Peroxynitrite is the major species formed from different flux ratios of co-generated nitric oxide and superoxide: direct reaction with boronate-based fluorescent probe.

There is much interest in the nitration and oxidation reaction mechanisms initiated by superoxide radical anion (O(2)()) and nitric oxide (()NO). It is well known that O(2) and ()NO rapidly react to form a potent oxidant, peroxynitrite anion (ONOO(-)). However, indirect measurements with the existing probes (e.g. dihydrorhodamine) previously revealed a bell-shaped response to co-generated ()NO and O(2) fluxes, with the maximal yield of the oxidation or nitration product occurring at a 1:1 ratio. These results raised doubts on the formation of ONOO(-) per se at various fluxes of ()NO and O(2). Using a novel fluorogenic probe, coumarin-7-boronic acid, that reacts stoichiometrically and rapidly with ONOO(-) (k = 1.1 x 10(6) m(-1)s(-1)), we report that ONOO(-) formation increased linearly and began to plateau after reaching a 1:1 ratio of co-generated ()NO and O(2) fluxes. We conclude that ONOO(-) is formed as the primary intermediate during the reaction between ()NO and O(2) co-generated at different fluxes.