Staff PublicationUnderstanding the reaction rate constant of a probe compound with its target molecule is essential for its
selection and use in biological and non-biological systems. Over the past decade, the probe, 4, 5-diaminofluorescein
(DAF-2) has been widely employed for the detection and imaging of nitric oxide (NO•) in various media. However,
the rate constant for the nitrosation of DAF-2 in air-saturated nitric oxide solution is far from being understood. For
the first time, we have determined the second order rate constant for the reaction of DAF-2 with NO• (k2) in airsaturated solution using competition kinetics method. An alternative competition kinetics method which involves a
reciprocal plot of the reactant (DAF-2) and triazolofluorescein (DAF-2T), the product formed from the reaction of
DAF-2 with NO• was developed and compared to the standard competition kinetics method. Our particular approach
in this system is based on the use of oxy-hemoglobin (HbO2), a potent scavenger of NO• against the DAF-2 which
serves as the detector molecule. The product, DAF-2T, is separated from the reaction mixture by reversed-phase
high performance liquid chromatography and its fluorescence intensity signals were measured at excitation and
emission of 495 and 515 nm respectively. The results showed that the second order reaction rate constant of DAF-2
with NO• in air-saturated aqueous solution are comparable, with the average value of (6.28 ± 0.45) × 10 6
M-1s
-1
.
Also, DAF-2 can react with NO• directly thereby by-passing the N2O3-mediated formation of DAF-2T at low NO•
formation rate