Reaction Kinetics for Nitrosation of DAF-2 in Air Saturated Nitric Oxide Solution

Abstract

Understanding 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 rates