We are employing single-molecule methods to study the complex mechanistic details involved in protein chromatography.  Using fluorescence correlation spectroscopy, we observed differences of a-lactalbumin diffusion over glass, agarose, and charge-functionalized agarose. More recently, we have directly observed the adsorption/desorption dynamics of a-lactalbumin on agarose thin-film anion-exchange adsorbents using single-molecule super-resolution fluorescent imaging. We had hypothesized ion-exchange separations on classical ion-exchange matrices are dominated by adsorption to stochastically clustered sites with minimal contribution of the isolated charges, and we demonstrated that clustered-charge penta-argininamide adsorbents selectively favor biomolecules with inherent charge clustering, such as a-lactalbumin.  The obtained single molecule kinetics reveal that charge clustering greatly enriches events with long dwell times of protein on adsorbent.  The results of such studies could be used to improve adsorbent properties through better understanding of chromatographic transport, and to support a molecular-theoretic model of protein chromatography.

Studying protein transport and adsorption/desorption kinetics on ion-exchange adsorbents using a single-molecule approach

245th National Meeting (2013)

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