Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad, Pakistan.
Anal Biochem. 2011 Jul 15;414(2):226-31. doi: 10.1016/j.ab.2011.03.021. Epub 2011 Mar 23.
A simple and fast dynamically coated capillary electrophoretic method was developed for the characterization and inhibition studies of alkaline phosphatases(EC 3.1.3.1). An inside capillary enzymatic reaction was performed, and hydrolysis of the substrate 4-nitrophenylphosphate to 4-nitrophenol was measured. Fused-silica capillary surface was dynamically modified with polycationic polybrene coating. By reversal of the electroosmotic flow (EOF), analysis time was reduced up to 3 min as the anionic analytes were migrated in the same direction as the EOF. Furthermore, the sensitivity of the method was increased using electroinjection through high-field amplified injection. The baseline separation of 4-nitrophenylphosphate and 4-nitrophenol was achieved by employing 50 mM sodium phosphate as the running buffer (pH 8.5), 0.0025% polybrene, and a constant voltage of -15 kV, and the products were detected at 322 nm. Under the optimized conditions, a good separation with high efficiency was achieved. The new method was applied to study enzyme kinetics and inhibitor screening. K(m) and K(i) values obtained with the new CE method were compared well with the standard spectrophotometric method. Dynamic coating of fused-silica capillary gave fast and reproducible separation of substrate and product. The method can be easily optimized for inhibition studies of other isozymes.
一种简单快速的动态涂层毛细管电泳方法被开发用于碱性磷酸酶（EC 3.1.3.1）的特征描述和抑制研究。在毛细管内进行酶促反应，测量底物 4-硝基苯磷酸盐的水解产物 4-硝基苯酚。用聚阳离子聚集体对熔融石英毛细管表面进行动态修饰。通过反转电动流（EOF），分析时间缩短至 3 分钟，因为阴离子分析物与 EOF 相同方向迁移。此外，通过高场放大进样进行电注入，提高了方法的灵敏度。采用 50 mM 磷酸钠作为运行缓冲液（pH 8.5）、0.0025%聚集体和-15 kV 恒电压，可实现 4-硝基苯磷酸盐和 4-硝基苯酚的基线分离，产物在 322nm 处检测。在优化条件下，实现了高效的良好分离。该新方法被应用于研究酶动力学和抑制剂筛选。用新的 CE 方法获得的 K(m)和 K(i)值与标准分光光度法吻合良好。熔融石英毛细管的动态涂层可快速且重现地分离底物和产物。该方法可轻松优化用于其他同工酶的抑制研究。
