Abstract
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.
Highlights
Nitric oxide (NO) is involved in a variety of important biological functions in the cardiovascular system, the central and peripheral nervous system, the reproductive and the immune system [1,2,3,4,5,6]
capillary electrophoresis (CE)-laser-induced fluorescence (LIF) — Various solutions were assayed using CE-LIF detection to determine the specificity of the DAF-2 reaction with NO
In all DAF-2 standards, we found an impurity whose level directly correlated with DAF-2 concentration
Summary
4,5-diaminofluorescein (DAF-2) is widely used for detection and imaging of nitric oxide (NO) based on its sensitivity, non-cytotoxicity, 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). As reported here, the DAF-2 reaction to form a fluorescent product is not specific to NO as it reacts with dehydroascorbic acid (DHA) and ascorbic acid (AA) to generate new compounds that have similar fluorescence emission profiles as DAF-2T. 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 significant as DHA and AA often colocalize with nitric oxide synthase in the central nervous, cardiovascular and the immune systems, indicating the importance of understanding this chemistry
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