is present, extensive splitting of disulfide bonds ('an occur. Denatured or chemically modified proteins should be l)articularly susceptible to disulfide cleavage, since manv of the forces that stabilized the original "native" arrangement of disulfide bridges no longer exist. The technique used herein for monitoring the hydrolysis of disulfide bonds in bovine serum albumin should be applicable to other disulfide-containing proteins. The use of PHMB to force the hydrolysis equilibrium to the right, thereby generating at PH M B.. mercaptide which can be followed spectrophotometrically, might be a good approach to the stu(tv of the accessit)ilitv of and strain on disulfide bonds in proteins at alkaline plt. AC KN OV~q.E I ) ( ; I - M E N T S

The author wishes to thank Dr. K. O. PEI)ERSEN and Dr. D. EAKER for valuable discussions and Mr. S. CENTRING for performing the ultracentrifuge runs. R F F I- R E N C |".% :\. l'. l~VLl,: AND l:. SANGER, Biochem..]., 0 o (x955) 535. I). H . SP,XCK.~IAX, \V. I1. STEIX AND .% MOORF, J. Biol. Chem., 235 (x96o) 048. D. E. KOSHLAXD, Jr. A,~D S. *I. MOZERSKV, Federation P~oc., 23 (I964) 6o9. H . N. I:RE.','SDORFF, M. T. \VATSON AND \~'. KAUZMANN, J. Am. Chem. Soc., 75 (x953) 5 1 6 7 . H. :\..'MClg.ENZIF, 3I. l~. SMITH AYl) R. G. W A K E , Biochim. Biophys. Acta, 6 9 ( I 9 6 3 ) 222. kV. STRICKS AXD I. 31. KOLTHOFV, Anal. Chem., 25 (1953) I o 5 o . I..-(). :\NDERSSON, Bzochim. B~ophys. Acta, I 9 2 {106c)) 534. I,.-(). ANDERSSOX, Biochim. Biophys. Acta, I I7 (~966) I ~5. F. G. \VHI'r.~tORE AXD G. E. WOODWARD, in H . (;ILMAN AND A. 1t. BL:vr'r, Or eamc .gynthests, Coll. Vol. I, W i l e y , N e w York, 2 n d e(l., 1 9 6 I , p. I 5 9 a n d 5 1 9 . i o C. TANFORI), S. A..~WANSON ANt) \~'. S. SHORE, .]..4))?. Che;n. Not., 77 ( I 0 5 5 ) 6414" IS Z. B<)IIAK, .[. t¢iol. Chem., 239 (1004) 2~7'~.

i 2 3 -t 5 ~) 7 8 9

llmchim. Btophys. Acta, zoo ( t 0 7 o) 3~)3 -309