Kinetics of peroxynitrite reaction with amino acids and human serum albumin

An initial rate approach was used to study the reaction of peroxynitrite wi th human serum albumin (HSA) through stopped-flow spectrophotometry. At pH 7.4 and 37 degrees C, the second order rate constant for peroxynitrite reac tion with HSA was 9.1 +/- 1.1 x 10(3) M-1 s(-1). The rate constants for sul fhydryl-blocked HSA and for the single sulfhydryl were 5.9 +/- 0.3 and 3.8 +/- 0.8 x 10(3) M-1 s(-1), respectively. The corresponding values for bovin e serum albumin were also determined. The reactivity of sulfhydryl-blocked HSA increased at acidic pH, whereas plots of the rate constant with the sul fhydryl versus pH were bell-shaped. The kinetics of peroxynitrite reaction with all free L-amino acids were determined under pseudo first order condit ions. The most reactive amino acids were cysteine, methionine, and tryptoph an. Histidine, leucine, and phenylalanine (and by extension tyrosine) did n ot affect peroxynitrite decay rate, whereas for the remaining amino acids p lots of k(obs) versus concentration were hyperbolic. The sum of the contrib utions of the constituent amino acids of the protein to HSA reactivity was comparable to the experimentally determined rate constant, where cysteine a nd methionine (seven residues in 585) accounted for an estimated 65% of the reactivity. Nitration of aromatic amino acids occurred in HSA following pe roxynitrite reaction, with nitration of sulfhydryl-blocked HSA 2-fold highe r than native HSA, Carbon dioxide accelerated peroxynitrite decomposition, enhanced aromatic amino acid nitration, and partially inhibited sulfhydryl oxidation of HSA. Nitration in the presence of carbon dioxide increased whe n the sulfhydryl was blocked. Thus, cysteine 34 was a preferential. target of peroxynitrite both in the presence and in the absence of carbon dioxide.