High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study

摘要: 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 apparent heterogeneity lower at more-eluting, higher ionic strength. Here, we present an investigation into mechanism this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging Nanoscale Topography (mbPAINT). We observed number functional sites was smaller high strength these had reduced desorption kinetic heterogeneity, thus narrower predicted profiles, anion-exchange α-lactalbumin on agarose-supported, clustered-charge ligand stationary phase. Explanations narrowing population such inter-protein interactions protein or support structural changes were investigated through analysis, circular dichroism spectroscopy, microscopy agarose microbeads, respectively. results suggest reduction due to both electrostatic screening between tuning steric availability within support. Overall, have shown single molecule spectroscopy can aid understanding influence adsorbent participating chromatographic separation proteins.