Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin

Abstract

Thioredoxins and glutaredoxins, in their oxidized form, possess a single disulfide bridge located on an edge of the small compact molecules. In contrast to most other disulfide-containing proteins, this S-S bridge is cleaved by millimolar concentrations of sulfite in the absence of protein denaturing agents at pH 7-8 and ambient temperature; however, the reaction is not quantitative. Sulfitolysis of Escherichia coli thioredoxin was found to be associated .with an increase in fluorescence at 345 nm. A comparative study of sulfitolysis in 12 different thioredoxins and glutaredoxins of bacterial and plant origin has been made. Although they are all thought to be highly conserved in threedimensional structure, their reactivities towards sulfite and the effects of 6 M guanidinium chloride (not affecting, or enhancing sulfitolysis) vary strongly in the series, with E. coli thioredoxin being less reactive and plant thioredoxins and E. coli glutaredoxin being more susceptible molecules. Contrary to expectation, reaction with sulfite is not generally correlated with the presence of negatively or positively charged amino acid residues near the disulfide loop but is determined by individ-

Structure requirements for disulfide bridge sulfitolysis of oxidized Escherichia coli thioredoxin studied by fluorescence spectroscopy

“…In a previous study [6], we mentioned that sulfitolysis of E. coli thioredoxin causes a fluorescence increase similar to that observed upon reductive opening of the active-site disulfide bridge. The fluorescence spectrum of E. coli thioredoxin is dominated by Trp28 and its fluorescence is quenched in the oxidized state by the disulfide bridge [S].…”

“…In this process, the disulfide bridge of oxidized thioredoxin is cleaved by sulfite ions at physiological, millimolar concentrations, forming an inactive S-sulfonyl thioredoxin (Scheme 1). In previous experiments we measured sulfitolysis by labeling the cysteine residue generated in close proximity to the S-sulfonyl group with iod0[2-~H]acetic acid [6]. This method is limited by the accessibility of this cysteine residue for the alkylating agent.…”

“…To avoid such a limitation, we took advantage of the fact that the fluorescence of E. coli thioredoxin increases significantly upon reductive opening of the disulfide bridge [S], and that sulfitolytic cleavage induces a similar fluorescence change [6]. In this study, the sulfitolysis reaction of native and mutant E. coli thioredoxins measured by fluorescence spectroscopy are described.…”

“…Thioredoxin S-sulfonyl thioredoxin alkylation method (less than 5 % ) , may represent only a proportion of the actual yield [6]. To avoid such a limitation, we took advantage of the fact that the fluorescence of E. coli thioredoxin increases significantly upon reductive opening of the disulfide bridge [S], and that sulfitolytic cleavage induces a similar fluorescence change [6].…”

“…In previous experiments we measured sulfitolysis by labeling the cysteine residue generated in close proximity to the S-sulfonyl group with iod0[2-~H]acetic acid [6]. This method is limited by the accessibility of this cysteine residue for the alkylating agent.…”