A new spectrophotometric assay for dopachrome tautomerase.
The existence of a new enzyme involved in mammalian melanogenesis has been recently reported. The names dopachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nm on the basis of its ability to catalyze dopachrome decoloration. This method presents two major problems, derived from the instability of the substrate (dopachrome): (1) dopachrome must be prepared immediately before use, and (2) the rate of dopachrome decoloration in the absence of the enzyme is not negligible, and, furthermore, is enhanced by non-enzymatic agents. In order to overcome these problems, we present a new procedure that combines: (1) a quantitative, fast and easy way to prepare dopachrome from L-dopa by sodium periodate oxidation; (2) a spectrophotometric method in the UV region, at 308 nm, based on following the absorbance increase due to the enzyme-specific tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid as opposed to the absorbance decrease due to the spontaneous decarboxylative transformation of dopachrome into 5,6-dihydroxyindole. The advantages of these methods as compared to the previously used procedures are discussed.
Aroca
P
P
Departamento de Bioquimica y Biología Molecular, Facultad de Medicina, Universidad de Murcia, Spain.
Solano
F
F
García-Borrón
J C
JC
Lozano
J A
JA
D000818:Animals; D004156:Catechol Oxidase / Q000032:analysis*; D045563:Indolequinones; D007211:Indoles / Q000138:chemical synthesis; D019746:Intramolecular Oxidoreductases; D008297:Male; D008546:Melanoma, Experimental / Q000201:enzymology*; D051379:Mice; D011809:Quinones / Q000138:chemical synthesis; D013056:Spectrophotometry, Ultraviolet
D045563:Indolequinones; D007211:Indoles; D011809:Quinones; C001123:dopachrome; D004156:Catechol Oxidase; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase