Synthesis and characterization of a series of highly fluorogenic substrates for glutathione transferases, a general strategy.
Synthesis and characterization of a series of highly fluorogenic substrates for glutathione transferases, a general strategy.
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DOI：
10.1021/ja205500y
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发表时间：
2011-08
影响因子：
15
通讯作者：
Jie Zhang;Aya Shibata;Mika Ito;S. Shuto;Yoshihiro Ito;B. Mannervik;H. Abe;R. Morgenstern
中科院分区：
文献类型：
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作者：
Jie Zhang;Aya Shibata;Mika Ito;S. Shuto;Yoshihiro Ito;B. Mannervik;H. Abe;R. Morgenstern
谷胱甘肽转移酶 (GST) 在生物技术应用中用作易于纯化的融合伴侣，并且在某些肿瘤中也过度表达。因此，需要对酶进行灵敏检测。在这里，我们描述了 GST 新型荧光底物的合成和表征的一般策略。通过将亲电子磺酰胺键引入含有氨基的荧光分子[例如香豆素、甲酚紫和罗丹明的2,4-二硝基苯磺酰胺(DNs)衍生物]来合成底物。这些衍生物基本上是非荧光的，并且在 GST 催化二硝基苯磺酰胺裂解后，释放出游离荧光团（以及 1-谷胱甘肽-2,4-二硝基苯 + SO(2)）。所有基于香豆素、甲酚紫和罗丹明的荧光探针均被证明是大多数 GST 的良好底物，尤其是 GSTA(1-1)，其荧光强度增强（71-1200 倍）、高 k(cat)/K(m) 值 (10(4)-10(7) M(-1) s(-1)）和显着的速率增强（10（6）-10（9）倍）。该底物成功应用于定量细胞提取物中极低水平的 GST 活性，DNs-甲酚紫也成功应用于活细胞中微粒体 MGST(1) 活性的成像。甲酚紫染色的细胞在固定后保留其荧光，这是一个非常有用的特性。总之，我们描述了一种生成荧光 GST 底物的通用且通用的策略，其中一些提供了迄今为止针对 GST 描述的最灵敏的检测方法。
Glutathione transferases (GSTs) are used in biotechnology applications as fusion partners for facile purification and are also overexpressed in certain tumors. Consequently, there is a need for sensitive detection of the enzymes. Here we describe a general strategy for the synthesis and characterization of novel fluorogenic substrates for GSTs. The substrates were synthesized by introducing an electrophilic sulfonamide linkage to fluorescent molecules containing an amino group [e.g., 2,4-dinitrobenzenesulfonamide (DNs) derivatives of coumarin, cresyl violet, and rhodamine]. The derivatives were essentially nonfluorescent, and upon GST catalyzed cleavage of the dinitrobenzenesulfonamide, free fluorophore is released (and 1-glutathionyl-2,4-dinitrobenzene + SO(2)). All the coumarin-, cresyl violet- and rhodamine-based fluorogenic probes turned out to be good substrates for most GSTs, especially for GSTA(1-1), in terms of strong fluorescence increases (71-1200-fold), high k(cat)/K(m) values (10(4)-10(7) M(-1) s(-1)) and significant rate enhancements (10(6)-10(9)-fold). The substrates were successfully applied to quantitate very low levels of GST activity in cell extracts and DNs-cresyl violet was also successfully applied to the imaging of microsomal MGST(1) activity in living cells. The cresyl violet stained cells retained their fluorescence after fixation, which is a very useful property. In summary, we describe a general and versatile strategy to generate fluorogenic GST substrates, some of them providing the most sensitive assays so far described for GSTs.