Protocol abstract
Nature Protocols 3, - 8 - 21 (2008)
Published online: 20 December 2007 | doi:10.1038/nprot.2007.473
Subject Categories: Chemical modification | Isolation, purification and separation | Pharmacology and toxicology | Spectroscopy and structural analysis | Synthetic chemistry
Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine
Jacek Zielonka1,2, Jeannette Vasquez-Vivar1,2 & Balaraman Kalyanaraman1,2
Abstract
Various detection methods of the specific product of reaction of superoxide (O2
-
) with hydroethidine (HE), namely 2-hydroxyethidium (2-OH-E+), and with its mitochondria-targeted analog are described. The detailed protocol for quantification of 2-OH-E+, the unique product of HE/O2-
in cellular systems, is presented. The procedure includes cell lysis, protein precipitation using acidified methanol and HPLC analysis of the lysate. Using this protocol, we determined the intracellular levels of 2-OH-E+ and E+ in the range of 10 and 100 pmol per mg protein in unstimulated macrophage-like RAW 264.7 cells. In addition to HE, 2-OH-E+ and E+, we detected several dimeric products of HE oxidation in cell lysates. As several oxidation products of HE are formed, the superoxide-specific product, 2-OH-E+ needs to be separated from other HE-derived products for unequivocal quantification.
- Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
- Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Correspondence to: Balaraman Kalyanaraman1,2 e-mail: balarama@mcw.edu
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