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Relationship between rate of change of DAF‐2T fluorescence and nitric oxide (NO) concentration in vitro
Author(s) -
Carvalho Helena,
Pittman Roland N
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.927.3
Subject(s) - photobleaching , fluorescence , chemistry , kinetics , fluorescence recovery after photobleaching , analytical chemistry (journal) , nitric oxide , in vivo , biophysics , chromatography , optics , biology , physics , microbiology and biotechnology , organic chemistry , quantum mechanics
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 O 2 . In the present study we demonstrated the influence of light intensity that promoted photobleaching and the importance of [O 2 ] 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 N 2 (1200 nM NO); this procedure produced mixtures with varied PO 2 . 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 PO 2 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.