Studies on algal cytochromes. I. Purification and properties of cytochrome b-561 from Enteromorpha prolifera.

Cytochrome b-561 (Enteromorpha prolifera) was extracted from a green alga, E. prolifera, by immersion of the dried thalli in phosphate buffer solution. Purification was carried out by ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex column chromatographies, Bio-Gel gel filtration, and hydroxyapatite column chromatography. The reduced form of the cytochrome exhibited absorption maxima at 561 (alpha), 530.5 (beta), and 428.5 nm (gamma), and the oxidized form at 530.5, 417 (gamma), and 275 nm. The alpha-band of the reduced form was symmetric without any shoulder. The pyridine hemochrome showed absorption maxima at 556, 524, and 418 nm. The cytochrome does not combine with carbon monoxide or cyanide. The cytochrome showed little peroxidase activity. The cytochrome is oxidized by ferricyanide and reduced by cysteine, ascorbate, and hydrosulfite. Ferrocyanide and hydroquinone do not completely reduce it. Autoxidation of the cytochrome was found to be very slow. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferri-EDTA system to be +0.23 volt at pH 7.0 and 25 degrees C. The molecular weight of the cytochrome, estimated by Sephadex gel filtration, was 67 x 10(3).