Determination of Effector Binding Affinities Using Photoacoustic Calorimetry

Abstract

Determination of binding affinity and thermodynamic parameters of interactions between two molecules is a common procedure in biochemical contexts. Isothermal titration calorimetry and steady-state fluorescence titration are commonly used to determine the binding affinity of small effector compounds and ligands to large biomolecules such as proteins. These methods however have several disadvantages, such large quantity of protein in case of ITC and the presence of a fluorescence probe in case of fluorescence. Photoacoustic calorimetry (PAC) is often used to monitor volume and enthalpy change associated with a photo-triggered reaction. Here we have tested the application of PAC to measure equilibrium binding constant and thermodynamic parameters for small organic molecules binding to hemoglobin. This technique does not require a fluorescent probe to be present and instead takes advantage of the fact that the association of small organic molecules to hemoglobin modulates the amplitude of PAC signal for O2 photo-dissociation from this protein. From the temperature dependence of the equilibrium constants, the enthalpy changes were determined using Van’t Hoff plots. So far, we have determined the dissociation constant (KD) and enthalpy change (ΔH) for binding of inositol hexakisphosphate (IHP) (KD = 73.5 μM at 20 °C, ΔH = −9.8 kJ mol−1) and 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) (KD = 46.7 µM at 20 °C, ΔH = 13.2 kJ mol−1) to fully-oxygenated adult human hemoglobin; and the determined parameters are in excellent agreement with those published previously. Being able to determine the binding affinity and thermodynamic parameters for ligand binding to proteins is extremely useful for understanding the environment of the ligand binding site, potentially allowing us to create new and more effective drugs. The use of PAC may provide a new approach of evaluating ligand protein interactions quickly and with smaller quantities of protein.