Membrane protein changes induced by tert-butyl hydroperoxide in red blood cells

Abstract

Red cells were incubated in the presence of t- Butyl hydroperoxide and effects on red cell membrane proteins were studied by SDS-polyacrylamide gel electrophoresis. t- Butyl hydroperoxide caused diminution in intensity of all major cytoskeletal bands with the concomitant formation of high molecular weight material. Membrane glycoproteins were unaffected. t- Butyl hydroperoxide increased hemoglobin binding to ghosts. After dissolution in SDS and β-mercaptoethanol, membrane-bound hemoglobin appeared on the gels in the form of monomers and crosslinked polymers of hemoglobin or globin chains. Crosslinking was partially prevented by metabolism of t- butyl hydroperoxide by the hexose monophosphate shunt except in methemoglobin-containing red cells where reaction with methemoglobin accounted for most of the consumption of t- butyl hydroperoxide. Metal chelators, deferoxamine mesylate and diethylenetriaminepentaacetic acid, had no effect on membrane protein changes. Butylated hydroxytoluene, diphenylamine and ascorbate, compounds that inhibit t- butyl hydroperoxide-induced red cell membrane lipid peroxidation, had no effect on t- butyl hydroperoxide-induced membrane protein changes. These results suggest that membrane proteins and membrane lipids have different mechanisms of peroxidant damage.