Hackemann Eva, Hasse Hans
Laboratory of Engineering Thermodynamics (LTD), University of Kaiserslautern, Kaiserslautern, Germany.
Laboratory of Engineering Thermodynamics (LTD), University of Kaiserslautern, Kaiserslautern, Germany.
J Chromatogr A. 2017 Oct 27;1521:73-79. doi: 10.1016/j.chroma.2017.09.024. Epub 2017 Sep 14.
Using salt mixtures instead of single salts can be beneficial for hydrophobic interaction chromatography (HIC). The effect of electrolytes on the adsorption of proteins, however, depends on the pH. Little is known on that dependence for mixed electrolytes. Therefore, the effect of the pH on protein adsorption from aqueous solutions containing mixed salts is systematically studied in the present work for a model system: the adsorption of bovine serum albumin (BSA) on the mildly hydrophobic resin Toyopearl PPG-600M. The pH is adjusted to 4.0, 4.7 or 7.0 using 25mM sodium phosphate or sodium citrate buffer. Binary and ternary salt mixtures of sodium chloride, ammonium chloride, sodium sulfate and ammonium sulfate as well as the pure salts are used at overall ionic strengths between 1500 and 4200mM. The temperature is always 25°C. The influence of the mixed electrolytes on the adsorption behavior of BSA changes completely with varying pH. Positive as well as negative cooperative effects of the mixed electrolytes are observed. The results are analyzed using a mathematical model which was recently introduced by our group. In that model the influence of the electrolytes is described by a Taylor series expansion in the individual ion molarities. After suitable parametrization using a subset of the data determined in the present work, the model successfully predicts the influence of mixed electrolytes on the protein adsorption. Furthermore, results for BSA from the present study are compared to literature data for lysozyme, which are available for the same adsorbent, temperature and salts. By calculating the ratio of the loading of the adsorbent for both proteins particularly favorable separation conditions can be selected. Hence, a model-based optimization of solvents for protein separation is possible.
使用盐混合物而非单一盐类对疏水作用色谱法（HIC）可能有益。然而，电解质对蛋白质吸附的影响取决于pH值。对于混合电解质的这种依赖性，人们了解甚少。因此，在本研究中，针对一个模型体系系统地研究了pH值对含混合盐水溶液中蛋白质吸附的影响：牛血清白蛋白（BSA）在轻度疏水树脂Toyopearl PPG - 600M上的吸附。使用25mM磷酸钠或柠檬酸钠缓冲液将pH值调节至4.0、4.7或7.0。使用氯化钠、氯化铵、硫酸钠和硫酸铵的二元和三元盐混合物以及纯盐，总离子强度在1500至4200mM之间。温度始终为25°C。混合电解质对BSA吸附行为的影响随pH值变化而完全改变。观察到混合电解质的正协同效应和负协同效应。使用我们小组最近引入的一个数学模型对结果进行分析。在该模型中，电解质的影响通过各离子摩尔浓度的泰勒级数展开来描述。使用本研究中确定的数据子集进行适当参数化后，该模型成功预测了混合电解质对蛋白质吸附的影响。此外，将本研究中BSA的结果与溶菌酶的文献数据进行比较，溶菌酶的这些数据可用于相同的吸附剂、温度和盐类。通过计算两种蛋白质在吸附剂上的负载量之比，可以选择特别有利的分离条件。因此，基于模型对蛋白质分离溶剂进行优化是可行的。
