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Date of Award
Doctor of Philosophy (PhD)
Paul L. Dubin
Peter C. Uden
Richard W. Vachet
Polyelectrolyte (PE)-protein interaction and its applications were investigated on protein aggregation and selective protein binding. More specifically, we studied the effect of protein charge anisotropy on the inhibition of protein aggregation and selective protein binding in the system with pure electrostatic interactions.
First of all, the aggregation mechanisms of three proteins (Zn/Zn-free insulin, bovine serum albumin (BSA), and β-lactoglobulin (BLG)) were studied by kinetic analysis of turbidimetric and dynamic light scattering (DLS) results. For insulin, zinc effects on multimerization and aggregation were also examined by size exclusion chromatography (SEC) and DLS. These proteins were found to undergo different aggregation mechanisms which results in different inhibition effects by bio-polyelectrolyte heparin under conditions of pH and ionic strength known to favor the formation of stable heparin-protein complexes through electrostatic interactions. The turbidimetric titration results in the presence of heparin reveal the heparin effects are dependent upon the heparin-protein binding affinity which is controlled by the charge anisotropy of proteins explored by DelPhi electrostatic modeling.
Secondly, the selective binding of protein isoforms (BLGA and B) were evaluated on different substrates to challenge the assumption that long range electrostatics lack of good selectivity as short range interactions. More specifically, we studied BLG A and B binding on different substrates with various geometries: cationic polymer, nanoparticle, and nanoparticle-coated surface by using a wide range of techniques including isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), turbidimetry, size exclusion chromatography (SEC), and ion exchange chromatography (IEC) etc. The selective protein binding by cationic PE was further demonstrated to be able to successfully enrich BLGA by a factor of 2 from normal BLG A/B mixture by PE coacervation, The relationship between protein charge anisotropy (patch) and binding affinity and between binding affinity and protein (BLGA and B) separation selectivity was elucidated by different separation techniques (SEC, IEC), ultrafiltration, and isothermal titration calorimetry (ITC).
Xu, Yisheng, "Selective Interactions of Proteins With Polyelectrolytes and Nanoparticles: Binding, Adsorption, Coacervation, and Inhibition of Aggregation" (2012). Doctoral Dissertations 1896 - February 2014. 398.