Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III.
By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron-sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification; D002417:Cattle; D002847:Chromatography, Agarose / Q000379:methods; D003575:Cytochromes c1 / Q000302:isolation & purification; D003902:Detergents; D014450:Electron Transport Complex III; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification; D008929:Mitochondria, Heart / Q000032:analysis*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D011808:Quinone Reductases / Q000302:isolation & purification*
D002352:Carrier Proteins; D003902:Detergents; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; C027208:ubiquinone-binding proteins; D003575:Cytochromes c1; D011808:Quinone Reductases; D014450:Electron Transport Complex III