First Advisor

Albert S. Benight

Term of Graduation

Winter 2021

Date of Publication


Document Type


Degree Name

Doctor of Philosophy (Ph.D.) in Chemistry






Ligand binding (Biochemistry), Serum albumin, Proteins -- Stability, Thermodynamics, Temperature measuring instruments, Calorimetry



Physical Description

1 online resource (xv, 342 pages)


Protein structure is integral to its function. For the past 70 years differential scanning calorimetry has been used to measure protein structural stability. More recently it has been used to study macromolecular interactions. Interactions between proteins and ligands can manifest on differential scanning calorimetry melting curves or thermograms. Utilizing differential scanning calorimetry thermograms to detect or diagnose diseases has been a major goal in disease diagnostics. However, correlating specific ligand-protein interactions, as manifested in a thermogram, with a disease-specific plasma thermogram, has proven elusive.

Modified human serum albumin was utilized to develop a process to capture and retrieve ligands from plasma. This process was demonstrated for two ligands that bind human serum albumin and subsequently perturb plasma thermograms. Human serum albumin was covalently modified by attachment of biotin to lysine residues. An investigation was performed to determine if modifications of human serum albumin affected the ability of the protein to bind ligands, in order to design better capture reagents.

An analytical differential scanning calorimetry method was devised for determining, quantitatively accurate, ligand binding constants to human serum albumin. This method was used to demonstrate that modifications of human serum albumin, either structurally by pH changes or covalently with attachment of biotin, significantly reduced the ability of albumin to bind ligands. To further validate our method, an additional 29 drug/ligands were examined to determine their binding constants to human serum albumin.

Drug/ligands were chosen to represent a wide variety of compound classes, including examples of regiochemical and functional derivatives. Within that group of compounds are those that are insoluble or sparingly soluble in aqueous solvent. A technique was conceived and implemented that allowed for the insoluble compounds to be analyzed in aqueous media without the use of any organic solvents. In all cases excellent agreement was found between measured binding constants and those reported in literature. From measurements of binding constants it was also possible to determine semi-quantitative ligand binding stoichiometries. The ability to determine binding constants and stoichiometries in a single set of experiments is unique among available techniques.

Discoveries that were made while developing capture reagents yielded novel insights into the link between ligand binding and global structural stability of human serum albumin. These insights, along with the new techniques that were developed, have broader applications than merely assaying ligand binding to human serum albumin. We have demonstrated applications of our methods to analyze ligand binding in complex mixtures of expressed proteins. An extension of this work would be to apply these methods in investigating viral infections in complex fluids.


© 2021 Matthew Walter Eskew

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