Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1987 Mar 1;242(2):417–423. doi: 10.1042/bj2420417

A new procedure for the purification of monodisperse highly active cytochrome c oxidase from bovine heart.

Y Li, A Naqui, T G Frey, B Chance
PMCID: PMC1147721  PMID: 3036090

Abstract

A simple and rapid method for the isolation of a large quantity of cytochrome c oxidase from bovine heart mitochondria was developed, based on selective solubilization of mitochondrial protein with first Triton and then lauryl maltoside. Gel filtration shows that the lauryl maltoside-solubilized oxidase preparation is in a hydrodynamically homogeneous state with a Stokes radius of 7.5 +/- 0.2 nm. It contains 8.0 mumol of haem (with an a/a3 ratio of 1)/g of protein. The catalytic constant (maximum turnover number) with respect to cytochrome c approaches 600 S-1. After further purification of the solubilized enzyme on a sucrose-gradient centrifugation, the purified enzyme has a haem content of 10.3 mumol/g of protein and eight major polypeptide bands shown on SDS/polyacrylamide-gel electrophoresis.

Full text

PDF
417

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Azzi A., Bill K., Broger C. Affinity chromatography purification of cytochrome c binding enzymes. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2447–2450. doi: 10.1073/pnas.79.8.2447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. CONRAD H., SMITH L. A study of the kinetics of the oxidation of cytochrome c by cytochrome c oxidase. Arch Biochem Biophys. 1956 Aug;63(2):403–413. doi: 10.1016/0003-9861(56)90055-8. [DOI] [PubMed] [Google Scholar]
  3. Capaldi R. A., Malatesta F., Darley-Usmar V. M. Structure of cytochrome c oxidase. Biochim Biophys Acta. 1983 Jul 15;726(2):135–148. doi: 10.1016/0304-4173(83)90003-4. [DOI] [PubMed] [Google Scholar]
  4. Einarsdóttir O., Caughey W. S. Bovine heart cytochrome c oxidase preparations contain high affinity binding sites for magnesium as well as for zinc, copper, and heme iron. Biochem Biophys Res Commun. 1985 Jun 28;129(3):840–847. doi: 10.1016/0006-291x(85)91968-0. [DOI] [PubMed] [Google Scholar]
  5. Ferguson-Miller S., Brautigan D. L., Margoliash E. Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase. J Biol Chem. 1976 Feb 25;251(4):1104–1115. [PubMed] [Google Scholar]
  6. Frey T. G., Chan S. H., Schatz G. Structure and orientation of cytochrome c oxidase in crystalline membranes. Studies by electron microscopy and by labeling with subunit-specific antibodies. J Biol Chem. 1978 Jun 25;253(12):4389–4395. [PubMed] [Google Scholar]
  7. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  8. Kadenbach B., Jarausch J., Hartmann R., Merle P. Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure. Anal Biochem. 1983 Mar;129(2):517–521. doi: 10.1016/0003-2697(83)90586-9. [DOI] [PubMed] [Google Scholar]
  9. Kuboyama M., Yong F. C., King T. E. Studies on cytochrome oxidase. 8. Preparation and some properties of cardiac cytochrome oxidase. J Biol Chem. 1972 Oct 25;247(20):6375–6383. [PubMed] [Google Scholar]
  10. Kuhn-Nentwig L., Kadenbach B. Immunological identification of four different polypeptides in 'subunit VII' of mammalian cytochrome c oxidase. FEBS Lett. 1984 Jul 9;172(2):189–192. doi: 10.1016/0014-5793(84)81123-0. [DOI] [PubMed] [Google Scholar]
  11. Kumar C., Naqui A., Chance B. The identity of pulsed cytochrome oxidase. J Biol Chem. 1984 Feb 25;259(4):2073–2076. [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. MINNAERT K. The kinetics of cytochrome c oxidase. I. The system: cytochrome c-cytochrome oxidase-oxygen. Biochim Biophys Acta. 1961 Jun 10;50:23–34. doi: 10.1016/0006-3002(61)91055-1. [DOI] [PubMed] [Google Scholar]
