Protein adsorption to poly(ethylenimine)-modified Sepharose FF: VI. Partial charge neutralization drastically increases uptake rate
Protein adsorption to poly(ethylenimine)-modified Sepharose FF: VI. Partial charge neutralization drastically increases uptake rate
复制标题
蛋白质对聚（氮丙啶）修饰的 Sepharose FF 的吸附：VI。
DOI：
10.1016/j.chroma.2015.11.084
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发表时间：
2016
影响因子：
4.1
通讯作者：
Yan Sun
中科院分区：
文献类型：
--
作者：
Yangyang Zhao;Xiaoyan Dong;Linling Yu;Yan Sun
研究了聚乙烯亚胺（PEI）接枝Sepharose FF树脂对牛血清白蛋白（BSA）的吸附和洗脱行为，发现了一个临界离子容量（cIC; 600 mmol/L），超过此离子容量后，BSA的吸附速率因发生明显的“链传递”效应而急剧增加。此外，高于cIC值，由于树脂的高电荷密度，蛋白质洗脱需要更高的盐浓度。在这项工作中，我们已经降低了PEI接枝树脂的PEI链上的电荷密度，通过用乙酸钠中和胺基。以IC为740 mmol/L的PEI改性树脂（FF-PEI-L740，IC > cIC）为原料，合成了3种IC分别为660、560和440 mmol/L的树脂（FF-PEI-R440）。研究了这些树脂对牛血清白蛋白的吸附和色谱行为。结果表明，当IC从740降至440 mmol/L时，吸附容量基本不变，有效扩散系数（De）随IC从740降至560 mmol/L变化不大（De/D 0 = 0.38 ± 0.04）。然而，有趣的是观察到FF-PEI-R440的贬值增加了三倍（De/D 0 = 1.23 ± 0.08）。认为FF-PEI-R440摄取速率的急剧增加是由于蛋白质分子可利用的结合位点减少，导致结合强度降低，从而促进了结合蛋白的“链传递”效应的发生。此外，对离子强度影响的研究表明，IC值越低，蛋白质结合对盐浓度的敏感性越高，这是由于在低表面电荷密度下容易屏蔽静电相互作用。洗脱峰处的离子强度也随着IC的降低而降低，与盐敏感性顺序一致。柱穿透研究表明，FF-PEI-R440的动态吸附容量远高于其他三种树脂在流速高于30 cm/h，因为它的高吸收速率。本研究结果为蛋白质与接枝聚合物之间的相互作用对吸附平衡和吸收动力学的影响提供了新的见解，这将有助于选择和设计合适的高效蛋白质色谱介质。
The adsorption and elution behaviors of bovine serum albumin (BSA) on poly(ethylenimine) (PEI)-grafted Sepharose FF resins were recently studied and a critical ionic capacity (cIC; 600 mmol/L) was found, above which the uptake rate increased drastically due to the occurrence of significant “chain delivery” effect. Moreover, above the cIC value, higher salt concentrations were required for protein elution due to the high charge density of the resins. In this work, we have reduced the charge density on the PEI chains of a PEI-grafted resin by neutralization of the amine groups with sodium acetate. PEI-modified resin with IC of 740 mmol/L (FF-PEI-L740, IC > cIC) was chosen as the starting material, and three resins with residual IC values of 660, 560 and 440 mmol/L (FF-PEI-R440) were obtained. The adsorption and chromatographic behaviors of these resins for BSA were investigated. It was found that, with IC decreasing from 740 to 440 mmol/L, the adsorption capacity kept almost unchanged; the effective protein diffusivity (De) also showed negligible variations as IC decreased from 740 to 560 mmol/L (De/D0= 0.38 ± 0.04). However, it was interesting to observe a three-fold increase of theDevalue for FF-PEI-R440 (De/D0= 1.23 ± 0.08). It is considered that the occurrence of the drastic uptake rate increase in FF-PEI-R440 was attributed to the decreased available binding sites for protein molecule, which led to the decrease of binding strength, thus facilitated the happenings of “chain delivery” effect of bound proteins. Besides, a study on the effect of ionic strength clarified that the lower the IC value, the higher the sensitivity of protein binding to salt concentration due to the easily screened electrostatic interactions at low surface charge densities. The ionic strength at the elution peak also decreased with decreasing IC in accordance with the salt sensitivity order. Column breakthrough studies demonstrated that the dynamic adsorption capacity of FF-PEI-R440 was much higher than the other three resins at flow rates higher than 30 cm/h because of its high uptake rate. The findings in this work provided new insights into the effects of the interactions between proteins and grafted polymers on adsorption equilibria and uptake kinetics, which would help the selection and design of suitable media for high-performance protein chromatography.