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 not, vert, similar 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.