High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: a single-molecule study.
High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: a single-molecule study.
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高离子强度缩小了参与蛋白质离子交换吸附的位点数量：单分子研究。
DOI：
10.1016/j.chroma.2014.03.075
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Landes,ChristyF
中科院分区：
文献类型：
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作者：
Kisley,Lydia;Chen,Jixin;Mansur,AndreaP;Dominguez-Medina,Sergio;Kulla,Eliona;Kang,MarciK;Shuang,Bo;Kourentzi,Katerina;Poongavanam,Mohan-Vivekanandan;Dhamane,Sagar;Willson,RichardC;Landes,ChristyF
通过调节移动的相盐浓度，常规控制离子交换色谱中蛋白质的保留和洗脱。从吸附等温线判断，已经反复观察到，在洗脱较多、离子强度较高时，吸附的表观不均匀性较低。在这里，我们提出了一个调查这种现象的机制，使用单分子，超分辨率成像技术，称为运动模糊点累积成像在纳米尺度地形（mbPAINT）。我们观察到，在高离子强度下，功能性吸附位点的数量较少，并且这些位点具有降低的解吸动力学不均匀性，因此对于α-乳清蛋白在琼脂糖支撑的电荷簇配体固定相上的阴离子交换吸附，预测洗脱曲线较窄。通过动力学分析、圆二色谱和琼脂糖微球显微镜分别研究了功能群体缩小的解释，如蛋白质间相互作用和蛋白质或支持物结构的变化。结果表明，异质性的降低是由于蛋白质和配体之间的静电屏蔽和调整琼脂糖载体内的空间可用性。总体而言，我们已经表明，单分子光谱可以帮助了解离子强度对参与蛋白质离子交换色谱分离的功能吸附剂位点的群体的影响。
The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique calledmotion-blur Points Accumulation for Imaging in Nanoscale Topography(mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.