Different mechanisms of formation of glutathione-protein mixed disulfides of diamide and tert-butyl hydroperoxide in rat blood

The mechanisms of glutathione-protein mixed disulfide (GSSP) formation caused by diamide and tert-butyl hydroperoxide were studied in rat blood after in vitro treatment in the 0.3–4 mM dose range. tert-Butyl hydroperoxide formed GSSP, via GSSG, according to the reaction, GSSG + PSH → GSSP + GSH, whereas diamide reacted first with protein SH groups, giving PS-diamide adducts and then, after reaction with GSH, GSSP. Moreover, after diamide treatment, GSSP patterns were characterized by a much slower or irreversible dose-related return to basal levels in comparison with those observed with tert-butyl hydroperoxide, always reversible. Experiments with purified hemoglobin revealed the existence of a large fraction of protein SH groups which formed GSSP and had a higher reactivity than GSH. Experiments on glucose consumption and role of various erythrocyte enzymes, carried out to explain the inertness of GSSP to reduction after treatment of blood with diamide, were substantially negative. Other tests carried out to confirm the efficiency of the enzymatic machinery of blood samples successively treated with diamide and tert-butyl hydroperoxide, indicated that GSSP preformed by diamide was difficult to reduce, whereas those generated by tert-butyl hydroperoxide were reversible as normal. Our results suggest that a fraction of GSSP generated by diamide is different and less susceptible to reduction than that obtained with tert-butyl hydroperoxide.