Department of Mechanical Engineering and Engineering Science, The University of North Carolina at Charlotte, Charlotte, North Carolina 28223, USA.
Phys Chem Chem Phys. 2012 Jan 14;14(2):790-801. doi: 10.1039/c1cp22965f. Epub 2011 Nov 16.
Ionic liquids are being intensely studied as promising media for the stabilization of proteins and other biomolecules. Choline dihydrogen phosphate (CDHP) has been identified as one of the most promising candidates for this application. In this work we have probed in more detail the effects that CDHP may have on the thermodynamics, structure, and stability of proteins, including one of therapeutic interest. Microcalorimetry and circular dichroism spectropolarimetry (CD) were used to assess the thermal stability of protein solutions in CDHP/water mixtures at various concentrations. Increasing thermal stability of lysozyme and interleukin-2 in proportion to CDHP concentration was observed. Isothermal titration calorimetry (ITC) was used to quantify binding interactions, and indicate that the mechanism for stability does not appear to be dependent upon CDHP binding to protein. CD and small angle X-ray scattering (SAXS) analyses were used to probe for structural changes due to the presence of CDHP. SAXS indicates charge effects on the surface of the protein play a role in protein stability in ionic liquids, and no significant alteration of the overall tertiary conformation of lysozyme was observed at 25 °C. However, after incubation at 37 °C or at higher concentrations of CDHP, small changes in protein structure were seen. Effects on protein activity were monitored using turbidity assays, and CDHP decreases protein activity but does not eliminate it. Protein solubility was also monitored using a turbidity assay and was found to be inversely proportional to the concentration of CDHP in solution.
离子液体作为稳定蛋白质和其他生物分子的有前途的介质，正受到广泛关注。磷酸二氢胆碱（CDHP）已被确定为该应用最有前途的候选物之一。在这项工作中，我们更详细地研究了 CDHP 可能对蛋白质（包括一种治疗性蛋白质）的热力学、结构和稳定性产生的影响。微量热法和圆二色光谱法（CD）用于评估蛋白质溶液在不同浓度的 CDHP/水中混合物中的热稳定性。观察到溶菌酶和白细胞介素-2在 CDHP 浓度下的热稳定性呈比例增加。等温滴定量热法（ITC）用于定量结合相互作用，并表明稳定性机制似乎不依赖于 CDHP 与蛋白质的结合。CD 和小角 X 射线散射（SAXS）分析用于探测由于 CDHP 的存在而导致的结构变化。CD 表明表面的电荷效应对蛋白质在离子液体中的稳定性起作用，并且在 25°C 时没有观察到溶菌酶整体三级构象的明显改变。然而，在 37°C 或更高浓度的 CDHP 孵育后，观察到蛋白质结构的微小变化。使用浊度测定法监测蛋白质活性的影响，发现 CDHP 降低了蛋白质的活性，但没有完全消除它。还使用浊度测定法监测蛋白质的溶解度，并发现其与溶液中 CDHP 的浓度成反比。
