Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an 710069, China.
Technology Center, China Tobacco Fujian Industrial Corporation, Xiamen 36021, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Sep 1;1125:121727. doi: 10.1016/j.jchromb.2019.121727. Epub 2019 Jul 19.
Characterization of the heterogeneity of a protein surface is important for understanding the binding mechanism of a drug to the protein. A systematic methodology of integrated adsorption energy distribution (AED) calculation with the Scatchard plot was used to characterize the heterogeneous binding of drugs to bovine serum albumin (BSA). Frontal affinity chromatography (FAC) was applied to generate the adsorption data of three drugs on the immobilized BSA column. The concave Scatchard plots cannot distinguish the heterogeneous model between Toth and bi-Langmuir. The calculation of AED profiles allowed the accurate selection of adsorption models to reveal the physical sense of the drugs. Warfarin-BSA complex proved to be the bi-Langmuir model with association constants of 6.6 × 10 M and 2.4 × 10 M, respectively. These results were highly consistent with traditional frontal analysis, ultrafiltration, and dynamic dialysis. Applying the AED related method, we achieved multiple and single kinds of binding sites for ephedrine-BSA and L-tryptophan-BSA complexes. Site-specific competitive studies exhibited non-competitive binding of ephedrine with warfarin or L-tryptophan on BSA, underlining that the location of ephedrine binding sites in the subdomain IIIB of BSA. This method permits an accurate heterogeneous characterization of drugs on a protein surface and has great potential in the reliable drug-protein interaction analysis.
蛋白质表面的异质性特征对于理解药物与蛋白质的结合机制非常重要。本研究采用整合吸附能分布（AED）计算与 Scatchard 图的系统方法，对药物与牛血清白蛋白（BSA）的异质结合进行了特征描述。采用前沿亲和色谱（FAC）生成三种药物在固定化 BSA 柱上的吸附数据。凹 Scatchard 图不能区分托特（Toth）和双朗缪尔（bi-Langmuir）之间的异质模型。AED 分布的计算可以准确选择吸附模型，揭示药物的物理意义。华法林-BSA 复合物被证明是双朗缪尔模型，其结合常数分别为 6.6×10 M 和 2.4×10 M。这些结果与传统的前沿分析、超滤和动态透析高度一致。应用 AED 相关方法，我们实现了麻黄碱-BSA 和 L-色氨酸-BSA 复合物的多种和单一结合位点。特异性竞争研究表明，麻黄碱与华法林或 L-色氨酸在 BSA 上呈非竞争性结合，这突出表明麻黄碱结合位点位于 BSA 的 IIIB 亚结构域。该方法可实现对蛋白质表面上药物的准确异质特征描述，在可靠的药物-蛋白质相互作用分析中具有很大的潜力。
