Differential effects of organic hydroperoxides and hydrogen peroxide on proteolysis in human erythrocytes

Publisher Website
Google Scholar
Abstract
The effects of tert-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide on proteolysis in human red blood cells have been examined. The organic hydroperoxides effectively stimulated the rate of protein degradation in red cells and in hemolysate; in contrast, H2O2 addition was without significant effect in either system. tert-Butyl hydroperoxide or cumene hydroperoxide (8 mM) increased the rate of protein degradation in red cells 2.3- and 4-fold, respectively, relative to control as monitored by tyrosine release. In hemolysate, tert-butyl hydroperoxide and cumene hydroperoxide, present at 8 mM, produced a 2- and 3-fold increase in the rate of protein degradation, respectively, as compared to controls. Hydroperoxide-stimulated proteolysis in red cells or in hemolysate was concentration-dependent and reached saturation at 8 mM hydroperoxide. The reaction was linear for 2 h after which a plateau was reached. In contrast to the results observed for the organic hydroperoxides, H2O2 (100 or 200 mM) addition either alone or in the presence of the catalase inhibitor 3-amino-1,2,4-triazole (50-200 mM), failed to stimulate proteolysis. N-Acetylcysteine (20 mM) and dimethylthiourea (50 mM) inhibited the rate of hydroperoxide-stimulated proteolysis in red cells by .apprx. 50 and .apprx. 35%, respectively, and in hemolysate by 25 and 40%, respectively. The hydroxyl radical scavengers methyl sulfoxide (50 mM) or dimethylfuran (50 mM), metal ion chelators, or spin traps failed to decrease significantly the rate of organic hydroperoxide stimulated proteolysis. In addition, inhibitors of the calpain/procalpain system in red cell or hemolysate incubations challenged by organic hydroperoxide were without significant effect on the rate of proteolysis. HPLC analysis of red cell incubations was employed to quantify the release of histidine and tyrosine. Whereas cumene hydroperoxide produced a concentration- and time-dependent release of these amino acids, H2O2 failed to stimulate amino acid release. These data confirm the findings of the fluorometric tyrosine assay. The reuslts of this study reveal that organic hydroperoxides produce protein damage in red cells and hemolysate as a result of alkyl or alkoxyl free radicals generated during hydroperoxide decomposition and that HPLC provides a quantitative approach for monitoring proteolysis in these systems. Proteolysis, which has been suggested as being a defense mechanism for the removal of protein damaged by xenobiotic insult, may also be a rapid and novel index of xenobiotic-mediated cellular injury.