Document Type
Article
Publication Date
5-23-2014
Abstract
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 called motion-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. © 2014 Elsevier B.V.
Keywords
bioseparations, heterogeneity, ion-exchange, mbpaint, optical nanoscopy
Language
English
Publication Title
Journal of Chromatography A
Grant
CBET-1134417
Rights
© 2014 Elsevier B.V. This peer reviewed Accepted Manuscript version and is published under a Creative Commons CC-BY-NC-ND licence (https://creativecommons.org/licenses/BY-NC-ND/4.0/), which permits non-commercial copying and redistribution of the material in any medium or format, provided the original work is not changed in any way and is properly cited.
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Kisley, L., Chen, J., Mansur, A. P., Dominguez-Medina, S., Kulla, E., Kang, M. K., ... & Landes, C. F. (2014). High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study. Journal of Chromatography A, 1343, 135-142.
Manuscript Version
Accepted Manuscript