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

Ming Tien, Barbara S. Berlett, Rodney L. Levine, P. Boon Chock, Earl R. Stadtman

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Abstract

The ability of peroxynitrite to modify amino acid residues in glutamine synthetase (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, but, in the absence of CO2, carbonyl formation increases (from 0.1- 1.2 equivalents/subunit) as the pH is raised from 7.2 to 10.5. This increase is attributable, in part but not entirely, to the increase in peroxynitrite (PN) stability with increasing pH. Of several amino acid polymers tested, only those containing lysine residues yielded carbonyl derivatives. In contrast, the nitration of tyrosine residues of both GS and BSA at pH 7.5 almost completely depends on the presence of CO2. However, the pH profiles of tyrosine nitration in GS and BSA are not the same. With both proteins, nitration decreases ≃65% with increasing pH over the range of 7.2- 8.4, but, then in the case of GS only, there is a 3.4-fold increase in the level of nitration over the range pH 8.4-8.8. The oxidation of methionine residues in both proteins and in the tripeptide Ala-Met-Ala was inhibited by CO2 at both high and low pH values. These results emphasize the importance of controlling the pH and CO2 concentrations in studies involving PN and indicate that PN is not likely to contribute appreciably to carbonyl formation or oxidation of methionine residues of proteins at physiological pH and CO2 concentrations.

Original languageEnglish (US)
Pages (from-to)7809-7814
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume96
Issue number14
DOIs
StatePublished - Jul 6 1999

All Science Journal Classification (ASJC) codes

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