title: Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide.
abstract: Hydroethidine (HE) or dihydroethidium (DHE), a redox-sensitive probe, has been widely used to detect intracellular superoxide anion. It is a common assumption that the reaction between superoxide and HE results in the formation of a two-electron oxidized product, ethidium (E+), which binds to DNA and leads to the enhancement of fluorescence (excitation, 500-530 nm; emission, 590-620 nm). However, the mechanism of oxidation of HE by the superoxide anion still remains unclear. In the present study, we show that superoxide generated in several enzymatic or chemical systems (e.g., xanthine/xanthine oxidase, endothelial nitric oxide synthase, or potassium superoxide) oxidizes HE to a fluorescent product (excitation, 480 nm; emission, 567 nm) that is totally different from E+. HPLC measurements revealed that the HE/superoxide reaction product elutes differently from E+. This new product exhibited an increase in fluorescence in the presence of DNA. Mass spectral data indicated that the molecular weight of the HE/superoxide reaction product is 330, while ethidium has a molecular weight of 314. We conclude that the reaction between superoxide and HE forms a fluorescent marker product that is different from ethidium. Potential implications of this finding in intracellular detection and imaging of superoxide are discussed.
authors.affiliation: Biophysics Research Institute and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Hongtao
authors.initials: H
authors.lastname: Zhao
authors.forename: Shasi
authors.initials: S
authors.lastname: Kalivendi
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Joy
authors.initials: J
authors.lastname: Joseph
authors.forename: Kasem
authors.initials: K
authors.lastname: Nithipatikom
authors.forename: Jeannette
authors.initials: J
authors.lastname: Vásquez-Vivar
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006834:Hydrazines / Q000494:pharmacology; D013058:Mass Spectrometry; D009569:Nitric Oxide; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D009589:Nitrogen Oxides; D010084:Oxidation-Reduction; D010617:Phenanthridines / Q000737:chemistry / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000737:chemistry / Q000378:metabolism*; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D006834:Hydrazines; D020030:Nitric Oxide Donors; D009589:Nitrogen Oxides; D010617:Phenanthridines; D013481:Superoxides; C039316:potassium superoxide; D019820:Xanthine; D009569:Nitric Oxide; C058428:hydroethidine; C084012:1,1-diethyl-2-hydroxy-2-nitrosohydrazine; D004247:DNA; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D004996:Ethidium