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 reverse-phase HPLC was used for quantitative subunit analysis of bovine heart cytochrome c oxidase, a multisubunit membrane complex. By this approach normal cytochrome c oxidase 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 cytochrome c oxidase 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 reactivity of subunit VIa toward N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate and 4,4'-dipyridyl disulfide.
Liu
Y C
YC
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284-7760, USA.
Sowdal
L H
LH
Robinson
N C
NC
D000818:Animals; D000837:Anion Exchange Resins; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004220:Disulfides; D003576:Electron Transport Complex IV / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D008929:Mitochondria, Heart / Q000201:enzymology*; D009282:Naphthalenesulfonates; D011487:Protein Conformation; D011725:Pyridines; D012117:Resins, Synthetic; D013367:Submitochondrial Particles / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000737:chemistry
D000837:Anion Exchange Resins; D004220:Disulfides; D009282:Naphthalenesulfonates; D011725:Pyridines; D012117:Resins, Synthetic; D013438:Sulfhydryl Compounds; C007542:4,4'-dipyridyl disulfide; C006421:1,5-I-AEDANS; C042855:Mono Q; D003576:Electron Transport Complex IV