Hexokinase inhibitor screening based on adenosine 5′‐diphosphate determination by electrophoretically mediated microanalysis
Hexokinase inhibitor screening based on adenosine 5′‐diphosphate determination by electrophoretically mediated microanalysis
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DOI：
10.1002/elps.200800542
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发表时间：
2009-04
期刊：
影响因子：
2.9
通讯作者：
Tongdan Wang;Jingwu Kang
中科院分区：
文献类型：
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作者：
Tongdan Wang;Jingwu Kang
本文建立了一种基于毛细管电泳的己糖激酶抑制剂筛选方法。在该方法中，通过毛细管介导微量分析（EMMA）测定己糖激酶活性，该方法结合了柱上己糖激酶介导的反应和通过毛细管分离和UV检测测量产生的腺苷5′-二磷酸（ADP）。与不存在任何抑制剂时获得的参比电泳图相比，可直接从ADP的还原峰面积读出酶抑制。优化了柱上酶反应条件和5′-三磷酸腺苷（ATP）和ADP的分离条件。酶促反应的最佳缓冲液组成为含有5 mM MgCl 2的25 mM HEPES缓冲液（pH 7.5），而分离的最佳缓冲液组成为含有0.02%（m/v）溴化己二甲双胍（HDB）的100 mM Tris-磷酸盐缓冲液（pH 5.5）。幸运的是，在EMMA方法中，不连续缓冲系统可以很容易地适应。通过动态涂敷阳离子H2 O2 HDB使毛细管电泳方向反向，分离时间大大缩短到3 min以下。此外，HDB涂层也减少了ATP的峰拖尾。Z′因子高达0.98，表明筛选数据的质量较高。本方法简单、稳健且具有成本效益。
A CE‐based method for hexokinase inhibitor screening was developed in the present paper. In this method, hexokinase activity was assayed via electrophoretically mediated microanalysis (EMMA), which combines on‐column hexokinase‐mediated reaction and measurement of produced adenosine 5′‐diphosphate (ADP) via electrophoretical separation and UV detection. Enzyme inhibition can be read out directly from the reduced peak area of ADP in comparison with a reference electropherogram obtained in the absence of any inhibitor. Conditions for on‐column enzyme reaction and separation of adenosine 5′‐triphosphate (ATP) and ADP were optimized. The optimal buffer composition for enzymatic reaction was 25 mM HEPES buffer (pH 7.5) containing 5 mM MgCl2, whereas the optimal buffer composition for separation was 100 mM Tris‐phosphate buffer (pH 5.5) containing 0.02% (m/v) hexadimethrine bromide (HDB). Fortunately, discontinuous buffer system can be adapted easily in the EMMA method. The time for separation was reduced dramatically to less than 3 min by reversing the direction of EOF via dynamically coating the capillary wall with the cationic polyelectrolyte HDB. Moreover, the peak tailing of ATP was also reduced by HDB coating. The Z′ factor as high as 0.98 was obtained, indicating a high quality of the screening data. The present method is simple, robust and cost‐effective.