Separation and Quantitation of CytochromecOxidase Subunits by Mono-Q Fast Protein Liquid Chromatography and C18 Reverse-Phase High-Performance Liquid Chromatography

Mono-Q fast protein liquid chromatography (FPLC) combined with C18 reverse-phase HPLC was used for quantitative subunit analysis of bovine heart cytochromecoxidase, a multisubunit membrane complex. By this approach normal cytochromecoxidase preparations were shown to be a mixture of enzyme that has all 13 subunits and complexes that are missing 1–3 subunits. A distinct advantage of this procedure is that homogeneous 13- or 11-subunit enzyme can be easily isolated from heterogeneous cytochromecoxidase mixtures. The method involves: (1) separation of complexes that are depleted of subunits using Mono-Q FPLC and (2) quantitative subunit analysis of the purified complexes by C18 reverse-phase HPLC with a water/acetonitrile gradient in 0.1% trifluoroacetic acid. The approach has four distinct advantages over other methods of analysis, e.g., sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) or C4 reverse-phase HPLC. (1) The reproducible yield and the baseline resolution between each eluting subunit permits quantitative determination of the subunit content with an accuracy of ± 5%. (2) Subunits that are very difficult to separate by SDS–PAGE, e.g., subunits VIa, VIb, and VIc, are completely resolved by this system. (3) The combination of Mono-Q purification and C18 reverse-phase HPLC analysis permits an accurate assessment of both homogeneity and subunit content. (4) The quantitative nature of the reverse-phase HPLC system also makes it a powerful method for analyzing the specificity and extent of chemical modification of specific subunits as is shown by the difference in reac tivity of subunit VIa towardN-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate and 4,4′-dipyridyl disulfide.