title: Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: identification of homo- and heterodimers.
abstract: Here we report that ferricytochrome c (cyt c(3+)) induces oxidation of hydroethidine (HE) and mitochondria-targeted hydroethidine (Mito-HE or MitoSOX Red) forming highly characteristic homo- and heterodimeric products. Using an HPLC-electrochemical (EC) method, several products were detected from cyt c(3+)-catalyzed oxidation of HE and Mito-HE and characterized by mass spectrometry and NMR techniques as follows: homodimers (HE-HE, E(+)-E(+), Mito-HE-Mito-HE, and Mito-E(+)-Mito-E(+)) and heterodimers (HE-E(+) and Mito-HE-Mito-E(+)), as well as the monomeric ethidium (E(+)) and mito-ethidium (Mito-E(+)). Similar products were detected when HE and Mito-HE were incubated with mitochondria. In contrast, mitochondria depleted of cyt c(3+) were much less effective in oxidizing HE or Mito-HE to corresponding dimeric products. Unlike E(+) or Mito-E(+), the dimeric analogs (E(+)-E(+) and Mito-E(+)-Mito-E(+)) were not fluorescent. Superoxide (O(2)(*-)) or Fremy's salt reacts with Mito-HE to form a product, 2-hydroxy-mito-ethidium (2-OH-Mito-E(+)) that was detected by HPLC. We conclude that HPLC-EC but not the confocal and fluorescence microscopy is a viable technique for measuring superoxide and cyt c(3+)-dependent oxidation products of HE and Mito-HE in cells. Superoxide detection using HE and Mito-HE could be severely compromised due to their propensity to undergo oxidation.
authors.affiliation: Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
authors.forename: Jacek
authors.initials: J
authors.lastname: Zielonka
authors.forename: Satish
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Micael
authors.initials: M
authors.lastname: Hardy
authors.forename: Olivier
authors.initials: O
authors.lastname: Ouari
authors.forename: Marcos
authors.initials: M
authors.lastname: Lopez
authors.forename: Jeannette
authors.initials: J
authors.lastname: Vasquez-Vivar
authors.forename: Narayan G
authors.initials: NG
authors.lastname: Avadhani
authors.forename: B
authors.initials: B
authors.lastname: Kalyanaraman
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D045304:Cytochromes c / Q000378:metabolism*; D019281:Dimerization; D005453:Fluorescence; D005456:Fluorescent Dyes; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008930:Mitochondria, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010617:Phenanthridines / Q000378:metabolism*; D051381:Rats; D013481:Superoxides / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010617:Phenanthridines; D013481:Superoxides; C058428:hydroethidine; D045304:Cytochromes c