A Fluorophore-Containing Nitroxide as a Probe to Detect Superoxide and Hydroxyl Radical Generated by Stimulated Neutrophils

Abstract

Toward the development of a fluorescence assay in combination with confocal microscopy to image free radicals generated by cells, we synthesized a fluorophore-nitroxide, 5-((2-carboxy)phenyl)-5-hydroxy-1-((2,2,5,5-tetramethyl-1-oxypyrrolidin-3-yl)methyl)-3-phenyl-2-pyrrolin-4-one sodium salt, and tested the applicability of this probe to detect oxygen-centered free radicals. The reaction of the fluorophore-nitroxide with superoxide (10 μM/min) generated either by the reaction of xanthine oxidase on xanthine or by PMA-activated neutrophils in the presence of cysteine (200 μM) resulted in a loss of electron spin resonance (ESR) signal intensity concurrent with an increase in fluorescence emission. The decrease in ESR signal and the augmentation in fluorescence emission were inhibited by the addition of superoxide dismuatse. This fluorophore-nitroxide also reacted with methyl radical generated by the reaction of hydroxyl radical with DMSO (0.14 M). In this case a loss in ESR signal intensity concomitant with an increase in fluorescence emission which were inhibited by catalase (300 U/ml), was recorded. These results clearly demonstrated the feasibility of using fluorescence methodology in conjunction with a fluorophore-nitroxide to detect oxygen-centered free radicals in biological systems.