Identification of proteins containing cysteine residues that are sensitiveto oxidation by hydrogen peroxide at neutral pH

A procedure for detecting proteins that contain H2O2-sensitive cysteine (or selenocysteine) residues was developed as a means with which to study prot ein oxidation by H2O2 in cells. The procedure is based on the facts that H2 O2 and biotin-conjugated iodoacetamide (BIAM) selectively and competitively react with cysteine residues that exhibit a low pK(a), and that the decrea se in the labeling of cell lysate proteins with BIAM caused by prior exposu re of cells to H2O2 or to an agent that induces H2O2 production can be moni tored by streptavidin blot analysis. This procedure was applied to rat pheo chromocytoma PC12 cells directly treated with H2O2, mouse hippocampal HT22 cells in which H2O2 production was induced by glutamate, and human erythrol eukemia K562 cells in which H2O2 production was induced by phorbol myristat e acetate. It revealed that several cell proteins contain cysteine or selen ocysteine residues that are selectively oxidized by H2O2. Three of these H2 O2-sensitive proteins were identified as a member of the protein disulfide isomerase family, thioredoxin reductase, and creatine kinase, all of which were previously known to contain at least one reactive cysteine or selenocy steine at their catalytic sites. This procedure should thus prove useful fo r the identification of proteins that are oxidized by H2O2 generated in res ponse to a variety of extracellular agents. (C) 2000 Academic Press.