Ligand binding constants for human serum albumin evaluated by ratiometric analysis of DSC thermograms

Abstract

This paper describes an expanded application of our recently reported method (Eskew et al., Analytical Biochemistry 621,1 2021) utilizing thermogram signals for thermal denaturation measured by differential scanning calorimetry. Characteristic signals were used to quantitatively evaluate ligand binding constants for human serum albumin. In our approach the ensemble of temperature dependent calorimetric responses for various protein-ligand mixtures and native HSA were compared, in a ratiometric manner, to extract binding constants and stoichiometries. Protein/ligand mixtures were prepared at various ligand concentrations and subjected to thermal denaturation analysis by calorimetry. Measurements provided the melting temperature, Tm, and free-energy ΔGcal(37°C) for melting ligand-bound Albumin as a function of ligand concentration. Concentration dependent behaviors of these parameters derived from protein/ligand mixtures were used to construct dose-response curves. Fitting of dose-response curves yielded quantitative evaluation of the ligand binding constant and semi-quantitative estimates of the binding stoichiometry. Many of the ligands had known binding affinity for Albumin with binding constants reported in the literature. Evaluated binding parameters for the ligands impressively agreed with reported literature values determined using other standard experimental methods. Results are reported for 29 drug ligands binding to Albumin. These validate our calorimetry-based process for applications in pre-clinical drug screening.