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 (H(2)O(2)) oxidized DAF-2 to a relatively stable nonfluorescent intermediate (t(1/2) approximately 90 s). Oxidation of DAF-2 prior to the addition of the z.rad;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 (O(2)(*-)) also increased the yields of DAF-2 triazole formation, which was completely inhibited when O(2)(*-) 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.
Jourd'heuil
David
D
Center for Cardiovascular Sciences, Albany Medical College, Albany, NY 12208, USA . jourdhd@mail.amc.edu
D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D019793:Fluorescein / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005609:Free Radicals; D007202:Indicators and Reagents / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D009584:Nitrogen / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays
C111205:4,5-diaminofluorescein; D005456:Fluorescent Dyes; D005609:Free Radicals; D007202:Indicators and Reagents; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D009584:Nitrogen; D010100:Oxygen; D019793:Fluorescein