Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography.
Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.
Robinson
N C
NC
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Dale
M P
MP
Talbert
L H
LH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003576:Electron Transport Complex IV / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008969:Molecular Sequence Data
D000596:Amino Acids; D046911:Macromolecular Substances; D003576:Electron Transport Complex IV