The Determination of Nitrotyrosine Residues in Proteins

Nitration of the ortho position of tyrosine results in the formation of 3-nitrotyrosine. Nitration of tyrosine residues in proteins using tetranitromethane has been used extensively to investigate the role of tyrosine residues in the function of many proteins ( 1 ). However, the existence of tyrosine-nitrated proteins in vivo was not investigated until the discovery of a potential nitrating agent. It was shown that the major protein modification following the reaction of peroxynitrite with proteins is 3-nitrotyrosine ( 2 ). Peroxynitrite is an oxidant formed by the near diffusion-limited reaction between two free radicals, nitric oxide (NO) and superoxide ( 3 ). Inflammatory cells, endothelium, and, potentially, other cells generate peroxynitrite upon stimulation of NO and superoxide production ( 4 – 6 ). Therefore, we and others have explored the possibility of using 3-nitrotyrosine as a marker for peroxynitrite-mediated oxidative stress. Published data has provided evidence that 3-nitrotyrosine is formed in a variety of human diseases and animal models of disease ( 7 – 17 ). Endogenous-tyrosine nitration is almost certainly derived via enzymatically produced NO although NO itself is not a nitrating agent ( 7 – 9 ). Chemically, protein-tyrosine residues can be nitrated by tetranitromethane ( 1 ), nitric acid plus sulfuric acid ( 18 ), nitrogen dioxide ( 19 ), the acidification of nitrite ( 20 ), and the reaction of nitrite with hypochlorous acid ( 21 ). However, under pathophysiological conditions, it appears that peroxynitrite is the proximal species for the formation of protein nitrotyrosine in vivo ( 22 ) and that CO ₂ is a catalyst for peroxynitrite-mediated nitration of tyrosine residues ( 22 – 24 ) ( see Fig. 1 ).

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