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.