Enzymatic Reduction of 5,5'-dithiobis-(2-nitrobenzoic Acid) by Lysate of Rat Liver Mitochondria

The oxidation of mitochondrial sulphydryl groups is known to increase the permeability of mitochondrial membranes and to be a key event in oxidative stress. Resistance to this damage is thought to involve thioredoxin reductase. In this study, the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) by a lysate of rat liver mitochondria was used to assay the mitochondrial disulphide reducing capacity. NADPH-dependent reduction of DTNB was used to distinguish enzymatic reduction from the non-enzymatic reduction. Enzymatic reduction by the mitochondrial lysate was suppressed by DTNB at concentrations exceeding 0.25 mM and by pH above 7.0. It was strongly inhibited by Zn2+ and Mn2+ (IC50 about 2.5 and 20 microM, respectively) and was more weakly inhibited by Mg2+ and Ca2+ (IC50 about 1.8 and 2.1 mM, respectively). As a consequence of inhibition by divalent cations, the reaction was stimulated by both physiological (ATP, ADP, pyrophosphate and citrate) and non-physiological (EDTA and EGTA) chelators. Reduction of insulin disulphides by the mitochondrial lysate was dependent on the presence of a divalent cation chelator during the isolation of mitochondria and during the enzyme assay. Our results suggest that stimulation of mitochondrial disulphide reducing activity by lowered pH, as well as by increased levels of ATP, ADP and citrate, has the potential to contribute to the maintenance of mitochondrial sulphydryl groups under oxidative conditions.