ANALYTICAL
75, 674-675
BIOCHEMISTRY
(1976)
Reduction of Disulfide-Containing Amines, Amino Acids, and Small Peptides A simple method for the preparation of pure amine-containing thiol compounds of low molecular weight free of their oxidized counterparts and reducing agents has been developed. Disulfide bonds are reduced with dithiothreitol (DTT) and excess DTT is extracted with ethyl acetate. Methods previously described have required the use of a special electrolytic desalting apparatus (1) or NaBH, (2). Borohydride is a powerful reducing agent lacking selective effect on disulfides, and borate remains in solution following reduction. The thiol reducing agents dithiothreitol (DTT) and dithioerythritol (DTE) are much more specific for reduction of disulfides and the reaction goes to completion according to the following equation’: DTT or DTE + RSSR ’ -+ DTT (oxidized)
or
DTE (oxidized) The driving force of the reaction is provided disulfide formed (3).
+ RSH
by the stability
+ R’SH.
of the ring
METHODS
DTT, cysteine, cysteamine, glutathione, cystine, cystamine, 5,5’dithiobis(2-nitrobenzoic acid) (DTNB), and oxidized glutathione were obtained from Calbiochem, solvents and other chemicals from Fisher Scientific. Free thiols were assayed by the method of Ellman (4). Disulfides were determined by the Zahler and Cleland method (5) in which excess DTT or DTE in the reaction mixture is bound by arsenite ion and free monothiol is assayed calorimetrically with DTNB. Reduction is carried out as follows: Disulfides were dissolved in 50 mM Tris buffer (final pH 8-9), containing 2- to 5-fold molar excess of DTT, and flushed with nitrogen. After several minutes to one hour at room temperature the reaction mixture was acidified with HCl to a pH of 2 and the DTT extracted at least twice (shaking 2 min + brief centrifugation) with a lo-fold volume of ethyl acetate. The aqueous phase was then assayed for thiols and disulfides. 1 Abbreviations not R’SH = monothiols.
defined
in text:
RSSR
674 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction in any form reserved.
= disulfide
(mixed
or symmetrical):
RSH,
675
SHORT COMMUNICATIONS TABLE PERCENTAGES
RECOVERED AS MONOTHIOLS TWICE WITH 10 VOLUMES
Sulfur compound Cysteine Cysteamine Glutathione (GSH) Cystine” Cystamine” Oxidized glutathione” Dithiothreitol (DTT)
1 IN AQUEOUS OF ORGANIC
PHASE AFTER SOLVENT
EXTRACTION
Chloroform
Ethyl acetate
104 94 7
(GSSG)
108 114 105 112 98 96 0.4
R Previously reduced with D’IT as described.
RESULTS AND DISCUSSION
Essentially only amine-containing monothiols remain in the aqueous phase (Table 1). The monothiols investigated, cysteine, cysteamine and glutathione, were insoluble in chloroform and ethyl acetate at acid pH, while DTT is removed from the water phase by extraction with ethyl acetate. Chloroform or other solvents tested were less effective in extracting DTT. The disulfides tested, cystine, cystamine, and oxidized glutathione, were quantitatively reduced by DTT and gave close to 100% recoveries as monothiols when assayed by solvent extraction and Ellman’s method or by the Zahler and Cleland method. The purified monothiols may be used directly in further experiments or recovered as the hydrochloride contaminated with Tris-HCl following lyophilization. With this method many biologically significant thiol compounds that have become oxidized during storage or are available only as disulfides can be easily reduced and recovered in aqueous solution free of toxic materials and suitable for use in a variety of experiments. REFERENCES 1. Smith, I. (1969) in Chromatographic and Electrophoretic Techniques, 3rd ed., Vol. 1, pp. 41-52, Interscience, New York. 2. Brown, W. D. (l%O) Biochem. Biophys. Acta 44, 365-367. 3. Cleland, W. W. (1964) Biochemistry 3, 480-482. 4. Ellman, G. L. (1959) Arch. Biochem. Biophys., 82, 70-77. 5. Zahler, W. L., and Cleland, W. W. (1968)J. Viol. Chem. 243, 716-719. JEAN
BUTLER
P.
STEPHEN JOSEPH National Insritutes Bethesda, Maryland Received March
of Health 20014 20, 1976: accepted
June 4. 1976
D.
SPIELBERG
SCHULMAN