Chemistry Department, University of Nebraska, Lincoln, NE 68588-0304, USA.
J Chromatogr A. 2011 Dec 9;1218(49):8915-24. doi: 10.1016/j.chroma.2011.04.078. Epub 2011 May 6.
This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous binding in biomolecular interactions, using the binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of binding heterogeneity. The methods developed in this work for the detection of binding heterogeneity are not limited to drug interactions with HSA but could be applied to other types of drug-protein binding or to additional biological systems with heterogeneous binding.
本研究以乙酰己脲与人血清白蛋白（HSA）的结合为模型，考察了使用前沿分析和高效亲和色谱法检测生物分子相互作用中异质结合的情况。通过使用该模型系统和色谱理论，发现与传统等温线相比，双倒数图更容易用于初步检测结合位点的异质性。由于异质性，双倒数图中出现的线性偏离是分析物浓度、系统中结合位点的相对亲和力以及每种类型的结合位点数量的函数。在各种条件下确定并比较了这些偏差的大小。还生成了图，以显示在一般异质体系或在有关于结合异质性程度的初步信息的情况下，需要哪些实验条件来观察这些偏差。本工作中开发的用于检测结合异质性的方法不仅限于 HSA 与药物的相互作用，还可应用于其他类型的药物-蛋白质结合或具有异质结合的其他生物系统。
