Direct Determination of High-Affinity Binding Constants by Continuous Injection Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is a method to determine thermodynamic values (Δ<i>G</i>, Δ<i>H</i>, and Δ<i>S</i>) for ligand–receptor binding in biological and abiological systems. It is challenging to directly determine subnanomolar dissociation constants using a standard incremental injection approach ITC (IIA-ITC) measurement. We recently demonstrated a continuous injection approach ITC (CIA-ITC) [J. Phys. Chem. B 2021, 125, 8075–8087]enables the estimation of thermodynamic parameters in situ. In this work, we demonstrate a label-free and surface modification-free CIA-ITC to determine the complete binding thermodynamics of a ligand with a subnanomolar dissociation constant <i>K</i>_D. The <i>K</i>_D for desthiobiotin (DTB)–avidin binding was determined to be 6.5 pM with respect to the ligand by CIA-ITC, a quantity unsuccessfully measured with IIA-ITC and surface plasmon resonance spectroscopy (SPR). This value compares well with literature-reported spectroscopic determination of DTB–avidin binding. Criteria with respect to the concentration of the ligand and receptor and flow rate for obtaining true equilibrium dissociation constants without displacement titration are presented.