Interfering with nitric oxide measurements. 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid

Area of Science:

• Biochemistry
• Analytical Chemistry
• Chemical Biology

Background:

• 4,5-Diaminofluorescein (DAF-2) is a widely used fluorescent probe for nitric oxide (NO) detection.
• DAF-2 reacts with NO and reactive nitrogen species in the presence of oxygen to form a fluorescent triazole product, DAF-2T.
• The specificity of DAF-2 for NO detection has been questioned due to potential cross-reactions.

Purpose of the Study:

• To investigate the specificity of DAF-2 for nitric oxide detection.
• To characterize the reaction products of DAF-2 with dehydroascorbic acid (DHA) and ascorbic acid (AA).
• To assess the implications of these reactions in biological systems where NO, DHA, and AA may coexist.

Main Methods:

• Capillary electrophoresis with laser-induced fluorescence detection.
• Electrospray mass spectrometry for characterizing reaction products.
• In vitro studies of DAF-2 reactions with NO, DHA, and AA.

Main Results:

• DAF-2 reacts with DHA and AA to form fluorescent compounds with emission profiles similar to DAF-2T.
• DHA competes with DAF-2, attenuating DAF-2T formation.
• AA significantly decreases DAF-2T formation, likely due to its reducing activity impacting available N(2)O(3).

Conclusions:

• The DAF-2 reaction is not specific to NO, as it yields fluorescent products with DHA and AA.
• These cross-reactions are significant in biological contexts where DHA and AA colocalize with nitric-oxide synthase.
• Understanding these interactions is crucial for accurate NO measurements in complex biological samples.