1. Overview
Isothermal Titration Calorimetry (ITC) is a highly sensitive, label-free analytical technique that directly measures the heat released or absorbed during molecular interactions. ITC provides a complete thermodynamic characterization of binding events, including the binding constant (Ka), enthalpy change (ΔH), entropy change (ΔS), Gibbs free energy (ΔG⁰), and binding stoichiometry (n). Because it does not require molecular labeling or immobilization, ITC is widely regarded as a gold-standard method for quantitative analysis of biomolecular interactions in solution.
Features
2. Principle
All molecular interactions possess a unique thermodynamic signature defined by four key parameters: binding constant (Ka), enthalpy (ΔH), entropy (ΔS), and stoichiometry (n). ITC directly measures the heat change associated with these interactions under isothermal conditions.
During an ITC experiment, small aliquots of a titrant (e.g., ligand) are injected into a sample cell containing the analyte (e.g., protein). Each injection produces a heat pulse corresponding to the binding reaction between the two molecules. The cumulative heat signal reflects the progression of binding until saturation is reached.
Key Thermodynamic Relationships
-
Gibbs Free Energy
ΔG⁰ = –RT ln Ka -
Entropy
ΔG⁰ = ΔH – TΔS
→ ΔS = (ΔH – ΔG⁰) / T -
Dissociation Constant
Kd = 1 / Ka
Together, these parameters provide a complete thermodynamic description of molecular binding and reveal whether the interaction is driven primarily by enthalpy, entropy, or a combination of both
3. Data Interpretation
The raw ITC output consists of a series of heat flow peaks following each titrant injection.
- Peak Integration
The area under each heat flow peak is integrated to obtain the heat released or absorbed per injection. - Binding Isotherm Construction
Integrated heat values are plotted as a function of the molar ratio of titrant to analyte. - Model Fitting
The binding isotherm is fitted using an appropriate binding model (commonly a single-site or independent-site model) to extract: - Binding enthalpy (ΔH)
- Binding constant (Ka)
- Stoichiometry (n)
- Thermodynamic Analysis
Using ΔH and Ka, ΔG⁰ and ΔS are calculated, enabling interpretation of the molecular driving forces underlying the interaction.
4. Example Applications
ITC is widely used in biochemical, biophysical, and pharmaceutical research due to its quantitative and label-free nature.
Typical Applications Include:
- Drug discovery and validation (ligand–target binding affinity and thermodynamics)
- Protein–protein and protein–nucleic acid interaction studies
- Characterization of molecular variants, mutants, or binding-site modifications