Zhang Lin, Zhao Guofeng, Sun Yan
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
J Phys Chem B. 2009 May 14;113(19):6873-80. doi: 10.1021/jp809754k.
Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. The method has been successfully applied in the separation and purification of antibodies and other proteins. However, little is understood about protein conformational transition and the dynamic process within adsorbent pores. In the present study, a pore model is established to represent the realistic porous adsorbent composed of matrix and immobilized HCIC ligands. Protein adsorption, desorption, and conformational transition in the HCIC pore and its implications to the separation performance are shown by a molecular dynamics simulation of a 46-bead beta-barrel coarse-grained model protein in the adsorbent pore. Repeated adjustment of both protein position and orientation is observed before reaching a stable adsorption. Once the protein is adsorbed, there is a dynamic equilibrium between unfolding and refolding. The effect of hydrophobic interaction strength between protein and ligands on adsorption phenomena is then examined. Strong hydrophobic interaction, representing the presence of high-concentration lyotropic salt in mobile phase, can speed up the adsorption but cause protein unfolding more significantly. On the contrary, weak hydrophobic interaction, representing the absence of a lyotropic salt or the presence of a chaotropic agent, can reserve native protein conformation but does not lead to stable adsorption. In the elution, protein unfolding occurs due to simultaneous hydrophobic adsorption and electrostatic repulsion in the opposite directions. When the protein has been desorbed, the conformational transition between unfolded and native protein is still observed due to the long-range nature of electrostatic interaction. The simulation has provided molecular insight into protein conformational transition in the whole HCIC process, and it would be beneficial to the rational design of ligands and parameter optimizations for high-performance HCIC.
疏水电荷诱导色谱法（HCIC）是一种吸附色谱法，它结合了吸附过程中的疏水相互作用和洗脱过程中的静电排斥作用。该方法已成功应用于抗体和其他蛋白质的分离与纯化。然而，对于蛋白质构象转变以及吸附剂孔内的动态过程，人们了解甚少。在本研究中，建立了一个孔模型来代表由基质和固定化HCIC配体组成的实际多孔吸附剂。通过对吸附剂孔内一个46珠β桶状粗粒度模型蛋白进行分子动力学模拟，展示了HCIC孔内蛋白质的吸附、解吸和构象转变及其对分离性能的影响。在达到稳定吸附之前，观察到蛋白质的位置和取向会反复调整。一旦蛋白质被吸附，就会在展开和重新折叠之间存在动态平衡。然后研究了蛋白质与配体之间疏水相互作用强度对吸附现象的影响。强疏水相互作用代表流动相中存在高浓度的离液盐，它可以加速吸附，但会更显著地导致蛋白质展开。相反，弱疏水相互作用代表不存在离液盐或存在变性剂，它可以保留天然蛋白质构象，但不会导致稳定吸附。在洗脱过程中，由于相反方向的同时疏水吸附和静电排斥，蛋白质会发生展开。当蛋白质被解吸时，由于静电相互作用的长程性质，仍可观察到未折叠和天然蛋白质之间的构象转变。该模拟为整个HCIC过程中蛋白质构象转变提供了分子层面的见解，这将有助于高性能HCIC配体的合理设计和参数优化。
