Effect of phosphate buffer solutions on the reactions of glutathione with hydrogen peroxide and peroxyl radicals

Abstract

Differences in the kinetics and mechanism of the reaction of glutathione (GSH) with hydrogen peroxide (H₂O₂) in deionized water and in phosphate buffer systems with pH ≥ 7 frequently used in biochemical studies were revealed. The formation of GSH dimers and complexes with H₂O₂ in water plays a substantial role in the kinetics of the process, which is manifested as nonlinear dependences of the rate of GSH consumption (W_GSH) and the rate of radical formation (W_i) on the reagent concentrations. In phosphate buffer solutions (PBS), the oxidation of GSH by air oxygen is enhanced and the radical formation rate decreases sharply. An effect of NaCl and KCl in PBS on W_GSH and W_i was observed, unlike a sodium—potassium phosphate buffer mixture (PB). Under other equivalent conditions, W_GSH PBS is several times lower and W_i is higher than those in PB containing no chlorides. It was found that the rate of the thiol-ene reaction of unsaturated phenol resveratrol (RVT) with GSH initiated by the radicals formed in the presence of H₂O₂ in PBS is nearly three times lower than that in water, whereas in PB resveratrol is not consumed under the same conditions. However, in the reactions with peroxyl radicals formed upon the decomposition of 2,2′-azobis(2-methylpropionamidine) dihydrochloride the GSH consumption rate is the same in both phosphate buffer systems.