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.