A Continuous Spectrophotometric Method for the Determination of Monophenolase Activity of Tyrosinase Using 3-Methyl-2-benzothiazolinone Hydrazone

Unification for the Expression of the Monophenolase and Diphenolase Activities of Tyrosinase

“…The acidic character of MBTH required the use of 50 mM buffer in the assay medium. To dissolve the MBTH-quinone adducts, 2% (v/v) N,N -dimethylformamide (DMF) was added to the assay medium (9,10,(15)(16)(17)(18)(19)(20)(21). Milli-Q system (Millipore Corp.) ultrapure water was used throughout this research.…”

“…This assay method is highly sensitive, reliable, and precise (9,10,(15)(16)(17)(18)(19)(20)(21). MBTH traps the enzyme-generated o-quinones to render a soluble MBTH-quinone adduct with high molar absorptivity.…”

“…At pH 6.5 these adducts were soluble and evolved showing an isosbestic point (10,(15)(16)(17)(18)(19)(20)(21). The stability of the MBTHquinone adducts and the rapid assays provide a reliable method for determining the monophenolase and diphenolase activities of tyrosinase from several sources (9,10,(15)(16)(17)(18)(19)(20)(21).…”

“…c) Diphenolase activity can be measured easily with substrates. These give rise to o-quinones, which are stabilised by nucleophilic addition and redox reaction (9,10,16,18,26). However, when substrates like catechol and 4-methylcatechol are used, the high degree of o-quinones instability hinders their measurement, since o-quinones react with water, any nucleophile present, its corresponding o-diphenol, and/or themselves (9,10,16,18,26).…”

“…For example, in the case of the physiological substrates of PPO in mammals, L-tyrosine/L-dopa, the amino group of the side chain is involved in an intramolecular Michael 1,4-addition into the benzene ring to generate a colourless diphenolic adduct, which is oxidised to a coloured quinonic product (5,6). A similar process occurs after the intermolecular addition originated by a powerful nucleophile incorporated in the assay medium, such as 3-methyl-2-benzothiazolinone hydrazone or MBTH (9,10). Obviously, this process does not occur in the presence of strong coupled reductants, such as ascorbic acid, due to the o-quinone reduction to its corresponding o-diphenol.…”