A Triton X-100-hydroxyapatite procedure for a rapid purification of bovine heart cytochrome-c oxidase. Characterization of the cytochrome-c oxidase/Triton X-100/phospholipid mixed micelles by laser light scattering.
Cytochrome-c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1) has been isolated from bovine heart mitochondria by the combined use of the non-ionic detergent Triton X-100 as solubilizing agent and hydroxyapatite chromatography as the most important step in the purification of this membrane protein. This method is fast and very reproducible. The enzymic complex, purified in the form of protein/detergent/phospholipid mixed micelles, contains 9.7 mumol heme a per mg protein, and has a high molecular activity (500 mol cytochrome c per s per mol enzyme). These mixed micelles have been studied by laser light scattering, which has shown that the average molecular weight of the micelles is 540 +/- 80 kDa. This implies that cytochrome-c oxidase is purified in the form of a dimer. The average quadratic radius of gyration of the micelles is 40 +/- 10 nm, corresponding in our case to an approximately spherical shape.
Valpuesta
J M
JM
Department of Biochemistry, Faculty of Science, University of the Basque Country, Bilbao, Spain.
Barbón
P G
PG
D000818:Animals; D002417:Cattle; D017886:Durapatite; D003576:Electron Transport Complex IV / Q000302:isolation & purification*; D006882:Hydroxyapatites; D007834:Lasers; D008722:Methods; D008823:Micelles; D009206:Myocardium / Q000201:enzymology*; D017830:Octoxynol; D010743:Phospholipids; D011092:Polyethylene Glycols; D012542:Scattering, Radiation
D006882:Hydroxyapatites; D008823:Micelles; D010743:Phospholipids; D011092:Polyethylene Glycols; D017830:Octoxynol; D017886:Durapatite; D003576:Electron Transport Complex IV