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

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dc.citation.firstpage135en_US
dc.citation.journalTitleJournal of Chromatography Aen_US
dc.citation.lastpage142en_US
dc.citation.volumeNumber1343en_US
dc.contributor.authorKisley, Lydiaen_US
dc.contributor.authorChen, Jixinen_US
dc.contributor.authorMansur, Andrea P.en_US
dc.contributor.authorDominguez-Medina, Sergioen_US
dc.contributor.authorKulla, Elionaen_US
dc.contributor.authorKang, Marcien_US
dc.contributor.authorShuang, Boen_US
dc.contributor.authorKourentzi, Katerinaen_US
dc.contributor.authorPoongavanam, Mohan-Vivekanandanen_US
dc.contributor.authorDhamane, Sagaren_US
dc.contributor.authorWillson, Richard C.en_US
dc.contributor.authorLandes, Christy F.en_US
dc.date.accessioned2015-07-10T15:47:01Zen_US
dc.date.available2015-07-10T15:47:01Zen_US
dc.date.issued2014en_US
dc.description.abstractThe 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 ?-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.en_US
dc.identifier.citationKisley, Lydia, Chen, Jixin, Mansur, Andrea P., et al.. "High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study." <i>Journal of Chromatography A,</i> 1343, (2014) Elsevier: 135-142. http://dx.doi.org/10.1016/j.chroma.2014.03.075.en_US
dc.identifier.doihttp://dx.doi.org/10.1016/j.chroma.2014.03.075en_US
dc.identifier.urihttps://hdl.handle.net/1911/80885en_US
dc.language.isoengen_US
dc.publisherElsevieren_US
dc.rightsThis is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by Elsevier.en_US
dc.subject.keywordmbPAINTen_US
dc.subject.keywordbioseparationsen_US
dc.subject.keywordion-exchangeen_US
dc.subject.keywordheterogeneityen_US
dc.subject.keywordoptical nanoscopyen_US
dc.titleHigh ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule studyen_US
dc.typeJournal articleen_US
dc.type.dcmiTexten_US
dc.type.publicationpost-printen_US
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