ISOLATION, PURIFICATION, AND CHARACTERIZATION OF CRITHIDIA FASCICULATA CYTOCHROME C555

Cytochrome c555 was isolated from Crithidia fasciculata (Anopheles strain; A.T.C.C. No. 11745), and was purified by salt fractionation and chromatography on Amberlite CG-50 cation-exchange resin. The cellular content of cytochrome c555 was estimated to be 1.56 × 105 molecules per cell, accounting for about 25% of the hemin requirement of the organism. Absorbance maxima occurred at 413 mμ for the oxidized form, and at 420, 525, and 555.5 mμ for the reduced form, the extinction coefficient of the latter peak having been determined to be 29.7 mm-1 cm-1. The α-maximum of the pyridine hemochromogen was at 553 mμ. Acid acetone failed to remove the heme moiety from the cytochrome. The mono-heme protein had a minimum molecular weight of 12,051, calculated from the amino acid composition. The value calculated from ultracentrifugation data was 13,200. The mobility of the protein on Amberlite columns and upon electrophoresis in polyacrylamide gels was consistent with that which would have been expected from the amino acid composition. The isoelectric point was at pH 9.9, while the midpoint of the oxidation-reduction potential was +280 mV at pH 7.0. The cytochrome was not autooxidizable, nor did it combine with carbon monoxide at neutral pH. Comparison with other purified cytochromes c from a variety of sources showed that, in most of its properties, cytochrome c555 was similar to cytochromes from certain yeasts. © 1969.