An Indirect Free Radical-based Assay for the Enzyme Cellobiose:quinone Oxidoreductase

A sensitive and quantitative assay for the detection of cellobiose:quinone oxidoreductase (CBQase) is described. The assay is based on the ability of CBQase to reduce the cation radicals formed by the laccase-mediated oxidation of chlorpromazine (CPZ). Formation of the CPZ radical cation is readily followed at 530 nm, and the net rate of its formation is decreased in proportion to the amount of CBQase activity present. Advantages of this assay are its increased sensitivity due to the high extinction coefficient of the CPZ radical, the high solubility of the substrate in water, and the assay's ability to detect reductive activity in the presence of laccase and other oxidative enzymes. The assay also detects other enzymes, such as glucose oxidase, which have CPZ radical-reducing activity.