Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.
Mobility shift-based electrophoresis coupled with fluorescent detection enables real-time enzyme analysis of carbohydrate sulfatase activity.
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DOI：
10.1042/bcj20200952
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发表时间：
2021-02-26
期刊：
影响因子：
--
通讯作者：
Cartmell A
中科院分区：
文献类型：
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作者：
Byrne DP;London JA;Eyers PA;Yates EA;Cartmell A
硫代碳水化合物代谢是一个基本的过程，发生在生命的各个领域。碳水化合物硫酸酯酶是从碳水化合物中去除硫酸盐基团的酶，对复杂多糖的解聚合至关重要。尽管碳水化合物硫酸酯酶具有重要的生物学意义，但对其研究甚少，在准确评估酶活性、特异性和动力学参数方面仍存在挑战。最值得注意的是，从硫酸盐基质中分离脱硫产物目前是一个耗时的过程。在本文中，我们描述了快速毛细管电泳耦合底物荧光检测作为一种高通量和简便的方法分析碳水化合物硫酸酯酶活性的发展。该方法基于现有的微流控技术，结合一种新的合成荧光6S-GlcNAc碳水化合物底物，可用于测定动力学和抑制参数。此外，在时间和资源方面，我们将该技术与高性能阴离子交换色谱法和基于核磁共振的方法进行了比较，这两种方法是目前酶促碳水化合物硫酸化分析的两种“金标准”。我们的研究清楚地证明了迁移率转移测定法用于定量纯化的磺化酶近实时碳水化合物脱硫的优势，并将支持寻找这些疾病相关酶的小分子抑制剂。
Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the enzymatic activity, specificity and kinetic parameters. Most notably, the separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique, in terms of both time and resources, to high-performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and will support the search for small molecule inhibitors of these disease-associated enzymes.