SEPARATION AND QUANTITATION OF CYTOCHROME-C-OXIDASE 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 rev erse-phase HPLC was used for quantitative subunit analysis of bovine h eart cytochrome c oxidase, a multisubunit membrane complex, By this ap proach normal cytochrome c oxidase preparations were shown to be a mix ture of enzyme that has all 13 subunits and complexes that are missing 1-3 subunits, A distinct advantage of this procedure is that homogene ous 13- or Il-subunit enzyme can be easily isolated from heterogeneous cytochrome c oxidase mixtures, The method involves: (1) separation of complexes that are depleted of subunits using Mono-Q FPLC and (2) qua ntitative subunit analysis of the purified complexes by C18 reverse-ph ase HPLC with a water/acetonitrile gradient in 0.1% trifluoroacetic ac id, The approach has four distinct advantages over other methods of an alysis, e,g,, sodium dodecyl sulfate polyacrylamide gel electrophoresi s (SDS-PAGE) or C4 reverse-phase HPLC, (1) The reproducible yield and the baseline resolution between each eluting subunit permits quantitat ive determination of the subunit content with an accuracy of +/- 5%. ( 2) Subunits that are very difficult to separate by SDS-PAGE, e.g., sub units Via, VIb, and VIc, are completely resolved by this system, (3) T he combination of Mono-Q purification and C18 reverse-phase HPLC analy sis permits an accurate assessment of both homogeneity and subunit con tent. (4) The quantitative nature of the reverse-phase HPLC system als o makes it a powerful method for analyzing the specificity and extent of chemical modification of specific subunits as is shown by the diffe rence in reactivity of subunit VIa toward iodoacetylamidoethy-1-aminon aphthalene-5-sulfonate and 4,4'-dipyridyl disulfide, (C) 1995 Academic Press, Inc.