利用去甲肾上腺素作为酶促底物以及带有荧光检测的反相高效液相色谱法改进儿茶酚-O-甲基转移酶活性测定法。

Improved assay for catechol-O-methyltransferase activity utilizing norepinephrine as an enzymatic substrate and reversed-phase high-performance liquid chromatography with fluorescence detection.

作者信息

Aoyama Nozomi, Tsunoda Makoto, Imai Kazuhiro

机构信息

Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

出版信息

J Chromatogr A. 2005 May 13;1074(1-2):47-51. doi: 10.1016/j.chroma.2005.03.037.

DOI:10.1016/j.chroma.2005.03.037

PMID:15941038

Abstract

We have previously established a rapid catechol-O-methyltransferase (COMT) assay using norepinephrine (NE) as a natural substrate and flow-injection analysis. In this study, the method is improved for screening of COMT inhibitors or activators using reversed-phase high-performance liquid chromatographic separation with fluorescence detection. The excess substrate, NE, was removed by the addition of borate in the eluent for HPLC to make an ionic complex with NE, which was eluted faster than the enzymatic product, normetanephrine. The method had good precision and accuracy, and was able to assay one sample in 5 min, showing the usability for screening of COMT inhibitors or activators.

摘要

我们之前建立了一种快速儿茶酚-O-甲基转移酶（COMT）测定法，该方法使用去甲肾上腺素（NE）作为天然底物并结合流动注射分析。在本研究中，对该方法进行了改进，采用反相高效液相色谱分离结合荧光检测来筛选COMT抑制剂或激活剂。通过在高效液相色谱（HPLC）洗脱液中加入硼酸盐来去除过量的底物NE，使其与NE形成离子复合物，该复合物的洗脱速度比酶促产物去甲变肾上腺素快。该方法具有良好的精密度和准确性，能够在5分钟内测定一个样品，显示出可用于筛选COMT抑制剂或激活剂。