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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.

A tryptic resistant heme peptide has been prepared and purified from cardiac cytochrome c1. This purified peptide is not further hydrolyzed by reactions of other proteolytic enzymes, such as pronase. The peptide contains 2 residues each of serine, cysteine and valine, and 1 residue each of alanine, methionine, tyrosine, histidine, arginine, proline, glutamic acid (glutamine) and aspartic acid. The intensity of the absorption spectrum of the peptide has been found to be dependent upon, but the positions of the absorption maxima do not vary with, concentration. The heme peptide does not show multiple splitting of absorption peaks at liquid N2 temperatures as does the intact cytochrome C1. However, cyanide rapidly reacts with the peptide and causes significant spectral changes. CD spectra of the peptide exhibit a typical profile of a non-structured heme peptide with positive CD bands in the Soret region and around 250 nm, and a broad negative extreme of 320-360 nm. The similarities and differences between the tryptic resistant heme peptides from cytochromes c1 and c have been compared.

Proton ENDOR resonances have been found from at least two different protons with fairly large and isotropic couplings of about 12 and 19 MHz. It is possible that such protons are attached to carbons that are one bond removed from the point of ligation to copper. A number of weakly coupled protons with anisotropic couplings have also been seen. None of the protons, either weakly or strongly coupled, appears to exchange with 2H2O. We have obtained nitrogen ENDOR from at least one nitrogen with a hyperfine coupling large enough for the nitrogen to be a ligand of copper. We have not yet demonstrated experimentally ENDOR characteristic of the copper nucleus itself.