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

Abstract

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.

Characterization of cross-linked cellulosic ion-exchange adsorbents: 2. Protein sorption and transport

“…Another noteworthy feature is the sharper decline of the maximum capacity for lactoferrin with increasing salt. This may reflect an exclusion effect similar to that seen on polymer-modified media for larger proteins (Bowes and Lenhoff, 2011a) or could be due, as above, to the surface charge distribution on lactoferrin, which may make it more susceptible to electrostatic screening (Kisley et al, 2014). …”

Pilot-scale purification of α-lactalbumin from enriched whey protein concentrate by anion-exchange chromatography and ultrafiltration

“…At neutral pH, β-Lg has a higher negative net charge than α-La (−17.6 and −15.6 for β-Lg A and β-Lg B, vs −3.7 for α-La; Lucas et al 1998). At lower concentrations of NaAc (0.20, Table 3 The volume fraction of particles with size between 0.1 and 1 μm in solutions with various concentrations of α-La-enriched whey protein concentrate ( Values are expressed as means±SD from three independent replicates Table 4 Purity of α-La, when using different NaAc concentrations in the α-La-enriched whey protein concentrate (αWPC) applied to the ion exchange column NaAc (mol.L , excess Ac − may compete with β-Lg for the binding sites, thus starting to displace β-Lg from the ion exchanger (Kisley et al 2014). A concentration of 0.3 mol.L −1 NaAc was then chosen for the pilot-scale separation.…”

Ensemble and single-molecule biophysical characterization of D17.4 DNA aptamer–IgE interactions

“…A home-built total internal reflectance fluorescence microscope that has been described previously [34,50] was used for fluorescence imaging. Briefly, the beam from a solid state 532 nm laser was used for excitation and passed through an acousto-optic modulator (AOM) controlled by frequency generators to synchronize excitation and detection rate enabling slower kinetics (otherwise masked by photobleaching of fluorophores) to be observable.…”

“…shorter time scales and free of ensemble averaging, as we have previously applied to aptamer and protein adsorption processes [33,64,65]. Fig.…”

“…The study of dynamic biological events at the nanoscale has been greatly advanced in recent years by the advent of single molecule spectroscopy (SMS). Recent advances in SMS have made it possible to discern individual dynamical events within complex biological processes such as binding [31][32][33][34] and structural fluctuations [35][36][37][38]. Single molecule imaging by Total Internal Reflectance Fluorescence (TIRF) microscopy and fluorescence resonance energy transfer are useful in measuring single association/dissociation events without the ensemble averaging that occurs in bulk measurements [39][40][41][42][43][44].…”