Subunit structure of the reconstitutively active cytochrome b-c1 complex. Determination of amino acids and molar distribution of subunit fractions from gel electrophoresis.

A quantitative method has been developed to analyze the amino acid composition of protein subunits directly from the Coomassie Blue-stained band of polyacrylamide gel columns after electrophoresis. It is an improved method originally reported by Houston (Houston, L. L. (1971) Anal. Biochem. 44, 81--88). The results obtained can be thus used for the calculation of the molar ratios of subunit components of protein. The manipulation of the method and computation of the results are illustrated by a very complicated lipoprotein complex. The subunit molar ratios of the reconstitutively active cytochrome b-c1 complex were determined to be 2, 2, 2, 3, 2, 2, and 5 among the seven bands of the corresponding molecular weights of 53 000, 50 000, 37 000, 30 000, 28 000, 17 000, and 15 000, from gel electrophoretic columns. The amino acid composition of each subunit fraction determined directly from hydrolysis of gel was comparable with that obtained by actual isolation of each subunit.