Radiochemical high-performance liquid chromatographic assay for the determination of catechol O-methyltransferase activity towards various substrates

A new chromatographic catechol O-methyltransferase (COMT) assay based on S- adenosyl-L-[methyl-C-14]methionine and on-line radioactivity detection was developed. With minor modifications in the mobile phase composition the met hylation velocities for 30 structurally diverse compounds including simple catechols, neurotransmitters, catecholestrogens and catecholic drugs could be measured using human and rat recombinant soluble COMT. The enzymes showe d very similar substrate selectivities. The radiochemical method was valida ted using 3,4-dihydroxybenzoic acid as a model substrate and it was shown t hat accurate and reproducible methylation velocity values could be achieved for both of the catecholic hydroxyls. The method proved to be suited for d etermining the enzyme kinetic parameters and can probably be further used f or gathering enzyme kinetic data on differentially substituted catechols in order to construct proper structure-activity relationships for COMT. (C) 1 999 Elsevier Science B.V. All rights reserved.