Reactions of unsymmetrical disulfides

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

Reactions II. Sulfitolysis

South

:ljrican Wool and Industrial Received

(1967)

of Unsymmetrical

of Disulfide

XL’. J. J. VAN

121, 729-731

Bonds

Linking

REXSBURG

AND

Textile Research Research, Port March

22, 1967;

Disulfides Proteins 0.

Institute, Elizabeth, accepted

A.

to Small Molecules SWANEPOEI,

Council South April

for Scientijk Africa

7, 1967

The reactions of sulfite on the mixed disulfides of glutathione and cysteine and on those of thiol-proteins and cysteine have been investigated. It, was folmd that substitution at the S-S bond of the glutathione-cysteine disulfide takes place at random but that a specific sulfur atom (that of the “free” cysteine part of the molecule) in the protein-cysteine mixed disulfides is attacked more frequently than its mate. This directed substitution is eliminated when the reaction is carried out in a concentrated solution of urea.

atom in the unsymmet’rical was determined.

The existence of naturally occurring compounds containing unsymmetrical disulfide linkages between a small molecule and a peptide chain which forms part of a protein has been established by several authors. Thus, streptococcal proteinase (1) is a mixed disulfide of an unidentified volatile mercaptan and the cysteinyl residue of a protein, and nonmercaptalbumin (2) is a mixed disulfide compound of mercapt.albumin with cysteine and, to a lesser extent, glutathione. It is known (3) that the nucleophilic attack of sulfite on the sulfur-sulfur bond of small unsymmetrical disulfide compounds frequently occurs preferentially at a specific sulfur atom rather than at random. It is also considered (3) that substituents on the carbon atoms in the alpha and beta positions to the disulfide bond influence the approach of t,he entering sulfite group and that this steric hindrance is primarily responsible for t.he preferential attack at the specific sulfur atom. In the present investigation several natural proteins which contain free thiol groups were induced to form unsymmetrical disulfides with cysteine. These compounds were reacted wit’h sulfite, and the extent of t,he preference for attack at a specific sulfur

EXPERIMEKTSL

disulfide linkage

PROCEDURE

Preparation of the ,mixed d&&de of glutathione and cylsteine (G-SS-Cy). Equal volumes of 1OW Y solutions of cystine-3sS and glutathione in 0.1 11 Tris buffer, pH 7.2, were mixed and left to stand at room temperature for 30 minut.es. The solution was then acidified to about pH 2.5 by the addition of HCI, and the various components were separated by chromatography on a column (0.9 X 50 cm) of sulfonated polystyrene resin (Beckman automatic amino acid analyzer, model 120B). The products were eluted wit’h citrate buffer at pH 2.98 at 50”. The unsymmetrical disulfide was identified by its yield of cysteine and glutathione when reacted with excess mercaptoethanol at pII 7. Preparation of mixed disuljides of thiol-proteins and cysteine. Cyst’ine-35S (0.1 M, 0.025 ml) was added to 10 ml of the solution of the appropriate protein (1%) in 0.1 M Tris buffer (pH 7.2). After the solution was stirred for 30 minutes at room temperature, it was exhaustively dialyzed in Visking No. 23 dialysis tubing and then freeze-dried. Electrophoresis of the product on paper saturated with 10% acetic acid gave only a single radioactive band and showed that no residual cystine (or cysteine) was present. The following unsymmetrical disulfides were 729

730

VAN

RESSBURG

prepared by this method: Bovine plasma albumirl~S~S~cysteine (BPAs-s-Cy) UreaseeS-S-Cy (IJr-S-S-Q) Hemoglobin-S%-Cy (Hm-S-S-Cy) Lactalbumin-S-S-Q (La-S-S-Cy) Papa&S-S-Cy (Pa&-S-Cy). Sul$folysis in fhe absence oj urea. The entire sample of the freeze-dried protein containing the unsymmetrical disulfide, or 0.2 ml of the solution containing the isolated disulfide GSX-Cy, was dissolved in 5 ml Tris buffer (1.0 M, pH 7.2). 9 l-ml aliquot, of a solution containing PiapSOI (0.4 M) and CH3HgI (0.013 M) was then added. After stirring gently at room temperature for 20 minutes the pH was lowered to about 2.5 by the addition of glacial acetic acid. An aliquot of this solution (0.05 ml) was subjected to high-voltage electrophoresis (50 V/cm, 90 minrltes) with a solution of 10% acet.ic acid as the electrolyte. After drying, the paper strips were scanned on a Packard radiochromatogram scanner. Invariably only two radioactive bands were obtained which were identified as those of S-srdfo-cysteine and S-methylmercuric cysteine. The concent,rations of the radioactive substances were determined by integration of the peaks on the recorded scans. Checks were carried out to ensure that the sumtotal of the activity of the separated bands corresponded to that of the aliquot taken before electrophoresis Sulfitolysis in the presence of 6.Y II urea. The freeze-dried sample of protein containing the unsymmetrical disulfide was dissolved in 5 ml of a 1.0 M Tris buffer (pH 7.2) containing llrea (8 M). The rest of the procedure remained the same as described in the previous paragraph.