  14. Naqui A., Chance B., Cadenas E. Reactive oxygen intermediates in biochemistry. Annu Rev Biochem. 1986;55:137–166. doi: 10.1146/annurev.bi.55.070186.001033. [DOI] [PubMed] [Google Scholar]
  15. Naqui A., Kumar C., Ching Y. C., Powers L., Chance B. Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding. Biochemistry. 1984 Dec 4;23(25):6222–6227. doi: 10.1021/bi00320a051. [DOI] [PubMed] [Google Scholar]
  16. Nałecz K. A., Bolli R., Azzi A. Preparation of monomeric cytochrome C oxidase: its kinetics differ from those of the dimeric enzyme. Biochem Biophys Res Commun. 1983 Jul 29;114(2):822–828. doi: 10.1016/0006-291x(83)90855-0. [DOI] [PubMed] [Google Scholar]
  17. Nozaki Y., Schechter N. M., Reynolds J. A., Tanford C. Use of gel chromatography for the determination of the Stokes radii of proteins in the presence and absence of detergents. A reexamination. Biochemistry. 1976 Aug 24;15(17):3884–3890. doi: 10.1021/bi00662a036. [DOI] [PubMed] [Google Scholar]
  18. Sinjorgo K. M., Steinebach O. M., Dekker H. L., Muijsers A. O. The effects of pH and ionic strength on cytochrome c oxidase steady-state kinetics reveal a catalytic and a non-catalytic interaction domain for cytochrome c. Biochim Biophys Acta. 1986 Jun 10;850(1):108–115. doi: 10.1016/0005-2728(86)90014-9. [DOI] [PubMed] [Google Scholar]
  19. Suarez M. D., Revzin A., Narlock R., Kempner E. S., Thompson D. A., Ferguson-Miller S. The functional and physical form of mammalian cytochrome c oxidase determined by gel filtration, radiation inactivation, and sedimentation equilibrium analysis. J Biol Chem. 1984 Nov 25;259(22):13791–13799. [PubMed] [Google Scholar]
  20. Sun F. F., Prezbindowski K. S., Crane F. L., Jacobs E. E. Physical state of cytochrome oxidase. Relationship between membrane formation and ionic strength. Biochim Biophys Acta. 1968 May 28;153(4):804–818. doi: 10.1016/0005-2728(68)90008-x. [DOI] [PubMed] [Google Scholar]
  21. Thompson D. A., Ferguson-Miller S. Lipid and subunit III depleted cytochrome c oxidase purified by horse cytochrome c affinity chromatography in lauryl maltoside. Biochemistry. 1983 Jun 21;22(13):3178–3187. doi: 10.1021/bi00282a022. [DOI] [PubMed] [Google Scholar]
  22. Vanneste W. H. The stoichiometry and absorption spectra of components a and a-3 in cytochrome c oxidase. Biochemistry. 1966 Mar;5(3):838–848. doi: 10.1021/bi00867a005. [DOI] [PubMed] [Google Scholar]
  23. Vanneste W. H., Ysebaert-Vanneste M., Mason H. S. The decline of molecular activity of cytochrome oxidase during purification. J Biol Chem. 1974 Dec 10;249(23):7390–7401. [PubMed] [Google Scholar]
  24. Weiss H., Kolb H. J. Isolation of mitochondrial succinate: ubiquinone reductase, cytochrome c reductase and cytochrome c oxidase from Neurospora crassa using nonionic detergent. Eur J Biochem. 1979 Aug 15;99(1):139–149. doi: 10.1111/j.1432-1033.1979.tb13240.x. [DOI] [PubMed] [Google Scholar]
  25. YONETANI T. Studies on cytochrome oxidase. II. Steady state properties. J Biol Chem. 1960 Nov;235:3138–3143. [PubMed] [Google Scholar]
  26. YONETANI T. Studies on cytochrome oxidase. III. Improved preparation and some properties. J Biol Chem. 1961 Jun;236:1680–1688. [PubMed] [Google Scholar]
  27. Yu C., Yu L., King T. E. Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c. J Biol Chem. 1975 Feb 25;250(4):1383–1392. [PubMed] [Google Scholar]
  28. van Buuren K. J., Nicholis P., van Gelder B. F. Biochemical and biophysical studies on cytochrome aa 3 . VI. Reaction of cyanide with oxidized and reduced enzyme. Biochim Biophys Acta. 1972 Feb 28;256(2):258–276. doi: 10.1016/0005-2728(72)90057-6. [DOI] [PubMed] [Google Scholar]
  29. van Gelder B. F. On cytochrome c oxidase. I. The extinction coefficients of cytochrome a and cytochrome a3. Biochim Biophys Acta. 1966 Apr 12;118(1):36–46. doi: 10.1016/s0926-6593(66)80142-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES