Dopamine (DA) degradation in cells generates H2O2, which can be further reduced to hydroxyl radicals (OH ) in the presence of iron. Cellular damages inflicted by DA-derived OH have been studied extensively in relation to the pathogenesis of Parkinsonian's disease. We established a procedure that detects proteins with H2O2-sensitive Cys (or Secys) residues and studied protein oxidation by DA-derived H2O2 in PC12 cells. The procedure is based on the fact that at pH 6.5, H2O2 and biotin-conjugated iodoacetamide (BIAM) selectively and competiviely react with Cys residues with low pKa value. Decrease in BIAM-labeling due to prior exposure of cells to DA was followed by blot analysis with horseradish peroxidase-conjugated streptavidin after separation on SDS-gels. Using the procedure, a number of proteins in PC12 cells were shown to have Cys residues that are sensitive to oxidation by DA-derived H2O2. Notable targets include ERP72 and ERP60, two members of protein disulfide isomerase family and creatine kinase. Phospholipase C-gamma1 and thioredoxin reductase, two minor components of PC12 cells, were also oxidized by DA-derived H2O2. By using [14C]iodoacetamide instead of BIAM, glyceraldehyde 3-phosphate dehydrogenase was identified as a target. In vitro studies with purified enzymes suggest that the essential residues, Cys283 of creatine kinase, Cys151 of glyceraldehyde 3-phosphate dehydrogenase, Secys498 of thioredixin reductase are specifically oxidized by H2O2. Although the identified targets represent only a portion of proteins that are modified by DA-derived H2O2, impairment of their functions have previously been closely related to cell death. We propose therefore that oxidation of proteins with reactive cysteine reisdues by DA-derived H2O2 might be largely responsible for the DA-induced apoptosis of neuronal cells and a central player in the pathogenesis of Parkinson's disease.
