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, peroxynit rite appears to directly react with aromatic and sulfhydryl nucleophil es. At physiological pH, peroxynitrite rapidly decomposes to species w ith . OH and NO2 . character. These reactive species are shown to init iate lipid peroxidation, hydroxylate aromatic residues, and nitrate ar omatic residues. Recently, peroxynitrite was found to inactivate the p olyclonal 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 t he kinetics of migrational change with peroxynitrite could be describe d by two mathematical models. The first model described high affinity, first-order reactivity at one site, and lower affinity, first-order r eactivity 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 w as exposed to increasing concentrations of peroxynitrite indicated tha t the second model was correct. Only tyrosine residues were lost at th e lowest concentration of peroxynitrite, which is consistent with high affinity, first-order reactivity at a single site. At higher concentr ations of peroxynitrite, several other primarily polar amino acids wer e lost at a more or less linear rate. This was consistent with the sec ond component of the model. If tyrosine residues were lost with high a ffinity, then perhaps a known product of tyrosine nitration by peroxyn itrite, namely nitrotyrosine, might be found. A reductive/oxidative el ectrochemical 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 anti body protein samples that had been previously exposed to various conce ntrations of peroxynitrite dose-dependently. The results show that the formation of nitrotyrosine from peroxynitrite roughly parallels the l oss of tyrosine. Previously, we have shown peroxynitrite to be toxic t o cerebellar granule cells and mouse spinal neurons in cell culture. T hese results indicate that proteins may be important targets related t o the toxicity of peroxynitrite. This data suggest that nitrotyrosine may be an important marker of peroxynitrite reactivity in vivo. (C) 19 97 Academic Press.