TY - JOUR
T1 - High ionic strength narrows the population of sites participating in protein ion-exchange adsorption
T2 - A single-molecule study
AU - Kisley, Lydia
AU - Chen, Jixin
AU - Mansur, Andrea P.
AU - Dominguez-Medina, Sergio
AU - Kulla, Eliona
AU - Kang, Marci K.
AU - Shuang, Bo
AU - Kourentzi, Katerina
AU - Poongavanam, Mohan Vivekanandan
AU - Dhamane, Sagar
AU - Willson, Richard C.
AU - Landes, Christy F.
N1 - Funding Information:
C.F.L. thanks the NSF [ CBET-1134417 ] and [ CHE-1151647 ]; the Welch Foundation [ C-1787 ], and the NIH [ GM94246-01A1 ], for support of this work. R.C.W. thanks the NSF [ CBET-1133965 ] and the Welch Foundation [ E-1264 ]. L. K. thanks the NSF for Graduate Research Fellowship 0940902. We thank S. Link and research group for discussions.
PY - 2014/5/23
Y1 - 2014/5/23
N2 - 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.
AB - 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.
KW - Bioseparations
KW - Heterogeneity
KW - Ion-exchange
KW - MbPAINT
KW - Optical nanoscopy
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U2 - 10.1016/j.chroma.2014.03.075
DO - 10.1016/j.chroma.2014.03.075
M3 - Article
C2 - 24751557
AN - SCOPUS:84899970862
SN - 0021-9673
VL - 1343
SP - 135
EP - 142
JO - Journal of Chromatography A
JF - Journal of Chromatography A
ER -