Oxidative chemistry of fluorescent dyes: implications in the detection of reactive oxygen and nitrogen species |
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Authors: | Kalyanaraman Balaraman |
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Institution: | Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. balarama@mcw.edu |
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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. |
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