Isothermal Titration Calorimetry
This technique has the advantage of offering a full thermodynamic characterisation in a single experimental procedure.
Figure 1: Picture of ITC Instrumentation available with Avacta.
The diverse interactions of macromolecules in biological systems, for example, the adsorption and interaction of a virus or protein with an adjuvant such as aluminium hydroxide, protein-DNA interactions, antibody-antigen and hormone-receptor interactions, can be fully thermodynamically characterised.
Measurements of enthalpy (
H), entropy (S), and calculated binding constants (k), as well as the reaction stoichiometry (n) can be established.
(1) Where Gº, Hº and Sº are the free energy, enthalpy and entropy change respectively.
Essentially calorimetry titrations work by gradual addition of a specific 'ligand' to a 'macromolecule' which it binds to over time. The binding event will result in absorbed heat or evolved heat which is measured as the differential power (DP) between the sample and reference cell. The graph below represents the data collected and generated from such a titration. The upper of the two graphs shows the DP measurement as the ligand is added to the sample cell, containing the macromolecule. Each of the spikes represents an individual ligand addition and the integral of the area of the peak can be used to calculate H. The calculation of H with increasing injectant number is shown in the lower of the two graphs.
Figure 2 Isothermal Titration Calorimetry of Protein-Protein Interactions. Example of a typical calorimetric titration data. Upper graph showing the differential power recorded directly over time and the lower figure showing the H over molar ratio.
Should the injectant cause an exothermic reaction where there is evolution of heat, there will be a negative DP signal recorded as in the upper panel in Figure 2 above. This negative change is due to the heat evolved chemically meaning that the instrument does not need to provide heat to maintain the equilibrium between the cells. The opposite is true for an endothermic reaction. The DP has units of power therefore the integral of the peak yields a measurement of thermal energy, H. The heat evolved or absorbed is directly proportional to the amount of binding that results from the mixing of the 'macromolecule' to the 'ligand'. As the system reaches saturation, the signal diminishes until only heats of dilution are observed. A binding curve (as in the lower panel for Figure 2) is then obtained from a plot of the heats from each injection against the ratio of ligand and binding partner in the cell.
ITC can be used in the development of formulations and assessing the effects of different pH and solvents on the molecular interactions as well as a measuring the unfolding of proteins by denaturants or refolding of proteins in refolding buffer.
Current facilities include a Microcal VP-ITC microcalorimeter which can perform experiments between ~10°C and 60°C.