The rate, stoichiometry, and products for the oxidation of reduced glutathione (GSH) by 2,6-dichloroindophenol are dependent in part on the hydrogen-ion concentration and the presence of metal ions. In the absence of added metal ions, the major products of the reaction are the mono- and disubstituted S-glutathionyl-2,6-dichloroindophenols. These new dye derivatives were separated by chromatographic techniques and characterized by utilization of 14C-labeled GSH. The maximum rate of formation of these substituted dye derivatives was observed in the pH range 6-7. Maximal yields of these derivatives were also obtained in this pH range. In this process GSH and indophenol react in the molar ratio 1:1. In acid and alkaline pH zones there is some suppression of the substitution reaction and a concomitant increase in the formation of oxidized glutathione (GSSG) as an end product. However under these conditions GSSG has not been obtained as a major product. Kinetic studies over a broad pH range indicate a common rate-limiting step resulting in the initial formation of an intermediate, GS: dye through nucleophilic attack of the GS- anion on the indophenol component, effecting partial transfer of an electron. The mechanism of the competing pathways leading to the formation of substituted indophenols and GSSG is considered briefly. Added Cu2+ ions suppress the nucleophilic behavior of the thiol anion with formation of a thiol-copper complex which transfers electrons directly to the dye. In this case the stoichiometry is 2 moles GSH oxidized per mole dye reduced and, with sufficient Cu2+, GSSG is formed in theoretical yield, as revealed by specific enzymatic assay with the enzyme glutathione reductase.
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