Methods to Measure the Reactivity of Peroxynitrite-derived Oxidants Toward Reduced Fluoresceins and Rhodamines

The commonest probes for "reactive oxygen and nitrogen species" are reduced fluorescein and rhodamine dyes that fluoresce when oxidized. The reduced dyes are reactive toward peroxynitrite, although probably not directly but via free radical oxidants derived from it: hydroxyl, carbonate, and nitrogen dioxide free radicals. The reaction with peroxynitrite can be monitored by rapid mixing and stopped-flow spectrophotometry, but reliable measurement of reactivity of the peroxynitrite-derived radicals requires specialized techniques such as flash photolysis or pulse radiolysis to monitor the fast reactions in real time. A key feature of oxidation by radicals is that the reaction produces an intermediate fluorescein or rhodamine radical, which normally is oxidized further by oxygen to yield the fluorescent, stable product. Susceptibility of the yield of fluorescence to interference by antioxidants can be assessed from kinetic parameters, which reflect reactivity. This chapter outlines methods for estimation of key rate constants involving peroxynitrite-derived oxidants.