Differential Binding Models for Direct and Reverse Isothermal Titration Calorimetry.

Area of Science:

• Biophysical Chemistry
• Thermodynamics
• Analytical Chemistry

Background:

• Isothermal titration calorimetry (ITC) is crucial for measuring binding stoichiometry and thermodynamics.
• Evaluating titration curves for receptors with multiple binding sites is complex, especially without prior knowledge of stoichiometry or binding mechanisms.
• A previous theoretical study introduced a differential binding model (DBM) to analyze calorimetry titrations independently of site interactions.

Purpose of the Study:

• To demonstrate the practical application of the differential binding model (DBM) for evaluating calorimetry titrations of multi-site receptors.
• To enable analysis independent of the titration direction (direct or reverse).
• To provide a method for calculating equilibrium concentrations and heat signals using ordinary differential equations (ODEs).

Main Methods:

• Developed a set of ordinary differential equations (ODEs) with the general form d[S]/dV for numerical integration.
• Calculated equilibrium concentrations of free and bound species at each injection step.
• Applied the updated DBM with global regression analysis to direct and reverse calorimetric titrations of gadolinium ions with multidentate ligands (DGA, CIT, NTA).

Main Results:

• The updated DBM successfully evaluated direct and reverse calorimetry titrations of gadolinium ions with varying denticity ligands.
• Identified factors influencing titration curve shape, enabling optimization of titrant and analyte concentrations.
• Statistical tests validated the determined stoichiometries for the studied metal-ligand pairs.

Conclusions:

• The enhanced differential binding model (DBM) provides a flexible and robust approach for analyzing complex binding events in calorimetry titrations.
• The model's ability to handle multi-site interactions and varying titration directions simplifies thermodynamic and stoichiometric analysis.
• This method offers a valuable tool for researchers studying molecular interactions without pre-defined binding mechanisms.