Isothermal titration calorimetry (ITC) is a biophysical technique that allows the thermodynamic study of two interacting species. When these two species interact, heat is either generated or absorbed. By measuring these interaction heats, binding constants (K), reaction stoichiometry (n), and thermodynamic parameters including enthalpy (ΔH) and entropy (ΔS) can be accurately determined. In addition, varying the temperature of the experiment allows the determination of the heat capacity (ΔCp) for the reaction. Thermodynamic data provides a molecular level insight into the forces that drive complex formation, including hydrogen bonding, hydrophobic interactions, charge-charge interactions, etc.
Isothermal titration calorimeter (ITC) is a micro calorimeter which is to measure micro calories of heat evolved or absorbed by a chemical reaction. ITC experiments can yield binding constants (K), reaction stoichiometry (n), enthalpy (ΔH) and entropy (ΔS), and activation energy (A) for a reaction. Thermodynamic data provides a molecular level insight into the forces that drive complex formation, including hydrogen bonding, hydrophobic interactions, charge-charge interactions, etc.
The ITC4200 microcalorimeter (Calorimetry Sciences Corporation (now part of TA Instruments)) is used to study almost any kind of interaction, including solutes with immobilized enzymes, tissue samples, or other solid materials in suspension. Titration experiments are typically fast (approximately 1-2 hours) yielding accurate values of K (in the range of 102 to 108 M-1), n, ΔH, and ΔS. In addition, analytical data (e.g. enzyme assay) or kinetic data (for slow reactions) may be obtaind in a batch injection experiment.
The CSC ITC may also be used to study the decomposition/stability of organic (ex. drugs) and inorganic materials over time (days to weeks). This is particularly useful for determining shelf/storage life of drugs, etc.
Types of Experiments:
Types of Biomaterials:
|Affinity Constant (Ka)||102 to 109 M-1|
|Competitive Binding Affinity Constant (Ka)||109 to 1012 M-1|
|Minimum Detectable Heat||0.1 μcal (0.4 μJ)|
|Baseline Stability||± 0.2 μcal s-1 hr (±0.08 μW hr-1)|
|Cell Volume||0.5-1.3 mL (full cells are recommended)|
|Precision Buret||25-250 μL|
|Volume Increment||1-20 μL (± 0.01 μL delivery precision)|
|Stir Rate||0-500 rpm|
|Temperature Range||0-100 °C (Bath stability ±0.0005 °C at 25 °C)|
The heat produced from each injection of ligand solution into the macromolecule solution is a combination of all reactions in the cell. This includes the ligand-macromolecule complexation, ligand dilution heat, macromolecule dilution heat, buffer ionization, and heat of mixing. To reduce heats from secondary interactions, a few guidelines on sample preparation should be followed
ITC experiments are known to consume great quantities of sample, especially for protein interactions. To achieve best results, it is highly recommended to fill the ITC reaction cell to capacity (1.3 mL). In order to use the minimum sample necessary, it is important to use simulation software to plan your experiments. Knowledge of the K and ΔH is advantageous.
Weismann (Wiseman et al. Analytical Biochemistry, 179, 1989, 131-137) recognized that the binding isotherm was dependent upon K and Cmacromolecule. It is widely accepted that the Wiseman constant, c, in the 10-500 range in order to obtain reliable n, K, ΔH simultaneously. The starting Cmacromolecule can be calculated from the following equation:
Cmacromolecule = (10 to 50)/K
Assuming the intereaction is 1:1, the ligand concentration, Cligand, should be approximately 10X higher than Cmacromolecule. For protein interactions, generally a minimum of 1mg mL-1 is needed, and very often, much higher concentrations may be required.