Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Anal Chem. 2009 Oct 15;81(20):8644-8. doi: 10.1021/ac901419x.
The interplay between biophysical characteristics such as protein size and shape and protein function is difficult to ascertain using simple methods. Here, we present an approach for characterizing both protein-ligand binding as well as protein hydrodynamic radius in one operation combining electrophoresis and size measurement by dispersion using capillaries. The methodology is based on the integration of Taylor dispersion analysis and capillary electrophoresis and is here demonstrated using commercially available capillary electrophoresis instrumentation modified with a pixel sensor UV area imager, allowing two detection points along the capillary. Analytes are the human serum proteins alpha(1)-acid glycoprotein and albumin interacting with the drug propranolol in a frontal analysis mode. Upon introduction of the propranolol-protein sample, voltage is initially applied to facilitate electrophoretically mediated separation of ligand and protein and frontal analysis. Then a pressure mobilization step is used whereby Taylor dispersion can be characterized online based on the signal from the UV area imager. Estimates of ligand binding and values for hydrodynamic radii agree with values obtained by independent methods.
生物物理特性（如蛋白质大小和形状）与蛋白质功能之间的相互作用很难用简单的方法确定。在这里，我们提出了一种方法，可将电泳和通过毛细管分散进行的尺寸测量结合在一个操作中，同时对蛋白质-配体结合和蛋白质流体力学半径进行特征描述。该方法基于泰勒分散分析和毛细管电泳的整合，这里使用经过改进的带有像素传感器紫外面积成像仪的商业毛细管电泳仪器进行了演示，允许在毛细管上进行两个检测点。分析物是人血清蛋白α(1)-酸性糖蛋白和白蛋白，它们以前沿分析模式与药物普萘洛尔相互作用。在引入普萘洛尔-蛋白质样品后，最初施加电压以促进配体和蛋白质的电泳介导分离和前沿分析。然后使用压力迁移步骤，可根据紫外面积成像仪的信号在线表征泰勒分散。配体结合的估计值和流体力学半径的值与通过独立方法获得的值一致。
