Journal of Chromatography B: Biomedical Sciences and Applications
Radiochemical high-performance liquid chromatographic assay for the determination of catechol O-methyltransferase activity towards various substrates
Introduction
Catechol O-methyltransferase (EC 2.1.1.6, COMT) catalyses the transfer of the methyl group of S-adenosyl-l-methionine (AdoMet) to one of the catecholic hydroxyls of various endogenous and exogenous catechols including catecholamines, catecholestrogens and many drugs [1]. COMT appears in two forms, of which the soluble form (S-COMT) predominates over the membrane-bound form (MB-COMT) in most tissues. During the last decade, there has been a remarkable interest in COMT both as a possible drug target and regarding its role in drug metabolism and interactions. Recently, two COMT inhibitors, entacapone and tolcapone, have been introduced in order to enhance the l-dopa/dopa decarboxylase inhibitor therapy in the treatment of Parkinson’s disease [2], [3]
Although the structure-activity relationships of COMT inhibition has been determined for S-COMT isolated from rat liver [4] and brain [5], the substrate selectivity of COMT has not been studied extensively. Information about the structure-activity relationships of COMT, and other catechol conjugation enzymes could, however, greatly benefit drug design in evaluating the metabolism and metabolic interactions of catecholic drugs and drug candidates. The two forms of human and rat COMT have been successfully cloned and expressed [6], [7], [8], [9], [10], [11], [12], and the recombinant proteins are very useful in gathering biological data for structure-activity analysis. However, analytical methods suitable for measuring the in vitro methylation velocity of structurally diverse compounds are needed. Catechols provide two methylation sites and when mechanisms underlying the regioselectivity are investigated, methods also capable of separating the possible regioisomeric products must be available. Earlier high-performance liquid chromatographic methods with different detection systems including UV- [13], electrochemical [14], [15], fluorimetric [16], [17], [18] and radiochemical [19] detectors, have been developed to measure COMT activity using a specific substrate or to determine the methylation velocity for a small number of substrates. The use of 14C- or 3H-AdoMet in the reaction is a method of choice when the methylation velocities of many structurally diverse compounds have to be determined. However, methods commonly used in the separation of radioactively labelled AdoMet and the products, like liquid–liquid extraction and thin-layer chromatography [20], [21], [22], are time-consuming and do not allow the separation of the regioisomeric methylated products from each other.
The present paper describes a simple COMT assay based on HPLC with on-line radioactivity detection. The developed method was shown to be applicable for the screening of 30 structurally diverse catechols for their methylation velocity catalysed by recombinant human and rat soluble COMT. The method was validated using 3,4-dihydroxybenzoic acid as a model substrate and further shown to be suitable for enzyme kinetic measurements.
Section snippets
Chemicals
The catechols tested and the other reagents used were purchased from Aldrich (Steinheim, Germany), Boehringer–Mannheim (Mannheim, Germany), Fluka (Buchs, Switzerland), ICN (Costa Mesa, CA, USA), Merck (Darmstadt, Germany), Rathburn Chemicals (Scotland, UK), Riedel-de Haën (Seelze, Germany), and Sigma (St. Louis, MO, USA), and were of the highest grade available. 3,5-Dinitrocatechol was kindly supplied from Orion Pharma (Espoo, Finland). S-adenosyl-l-[methyl-14C]methionine was obtained from NEN
Chromatographic method
A new COMT assay was developed, which is based on measurement of the products formed utilising 14C-labelled co-substrate S-adenosyl-l-methionine and HPLC with an on-line radioactivity detector. The great advantage of the method compared to previous methods is that the methylation velocity of structurally diverse catechols catalysed by S-COMT could be determined only with simple modifications in the mobile phase. The set of 30 studied compounds included catecholamines, catecholestrogens and
Conclusions
An accurate and reproducible HPLC method with on-line radioactivity detection for the assay of COMT was developed. The method was proved to be suited for the determination of methylation velocity for 30 structurally diverse catechols. The method was also shown to be applicable for the determination of enzyme kinetic parameters for the reaction of each catecholic hydroxyl. Using this method it will be probably possible to determine enzyme kinetic parameters for a large variety of substrates
Acknowledgements
This work was supported by the BIOMED 2 Program (BMH4-97-2621) of the European Union, which is gratefully acknowledged. We also thank Mrs. Raija Savolainen (Orion Pharma) for excellent technical assistance.
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Present address: National Public Health Institute, Laboratory of Human Molecular Genetics, Mannerheimintie 166, 00300 Helsinki, Finland.