bc1-Complex from beef heart. One-step purification by hydroxyapatite chromatography in Triton X-100, polypeptide pattern and respiratory chain characteristics.
A new simple method for the purification of the bc1-complex has been developed. The polypeptide composition of the complex was analysed by dodecyl sulfate-polyacrylamide gel electrophoresis. The content of chain components and phospholipids was determined. The b-type cytochromes were further characterized by their absorbance spectra and midpoint potentials. (1) Starting from a Triton X-100 extract of submitochondrial particles supplemented with antimycin, the bc1-complex is purified by adsorption chromatography on hydroxyapatite with citrate as specific eluant. (2) The complex splits in dodecyl sulfate into five main polypeptides with apparent molecular weight of 47, 44, 31, 11 and less than 10 kdalton. (3) The  purified complex has a heme-b content of 8.0 mumol/g protein and a cytochrome c1 content of 3.8 mumol/g protein. (4) The cytochromes show the typical absorbance spectra of cytochromes b-562 and b-565 and are present in approximately equal amounts with midpoint potentials of Em7 = + 100 mV and Em7 = + mV respectively. Carbon monoxide does not bind to the cytochromes. (5) The nonheme iron protein content of the complex is diminished to 0.6 mumol/g protein. (6) The use of the nonionic surfactant Triton X-100 leads to a complete loss of lipids and ubiquinone of the bc1-complex. (7) The complex contains no succinate dehydrogenase as indicated by the absence of the 69 kdalton subunit in the dodecyl sulfate gel electrophoresis. In addition, it lacks an ubiquinone cytochrome c reductase activity and other electron transferring activities. This may be inferred from an inhibition by antimycin and depletion of ubiquinone and phospholipids. The highly purified and relative stable complex can be prepared giving 50% yield and may be suitable for protein chemistry studies.
D000818:Animals; D002417:Cattle; D002845:Chromatography; D003574:Cytochrome c Group / Q000302:isolation & purification; D003580:Cytochromes / Q000302:isolation & purification / Q000378:metabolism; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D008931:Mitochondria, Muscle / Q000378:metabolism*; D009206:Myocardium; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D011092:Polyethylene Glycols; D013053:Spectrophotometry; D014451:Ubiquinone / Q000032:analysis
D003574:Cytochrome c Group; D003580:Cytochromes; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D010455:Peptides; D010743:Phospholipids; D014451:Ubiquinone; D011092:Polyethylene Glycols