Browsing by Author "Kourentzi, Katerina"

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    Fluorescence Correlation Spectroscopy Study of Protein Transport and Dynamic Interactions with Clustered-Charge Peptide Adsorbents
    (Wiley-Blackwell, 2012-08) Daniels, Charlisa R.; Kisley, Lydia; Kim, Hannah; Chen, Wen-Hsiang; Poongavanam, Mohan- Vivekanandan; Reznik, Carmen; Kourentzi, Katerina; Willson, Richard C.; Landes, Christy F.
    Ion-exchange chromatography (IEX) relies on electrostatic interactions between the adsorbent and the adsorbate, and is used extensively in protein purification. Conventional IEX utilizes ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clusteredcharge adsorbent surfaces using Fluorescence Correlation Spectroscopy. We can resolve protein free diffusion, hindered diffusion and association with bare glass, agarose-coated, and agaroseclustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered charge adsorbent and other surface interactions at the molecular scale.
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    High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study
    (Elsevier, 2014) Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Dominguez-Medina, Sergio; Kulla, Eliona; Kang, Marci; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.
    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 ?-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.