Faculty Collaboration Database

Detection of 2-hydroxyethidium in cellular systems: a unique marker product of superoxide and hydroethidine. Nat Protoc 2008;3(1):8-21

Date

01/15/2008

Pubmed ID

18193017

DOI

10.1038/nprot.2007.473

Scopus ID

2-s2.0-38149142769 (requires institutional sign-in at Scopus site)   326 Citations

Abstract

Various detection methods of the specific product of reaction of superoxide (O(2)(*-)) 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/O(2)(*-) 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.

Author List

Zielonka J, Vasquez-Vivar J, Kalyanaraman B

Authors

Balaraman Kalyanaraman PhD Professor in the Biophysics department at Medical College of Wisconsin
Jeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin
Jacek M. Zielonka PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Cell Line
Chromatography, High Pressure Liquid
Ethidium
Fluorescent Dyes
Mice
Phenanthridines
Spectrometry, Fluorescence
Spectrophotometry, Ultraviolet
Superoxides