Fluorometric assay for the determination of glutathione reductase activity.
Fluorometric assay for the determination of glutathione reductase activity.
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DOI：
10.1021/ac071518p
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发表时间：
2007-10
影响因子：
7.4
通讯作者：
Andrew M. Piggott;P. Karuso
中科院分区：
文献类型：
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作者：
Andrew M. Piggott;P. Karuso
游离巯基的测定在生物技术的许多方面是重要的，例如测量巯基反应性探针的偶联效率、测定含半胱氨酸的半抗原和测定还原酶/巯基转移酶活性，以及在食品工业的各个方面。这通常使用Ellman试剂通过比色法实现，尽管该测定相对不敏感并且Ellman试剂不稳定。在本文中，我们描述了一个高灵敏度的荧光测定游离巯基的基础上FRET，我们已经用来开发一个灵敏的谷胱甘肽还原酶活性测定。该测定利用了当含有通过二硫键连接的两个荧光素分子的探针被谷胱甘肽切割时在520 nm处发生的荧光强度的特异性增加。该测定比常用的比色谷胱甘肽还原酶测定灵敏2个数量级，并具有更大的动态范围。
The determination of free sulfhydryl groups is important in many aspects of biotechnology, such as measuring the coupling efficiency of thiol-reactive probes, assaying cysteine-containing haptens, and assaying reductase/thiol transferase activity, as well as in various aspects of the food industry. This is generally achieved colorimetrically using Ellman's reagent, although the assay is relatively insensitive and Ellman's reagent is unstable. In this paper, we describe a highly sensitive fluorometric assay for free sulfhydryl groups based on FRET, which we have used to develop a sensitive assay for glutathione reductase activity. The assay exploits the specific increase in fluorescence intensity that occurs at 520 nm when a probe containing two molecules of fluorescein linked via a disulfide group is cleaved by glutathione. The assay is 2 orders of magnitude more sensitive than the commonly used colorimetric glutathione reductase assay and has a greater dynamic range.