New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase.
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 L-[carboxy-14C]tyrosine as the substrate, 14CO2 is released from the products of the hydroxylation and further metabolism of L-[carboxy-14C]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.
Sir William Dunn School of Pathology, University of Oxford, England.
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Winder
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Harris
D000818:Animals; D002250:Carbon Radioisotopes; D002460:Cell Line; D004295:Dihydroxyphenylalanine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D013816:Thermodynamics; D014443:Tyrosine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D002250:Carbon Radioisotopes; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase; D014442:Monophenol Monooxygenase