Improved assay of reaction products to quantitate catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection |
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| Affiliation: | 1. Center for Advanced Studies in Chemistry, North-Eastern Hill University, Shillong 793 022, India;2. Department of Biotechnology & Bio-informatics, North-Eastern Hill University, Shillong 793 022, India;1. Cincinnati Children''s Hospital Medical Center, Division of Pediatric Rehabilitation, United States;2. University of Cincinnati, Department of Psychiatry and Behavioral Neuroscience, United States |
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| Abstract: | We applied coulometric detection (three electrochemical electrodes in series) to quantitate vanillic acid and isovanillic acid using reversed-phase HPLC. The formation of these reaction products from dihydroxybenzoic acid was used as a precise and reproducible measure of catechol-O-methyltransferase (COMT) activity in striatal homogenates and recombinant membrane-bound COMT protein. This detection system has a higher sensitivity (0.5 pmol per injection) than a single-cell amperometric detection. As in a previous method, the deproteinized supernatants of the COMT assay could be injected directly onto the HPLC system allowing the handling of a large number of samples in one day. |
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