THE OXIDATIVE MODIFICATION of proteins is a natural consequence of aerobic life and is also recognized to play a crucial role in the pathological response of cells to increased oxidative stress. Several oxidative modifications of a protein can occur as a result of oxidative stress. These can range from the facile oxidation of cysteine residues to changes caused by higher levels of oxidative stress, resulting in covalent crosslinking with other proteins (whether by SS linkage or by 2-2!-biphenyl crosslink of 2 tyrosyl radicals), or the formation of noncovalent aggregates or even formation of adducts of proteins with other lipid, carbohydrate, or nucleic acid radicals. Peroxynitrite (ONOO J), the reaction product of nitric oxide and superoxide, is implicated as a key oxidant species in several pathologies and is well known to oxidize proteins. The range of possible modifications begins from the mild oxidation of susceptible cysteine sulfhydryls (resulting in their S-nitrosylation and S-glutathiolation) to what is likely the result of a higher concentration and/or longer duration of exposure to ONOO J to cause direct nitration of tyrosine residues. The nitration of proteins by oxidant species other than ONOO J is also recognized under certain conditions (6). These modifications often result in the alteration of protein function or structure and, usually, inhibition of enzyme function. Proteins containing nitrotyrosine residues have been detected in different pathologies, including diabetes, hypertension, and atherosclerosis, all associated with enhanced oxidative stress, including that caused by increased production of ONOO J. The quest to identify nitrated proteins as a reliable in vivo marker of oxidative stress, particularly to that caused by ONOO J, has proceeded with gusto. Early attempts utilized a polyclonal anti-nitrotyrosine antibody raised originally against nitrated keyhole limpet hemocyanin. In principle, this antibody detects several proteins containing tyrosine residues modified in position 3 of the phenyl ring by a nitro group (3). This antibody is most sensitive in its ability to detect nitrated proteins in situ by immunohistochemistry but has also been used to detect specific nitrated proteins isolated from cells by immunoblotting. Recently, a proteomic approach using this pan-nitrotyrosine antibody showed that a total of 48 putative proteins containing nitrotyrosine were identified in whole heart homogenates of aged rats (8). Two such proteins, a key mitochondrial antioxidant enzyme, Mn2+ superoxide dismutase (MnSOD), or the sarcoplasmic reticulum calcium ATPase type 2 (SERCA2), are nitrated at one or more tyrosine residues in disease states, resulting in a loss of activity. MnSOD is nitrated by ONOO J in its active catalytic site and inactivated by nitration of a single tyrosine, Y-34 (10).

SERCA2 has been shown to be nitrated at two adjacent tyrosine residues (Y-294, Y-295) in skeletal and cardiac muscle from aged animals (9, 15), and tyrosine nitration of this enzyme results in decreased activity (1). Because these enzymes regulate critical components of the cell’s ability to deal with oxidative stress and calcium handling, it is likely that an inactivation of these enzymes will contribute to the pathogenesis of disease and aging. Indeed, a study in this issue of the