Yoon Hey Young, Hong Jong-In
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea.
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea.
Anal Biochem. 2017 Jun 1;526:33-38. doi: 10.1016/j.ab.2017.03.012. Epub 2017 Mar 16.
Sulfatases catalyze the hydrolysis of sulfate esters that are present in a range of biomolecules. This is an important step in several biological processes such as cellular degradation, hormone regulation, and cell signaling. We have developed a new activity-based sulfatase probe (probe 1) that generates a fluorescent N-methylisoindole upon hydrolysis by sulfatase. Because of the autoxidation of N-methylisoindole, the sulfatase activity was also tested under reducing conditions, containing either glutathione (GSH) or tris(2-carboxyethyl)phosphine (TCEP), exhibiting little change in kinetic parameters compared to non-reducing conditions. Probe 1 displayed reasonable kinetic parameters under both non-reducing and reducing conditions, among which the use of Tris buffer and Tris buffer containing GSH appeared to be appropriate conditions for inhibitor screening. Probe 1 showed stronger intensity upon treatment with sulfatase under neutral conditions than under acidic conditions, but it still has limitations in the selectivity for a specific sulfatase. Nevertheless, the fluorescent signal generated as a result of the release of N-methylisoindole after treatment of probe 1 with sulfatase provides a new assay for measuring sulfatase activity that could be adapted for high throughput screening.
硫酸酯酶催化一系列生物分子中存在的硫酸酯的水解。这是细胞降解、激素调节和细胞信号传导等多个生物过程中的重要步骤。我们开发了一种基于活性的新型硫酸酯酶探针（探针1），它在被硫酸酯酶水解后会生成荧光N-甲基异吲哚。由于N-甲基异吲哚的自氧化作用，硫酸酯酶活性也在含有谷胱甘肽（GSH）或三（2-羧乙基）膦（TCEP）的还原条件下进行了测试，与非还原条件相比，动力学参数几乎没有变化。探针1在非还原和还原条件下均表现出合理的动力学参数，其中使用Tris缓冲液和含有GSH的Tris缓冲液似乎是进行抑制剂筛选的合适条件。探针1在中性条件下用硫酸酯酶处理后的强度比在酸性条件下更强，但在对特定硫酸酯酶的选择性方面仍存在局限性。然而，探针1用硫酸酯酶处理后由于N-甲基异吲哚的释放而产生的荧光信号为测量硫酸酯酶活性提供了一种新的检测方法，可适用于高通量筛选。
