Isolation and purification of membrane-bound cytochrome b-560 from photosynthetic bacterium Chromatium vinosum

A membrane-bound cytochrome of the b-type (cytochrome b-560) was success-fully purified from chromatophores of the photosynthetic purple sulfur bacterium Chromatium vinosum by treatment with sodium cholate, sodium deoxycholate, sodium thiocyanate, and bacterial alkaline protease (EC 3·4·21·14) followed by gel filtration. The purified cytochrome b-560 showed the absorption maxima at 279, 412.5 and 533 nm in the oxidized form, and 427, 530 and 560 nm in the reduced form. Reduced-minus-oxidized difference millimolar absorption coefficient was 14.0 for a wavelength pair, 560 minus 540 nm. Isolated cytochrome b-560 was electrophoretically homogeneous, and its minimal molecular weight was estimated to the 13,000 by SDS polyacrylamide gel electrophoresis. The midpoint potential at pH 8.0 was -110mV, and was not dependent on the ambient pH in the pH range of 6.8 to 8.8.