Photoreduction of the fluorescent dye 2′-7′-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

• Published in: Free radical biology & medicine 1999, Vol.26 (1), p.148-161
• Main Authors: Marchesi, Emanuela, Rota, Cristina, Fann, Yang C., Chignell, Colin F., Mason, Ronald P.
• Format: Article
• Language: English
• Subjects: 2′-7′-Dichlorofluorescein; Aerobiosis; Anaerobiosis; Catalase - metabolism; Catalase - pharmacology; Electron Spin Resonance Spectroscopy; Fluoresceins - chemistry; Fluoresceins - metabolism; Fluoresceins - radiation effects; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Fluorescent Dyes - radiation effects; Free radical; Free Radicals - chemistry; Free Radicals - metabolism; Free Radicals - radiation effects; Glutathione - metabolism; In Vitro Techniques; NAD - metabolism; Oxidation-Reduction; Oxidative Stress; Oxygen Consumption - drug effects; Photochemistry; Photoreduction; Solutions; Spin-trapping; Superoxide; Superoxide Dismutase - metabolism; Superoxide Dismutase - pharmacology
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/S0891-5849(98)00174-9

The photoreduction of 2′-7′-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/ ·OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.