Detergent-solubilized monomeric and dimeric cytochrome bc1 isolated from bovine heart.
Mitochondrial cytochrome bc1 complex, isolated from frozen bovine heart, was solubilized with five different "non-denaturing" detergents: dodecyl maltoside, octaethylene glycol monododecyl ether (C12E8), Triton X-100, Tween 20, and sodium cholate. The hydrodynamic properties of the solubilized complex III's were then investigated by sedimentation analysis. Complex III exists as a stable and monodisperse dimer when it is solubilized in low ionic strength buffer with a low concentration of any of the five detergents. At pH 7.8, 20 degrees C, the protein sediments as a homogeneous species with an s(obs) of about 14 S. The protein molecular weight of the 14S particle, after correction for bound detergent, is 465,000 +/- 30,000 as measured by sedimentation equilibrium analysis. The aggregation state and/or homogeneity of cytochrome bc1 is strongly dependent upon the concentration of the solubilizing detergent and ionic strength. The enzyme becomes a homogeneous, monomeric complex with a protein molecular weight of 235,000 +/- 20,000 and an s(obs) of 10-10.5 S after it is solubilized in high concentrations of Tween 20 (more than 5 mg/mg of protein) and sodium chloride (more than 0.5 M). However, a heterogeneous mixture of subcomplexes is produced upon solubilization of the complex with high concentrations of the other detergents and 0.5 M NaCl. Monomerization of cytochrome bc1 by Tween 20 and 0.5 M NaCl has no effect on either the spectral properties, the subunit composition, or the enzymatic activity and is reversible since the dimeric 14S particle is regenerated upon removal of the high concentration of salt.(ABSTRACT TRUNCATED AT 250 WORDS)
Musatov
A
A
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Robinson
N C
NC
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002417:Cattle; D019826:Cholic Acid; D002793:Cholic Acids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000737:chemistry*; D014450:Electron Transport Complex III / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005629:Frozen Sections; D005960:Glucosides / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D017830:Octoxynol / Q000737:chemistry; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers; D011136:Polysorbates / Q000737:chemistry; D012995:Solubility; D014021:Tissue Preservation; D014461:Ultracentrifugation
D002793:Cholic Acids; D003902:Detergents; D005960:Glucosides; D011108:Polymers; D011136:Polysorbates; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D000968:Antimycin A; C040358:dodecyl maltoside; D017830:Octoxynol; D014450:Electron Transport Complex III; D019826:Cholic Acid