Pseudomonas cytochrome C-551 peroxidase. A purification procedure and study of CO-binding kinetics

Abstract

A procedure is described for the purification of cytochrome c peroxidase from P. aeruginosa involving extraction by sonication, followed by acid precipitation and chromatography on only 2 types of gel. The final preparation had a purity ratio A407/A280 of 4.2 and was found to be essentially pure by isoelectric focusing. The enzyme was unstable during degassing under vacuum except in the presence of detergent. The kinetics of CO binding to dithionite-reduced peroxidase were studied with stopped-flow and flash-photolysis techniques; the results obtained between pH 5-7 suggest the existence of 2 forms of dithionite-reduced enzyme in slow equilibrium.