PEAQ-ITC Automated

Isothermal titration calorimetry is a physical technique used to measure the heat change that occurs when two molecules interact with each other. It can be used to characterize the interactions of small molecules, proteins, antibodies, nucleic acids, lipids and other biomolecules, but also to measure enzyme kinetics.

The MicroCal PEAQ-ITC Automated system is a highly sensitive isothermal titration calorimeter, which offers the convenience of unattended operation for high productivity. It has a sample capacity of 384 samples, which corresponds to four 96-well plates. As it is a label-free in solution technique, it ensures the analysis of unaltered biomolecules in their native state over a broad dynamic range. Dissociation constants can be measured down to 10^-12 M (using direct or competitive binding techniques). The instrument operates in a temperature range between 2-80 °C.

ITC consists of two cells surrounded by an adiabatic jacket. Sample and reference cell of the MicroCal PEAQ-ITC Automated system in our facility are made from Hastelloy, a highly inert material. As the binding partners are mixed within the sample cell, heat is either released or absorbed as a result of the redistribution of non-covalent bonds, for example, when the interacting molecules go from the free to the bound state. ITC monitors the heat changes by measuring the differential power that is applied to the cell heaters in order to maintain zero temperature difference between sample and reference cell. The reference cell usually contains water, which has the same heat capacity as most of the sample buffers. The sample cell contains one of the binding partners (often, but not necessarily a macromolecule) and a stirring syringe which holds the other binding partner (often, but not necessarily the ligand). During the experiment, ligand is injected into the sample cell, typically in 0.5-3 µL aliquots. Each injection results in a heat pulse that is plotted against time and normalized for concentration to generate a titration curve of kcal/mol vs. molar ratio (ligand/sample). The resulting isotherm is fitted to a binding model and analysed to give thermodynamic parameters like affinity (K_D), stoichiometry (N), entropy (∆S) and enthalpy of interaction (∆H). Binding affinity is a combined function of the binding enthalpy (∆H) and the binding entropy (∆S). Binding enthalpy reflects the strength of the interaction due to hydrogen bond formation and van der Waals interactions. Binding entropy is a combination of the change in entropy from desolvation and conformational changes upon complex formation.