Journal
ANALYTICAL BIOCHEMISTRY
Volume 312, Issue 1, Pages 23-32Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0003-2697(02)00408-6
Keywords
tyrosinase; direct assay; monooxygenase; cresolase; oxidase; catecholase; diazo derivatives; phenol; catechol; p-coumaric acid; caffeic acid; kinetic parameters
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Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (NIT). 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 lambda(max)(s) with the least interference of the intermediates' or products' absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of NIT 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. (C) 2003 Elsevier Science (USA). All rights reserved.
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