Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, NY 12180, USA.
J Chromatogr A. 2010 Nov 19;1217(47):7393-400. doi: 10.1016/j.chroma.2010.09.052. Epub 2010 Sep 29.
Urea is widely employed to facilitate protein separations in ion exchange chromatography at various scales. In this work, five model proteins were used to examine the chromatographic effects of protein conformational changes induced by urea in ion exchange chromatography. Linear gradient experiments were carried out at various urea concentrations and the protein secondary and tertiary structures were evaluated by far UV CD and fluorescence measurements, respectively. The results indicated that chromatographic retention times were well correlated with structural changes and that they were more sensitive to tertiary structural change. Steric Mass Action (SMA) isotherm parameters were also examined and the results indicated that urea induced protein conformational changes could affect both the characteristic charge and equilibrium constants in these systems. Dynamic light scattering analysis of changes in protein size due to urea-induced unfolding indicated that the size of the protein was not correlated with SMA parameter changes. These results indicate that while urea-induced structural changes can have a marked effect on protein chromatographic behavior in IEX, this behavior can be quite complicated and protein specific. These differences in protein behavior may provide insight into how these partially unfolded proteins are interacting with the resin material.
尿素在各种规模的离子交换色谱中被广泛用于促进蛋白质分离。在这项工作中，使用了五种模型蛋白来研究尿素在离子交换色谱中诱导的蛋白质构象变化对色谱的影响。在不同的尿素浓度下进行线性梯度实验，通过远紫外 CD 和荧光测量分别评估蛋白质的二级和三级结构。结果表明，色谱保留时间与结构变化密切相关，且对三级结构变化更为敏感。还研究了立体质量作用（SMA）等温线参数，结果表明，尿素诱导的蛋白质构象变化会影响这些体系中的特征电荷和平衡常数。由于尿素诱导的去折叠引起的蛋白质大小变化的动态光散射分析表明，蛋白质的大小与 SMA 参数变化无关。这些结果表明，虽然尿素诱导的结构变化会对 IEX 中的蛋白质色谱行为产生显著影响，但这种行为可能非常复杂且具有蛋白质特异性。这些蛋白质行为的差异可能为我们了解这些部分展开的蛋白质与树脂材料的相互作用提供了线索。
