Isothermal titration calorimetry

Isothermal titration calorimetry (ITC, Eng. ' Isothermal titration calorimetry ') is a biophysical technique that is used for the determination of thermodynamic parameters of biochemical binding processes. In most cases, the binding of small molecules, such as medically active substances in large macromolecules ( proteins, DNA, etc.) is analyzed and characterized thermodynamically. This allows us to draw conclusions about the energetics of binding and the number and the ratio of the particles involved.

Thermodynamic measurements

The ITC is a quantitative measurement method, which directly binding affinity Ka ( binding strength ), binding enthalpy AH ( latent heat ) and the binding stoichiometry n (ratio of involved particles ) can measure the studied interaction. These measurements allow calculation of the change in Gibbs enthalpy and entropy? G? S by the following relation to:

( wherein R is the gas constant and T is the absolute temperature ).

Measuring device

An isothermal titration calorimeter consists of two identical, high wärmeleitdenden materials ( gold or Hastelloy), which are incorporated in adiabatic coated cells. Sensitive thermocouples detect the temperature of the liquids within the cells with high accuracy. One of the two cells is used as a reference cell ( with water or buffer solution), and the other serves as a sample cell, in which the macromolecule is in the exact same solution ( water or buffer ) is present. The liquid volume per cell is usually a few ml Both cells are heated at the beginning with constant power (usually less than 1 mW ), wherein a feedback mechanism controls the heating mechanism of the sample cell, depending on the temperature in the cell.

Implementation

During the experiment exactly known amounts of the ligands are added, resulting in either a recording or a release of heat to the liquid within the sample cell. Measuring the time-dependent supply of power by the heating mechanism of the sample cell, which is required to maintain the same temperature as in the reference cell.

In exothermic reactions (when in the binding of the ligand by the macromolecules in the solution, releasing heat ) increases the temperature of the solution in the sample cell, so that less energy needs to be supplied in order to precisely heat the sample cell the same as the reference cell. Conversely, more energy must be supplied in an endothermic reaction which cools the solution to the sample cell. The adjustment of the supplied quantity of energy is effected in both cases by means of the feedback mechanism described above.

Evaluation

The measurement results are as needed to maintain the same temperatures required power in μcal / s plotted as a function of time in s. Also mu.J (instead μcal ) as an energy unit is in use. The diagram thus obtained the raw data contains a number of " points", so-called spikes, each spike of a ligand injection with the following temperature change and reinstatement of the temperature is due to the feedbacker. The area under each spike corresponding to the amount of heat which, in this free injection or is absorbed and therefore can be determined by integrating as a function of time. The pattern of this latent heat effects can be analyzed as a function of the molar ratio ligand / macromolecule, whereby the thermodynamic parameters of the investigated interaction can be determined. The samples should be degassed prior to the start of the experiment under vacuum, since air bubbles interfere with measurements within the cells and can lead to abnormal spectra.