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

Isothermal
titration calorimetry (ITC) is a method to determine
thermodynamic values (ΔG, ΔH, and ΔS) 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 KD. The KD 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.