Abstract
Cytochrome c oxidase (ferrocytochrome c oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria by affinity chromatography. Phospholipids were removed by washing the oxidase with detergent on the affinity column; 1 mole of cardiolipin remained per mole of heme a. The oxidase was mixed with excess cytochrome c in 1.5% (wt/vol) cholate to form a complex. Slow removal of the detergent from the mixture by dialysis resulted in crystallization of cytochrome oxidase in the form of a 1:1 complex with cytochrome c. The chemical composition and spectrophotometric properties of the crystal are described. Increasing the solubility of a hydrophobic membrane protein by combination with hydropholic ligand is demonstrated as a maneuver for crystallizing the membrane protein.
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- FOWLER L. R., RICHARDSON S. H., HATEFI Y. A rapid method for the preparation of highly purified cytochrome oxidase. Biochim Biophys Acta. 1962 Oct 8;64:170–173. doi: 10.1016/0006-3002(62)90770-9. [DOI] [PubMed] [Google Scholar]
- GRIFFITHS D. E., WHARTON D. C. Studies of the electron transport system. XXXVI. Properties of copper in cytochroma oxidase. J Biol Chem. 1961 Jun;236:1857–1862. [PubMed] [Google Scholar]
- HATEFI Y., HAAVIK A. G., FOWLER L. R., GRIFFITHS D. E. Studies on the electron transfer system. XLII. Reconstitution of the electron transfer system. J Biol Chem. 1962 Aug;237:2661–2669. [PubMed] [Google Scholar]
- LUBOCHINSKY B., ZALTA J. P. Microdosage colorimétrique de l'azote ammoniacal. Bull Soc Chim Biol (Paris) 1954;36(9):1363–1366. [PubMed] [Google Scholar]
- MARGOLIASH E., FROHWIRT N. Spectrum of horse-heart cytochrome c. Biochem J. 1959 Mar;71(3):570–572. doi: 10.1042/bj0710570. [DOI] [PMC free article] [PubMed] [Google Scholar]
- ORII Y., SEKUZU I., OKUNUKI K. Studies on cytochrome cl. II. Oxidation mechanism of cytochrome c1 in the presence of cytochromes a and c1. J Biochem. 1962 Mar;51:204–215. doi: 10.1093/oxfordjournals.jbchem.a127522. [DOI] [PubMed] [Google Scholar]
- Ozawa T., Okumura M., Yagi K. Purification of cytochrome oxidase by using Sepharose-bound cytochrome c. Biochem Biophys Res Commun. 1975 Aug 4;65(3):1102–1107. doi: 10.1016/s0006-291x(75)80499-2. [DOI] [PubMed] [Google Scholar]
- ROUSER G., KRITCHEVSKY G., GALLI C., HELLER D. DETERMINATION OF POLAR LIPIDS: QUANTITATIVE COLUMN AND THIN-LAYER CHROMATOGRAPHY. J Am Oil Chem Soc. 1965 Mar;42:215–227. doi: 10.1007/BF02541135. [DOI] [PubMed] [Google Scholar]
- Skipski V. P., Peterson R. F., Barclay M. Quantitative analysis of phospholipids by thin-layer chromatography. Biochem J. 1964 Feb;90(2):374–378. doi: 10.1042/bj0900374. [DOI] [PMC free article] [PubMed] [Google Scholar]
- WAGNER H., HOERHAMMER L., WOLFF P. [Thin layer chromatography of phosphatides and glycolipids]. Biochem Z. 1961;334:175–184. [PubMed] [Google Scholar]
- WILLIAMS J. N., Jr A METHOD FOR THE SIMULTANEOUS QUANTITATIVE ESTIMATION OF CYTOCHROMES A, B, C1, AND C IN MITOCHONDRIA. Arch Biochem Biophys. 1964 Sep;107:537–543. doi: 10.1016/0003-9861(64)90313-3. [DOI] [PubMed] [Google Scholar]
- YAGI K., OZAWA T. CRYSTALLIZATION OF A HOLO-ENZYME-PRODUCT COMPLEX OF D-AMINOACID OXIDASE. J Biochem. 1963 Aug;54:204–205. [PubMed] [Google Scholar]
- YONETANI T. Studies on cytochrome oxidase. III. Improved preparation and some properties. J Biol Chem. 1961 Jun;236:1680–1688. [PubMed] [Google Scholar]