The Use of Disc Gel Electrophoresis with Nonionic Detergent in the Purification of Cytochrome f from Spinach Grana Membranes

Abstract

Abstract Cytochrome f was purified from spinach grana membranes with a final yield of 42%. Using technology of disc gel electrophoresis with and without nonionic detergent, it was found that the cytochrome was intrinsically hydrophobic and existed as soluble aggregates at pH 8 which could be disaggregated by 0.1% nonionic detergent to a single, stable, electrophoretic species. Disc gel electrophoresis with non-ionic detergent was indispensable both in designing and in monitoring the purification procedure and served as a final preparative chromatographic step. The cytochrome moved as a single electrophoretic species throughout the purification. Cytochrome f was inactivated by urea, guanidine, and sodium dodecyl sulfate, which have commonly been used as disaggregating agents. Satisfactory chromatography of cytochrome f in nonionic detergent solution was found possible in uncharged support media (disc gel electrophoresis or, alternatively, on Sephadex G-200), but not on DEAE-cellulose. Proof of homogeneity of cytochrome f is presented. The cytochrome appears to contain one heme-containing polypeptide chain and one non-heme chain, each about 31,000. The absorbance spectra of cytochrome f (reduced, oxidized, and reduced minus oxidized) at 22° and at -199° are presented. The cytochrome was not autoxidizable, did not react with carbon monoxide, and contained no non-heme iron. The cytochrome was originally tightly bound, perhaps hydrophobically, to grana membranes.