Abstract
This chapter describes the assay, purification, and properties of phenoxazinone synthase. Phenoxazinone synthase is possibly involved in the synthesis of actinocin (2-amino-4,6-dimethyl-3-phenoxazinone-1,9-dicarboxylic acid), the chromophore of actinomycin. The enzyme catalyzes the oxidative condensation of two moles of an o -aminophenol to form one mole of phenoxazinone. The enzyme assay is based on the increase in optical density in the 430–455 m μ region due to the formation of the phenoxazinone pigment. Incubations are performed in a Beckman DU spectrophotometer in which the cuvette chamber is maintained at 37°. The reaction mixture contains two micromoles of substrate, 250 micromoles of sodium acetate buffer, pH 5.3, enzyme, and water to a total volume of 3 ml. Because of the availability of 3-hydroxyanthranilic acid, it is used as the substrate for routine studies. With both the sonic extract and the purified enzyme preparation, a significant lag period is observed in phenoxazinone formation during the initial two minutes of incubation. After this lag phase, the rate of reaction is linear; enzyme activity units are based on the optical density change during the 5- to 10-minute period. o -, m -, and p -anisidine; o -, m -, and p -aminobenzoic acid; m -aminophenol; and o -aminobenzenethiol are good to excellent inhibitors of enzyme activity.
Published Version
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