Dual fluorophore-nitronyl probe for investigation of superoxide dynamics and antioxidant status of biological systems

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was synthesized and used to detect superoxide radicals and to quantitatively estimate the antioxidant activity of biological compounds in a wide range of antioxidant redox potential. This approach is based on the phenomenon of intramolecular fluorescence quenching of the pyrene fragment by the nitroxide in the dual fluorophore-nitronyl (FNO) molecule. The nitroxide fragment of the dual non-fluorescence molecule can be reduced by a variety of free radicals (e.g. superoxide) and antioxidants (e.g. ascorbic acid, quercetin, galangin, etc.). The reduction of FNO is accompanied by a drastic increase in fluorescence intensity and a corresponding decrease of the EPR signal. The results show that steady-state fluorescence measurements enable the detection of a very low flux of superoxide, starting from 10 nM/min. The fluorescence technique, which has been developed, allows detection of antioxidants at the submicromolar concentration scale while the EPR and light absorption techniques are limited to several micromolar. This method has also been applied to the determination of hydrogen peroxide in submicromolar concentrations. The specificity of this approach can be enhanced by the addition of superoxide dismutase and catalase.