Abstract:

This study presents a molecular dynamics simulation of protein separation by reverse phase liquid chromatography（RPLC）at all-atom level，using silica gel surface with C₄ compounds and cytochrome C as representative adsorbent and protein，respectively. The conformational transition of cytochrome C was monitored during the aqueous adsorption onto the adsorbent and elution with methanol. It was shown that during the adsorption，in which protein was attracted to the adsorbent surface，a simultaneous exclusion of water molecules occurred both at the adsorbent surface and the protein. A conformational transition of the protein conformation that led to the exposure of the hydrophobic amino acid residues，as compared to its native conformation in aqueous phase，was also displayed. During the elution by methanol，the replacement of water molecule by methanol occurred on both the surface of adsorbent and the adsorbed protein. As a result，the adsorbed protein was replaced by methanol. Above-mentioned simulation results reproduced the preferential hydration mechanism of the adsorption by RPLC and provided a molecular insight into the interaction among protein，adsorbent and the solvents at all-atom level，which is helpful for the design of new adsorbent and the optimization of RPLC process.

摘要：

采用全原子分子动力学方法模拟了反相色谱分离蛋白质的吸附和洗脱过程。采用表面键合C₄烷基链的硅胶基质作为反相色谱介质和细胞色素C作为蛋白质模型，模拟蛋白质在反相色谱分离过程中的构象变化。结果显示：在吸附过程中，蛋白质在释放出表面结合水分子的同时置换出色谱介质表面的水分子。与其在溶液中的天然构象相比，吸附态蛋白质的构象发生改变。在洗脱过程中，随着溶剂从水切换到甲醇，甲醇取代水分子包覆在介质和蛋白质表面，将蛋白质从介质表面置换下来。分子模拟的结果再现了有关反相色谱“优先水化”的吸附机制，并从分子水平上展现了吸附和洗脱过程中蛋白质、色谱介质和溶剂之间相互作用，对反相色谱介质设计和过程优化提供了参考。