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

Peroxynitrite, which is formed in biological systems by the reaction of nit ric oxide with superoxide anion, is a highly reactive molecule that can lea d to cell injury or cell death. Reactions of peroxynitrite under physiologi cal conditions include nitration of tyrosine-containing proteins or peptide s, and we have been investigating the behavior of human serum albumin follo wing exposure to peroxynitrite. Peroxynitrite, at relative concentrations r anging from 0.2 to 50 with respect to protein, was added to human serum alb umin in buffer at pH 7.2. The resulting mixtures were dialyzed to remove sm all molecules, dried under vacuum, and then digested with trypsin. The dige sts were analyzed by high performance liquid chromatography with W detectio n at 230 and 354 nm, the latter wavelength being selective for nitrotyrosin e. At the higher relative concentrations of peroxynitrite, the 354-nm chrom atograms contained a large number of peaks, including at least nine with mo lecular weights corresponding to nitration of nominal tryptic peptides. Fol lowing treatment with the lower relative concentrations of peroxynitrite, h owever, the 354-nm chromatograms were dominated by only two nitrated peptid es; these were identified by comparison of LC retention times and collision -induced decomposition mass spectra as nitro-(YTK413)-T-411 and nitro-Y(138 )LYEIAR(144). Each of these tyrosines resides in a known reactive site with in the protein, i.e., subdomains IIIA and IB, respectively. (C) 2001 Academ ic Press.