Studies on algal cytochromes. V. Purification and characterization of cytochrome c-552 from a red alga, Polysiphonia urceolata.

ABSTRACT

Cytochrome c-552 was extracted from a red alga, Polysiphonia urceolata, by immersion of the frozen trichomes in deionized water. Purification was carried out by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography, hydroxyapatite column chromatography, and Bio-Gel P-10 gel filtration. The ferrocytochrome c-552 has absorption maxima at 551.5(alpha), 522.5(beta), 416.3(gamma), 318(delta), 292, and 270 nm; those of the ferricytochrome are at 525, 408.5(gamma), and 358 nm. The pyridine ferrohemochrome showed absorption maxima at 550(alpha), 520(beta), and 414 nm(gamma). The alpha-band of the ferrocytochrome is symmetric without any shoulder at room temperature, and does not split even at liquid nitrogen temperature. The ferricytochrome showed a weak absorption shoulder at 695 nm, suggesting a methionine sulfur to be the sixth ligand of heme c iron. The cytochrome is oxidized by ferricyanide and reduced by ferrocyanide, cysteine, ascorbate, and hydrosulfite. Autoxidation was not observed. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferricyanide system to be 0.333 volt at pH 7.0 and 25 degrees C. The isoelectric points of the ferro- and ferricytochromes were determined to be at pH 3.85 and 4.02, respectively, by the isoelectric focusing method. The molecular weight was estimated to be about 12,000 from the results of gel filtration and SDS polyacrylamide gel electrophoresis.