ISOLATION AND PARTIAL CHARACTERIZATION OF THREE CYTOCHROMES C FROM P. CRUENTUM

PAULA KAY EVANS, Purdue University

Abstract

Three c-type cytochromes were isolated from the red alga, P. cruentum, and partially characterized. The cytochrome c-553 was an extrinsic membrane protein which was easily released to the aqueous phase with a yield of 10 to 15 mg of pure protein per 100g cells. The properties of this cytochrome were similar to other algal and cyanobacterial cytochromes which function as Photosystem I donors. Absorption maxima in reduced cytochrome c-553 were at 552.8, 521.8, and 415.6 nm. The molecular weight was 10 kilodaltons, the redox potential was +340 mV, and the pI was 4.3. The near infrared absorption band of ferricytochrome was found to titrate with a single pK of 9.5. In addition to the loss of the near infrared band at alkaline pH, other spectral changes were noted in the ferrocytochrome. The most obvious change was that of the alpha band shifting to 550 nm in the dithionite reduced cytochrome at alkaline pH. Small quantities of a cytochrome c-550 were detected in some preparations of P. cruentum. This cytochrome had absorption maxima in the reduced form of 550, 522, and 416 nm. The pI was 4.1. This cytochrome was isolated in the oxidized form and was not ascorbate reducible. The third c-type cytochrome isolated from P. cruentum was an intrinsic membrane protein probably analogus to higher plant cytochrome f. The absorption maxima in the reduced cytochrome were at 553.75, 522.5, and 421 nm. Cytochrome f was always isolated in the reduced form with a yield of 1 to 1.5 mg per 100g alga membranes. The cytochrome f was unstable at 4(DEGREES) or -20(DEGREES)C. The molecular weight was 26 kilodaltons, and the pI was 6.5. Photosynthetically active vesicles from P. cruentum were prepared. Exogenous cytochrome c-553 stimulated Photosystem I dependent activity but not reactions dependent on both Photosystems. Antibody to cytochrome c-553 had no effect on photochemical activity nor did it bind to vesicular cytochrome c-553.

Degree

Ph.D.

Subject Area

Biochemistry

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