Orthogonality of Calcium Concentration and Ability of 4,5-Diaminofluorescein to Detect NO

We have developed diaminofluoresceins (DAFs) and diaminorhodamines as fluorescent indicators for NO based on the specific reaction of the aromatic vicinal diamines with NO. Among them, 4,5-diaminofluorescein (DAF-2) is widely used for real-time biological imaging of NO in cultured cells or tissues by many researchers. Contrary to a recent report of divalent cation sensitivity and photoactivation of DAF-2 (Broillet, M. C., Randin, O., and Chatton, J. Y. (2001) FEBS Lett. 491, 227-232), our study using NO gas itself reveals that the reaction of DAF-2 and NO is completely independent of Ca2+ and Mg2+ at physiological concentrations. Ca2+ enhances not the conversion of DAF-2 into its fluorescent product (DAF-2 triazole) but the release of NO from NO donors. Therefore it is concluded that DAF-2 can provide reliable information on NO production in biological systems regardless of the dynamic changes of Ca2+ concentration.