Chapter 5
Isothermal Titration Calorimetry
Christopher M. Johnson
Abstract
Calorimetry is a classical biophysical method that by definition measures heat. In isothermal titration
calorimetry (ITC), the heat is the result of titrating interacting components together and allows direct
determination of the thermodynamics for this process. The measured heat reflects the enthalpy change
(ΔH ), and the prospect of determining this in biological systems where high-resolution structural information is available has led to the possibility of rational thermodynamics-guided design of ligands. Although
there are limitations to this approach due to the participation of solvent in the thermodynamics, ITC has
become an established technique in many labs providing a valuable tool with which to quantify protein–protein interactions. With careful use, ITC can also provide additional insights into the binding process or
be used in increasingly complex systems and where interaction is coupled to other molecular events.
Key words Isothermal titration calorimetry, ITC, Thermodynamics, Enthalpy, Entropy, Free energy,
Dissociation constant, Heat capacity, Stoichiometry, Binding affinity, Binding kinetics
1 Introduction
1.1 ITC: A
Measurement
Nirvana?
Many techniques for studying protein–ligand interactions discussed
elsewhere in this volume depend on reporter signals, such as fluorescence or absorbance, which change as a result of complex formation. Sometimes these changes can be a rather indirect consequence
of the binding event. Other techniques, such as fluorescence polarization, light scattering, or surface-based sensors using SPR or BLI,
report on a change in physical properties such as mass or size during
complex formation. Changes in these properties that produce adequate signal-to-noise measurements can often be large when compared to the changes expected for protein–ligand interactions.
In contrast, calorimetry simply measures directly the heat associated with making and breaking interactions that are intrinsic to
complex formation irrespective of any change in size or mass and in
the absence of any additional reporter labels required to give suitable spectroscopic properties. It is this “directness” of working with
unmodified materials and the ubiquitous nature of the predominantly non-covalent forces, with their associated heats, that drive
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
135
Isothermal Titration Calorimetry
Christopher M. Johnson
Abstract
Calorimetry is a classical biophysical method that by definition measures heat. In isothermal titration
calorimetry (ITC), the heat is the result of titrating interacting components together and allows direct
determination of the thermodynamics for this process. The measured heat reflects the enthalpy change
(ΔH ), and the prospect of determining this in biological systems where high-resolution structural information is available has led to the possibility of rational thermodynamics-guided design of ligands. Although
there are limitations to this approach due to the participation of solvent in the thermodynamics, ITC has
become an established technique in many labs providing a valuable tool with which to quantify protein–protein interactions. With careful use, ITC can also provide additional insights into the binding process or
be used in increasingly complex systems and where interaction is coupled to other molecular events.
Key words Isothermal titration calorimetry, ITC, Thermodynamics, Enthalpy, Entropy, Free energy,
Dissociation constant, Heat capacity, Stoichiometry, Binding affinity, Binding kinetics
1 Introduction
1.1 ITC: A
Measurement
Nirvana?
Many techniques for studying protein–ligand interactions discussed
elsewhere in this volume depend on reporter signals, such as fluorescence or absorbance, which change as a result of complex formation. Sometimes these changes can be a rather indirect consequence
of the binding event. Other techniques, such as fluorescence polarization, light scattering, or surface-based sensors using SPR or BLI,
report on a change in physical properties such as mass or size during
complex formation. Changes in these properties that produce adequate signal-to-noise measurements can often be large when compared to the changes expected for protein–ligand interactions.
In contrast, calorimetry simply measures directly the heat associated with making and breaking interactions that are intrinsic to
complex formation irrespective of any change in size or mass and in
the absence of any additional reporter labels required to give suitable spectroscopic properties. It is this “directness” of working with
unmodified materials and the ubiquitous nature of the predominantly non-covalent forces, with their associated heats, that drive
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
135