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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Conference Article|
September 21 2011
Oxidative chemistry of fluorescent dyes: implications in the detection of reactive oxygen and nitrogen species
Balaraman Kalyanaraman
Balaraman Kalyanaraman
1
1Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, U.S.A.
1email balarama@mcw.edu.
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Publisher: Portland Press Ltd
Received:
July 15 2011
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem Soc Trans (2011) 39 (5): 1221–1225.
Article history
Received:
July 15 2011
Citation
Balaraman Kalyanaraman; Oxidative chemistry of fluorescent dyes: implications in the detection of reactive oxygen and nitrogen species. Biochem Soc Trans 1 October 2011; 39 (5): 1221–1225. doi: https://doi.org/10.1042/BST0391221
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