Equilibrium and kinetic constants for the thiol-disulfide interchange reaction between glutathione and dithiothreitol.

Abstract

The equilibrium and rate constants for the reaction between oxidized and reduced glutathione and oxidized and reduced dithiothreitol have been determined at several pH values and temperatures. The measurements involve approach to equilibrium from both directions, quenching of the reaction by lowering the pH or by addition of methyl methanethiosulfonate, separation of reactants and products by reverse-phase HPLC, and determination of their concentrations. Analysis of reaction mixtures was carried out at various times to assure that equilibrium had been reached and to determine kinetic constants prior to the attainment of equilibrium.The thiol-disulfide interchange reaction is important in a variety of biological systems (1-3) and, in particular, in studies of the regeneration of disuffide-containing proteins from their reduced forms (4-6). The thiol/disulfide reagents in widest use in studies of the regeneration of proteins are oxidized glutathione (GSSG) and reduced glutathione (GSH), primarily because they are known to occur at significant concentrations in biological systems (7) and because they exhibit a suitable redox potential.The oxidation of monothiols such as GSH is a bimolecular process and is entropically different from the unimolecular process of disulfide formation that occurs in proteins. In contrast, the formation of cyclic disulfides such as oxidized dithiothreitol (DTToX) results from a unimolecular process. Glutathione forms stable mixed disulfides with protein thiols, which greatly complicates studies of regeneration pathways. On the other hand, while DTTOX is a much weaker oxidizing agent, it does not form stable mixed disulfides (because the cyclization reaction is very fast), making it a useful reagent for studies of protein regeneration (5, 8). It has been argued, though incorrectly, that, while these reagents differ in their entropies of disulfide formation, they are similar in their abilities to regenerate protein when concentrations are adjusted to give similar redox potentials (9).The role that these different types (mono-and cyclic thiol) of reagents play is a key feature of the detailed regeneration processes of proteins. Any attempt to distinguish these different roles requires a quantitative determination of their relative redox potentials. In order to make comparisons between the results of regeneration experiments using dithiothreitol and those using glutathione, it is necessary to know the equilibrium constant for the reaction between glutathione and dithiothreitol. The equations describing this equilibriumwhere the brackets indicate concentrations at equilibrium.Unfortunately, a wide range of values has been reported for this thiol-disulfide exchange equilibrium constant. Values of 8800 M at pH 7 and 300C (10) and 13,000 M at pH 7 and an unspecified temperature (11) have been reported, based on an indirect technique involving a lipoamide dehydrogenasemediated reaction between NAD+ and lipoamide. Use of intermediates populated during the regeneration of bovine pancreatic ...

Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

“…The redox potentials for the mitochondrial intermembrane space, GSH, and DTT were determined to be Ϫ255, Ϫ240, and Ϫ330 mV, respectively (31,32), and an even lower value for TCEP was predicted (33). Accordingly, the active site disulfide of Erv1p would be thermodynamically unstable in the intermembrane space or against any of above thiol reducing reagents if it was not well protected.…”

Site-Specific S-Glutathiolation of Mitochondrial NADH Ubiquinone Reductase

“…The equilibrium constant of thiol exchange between free cysteine and GSSG was reported to be 0.71 (24,25). The equilibrium constant between GSSG and thiol agent, dithiothreitol, was reported to be 2.00-2.20 × 10 2 (26). Presumably, protein conformation and localized environment of cysteine residue in the NQR limited the extent of protein Sglutathiolation even though the ratio of GSSG/NQR is high (350-1050).…”

Redox Regulation of Methionine Aminopeptidase 2 Activity

“…MetAP1 never acquired both of the corresponding cysteines. using a value of Ϫ307 mV for the redox potential of the DTT disulfide bond (22) and Ϫ270 mV for the redox potential of the thioredoxin disulfide bond (23).…”