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Document Type

Campus-Only Access for Five (5) Years

Degree Name

Doctor of Philosophy (PhD)

Degree Program


Year Degree Awarded


Month Degree Awarded


First Advisor

Paul L. Dubin

Second Advisor

Michael J. Knapp

Third Advisor

Igor A. Kaltashov

Fourth Advisor

Sarah L. Perry

Subject Categories

Analytical Chemistry | Physical Chemistry | Polymer Chemistry


Protein charge anisotropy results from the asymmetric distribution of charged residues on the exterior of a particular protein. Interactions between proteins and other macromolecules can be described in terms of attractive electrostatics; since electrostatic free energies, at optimal I, are on the order of kT, it is unlikely that such associations would result in desolvation, thus it is reasonable to consider such intermolecular attractions as being mediated by hydrated protein surfaces. Such interactions can be broken down in terms of a single protein interacting with a range of “binding partners”, including (1) protein-protein interactions, (2) protein-polymer interactions, and (3) protein-surface interactions. Protein-protein interactions can be divided into two types, self-interaction, where two monomers of the same protein interact as is the case with either multimerization or aggregation; or heteroprotein interactions where two different proteins associate, such as is seen with heteroprotein coacervation. Protein-polyelectrolyte (PE) interactions, have been shown to result in formation of either soluble complexes, or in the case of protein-PE coacervation, liquid-liquid phase separation (LLPS). The final case becomes useful when examining non-ideal chromatographic behavior of proteins, where interactions between protein and stationary phase can alter elution times, relative to those seen for the purely hydrodynamic (or ideal) case. In all cases it is the hydrated protein that is key to such interactions.

The aggregation of model protein β-lactoglobulin (BLG) near its isoelectric point was studied as a function of ionic strength and pH, where rates of aggregation were obtained through a highly precise and convenient pH/turbidimetric titrations. A similar titration procedure was used to obtain self-association rates of the more pharmaceutically relavent protein, monoclonal antibody (mAb), at low temperature. Antibody (mAb) centrifuged at T < 0oC underwent LLPS upon thawing. The dense-phase was clearly identified, and a sharp interface between mAb-enriched and mAb-depleted phases was confirmed. In cases where LLPS was induced in the presence of a dye-labeled protein of opposite charge to mAb, the two proteins were colocalized in the lower phase.

Heteroprotein coacervation of BLG and lactoferrin (LF) was examined by SANS and rheology; the molar stoichiometry in coacervate has been reported to be LF(BLG2)2 assumed to be the primary structural unit of the coacervate. Surface-bound water in protein solutions was identified by a reduction in the heat of freezing of various coacervating systems, with a specific focus on non-freezing water (NFW) in protein-protein (heteroprotein) coacervates. These results are attributed to maximization of water-protein contacts, structural features that reflect the mode of sample assembly, as they are not seen in a non-coacervated LF-BLG solution with identical concentrations of all species.

A method was developed in order to determine the yield and selectivity of coacervates prepared from monoclonal antibody (mAb) and anionic polysaccharide hyaluronic acid (HA). The yield of mAb, in such coacervates, is shown to be as high as 80%, with final concentrations of MAb-A > 170 mg/mL; the yield of HA is ca. 75%; and a selectivity ratio, S, of ca. 490 was obtained for coacervates prepared at pHc BSA < pH < pHφmAb. Values corresponding to the start of complexation, and coacervation (pHc, pHφ) are reported over a range of ionic strengths, I <200mM.

Monoclonal antibody (mAb), a protein with a highly patchy (or anisotropic) surface, is shown to interact anomalously with chromatography columns at low I. Absolute molecular weight (MW) determination vis-à-vis light scattering detection, suggests that late eluting peaks, are larger in aggregation number than monomeric antibody. Such results suggest that despite the lower apparent size obtained from column calibration, chemical degradation cannot be responsible for these types of delayed elution volumes. This demonstrates the importance of equilibrium controlled protein-protein and protein-surface interactions even in single protein systems.

Protein self-interactions result in a type of LLPS with features similar to coacervation, but over a much less restricted range of conditions. Heteroprotein-interactions introduce a structural constraint that is reflected in a highly constrained set of conditions for heteroprotein coacervation. Protein-PE coacervates can be formed over a much more extended range of pH and I, possible due to the greater flexibility of the PE chain; and finally, direct interactions with chromatography columns may involve attractive electrostatic interactions, analogous to those seen in coacervating systems.