title: Quinol type compound in cytochrome c preparations leads to non-enzymatic reduction of cytochrome c during the measurement of complex III activity.
abstract: Measurement of complex III activity is critical to the diagnosis of human mitochondrial disease and the study of mitochondrial pathobiology. Activity is measured as the maximal rate of antimycin A-sensitive reduction of exogenous cytochrome c by detergent-solubilized mitochondria. Complex III activity exhibited an unexpected variation based upon the commercial source of cytochrome c owing to an increase in the antimycin A-insensitive background reduction of cytochrome c and variable increases in total activity. Analysis of cytochrome c (producing a high-background) by fast protein liquid chromatography yielded a contaminant peak containing a lipid extractable component with redox spectra and mass spectroscopy fragmentation suggestive of a quinol. Measurement of inhibitor-sensitive rates are critical for the accurate and reproducible measurement of complex III activity and serve as a key quality control to screen for non-enzymatic reactions that obscure complex III activity.
authors.affiliation: Department of Medicine, Division of Cardiology, Case Western Reserve University, Cleveland, OH 44106, USA. smoghaddas@emh.org <smoghaddas@emh.org>
authors.forename: Shadi
authors.initials: S
authors.lastname: Moghaddas
authors.forename: Anne M
authors.initials: AM
authors.lastname: Distler
authors.forename: Charles L
authors.initials: CL
authors.lastname: Hoppel
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Lesnefsky
mesh_terms: D000968:Antimycin A / Q000494:pharmacology; D002853:Chromatography, Liquid / Q000379:methods; D045304:Cytochromes c / Q000737:chemistry* / Q000592:standards; D004340:Drug Contamination; D014450:Electron Transport Complex III / Q000032:analysis* / Q000378:metabolism; D006873:Hydroquinones / Q000032:analysis*; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism*; D010084:Oxidation-Reduction; D015203:Reproducibility of Results
chemical_list: D006873:Hydroquinones; D000968:Antimycin A; D045304:Cytochromes c; D014450:Electron Transport Complex III