A new procedure for the purification of monodisperse highly active cytochrome c oxidase from bovine heart.
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.
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003576:Electron Transport Complex IV / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D007700:Kinetics; D008722:Methods; D009206:Myocardium / Q000201:enzymology*; D011092:Polyethylene Glycols; D012995:Solubility; D013053:Spectrophotometry
D005960:Glucosides; D011092:Polyethylene Glycols; C040358:dodecyl maltoside; D003576:Electron Transport Complex IV