Protein nitration by peroxynitrite: A method for monitoring nitric oxide neurotoxicity

During ischemia, peroxynitrite may be a toxic intermediate which forms in vivo when nitric oxide condenses with superoxide. Alone, peroxynitrite appears to directly react with aromatic and sulfhydryl nucleophiles. At physiological pH, peroxynitrite rapidly decomposes to species with . OH and NO2 . character. These reactive species are shown to initiate lipid peroxidation, hydroxylate aromatic residues, and nitrate aromatic residues. Recently, peroxynitrite was found to inactivate the polyclonal antibody to cAMP. This functional loss of activity was also correlated to a loss of structural integrity as verified by capillary electrophoresis. Here, the dose-dependent effects of peroxynitrite on the electrophoretic migration of a monoclonal antibody (monoclonal to plasminogen activator inhibitor-1) were studied. Results showed that the kinetics of migrational change with peroxynitrite could be described by two mathematical models. The first model described high affinity, first-order reactivity at one site, and lower affinity, first-order reactivity at a second site. The second model described high affinity, first-order reactivity at one site, and nonspecific, linear reactivity at multiple sites. Amino acid analysis of the antibody protein that was exposed to increasing concentrations of peroxynitrite indicated that the second model was correct. Only tyrosine residues were lost at the lowest concentration of peroxynitrite, which is consistent with high affinity, first-order reactivity at a single site. At higher concentrations of peroxynitrite, several other primarily polar amino acids were lost at a more or less linear rate. This was consistent with the second component of the model. If tyrosine residues were lost with high affinity, then perhaps a known product of tyrosine nitration by peroxynitrite, namely nitrotyrosine, might be found. A reductive/oxidative electrochemical detection scheme of nitrotyrosine was found to be 50-100 times more sensitive than standard spectrophotometric detection. This redox HPLC assay was used to measure nitrotyrosine in hydrolyzed antibody protein samples that had been previously exposed to various concentrations of peroxynitrite dose-dependently. The results show that the formation of nitrotyrosine from peroxynitrite roughly parallels the loss of tyrosine. Previously, we have shown peroxynitrite to be toxic to cerebellar granule cells and mouse spinal neurons in cell culture. These results indicate that proteins may be important targets related to the toxicity of peroxynitrite. This data suggest that nitrotyrosine may be an important marker of peroxynitrite reactivity in vivo. (C) 1997 Academic Press.