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

Yuan Chao Liu, Linda H. Sowdal, Neal C. Robinson

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Original languageEnglish
Pages (from-to)135-142
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume324
Issue number1
DOIs
StatePublished - Dec 1 1995

Fingerprint

Liquid chromatography
High performance liquid chromatography
Reverse-Phase Chromatography
Liquid Chromatography
High Pressure Liquid Chromatography
Electrophoresis
Sodium Dodecyl Sulfate
Trifluoroacetic Acid
Proteins
Chemical modification
Enzymes
Polyacrylamide Gel Electrophoresis
Disulfides
Purification
Membranes
Water
Mono Q
polyacrylamide gels

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

Cite this

Separation and Quantitation of CytochromecOxidase Subunits by Mono-Q Fast Protein Liquid Chromatography and C18 Reverse-Phase High-Performance Liquid Chromatography. / Liu, Yuan Chao; Sowdal, Linda H.; Robinson, Neal C.

In: Archives of Biochemistry and Biophysics, Vol. 324, No. 1, 01.12.1995, p. 135-142.

Research output: Contribution to journalArticle

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title = "Separation and Quantitation of CytochromecOxidase Subunits by Mono-Q Fast Protein Liquid Chromatography and C18 Reverse-Phase High-Performance Liquid Chromatography",
abstract = "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.",
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N2 - 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.

AB - 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.

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