Enhanced Protein Affinity and Selectivity of Clustered-Charge Anion-Exchange Adsorbents

Abstract

In this work, we examined the possibility of improving ion-exchange adsorbent performance by nanoscale structuring of ligands into clusters of fixed size rather than a random distribution of individual charges. The calcium-depleted form of the protein alpha-lactalbumin, which displays a cluster of acidic amino acid residues, showed enhanced adsorption affinity and capacity on clustered-charge pentalysinamide and pentaargininamide adsorbents as compared to single-charge lysinamide and argininamide adsorbents of matched total charge. Two differently charge-clustered mutants of rat microsomal cytochrome b(5), E11Q and E44Q, with the same total charge also were well differentiated by clustered-charge adsorbents. Thus, an organized rather than random distribution of charges may produce adsorbents with higher capacity and selectivity, especially for biomolecules with inherent charge clustering.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

“…Controls (SI Appendix, Fig. S3) showed that specific interactions were electrostatic, as expected for ion-exchange chromatography; this conclusion is further supported by the complete elution of α-lactalbumin from pentaargininamide adsorbents by 1 M NaCl in our previously reported ensemble studies of these systems (9). Nonspecific interactions with the porous agarose interface (distinguishable by statistical analysis as mentioned above) were observed with all supports because, at the low ligand loading used to achieve single-molecule-appropriate conditions, the majority of the surface is bare agarose.…”

“…Mechanistic detail is lost in ensemble analyses, reflecting the inherent heterogeneity of both the adsorbed biomolecules and the porous stationary phase supports (7). Ensemble adsorption isotherms, however, suggest the likelihood that protein and nucleic acid separations in ion-exchange columns may involve random ligand clustering (8)(9)(10). Additional support for such an assertion lies in the implementation of stationary phases of very high charge density by polymerization of charged monomers or layer-by-layer deposition (11)(12)(13), and in the demonstration that patches of high charge density on proteins often play a disproportionate role in their adsorption (4,6,(14)(15)(16)(17).…”

Biophysical characterization of DNA and RNA aptamer interactions with hen egg lysozyme

“…Jennissen and Heilmeyer [36] have shown that protein adsorption on hydrophobic interaction chromatography resins is observed only if the required critical hydrophobicity of the adsorbent for that protein is achieved, by use of larger clusters of hydrophobic alkyl groups on the resin. We have recently demonstrated superior performance of ion-exchange adsorbents synthesized so as to have clusters of charged groups, rather than individual charges (at the same total charge concentration) disposed in a randomly dispersed fashion [37]. In this regard, some aptamers could be envisaged as a cluster of anionic charges with strong general affinity towards cationic molecules, but with additional modest specificity for their target molecule.…”

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

“…Adsorbent heterogeneity can arise from a range of potential causes, including steric accessibility and constraint, surface entropy and mobility [13], and the stochastic clustering of ligands on the adsorbent surface [21]. We have found that while adsorbents based on pre-organized, penta-valent clustered-charge ligands show higher protein affinity and capacity than adsorbents of the same total density of charge randomly distributed, isotherms for protein adsorption even on these nominally-homogeneous adsorbents show heterogeneity [12]. This observation further implicates adsorbent steric and surface mobility properties as a ubiquitous source of heterogeneity; especially as our recent single-molecule observations highlighted the role of both ligand clustering and steric availability with the porous support [21] .…”

“…Ionic strength and (less frequently) pH often are used to tune adsorption characteristics [22], as they can influence the retention, resolution, and recovery of biomolecules [5-15] and can often provide non-denaturing elution. As discussed below, the observed heterogeneity often is lower at less-adsorptive conditions of higher ionic strength, as inferred from adsorption isotherm fit parameters [12,13]. Despite extensive and careful study, only limited mechanistic explanations of adsorption heterogeneity and its modulation by ionic strength are available.…”