Site-selective nitration of tyrosine in human serum albumin by peroxynitrite

Peroxynitrite, which is formed in biological systems by the reaction of nitric oxide with superoxide anion, is a highly reactive molecule that can lead to cell injury or cell death. Reactions of peroxynitrite under physiological conditions include nitration of tyrosine-containing proteins or peptides, and we have been investigating the behavior of human serum albumin following exposure to peroxynitrite. Peroxynitrite, at relative concentrations ranging from 0.2 to 50 with respect to protein, was added to human serum albumin in buffer at pH 7.2. The resulting mixtures were dialyzed to remove small molecules, dried under vacuum, and then digested with trypsin. The digests were analyzed by high performance liquid chromatography with UV detection at 230 and 354 nm, the latter wavelength being selective for nitrotyrosine. At the higher relative concentrations of peroxynitrite, the 354-nm chromatograms contained a large number of peaks, including at least nine with molecular weights corresponding to nitration of nominal tryptic peptides. Following treatment with the lower relative concentrations of peroxynitrite, however, the 354-nm chromatograms were dominated by only two nitrated peptides; these were identified by comparison of LC retention times and collision-induced decomposition mass spectra as nitro-Y(411)TK(413) and nitro-Y(138)LYEIAR(144). Each of these tyrosines resides in a known reactive site within the protein, i.e., subdomains IIIA and IB, respectively.