Isolation of a five-polypeptide cytochrome b-f complex from spinach chloroplasts.
A simple rapid purification of a cytochrome b-f complex from spinach chloroplasts is described. Novel features of the method include: 1) EDTA treatment of thylakoids prior to detergent extraction; 2) affinity chromatography over equine cytochrome c linked to Sepharose 4B; and 3) inclusion of the protease inhibitor phenylmethylsulfonyl fluoride. Cytochrome b-f complex is obtained in good yield, free of exogenous lipid, and with high plastoquinol:plastocyanin oxidoreductase activity. The complex contains 2 eq of cytochrome b-563 per eq of cytochrome f and a Rieske iron-sulfur center. Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate indicates that the complex is composed of five distinct polypeptides of Mr = 37,000, 33,500, 22,000, 19,000, and 16,500. Only the Mr = 33,500 and 22,000 polypeptides stain for heme. The Mr = 37,000 component in this preparation is absent from cytochrome b-f complex isolated by another procedure (Hurt, E., and Hauska, G. (1981) Eur. J. Biochem. 117, 591-599). The complex described here also differs in its spectrum at 77 K, its stability, and its buoyant density.
D002499:Centrifugation, Density Gradient; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000302:isolation & purification*; D003580:Cytochromes / Q000302:isolation & purification*; D045348:Cytochromes f; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D003573:Cytochrome b Group; D003580:Cytochromes; C027225:cytochrome b563; D045348:Cytochromes f