AND

SWSNEPOEL TABLE

I

BIDS OF THE ATTACK OF SCLFITE OS UNSYMMETRICAL DISI:I,FIDES Mixed

?;a I II III IV v VI

disulfide Description

G-S-S-Cy BPA-S-S-Q Ur-S-S-Cy Hm-S-S-Q La-S-S-Cy Pa-S-S-Cy

[CySSOs-l/[CySHgCHzl Without urea

In 6.7 M “Iea

1 9 4 2 9 2

1 1 1 1 1

tropherograms prepared as described under Experimental Procedure is therefore a measure of the bias. The values of this bias obtained for the various disulfides are summarized in Table I. The asymmetry of the disulfide compound in compound I is due to the free amino and carboxylic groups of one half-cystine comprising the disulfide and the incorporation of these groups of the other half-cystine int’o a polypeptide chain -CO-CH&H)-CHz--S I HOOC-CH(NHt)-CH*-H

This arrangement is common t#oall the disulfide compounds dealt, with in this paper. However, in contrast to compounds II through VI (see Table I), where the polyRESULTS AND DISCIJSSION peptide chains are very large molecules, When sulfite reacts with an unsymmet- the glutathione residue (in compound I) rical disulfide in the presence of MeHgI, the is only a tripeptide and has none of the following reaction takes place intricate three-dimensionally folded st’ructures associated with proteins. a R. S. S.R’ + a SOa-- + a CH,HgI+ The results in Table I show t’hat the two b R.SSO,+ b R’.SHgCHz + a Isulfur atoms in the disulfide linkage in compound I are attacked at random (bias = 1) + (u-b) R.SHgCH, + (a-b) It’.SSO,-. by the entering sulfite group. This is in agreeThe relative number of times that substitu- ment with an earlier conclusion (3) t#hat the tion takes place at t’he individual sulfur protection which t,ertiary substitution at the at’oms in the unsymmetrical disulfide (i.e., carbon atom beta to t,he sulfur atom offers the bias of the reaction) is given by the ratio against attack by sulfite is largely independ[RSSOS-],‘[R’SS03-I, which, according to ent of the nature of the substituents. Thus, the above equation, equals [RSSO,-]/ for a half-cystine residue the protective [RSHgCH,]. The ratio of t,he concentrations effect is the samewhether the amino and carof the radioactive X-sulfo-cysteine and S- boxylic groups are free or whether they are methylmercuric cysteine on any of the elec- incorporated as part’ of a peptide chain. This

evidence suggest,s t,hat it is improbable that the primary structures (the sequence of the amino acids in t’he peptidc chains) of the protein moieties of compounds II through VI are responsible for the tendency of sulfite to attack the sulfur atom belonging to the free cystcine group preferentially. However, it is clear from the results in Table I that’ in the mixed disulfides of c*ysteine and t#he various proteins, the sulfur atom belonging to the free cysteinc residue is invariably attacked more frequemly than the sulfur atom of the protein. Since by the argument expounded above the primary st,ructure of the prot’ein cannot be held responsible for this bias, it is concluded that the higher ordered st,ructures cause the attack to be directed to a specific sulfur atom. I;‘ava and his co-workers (4-6) considered the charge distribut’ion in the transition state when disulfide linkages are att.acked by nucleophilic reagents, and concluded that, similar to substitut,ion at saturated carbon, displacement at divalent sulfur takes place by a back-side attack. The t’ransition state t,herefore has the ent’ering and the exiting groups in a sbraight’ line. It follows that, for displacement to be possible, the approach of the entering group has to conform to rather strict geomet)rical conditions, the required angle of approach being 180” with respect to the disulfide linkage. A reasonable explanat,ion for the biased at8tack of sultit,e on protein-cysteine mixed disulfides seems to be that the various foldings of the higher ordered structures of the prot,ein mask the half-cystine residue which is part of the prolotein chain by hindering the approach of the at,tacking group. Since the sulfur atom belonging to t’he other half of the disulfide does not enjoy similar protect’ion by the protein, the 1)robabilit.v of

substitution taking place at the I&w sulfur atom is high. The result would be a biased attack such as has been observed in practice If the above argument is valid, one would ex1)ec.t the directed influence to disapljear if the ordered three-dimensional foldings of t,he protein wre dest,royed. One may of achicving such a destrucation without, disrupting the peptidc or the disulfide bonds in the protein is by denaturation with concentrated solutions of urea. In practice we found that. the bias was indeed dest.royed when the sulfit,olysis was carried out in solutions containing urea (see the last column of Table I). If the spatial arrangements of proteins can affect the approach of an attacking INcleophile in the way illustrated by the results of this investigation, it follows that there may be disulfides present wit,hin the contine~ of proteins which enjoy protection against attack from either side of the disultide. The react,ivity of such proteins xvould naturally be rather sluggish, and the addition of a denaturant such as urea would be a prerequisite for obtaining a reasonable reaction rat,e. That this is actually the case with at’ least a part of the disultide linkage in most ln+ot,eins is a n-ell-established fact.

1.

FERDINAND,

J.

2. Krsc,

Biol.

w.,

Chem.

STEIN, w. 240, 1150

ASI)

~fOORE,

6.,

Chem. (1961). J. J., ASD SWANEPOEL, 0. A., :I rch. Hiochem. Jjiophgs. 118, 531 (196Tj. 4. FAVA, h., ASH I'AJARR~, G., d. .I w. Chem. Sot. 78, 5203 (1956). 5. FAVA, A., ILICETO, A., ASD C.IMER.I, E., J. Am. C’hem. Sot. 79, 833 (1957). 6. F.sv.1, A., AW IIXET.\, A., J. .lm. C’hem. Sot. 80, 3478 (1958). 3.

VAS

T. P., J. Hid.

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(1965). 236, PC5

I~SSBURG,

S.