Biochemistry 1987, 26, 1955-1961
1955
Preparation and Properties of Cardiac Cytochrome cl+ Chong H. Kim* and Tsoo E. King Departments of Chemistry and Biology and Laboratory of Bioenergetics, State University of New York at Albany, Albany, New York 12222 Received August 20, 1986; Revised Manuscript Received December 9, 1986
A method for the large-scale isolation of beef heart mitochondrial cytochrome c1 in high purity was developed. This method gave higher yield of “one-band” cytochrome c1 than previously reported [Kim, C . H., & King, T. E. (1981) Biochem. Biophys. Res. Commun. 102, 607-6141. In addition, the present method was effective in the preparation of “two-band” cytochrome c1 which was used to prepare the hinge protein according to the principle of sequential resolution [Kim, C. H., & King, T:E. (1983) J. Biol. Chem. 258, 13543-135511. The isolation of one-band and two-band cytochrome c1 by this procedure could be completed within 3 or 4 days starting with succinate-cytochrome c reductase. One-band cytochrome c1 showed a molecular weight of 44 000 by sedimentation equilibrium and 29 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The disparities in these data from the actual value of 27 924 by amino acid sequence analysis, as previously reported [Wakabayashi, S.,Matsubara, H., Kim, C . H., & King, T. E. (1982) J . Biol. Chem. 257, 9335-93441, are most probably due to the formation of detergent or detergent-phosphate complex. A comparison of some properties of one-band cytochrome c1 with those of two-band cytochrome c1 clearly showed significant differences between the two preparations. These results suggest the hypothesis that one of the possible roles of the hinge protein in the mitochondrial respiratory chain is to stabilize the conformation of cytochrome cl, ABSTRACT:
C y t o c h r o m e c1 was independently discovered by Keilin and Hartree in Cambridge (Keilin, 1966) and Yakushiji and Okunuki in Japan (Yakusiji & Okunuki, 1940). However, the preparation of pure and active cytochrome c1 was not successful until King and co-workers reported a method in 1972 (King, 1978; Yu et al., 1972), and, more recently, van Gelder and co-workers reported another method in 1980 (Konig et al., 1980). However, the sample prepared even by the latter method was still heavily contaminated with the glutamic acid rich small protein known as the “hinge protein” [cf. Wakabayashi et al. (1982a) and Kim & King (1983)l. The evidence was the amino acid composition of this preparation. Our cytochrome c1 preparation reported in 1972 was actually cytochrome cI with the hinge protein in a 1 to 1 molar ratio, which we called “two-band” cl, and similarly, the cytochrome c1 preparation free of the hinge protein was called the “one-band” c1 (Kim & King, 1981, 1983). In our preliminary report (Kim & King, 1981), the yield of the preparation of one-band cytochrome cI free of the hinge protein was very low (
