高速液体クロマトグラフィーによるカテコール-O-メチルトランスフェラーゼ活性の測定

概要

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A method for high-performance liquid chromatographic (HPLC) determination of the m- and ρ-O-methylated products of 3,4-dihydroxybenzoic acid (DHBA) was investigated and applied to the assay of catechol-O-methyltransferase (COMT) of rat liver. The standard incubation mixture consisted of 0.5 ml of DHBA (600 μg/ml), 0.25 ml of S-adenosyl-L-methionine (4 mM), 0.05ml of MgCl_2 (0.1 M) and 0.2ml of COMT (10 mg of protein/ml) in 0.05 M phosphate buffer solution (pH 7.2). After incubation at 37 ℃ for 30 min, the reaction was stopped by adding 0.1 ml of 1 N HCl. The mixture was saturated with NaCl, and the products were extracted with 2.0 ml of ethyl acetate containing 4-hydroxybenzoic acid as internal standard. The extract was evaporated to dryness and the residue was redissolved in 1 ml of 1 % acetic acid, and 50 μl of it was applied onto HPLC with a column (15 cm×4 mm I.D., stainless steel) packed with LiChrosorb 5 RP 18 and a mobile phase consisting of acetic acid/acetonitrile/water (1:5:94v/v). Detection wavelength was 254 nm.
社団法人日本分析化学会の論文
1981-07-05