A stopped spectrophotometric assay for the dopa oxidase activity of tyrosinase

A stopped spectrophotometric assay for the dopa oxidase activity of tyrosinase has been developed to enable large numbers of samples to be screened very rapidly. The assay measures the pink pigment formed by the reaction of Besthorn's hydrazone (3-methyl-2-benzothiazoninone hydrazone, or MBTH) with dopaquinone, the product of oxidation of L-dopa by tyrosinase. Addition of perchloric acid stops the reaction and precipitates protein, enabling turbid as well as non-turbid samples to be assayed. Stability of the pink product is enhanced in acid solution and the pigment has a sharp absorbance maximum at 505 nm such that it is easily measured spectrophotometrically. Using the stopped assay, tyrosinase is detectable only in mammalian cell lines expected to express the enzyme, and the specificity of the assay has also been confirmed using tyrosinase inhibitors. The stopped MBTH assay is approx. 15-times more sensitive than the widely used dopachrome assay and can reliably detect the formation of as little as 350 pmol of product.