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 bl ood after in vitro treatment in the 0.3-4 mM dose range. tert-Butyl hy droperoxide formed GSSP, via GSSG, according to the reaction, GSSG + P SH --> GSSP + GSH, whereas diamide reacted first with protein SH group s, 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 r eversible. 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 rol e of various erythrocyte enzymes, carried out to explain the inertness of GSSP to reduction after treatment of blood with diamide, were subs tantially 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-buty l hydroperoxide were reversible as normal. Our results suggest that a fraction of GSSP generated by diamide is different and less susceptibl e to reduction than that obtained with tert-butyl hydroperoxide.