Purification and characterization of a serine protease in erythrocyte cytosol that is adherent to oxidized membranes and preferentially degrades proteins modified by oxidation and glycation

A serine protease that preferentially degrades oxidized and glycated proteins was shown to be present in erythrocyte cytosol. Human erythrocyte cytosol was labeled with [H-3] diiso- propyl fluorophosphate (DFP) and passed through a column of carboxymethyl-Sephadex to obtain radioactive fractions free of hemoglobin. The fractions contained a single radioactive 80-kDa protein, as analyzed by sodium dodecyl sulfate-polyacpylamide gel electrophoresis (PAGE)/fluorography. The radioactive 80-kDa protein bound to unoxidized erythrocyte membranes, and more effectively to oxidized membranes. The radioactive protein was purified through a column of diethylaminoethyl-cellulose and by preparative native-PAGE in a purity of 80%, Antibody against the cytosolic 80-kDa protein bound to 80-kDa protein of erythrocyte membranes, indicating the presence of the same protein in the membrane. The antibody bound more effectively to oxidized membranes than to unoxidized membranes, The 80-kDa protein partially purified from unlabeled cytosol degraded more effectively oxidized bovine serum albumin (BSA), oxidized Igf;, and glycated BSA more effectively than the corresponding unoxidized or unglycated proteins, Degradation of oxidized or glycated proteins was effectively inhibited by DFP, Hence, the protein is an 80-kDa serine protease that is adherent to oxidized membranes and is responsible for degradation of proteins modified by oxidation and glycation.