High yield purification of a four subunit caa3-type cytochrome oxidase from the thermophilic bacterium Bacillus PS3 using fast protein liquid chromatography.
The thermophilic bacterium, Bacillus PS3, was grown in a vigorously aerated nutrient broth at 65 degrees C with 100 mM glutamic acid serving as a supplemental carbon and nitrogen source. These growth conditions resulted in membranes highly enriched in cytochrome c oxidase (COX) [23.32 +/- 4.32 nmol heme a/g of cells (n = 5)], which is nearly a threefold higher concentration of COX (heme caa3-type) than previously reported for this organism. A new high-yield purification of COX was performed by extracting the bacterial membranes with Triton X-100 (7 mg/mg protein), followed by ion-exchange fast liquid protein chromatography using a QAE (trimethyl ammonium) resin with subsequent hydroxyapatite chromatography and ammonium sulfate fractionation. This purification regime resulted in a 16% yield of cytochrome c oxidase with 20 mg of pure caa3-type COX (13 nmol heme a/mg protein) isolated from 100 g of cells. SDS-PAGE showed that the isolated enzyme had four subunits with apparent Mr of 68, 38, 23, and 13 kDa. In addition, a new 34-kDa peptide was also detected in this preparation, which may represent the ORF1 gene product for this organism. Subunit II (Mr = 38 kDa) of the isolated enzyme was shown to contain covalently bound heme c by using both heme-staining of SDS-PAGE and immunoreactivity with an anti-cytochrome c antibody. The purified enzyme also exhibited high electron transfer activity (340s-1) when assayed at pH 6.5 in the presence of the nonionic detergent, beta-dodecyl maltoside.
Department of Biochemistry, School of Medicine, Wright State University, Dayton, Ohio 45435, USA.
R A
RA
Kirken
A J
AJ
Lincoln
P S
PS
Fink
L J
LJ
Prochaska
D001407:Bacillus / Q000166:cytology / Q000201:enzymology* / Q000254:growth & development; D002455:Cell Division; D002462:Cell Membrane / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006418:Heme / Q000737:chemistry; D007218:Industrial Microbiology / Q000379:methods; D010084:Oxidation-Reduction; D013054:Spectrophotometry, Atomic
D003574:Cytochrome c Group; D006418:Heme; D003576:Electron Transport Complex IV