title: Oxidative chemistry of fluorescent dyes: implications in the detection of reactive oxygen and nitrogen species.
abstract: HE (hydroethidine), a widely used fluorescent dye for detecting intracellular superoxide, undergoes specific oxidation and hydroxylation reactions. The reaction between HE and O2•- (superoxide radical) yields a diagnostic marker product, 2-hydroxyethidium. This is contrary to the popular notion that O2•- oxidizes HE to form ethidium. HE, however, undergoes a non-specific oxidation to form ethidium in the presence of other oxidants (hydroxyl radical, peroxynitrite and perferryl iron) and other dimeric products. The mitochondria-targeted HE analogue Mito-SOX® undergoes the same type of oxidative chemistry to form products similar to those formed from HE. On the basis of the oxidative chemical mechanism of HE and Mito-SOX®, we conclude that flurorescence microscopy or related techniques are not sufficient to measure the superoxide-specific hydroxylated products. HPLC methodologies are required to separate and identify these products. Peroxynitrite reacts rapidly and stoichiometrically with boronates to form specific products. Assays using fluorescent-based boronate probes will be more reliable for peroxynitrite determination than those using either dichlorodihydrofluorescein or dihydrorhodamine.
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. balarama@mcw.edu
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D001897:Boronic Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004996:Ethidium / Q000031:analogs & derivatives / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry; D015394:Molecular Structure; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry; D010617:Phenanthridines / Q000737:chemistry; D026361:Reactive Nitrogen Species / Q000032:analysis*; D017382:Reactive Oxygen Species / Q000032:analysis*; D012235:Rhodamines / Q000737:chemistry; D013481:Superoxides / Q000032:analysis
chemical_list: C506392:2-hydroxyethidium; C521281:5-(6'-triphenylphosphoniumhexyl)-5,6-dihydro-6-phenyl-3,8-phenanthridinediammine; D001897:Boronic Acids; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016877:Oxidants; D010617:Phenanthridines; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D012235:Rhodamines; D013481:Superoxides; D030421:Peroxynitrous Acid; C058428:hydroethidine; D004996:Ethidium