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

► We investigated the kinetics of DAF-FM activation by NO in aerated solutions. ► Data suggest NO2 rather than N2O3 as the reactive NO oxidation intermediate. ► The slope of DAF-T signal is related to [NO] according to ddt[FI]=2αk1[NO]2[O2][DAF-FM]k[NO]+[DAF-FM]. ► For [DAF-FM]>>[NO] the slope is proportional to [NO]2 and independent of [DAF-FM]. ► Recommendations for the calibration of DAF fluorescence are provided. 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: ddtFI=2αk1NO2O2DAF-FMkNO+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.