Tang Zhong-Mei, Kang Jing-Wu
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Fenglin Road 354, Shanghai, 200032, China.
Anal Chem. 2006 Apr 15;78(8):2514-20. doi: 10.1021/ac052030w.
A novel strategy for screening the enzyme inhibitors from the complex mixtures by capillary electrophoresis with an on-column immobilized enzyme microreactor created by an ionic binding technique is reported. The enzyme microreactor was prepared in two steps: First, the capillary wall was dynamically coated with a polycationic electrolyte hexadimethrine bromide (HDB) by simply flushing the column using the HDB solution. Subsequently, a plug of the enzyme solution was injected and incubated for 5 min to permit the enzyme molecules to immobilize on the positively charged coating via ionic binding. To demonstrate this strategy, angiotensin-converting enzyme (ACE) was employed as a model for the enzyme immobilization, inhibition study, and inhibitor screening. It has been proved that such a prepared immobilized ACE microreactor displays a high enough activity and stability. Furthermore, the immobilized enzyme microreactor could be easily renewed. The inhibition study or inhibitor screening was accomplished through the following procedure: (i) the substrate solution was injected and incubated within the microreactor for a short time span; (ii) subsequently, the voltage was applied to separate the product of the enzyme reaction from the unreacted substrate based on their different mobilities, the peak area of the product representing the enzyme activity; (iii) a certain amount of enzyme inhibitor or candidate compound was spiked into the substrate solution to assay the reduction of the immobilized enzyme activity. Thus, the inhibitors can be easily identified if the reduced peak area of the product is observed in electropherograms. Because the injection volume of the capillary was only 9.8 nL and the enzyme could be reusable, the assay cost could be dramatically reduced. The screening of a small compound library containing natural extracts and commercially available inhibitors was performed. The present approach has proved to be simple, rapid, and robust.
报道了一种通过毛细管电泳从复杂混合物中筛选酶抑制剂的新策略，该方法采用离子结合技术创建柱上固定化酶微反应器。酶微反应器的制备分两步进行：首先，通过用十六烷基二甲基溴化铵（HDB）溶液冲洗柱子，将聚阳离子电解质HDB动态涂覆在毛细管壁上。随后，注入一段酶溶液并孵育5分钟，以使酶分子通过离子结合固定在带正电荷的涂层上。为了验证该策略，使用血管紧张素转换酶（ACE）作为酶固定化、抑制研究和抑制剂筛选的模型。已证明这样制备的固定化ACE微反应器具有足够高的活性和稳定性。此外，固定化酶微反应器可以很容易地更新。抑制研究或抑制剂筛选通过以下步骤完成：（i）注入底物溶液并在微反应器中孵育短时间；（ii）随后，施加电压以根据未反应底物和酶反应产物的不同迁移率分离它们，产物的峰面积代表酶活性；（iii）将一定量的酶抑制剂或候选化合物加入底物溶液中，以测定固定化酶活性的降低。因此，如果在电泳图中观察到产物峰面积减小，就可以很容易地鉴定出抑制剂。由于毛细管的进样体积仅为9.8 nL且酶可重复使用，因此可以显著降低检测成本。对包含天然提取物和市售抑制剂的小分子化合物库进行了筛选。目前的方法已被证明是简单、快速且可靠的。
