Isothermal Titration Calorimetry
Also known as: ITC, isothermal calorimetry, microcalorimetry
Isothermal Titration Calorimetry (ITC) is a thermodynamic technique that measures heat released or absorbed during biomolecular binding events at constant temperature. Developed by Wiseman and colleagues in 1989, ITC directly determines binding affinity (Kd), enthalpy (ΔH), and entropy (ΔS) in a single experiment, making it one of the most comprehensive methods for characterizing molecular interactions without requiring labels or immobilization.
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When to use it
Apply ITC to characterize any bimolecular interaction: protein-ligand, protein-protein, protein-DNA, DNA-RNA, etc. ITC is optimal when Kd is in the micromolar to picomolar range (tighter binding requires very dilute solutions; weaker binding requires high concentrations). ITC does not require labels and directly measures thermodynamic parameters. Assumes binding reaches equilibrium within the experimental timescale.
Strengths & limitations
- Label-free: no fluorescent or radioactive tags needed; detects binding heat directly
- Comprehensive thermodynamic information: simultaneously measures Kd, ΔH, and ΔS from one titration
- Model-independent: does not assume a binding model; data often reveal binding behavior not apparent from kinetics alone
- Works for any binding interaction, including weak, non-specific interactions
- Slow kinetics: each titration point takes several minutes; a complete experiment may require 1-2 hours
- Sensitivity limited at very tight binding (Kd < 1 nM): requires extremely dilute solutions, increasing practical uncertainty
- Very weak binding (Kd > 1 mM): requires impractically high concentrations and large heats are difficult to measure precisely
- Aggregated or heterogeneous samples produce spurious heat and confound interpretation
Frequently asked
What does a negative ΔH mean in ITC?
Negative ΔH means the binding reaction releases heat (exothermic). This is typical for most protein-ligand interactions where favorable electrostatic and van der Waals interactions dominate. Positive ΔH indicates endothermic binding, less common but can occur in entropy-driven interactions.
How is Kd determined from ITC data?
The peak heat of each injection is plotted versus molar ratio (ligand/protein). Titration curves flatten as saturation is approached. The curve is fitted to a binding model; the Kd is the ligand concentration at which binding is 50% saturated. The shape and position of the titration curve reveal Kd and binding cooperativity.
Why is the first injection often discarded in ITC?
The first small injection (typically 2 microL) mixes poorly due to geometry of the cell and syringe. This creates anomalous heat. All subsequent 10 microL injections mix reproducibly, so data from injection 2 onward are typically used for analysis.
Sources
- Wiseman, T., Williston, S., Brandts, J. F., & Lin, L. N. (1989). Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Analytical Biochemistry, 179(1), 131-137. DOI: 10.1016/0003-2697(89)90213-3 ↗
- Garbett, N. C., & Chaires, J. B. (2012). Binding: A statistical thermodynamic model. Biophysical Journal, 86(6), 3493-3494. link ↗
How to cite this page
ScholarGate. (2026, June 3). Isothermal Titration Calorimetry. ScholarGate. https://scholargate.app/en/spectroscopy/isothermal-titration-calorimetry
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