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Advances in enzyme substrate analysis with capillary electrophoresis

机译：毛细管电泳酶底物分析的新进展

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Capillary electrophoresis provides a rapid, cost-effective platform for enzyme and substrate characterization. The high resolution achievable by capillary electrophoresis enables the analysis of substrates and products that are indistinguishable by spectroscopic techniques alone, while the small volume requirement enables analysis of enzymes or substrates in limited supply. Furthermore, the compatibility of capillary electrophoresis with various detectors makes it suitable for KM determinations ranging from nanomolar to millimolar concentrations. Capillary electrophoresis fundamentals are discussed with an emphasis on the separation mechanisms relevant to evaluate sets of substrate and product that are charged, neutral, and even chiral. The basic principles of Michaelis-Menten determinations are reviewed and the process of translating capillary electrophoresis electropherograms into a Michaelis-Menten curve is outlined. The conditions that must be optimized in order to couple off-line and on-line enzyme reactions with capillary electrophoresis separations, such as incubation time, buffer pH and ionic strength, and temperature, are examined to provide insight into how the techniques can be best utilized. The application of capillary electrophoresis to quantify enzyme inhibition, in the form of KI or IC50 is detailed. The concept and implementation of the immobilized enzyme reactor is described as a means to increase enzyme stability and reusability, as well as a powerful tool for screening enzyme substrates and inhibitors. Emerging techniques focused on applying capillary electrophoresis as a rapid assay to obtain structural identification or sequence information about a substrate and in-line digestions of peptides and proteins coupled to mass spectrometry analyses are highlighted.
机译：毛细管电泳为酶和底物表征提供了一个快速，经济高效的平台。毛细管电泳可实现的高分辨率可分析仅通过光谱技术无法区分的底物和产物，而小体积要求可在有限的供应量下对酶或底物进行分析。此外，毛细管电泳与各种检测器的兼容性使其适合于从纳摩尔浓度到毫摩尔浓度的KM测定。讨论了毛细管电泳的基本原理，重点是与评估带电，中性甚至手性的底物和产物对相关的分离机理。综述了Michaelis-Menten测定的基本原理，并概述了将毛细管电泳电泳图转换为Michaelis-Menten曲线的过程。为了将离线和在线酶反应与毛细管电泳分离耦合在一起，必须优化的条件（例如孵育时间，缓冲液pH和离子强度以及温度）经过检查，以提供最佳技术的见解。利用。详细介绍了毛细管电泳以KI或IC50形式量化酶抑制作用的方法。固定化酶反应器的概念和实现被描述为增加酶稳定性和可重复使用性的手段，以及筛选酶底物和抑制剂的强大工具。新兴技术集中在应用毛细管电泳作为一种快速分析方法来获得有关底物的结构鉴定或序列信息，并着重介绍了与质谱分析偶联的肽和蛋白质的在线消化方法。

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