New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase

Abstract

New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase (EC 1.14.18.1) have been developed. The tyrosine hydroxylase assay uses ~-[carboxy-'~C]tyrosine as the substrate. I4CO2 is released from the products of the hydroxylation and further metabolism of ~-[carboxy-'~C]tyrosine by incubation with ferricyanide, and measured radiometrically. D-Dopa is a preferable cofactor to L-dopa for the assay. Dopa oxidase activity is measured spectrophotometrically. Dopaquinone, produced on the oxidation of L-dopa, reacts with Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone) to form a pink pigment with an absorbance maximum at 505 nm. Details of the optimisation of conditions for the assays and their specificities for the two enzyme activities are described.It is well established that tyrosinase from lower organisms catalyses the first two steps of melanin synthesis: the hydroxylation of tyrosine to dopa, and the oxidation of dopa to dopaquinone [l]. The classical view is that this is also true for the mammalian enzyme (21; it has been suggested that mammalian tyrosinase may catalyse a third step in melanin synthesis: the oxidation of 5,6-dihydroxyindole [3, 41. There have, however, been reports of the separation of melanogenic tyrosine hydroxylase and dopa oxidase activities on purification of the mammalian enzyme [5, 61. To determine conclusively whether the tyrosine hydroxylase and dopa oxidase activities of tyrosinase are inherent in one protein molecule, satisfactory assays for these two activities are required. There are many problems associated with the existing assays so the aim of this work was to develop improved assays for both activities. Tyrosine hydroxylase assayThe tyrosine hydroxylase activity of tyrosinase requires Ldopa, the product of the reaction, as a cofactor. In the absence of cofactor, no activity [7] or very low activity [8] has been reported. In general, a cofactor is required at a low concentration relative to the substrate, but a relatively high concentration of L-dopa is required [9]. If tyrosinase does catalyse the first two steps of melanin synthesis, addition of L-dopa as a cofactor also represents addition of a competitive substrate. Oxidation of L-dopa by dopa oxidase activity will deplete the concentration of cofactor. The requirement for L-dopa as a cofactor thus makes the tyrosine hydroxylase activity of tyrosinase difficult to assay.The Pomerantz method [lo] is used most frequently to assay the tyrosine hydroxylase activity of tyrosinase. It uses ~-[3,5-~H]tyrosine as the substrate and measures the 3 H 2 0Correspondence to A.

Functional Conversion of Hemocyanin to Phenoloxidase by Horseshoe Crab Antimicrobial Peptides

“…An extinction coefficient of 4230 M Ϫ1 cm Ϫ1 at A 528 nm was used for the prolyl adduct of 4-methylcatechol (31). We assayed the activity for substrate specificity using the method of Winder and Harris (32), with the exception that the substrate solutions contained 5 mM substrate and 10 mM 3-methyl-2-benzothiazolinone hydrazone-HCl in the assay buffer. The activity was monitored at A 505 nm .…”

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.…”

“…The monophenolase and diphenolase activities of mushroom tyrosinase was determined spectrophotometrically by using MBTH (a potent nucleophile through its amino group), which attacks the enzyme-generated o-quinones (21). This assay method is highly sensitive, reliable, and precise (9,10,(15)(16)(17)(18)(19)(20)(21).…”

“…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.…”

Umbelliferone and Esculetin: Inhibitors or Substrates for Polyphenol Oxidases?

“…The coupling reaction between quinones and MBTH is often used for measuring PPO activity. 22,23) Also the quinone produced from esculetin oxidation reacted with MBTH leading to a better appearance of the spot. In Fig.…”