Interfering with nitric oxide measurements. 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid.
4,5-Diaminofluorescein (DAF-2) is widely used for detection and imaging of NO based on its sensitivity, noncytotoxicity, and specificity. In the presence of oxygen, NO and NO-related reactive nitrogen species nitrosate 4,5-diaminofluorescein to yield the highly fluorescent DAF-2 triazole (DAF-2T). However, as reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO because it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have fluorescence emission profiles similar to that of DAF-2T. When DHA is present, the formation of DAF-2T is attenuated because the DHA competes for DAF-2, whereas AA decreases the nitrosation of DAF-2 to a larger extent, possibly because of additional reducing activity that affects the amount of available N(2)O(3) from the NO. The reaction products of DAF-2 with DHA and AA have been characterized using capillary electrophoresis with laser-induced fluorescence detection and electrospray mass spectrometry. The reactions of DAF-2 with DHA and AA are particularly significant because DHA and AA often colocalize with nitric-oxide synthase in the central nervous, cardiovascular, and immune systems, indicating the importance of understanding this chemistry.
Zhang
Xin
X
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana 61801, USA.
Kim
Won-Suk
WS
Hatcher
Nathan
N
Potgieter
Kurt
K
Moroz
Leonid L
LL
Gillette
Rhanor
R
Sweedler
Jonathan V
JV
D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry*; D003683:Dehydroascorbic Acid / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry*; D009569:Nitric Oxide / Q000032:analysis*; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
C111205:4,5-diaminofluorescein; D009569:Nitric Oxide; D001205:Ascorbic Acid; D019793:Fluorescein; D003683:Dehydroascorbic Acid