Increased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide

4,5 Diaminofluorescein (DAF-2) is increasingly utilized as a fluorescent detector for nitric oxide (NO) in cells and tissues. In oxygenated solutions, reactive nitrogen species derived from NO autoxidation nitrosate DAF-2 to yield the highly fluorescent DAF-2 triazole. In the present study, we investigated the nitrosation of DAF-2 at a neutral pH by absorption and fluorescence spectroscopy using NONOates as chemical sources of NO. We found that both chemically synthesized peroxynitrite and horseradish peroxidase in the presence of hydrogen peroxide (H2O2) oxidized DAF-2 to a relatively stable nonfluorescent intermediate (t1/2 ∼ 90 s). Oxidation of DAF-2 prior to the addition of the NO donor DEA/NO resulted in an increase in fluorescence that was approximately 7-fold higher than treatment with DEA/NO alone. The increase in DAF-2 triazole formation upon oxidation of DAF-2 was confirmed by high performance liquid chromatography. Peroxynitrite generated in situ from the equimolar production of NO and superoxide (O2•−) also increased the yields of DAF-2 triazole formation, which was completely inhibited when O2•− was in excess of NO. We propose that DAF-2 is oxidized to a free radical intermediate that directly reacts with NO, thereby bypassing the requirement for NO autoxidation for the formation of DAF-2 triazole. Our findings indicate that DAF-2 fluorometric assays are quantitatively difficult to interpret in cells and in solution when oxidants and NO are co-generated.