Relationship between rate of change of DAF‐2T fluorescence and nitric oxide (NO) concentration in vitro

Abstract

DAF‐2 has been used for measurements of NO in vitro and in vivo, by virtue of its reaction with an auto‐oxidation product of NO to form the strongly fluorescent product DAF‐2T. Previously, we reported that the rate of change of DAF‐2T fluorescence was proportional to [NO]2, as predicted by the reaction kinetics of NO and DAF‐2 in the presence of O2. In the present study we demonstrated the influence of light intensity that promoted photobleaching and the importance of [O2] on the reaction between DAF‐2 and NO. The generation of DAF‐2T was evaluated in mixtures of 10 μM DAF‐2 with different concentrations of NO (range 100–1200 nM) made by mixing appropriate proportions of PBS equilibrated with room air with PBS equilibrated with 800 ppm NO/balance N2 (1200 nM NO); this procedure produced mixtures with varied PO2. Sequences of fluorescence images (at 10‐sec intervals over 300 sec) were acquired with a digital camera. Light intensities normally used in microscopic measurements induced strong photobleaching when the solutions were illuminated at intervals of 1‐10 sec. To avoid photobleaching, the incident light was attenuated using a 10% transmission neutral density filter. The non‐linear relationship between DAF‐2T fluorescence and [NO] was adequately described by the chemical kinetic model when the PO2 of the mixture was taken into account. These results are being used to interpret DAF‐2T fluorescence in terms of [NO] in vitro. Supported by NIH grants HL18292 and HL 79087.