Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation

The ability of peroxynitrite to modify amino acid residues in glutamine syn thetase (GS) and BSA is greatly influenced by pH and CO2. At physiological concentrations of CO2 (1.3 mM), the generation of carbonyl groups (0.2-0.4 equivalents/subunit) is little affected by pH over the range of 7.2-9.0, bu t, in the absence of CO2, carbonyl formation increases (from 0.1-1.2 equiva lents/subunit) as the pH is raised from 7.2 to 10.5. This increase is attri butable, in part but not entirely, to the increase in peroxynitrite (PN) st ability with increasing pH, Of several amino acid polymers tested, only tho se containing lysine residues yielded carbonyl derivatives. In contrast, th e nitration of tyrosine residues of both GS and BSA at pH 7.5 almost comple tely depends on the presence of CO2. However, the pH profiles of tyrosine n itration in GS and BSA are not the same. With both proteins, nitration decr eases approximate to 65% with increasing pH over the range of 7.2-8.4, but, then in the case of GS only, there is a 3.1 fold increase in the level of nitration over the range pH 8.4-8.8. The oxidation of methionine residues i n both proteins and in the tripeptide Ala-Met-Ala was inhibited by CO2 at b oth high and low pH values. These results emphasize the importance of contr olling the pH and CO2 concentrations in studies involving PN and indicate t hat PN is not likely to contribute appreciably to carbonyl formation or oxi dation of methionine residues of proteins at physiological pH and CO2 conce ntrations.