Monochlorobimane fluorometric method to measure tissue glutathione
Monochlorobimane fluorometric method to measure tissue glutathione
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DOI：
10.1006/abio.2000.4765
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发表时间：
2000-11-01
影响因子：
2.9
通讯作者：
Juurlink, BHJ
中科院分区：
文献类型：
--
作者：
Kamencic, H;Lyon, A;Juurlink, BHJ
谷胱甘肽（GSH）是细胞内主要的低分子量巯基，在细胞防御氧化应激因子中起着关键作用。测量GSH的常用技术使用反相高效液相色谱法（HPLC），然后用5，5 '-二硫代双（2-硝基苯甲酸）衍生，该技术虽然可靠且灵敏，但耗时且费力。测量培养细胞中GSH的常用技术是向培养基中加入一氯二亚胺，在培养基中它容易进入细胞以形成荧光GSH-一氯二亚胺加合物，该加合物可以用荧光法测量。该反应由谷胱甘肽S-转移酶催化。我们推断，加入谷胱甘肽S-转移酶和一氯二亚胺组织匀浆将允许一个快速可靠的方法来测量谷胱甘肽。在大鼠肝脏匀浆中评估了新试验的准确性。使用HPLC方法测定每种匀浆的一半的GSH，而使用一氯二亚胺方法测定另一半。发现这两种方法得到相同的结果。我们的结论是，一氯二亚胺荧光法是足够的具体可靠地测量组织GSH（C）2000学术出版社。
Glutathione (GSH) is the principal intracellular low-molecular-weight thiol and plays a critical role in the cellular defense against agents that impose oxidative stress. A common technique to measure GSH uses reversed-phase high-performance liquid chromatography (HPLC) following derivatization with 5,5'-dithiobis(2-nitrobenzoic acid), a technique, although reliable and sensitive, that is time consuming and laborious. A common technique to measure GSH in cultured cells is to add monochlorobimane to the culture medium where it readily enters cells to form a fluorescent GSH-monochlorobinane adduct that can be measured fluorometrically. This reaction is catalyzed by glutathione S-transferase. We reasoned that adding glutathione S-transferase and monochlorobimane to tissue homogenates would allow a rapid reliable method to measure GSH. The accuracy of the new test was assessed in homogenates of rat livers. One-half of each homogenate was assayed for GSH using a HPLC approach while the other half was assayed using the monochlorobimane approach. The two methods were found to give identical results. We conclude that the monochlorobimane fluorescent method is sufficiently specific to reliably measure tissue GSH (C) 2000 Academic Press.