Nitration and oxidation of a hydrophobic tyrosine probe by peroxynitrite in membranes: Comparison with nitration and oxidation of tyrosine by peroxynitrite in aqueous solution

It has been reported that peroxynitrite will initiate both oxidation and ni tration of tyrosine, forming dityrosine and nitrotyrosine, respectively. We compared peroxynitrite-dependent oxidation and nitration of a hydrophobic tyrosine analogue in membranes and tyrosine in aqueous solution. Reactions were carried out in the presence of either bolus addition or slow infusion of peroxynitrite, and also using the simultaneous generation of superoxide and nitric oxide. Results indicate that the level of nitration of the hydro phobic tyrosyl probe located in a lipid bilayer was significantly greater t han its level of oxidation to the corresponding dimer. During slow infusion of peroxynitrite, the level of nitration of the membrane-incorporated tyro syl probe was greater than that of tyrosine in aqueous solution. Evidence f or hydroxyl radical formation from decomposition of peroxynitrite in a dime thylformamide/water mixture was obtained by electron spin resonance spin tr apping. Mechanisms for nitration of the tyrosyl probe in the membrane are d iscussed. We conclude that nitration but,not oxidation of a tyrosyl probe b y peroxynitrite is a predominant reaction in the membrane. Thus, the local environment of target tyrosine residues is an important factor governing it s propensity to undergo nitration in the presence of peroxynitrite. This wo rk provides a new perspective on selective nitration of membrane-incorporat ed tyrosine analogues.