Kinetic analysis of DAF-FM activation by NO: Toward calibration of a NO-sensitive fluorescent dye

Abstract

Nitric oxide (NO) research in biomedicine has been hampered by the absence of a method that will allow quantitative measurement of NO in biological tissues with high sensitivity and selectivity, and with adequate spatial and temporal resolution. 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) is a NO sensitive fluorescence probe that has been used widely for qualitative assessment of cellular NO production. However, calibration of the fluorescent signal and quantification of NO concentration in cells and tissues using fluorescent probes, have provided significant challenge. In this study we utilize a combination of mathematical modeling and experimentation to elucidate the kinetics of NO/DAF-FM reaction in solution. Modeling and experiments suggest that the slope of fluorescent intensity (FI) can be related to NO concentration according to the equation: ddt[FI]=2αk1false[NOfalse]2[O2][DAF-FM]k[NO]+[DAF-FM] where α is a proportionality coefficient that relates FI to unit concentration of activated DAF-FM, k1 is the NO oxidation rate constant, and k was estimated to be 4.3 ± 0.6. The FI slope exhibits saturation kinetics with DAF-FM concentration. Interestingly, the effective half-maximum constant (EC50) increases proportionally to NO concentration. This result is not in agreement with the proposition that N2O3 is the NO oxidation byproduct that activates DAF-FM. Kinetic analysis suggests that the reactive intermediate should exhibit NO-dependent consumption and thus NO2• is a more likely candidate. The derived rate law can be used for the calibration of DAF-FM fluorescence and the quantification of NO concentration in biological tissues.

Mechanisms and kinetic profiles of superoxide-stimulated nitrosative processes in cells using a diaminofluorescein probe

“…(5)), DAF-2 must compete with the numerous cellular targets for these species. To test this expectation, we treated cells with different concentrations of DAF-2-DA (5, 10, and 20 mM), a procedure that has been shown to increase DAF-2 concentration intracellularly [10,48,49]. As expected, the rate of DAFT formation was greater for higher DAF-2-DA loading concentrations, and the effect was evident at 2 and 10 mM sper/NO (data not shown).…”

Generating S-Nitrosothiols from Hemoglobin

“…The fluorescein derivatives DAF-FM and DAF-2 were widely used to detect the presence of nitric oxide (for oxygen containing samples or N 2 O 3 (in the absence of oxygen) (27,61,(77)(78)(79)(80)(81)(82). A recent study (77) showed that in the presence of dioxygen, NO 2 rather than N 2 O 3 was likely to be the primary agent altering the DAF resulting in the enhanced fluorescence. In this study, the use of anaerobic conditions essentially eliminates that mechanism.…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…Choroidal capillary endothelial cells from the eyes of Thbs1 -/-mice had elevated phosphorylation of eNOS relative to WT cells, and intracellular NO in the null cells assessed using 4-amino-5-methylamino-2,7-difluorofluorescein (DAF) was sixfold higher than in WT cells. One should bear in mind that DAF is primarily detecting an oxidative product of NO rather than NO itself (176).…”