Purification and characterization of highly purified cytochrome b from complex III of baker's yeast.
A simple, high-yield purification procedure for cytochrome b from yeast Complex III has been developed. This procedure involves solubilization using chemical modification of the lysine residues with 3,4,5,6-tetrahydrophthalic anhydride followed by hydroxyapatite column chromatography. This purified cytochrome b has a heme content of 37.0 nmol cytochrome b/mg and a molecular weight on SDS gels of 25000-26000. Amino acid analysis indicates high hydrophobicity and is very comparable to the composition deduced from the gene sequence (Nobrega, F.G. and Tzagoloff, A. (1980) J. Biol. Chem. 255, 9828-9837). The latter data indicate a molecular weight of 42000 for the polypeptide; our heme analyses thus imply the presence of two hemes per polypeptide chain. Optical and MCD spectra are typical of a low-spin b-type cytochrome. MCD-potentiometric titration indicates a one-electron carrier with a single midpoint potential of -44 mV at pH 7.4 and 25 degrees C. The EPR spectrum of isolated cytochrome b has only one gz signal at 3.70, indicating that the 'strained' heme structure (Carter, K., T'sai, A. and Palmer, G. (1981) FEBS Lett. 132, 243--246) is still maintained. No indication of antimycin binding was demonstrated either by the direct-fluorescence method or binding-precipitation method although stoichiometric binding to the parent Complex III was readily demonstrated.
D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000302:isolation & purification* / Q000378:metabolism; D004227:Dithionite; D017886:Durapatite; D014450:Electron Transport Complex III; D006882:Hydroxyapatites; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D011808:Quinone Reductases / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000596:Amino Acids; D003573:Cytochrome b Group; D006882:Hydroxyapatites; D009097:Multienzyme Complexes; D004227:Dithionite; D017886:Durapatite; D009247:NADH, NADPH Oxidoreductases; D011808:Quinone Reductases; D014450:Electron Transport Complex III