- 作者：Balaraman Kalyanaraman ; Brian P. Dranka ; Micael Hardy ; Radoslaw Michalski ; Jacek Zielonka
- 关键词：2-OH-E+ ; 2-hydroxyethidium ; E+ ; ethidium ; HE ; hydroethidine ; HPr+ ; hydropropidine ; MitoSOXTM Red or Mito-HE ; HE conjugated to TPP+ group ; TPP+ ; triphenylphosphonium cation ; SOD ; superoxide dismutase
- 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
- 出版年：February, 2014
- 年：2014
- 卷：1840
- 期：2
- 页码：739-744
- 全文大小：1207 K
Background
Nearly ten years ago, we demonstrated that superoxide radical anion () reacts with the hydroethidine dye (HE, also known as dihydroethidium, DHE) to form a diagnostic marker product, 2-hydroxyethidium (2-OH-E+). This particular product is not derived from reacting HE with other biologically relevant oxidants (hydrogen peroxide, hydroxyl radical, or peroxynitrite). This discovery negated the longstanding view that reacts with HE to form the other oxidation product, ethidium (E+). It became clear that due to the overlapping fluorescence spectra of E+ and 2-OH-E+, fluorescence-based techniques using the 鈥渞ed fluorescence鈥?are not suitable for detecting and measuring in cells using HE or other structurally analogous fluorogenic probes (MitoSOXTM Red or hydropropidine). However, using HPLC-based assays, 2-OH-E+ and analogous hydroxylated products can be easily detected and quickly separated from other oxidation products.Scope of review
The principles discussed in this chapter are generally applicable in free radical biology and medicine, redox biology, and clinical and translational research. The assays developed here could be used to discover new and targeted inhibitors for various superoxide-producing enzymes, including NADPH oxidase (NOX) isoforms.
Major conclusions
HPLC-based approaches using site-specific HE-based fluorogenic probes are eminently suitable for monitoring in intra- and extracellular compartments and in mitochondria. The use of fluorescence-microscopic methods should be avoided because of spectral overlapping characteristics of -derived marker product and other, non-specific oxidized fluorescent products formed from these probes.
General significance
Methodologies and site-specific fluorescent probes described in this review can be suitably employed to delineate oxy radical dependent mechanisms in cells under physiological and pathological conditions. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.