A general fluorescence-based coupled assay for S-adenosylmethionine-dependent methyltransferases
A general fluorescence-based coupled assay for S-adenosylmethionine-dependent methyltransferases
复制标题
DOI：
10.1016/j.bbrc.2005.03.170
复制
发表时间：
2005-05-27
影响因子：
3.1
通讯作者：
Hrycyna, CA
中科院分区：
文献类型：
--
作者：
Wang, CH;Leffler, S;Hrycyna, CA
我们已经开发了一种简单而灵敏的基于荧光的两步偶联酶法来报告S-腺苷甲硫氨酸依赖性甲基转移酶的活性。该测定依赖于荧光素-胱胺-甲基红（FL-S-S-MR）报告分子，其可以被硫醇活化。在不存在硫醇的情况下，来自报道分子的荧光通过两个发色团之间的荧光共振能量转移而猝灭。在这份报告中，我们使用儿茶酚-O-甲基转移酶与添加的S-腺苷高半胱氨酸水解酶生产巯基高半胱氨酸。高半胱氨酸的存在导致胱胺系链中的二硫键裂解和荧光去猝灭，因为未偶联的发色团被稀释到周围介质中。FL-S-S-MR对巯基的敏感性和特异性使得能够检测
We have developed a simple and sensitive fluorescence-based two-step coupled enzyme assay to report the activity of S-adenosylmethionine-dependent methyltransferases. This assay relies on a fluorescein-cystamine-methyl red (FL-S-S-MR) reporter molecule that can be activated by thiols. In the absence of thiols, fluorescence from the reporter is quenched through fluorescence resonance energy transfer between the two chromophores. In this report, we use catechol-O-methyltransferase with the addition of S-adenosylhomocysteine hydrolase to produce the thiol homocysteine. The presence of homocysteine leads to disulfide bond cleavage in the cystamine tether and fluorescence dequenching as the uncoupled chromophores are diluted into the surrounding media. The sensitivity and specificity of FL-S-S-MR to thiols enabled detection of