A purification procedure for the soluble cytochrome oxidase and some other respiratory proteins from Pseudomonas aeruginosa.
The production of the soluble cytochrome oxidase/nitrite reductase in the bacterium Pseudomonas aeruginosa is favoured by anaerobic conditions and the presence of KNO3(20g/l) in the culture medium. Of three methods commonly used for the disruption of bacterial suspensions (ultrasonication, liquid-shear homogenization and glass-bead grinding), sonication proved the most efficient in releasing the Pseudomonas cytochrome oxidase. A polarographic assay of Pseudomonas cytochrome oxidase activity with sodium ascorbate as substrate and NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride as electron mediator is described. A purification procedure was developed which can be used on the small scale (40-litre cultures) or the large scale (400-litre cultures) and provides high yields of three respiratory-chain proteins, Pseudomonas cytochrome oxidase, cytochrome c551 and azurin, in a pure state. A typical preparation of 250g of Ps.aeruginosa cell paste yielded 180mg of Pseudomonas cytochrome oxidase, 81 mg of Pseudomonas cytochrome c551 and 275mg of Pseudomonas azurin.
D001400:Azurin / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003574:Cytochrome c Group / Q000302:isolation & purification*; D003576:Electron Transport Complex IV / Q000302:isolation & purification*; D009566:Nitrates; D009584:Nitrogen; D011550:Pseudomonas aeruginosa / Q000032:analysis* / Q000201:enzymology; D013010:Sonication; D013696:Temperature
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D009566:Nitrates; D001400:Azurin; D003576:Electron Transport Complex IV; D009584:Nitrogen