Tyrosinase Assay

ANALYTICAL BIOCHEMISTRY

Analytical Biochemistry 312 (2003) 23–32 www.elsevier.com/locate/yabio

Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates

Kamahldin Haghbeena,* and Eng Wue Tanb

a

National Research Center for Genetic Engineering and Biotechnology, P.O. Box 14155-6343, Tehran, Iran b Department of Chemistry, Otago University, P.O. Box 56, Dunedin, New Zealand Received 20 May 2002

Abstract Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their kmax (s) with the least interference of the intermediatesÕ or productsÕ absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver–Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented. Ó 2003 Elsevier Science (USA). All rights reserved.

Keywords: Tyrosinase; Direct assay; Monooxygenase; Cresolase; Oxidase; Catecholase; Diazo derivatives; Phenol; Catechol; p-Coumaric acid; Caffeic acid; Kinetic parameters...