title: Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and implications in redox signaling.
abstract: In this review, some of the recent developments in probes and assay techniques specific for superoxide (O2-) and hydrogen peroxide (H2O2) are discussed. Over the last decade, significant progress has been made in O2-and H2O2detection due to syntheses of new redox probes, better understanding of their chemistry, and development of specific and sensitive assays. For superoxide detection, hydroethidine (HE) is the most suitable probe, as the product, 2-hydroxyethidium, is specific for O2-. In addition, HE-derived dimeric products are specific for one-electron oxidants. As red-fluorescent ethidium is always formed from HE intracellularly, chromatographic techniques are required for detecting 2-hydroxyethidium. HE analogs, Mito-SOX and hydropropidine, exhibit the same reaction chemistry with O2-and one-electron oxidants. Thus, mitochondrial superoxide can be unequivocally detected using HPLC-based methods and not by fluorescence microscopy. Aromatic boronate-based probes react quantitatively with H2O2, forming a phenolic product. However, peroxynitrite and hypochlorite react more rapidly with boronates, forming the same product. Using ROS-specific probes and HPLC assays, it is possible to screen chemical libraries to discover specific inhibitors of NADPH oxidases. We hope that rigorous detection of O2-and H2O2in different cellular compartments will improve our understanding of their role in redox signaling.
authors: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. Electronic address: balarama@mcw.edu.
authors: Balaraman
authors: B
authors: Kalyanaraman
authors: Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille, France.
authors: Micael
authors: M
authors: Hardy
authors: Institute of Polymer and Dye Technology, Lodz University of Technology, Stefanowskiego 12/16, 90-924 Lodz, Poland.
authors: Radoslaw
authors: R
authors: Podsiadly
authors: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors: Gang
authors: G
authors: Cheng
authors: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors: Jacek
authors: J
authors: Zielonka
mesh_terms: D049109:Cell Proliferation; D002851:Chromatography, High Pressure Liquid; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D010617:Phenanthridines / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry; D015398:Signal Transduction; D013481:Superoxides / Q000737:chemistry*
chemical_list: D010617:Phenanthridines; D017382:Reactive Oxygen Species; D013481:Superoxides; C058428:hydroethidine; D010100:Oxygen
keywords: Fluorescent probes; Mitochondrial complex I; NADPH oxidase; Oxy radicals; Signal transducer