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Increased Nitric Oxide-Dependent Nitrosylation of 4,5-diaminofluorescein by Oxidants: Implications for the Measurement of Intracellular Nitric Oxide

Increased Nitric Oxide-Dependent Nitrosylation of 4,5-diaminofluorescein by Oxidants: Implications for the Measurement of Intracellular Nitric Oxide

David Jourd'heuil

Free Radic Biol Med. 2002 Sep 1;33(5):676-84.

PMID: 12208354

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4241314	4,5-Diaminofluorescein			Price

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