36] Hexopyranoside: Cytochrome c oxidoreductase from Agrobacterium

Abstract

This chapter describes the assay method, purification procedure, and properties of cytochrome c oxidoreductase from agrobacterium . Enzyme activity can be followed either manometrically as oxygen consumption, using 5-methyl phenazinium methylsulfate (PMS) as intermediate electron carrier, or spectrophotometrically, as decrease in optical density at 600 nm using 2,6-dichlorophenolindophenol (DIP) as terminal acceptor. The latter assay is described in the chapter. The reaction can be followed in any spectrophotometer at 600 nm. A cuvette of 1 cm light path receives 2.58 ml of DIP solution, 0.05–0.1 ml of enzyme, and water to a final volume of 2.7 ml. The amount of enzyme used depends on the possibilities of chart speed and scale expansion of the recorder. The enzyme is inducible and is known to occur only in most strains of Agrobacterium tumefaciens and A. radiobacter . The enzyme has also been called D-aldohexopyranoside dehydrogenase or D-glucoside 3-dehydrogenase. The enzyme is purified starting from a 150 1iter culture, but the amounts can be scaled down if required. The purification steps are: preparation of particle-free extract, removal of nucleic acids, ammonium sulfate fractionation, DEAE-cellulose chromatography, column electrophoresis on agarose, and gel filtration. The enzyme becomes increasingly labile upon purification, perhaps owing to gradual loss of FAD. The enzyme is stable for 2 weeks under nitrogen at –15%. The best pH range is 6.5–7.4. K m values measured with enzyme preparation are cellobiose, 0.2 m M ; lactobionate, 0.21 m M ; maltobionate, 0.36 m M ; lactose, 1.7 m M ; maltose, 2.8 m M ; glucose, 2.9 m M ; sucrose 4.1 m M , and galacrose, 25 m M .