Determination of catechol O-methyltransferase activity in relation to melanin metabolism using high-performance liquid chromatography with fluorimetric detection

A new sensitive method for the determination of catechol O-methyltransferase activity has been developed. The method is based on the O-methylation of the indolic intermediates of melanin metabolism. The substrate, 5,6-dihydroxyindole-2-carboxylic acid, is converted by the enzyme to two O-methylated products, which can be separated by high-performance liquid chromatography and measured with fluorimetric detection. The physiological presence of both substrate and products could be detected in crude melanoma cell extracts. The limit of sensitivity for detection of the O-methylated products is less than 0.5 pmol per injection. The method was compared with an earlier described HPLC method which makes use of uv detection of O-methylated products of 3,4-dihydroxybenzoic acid. The described method will be used to study the importance of catechol O-methyltransferase as a protective enzyme in (malignant) melanocytes