MICROCAL Isothermal Titration Calorimeter
The VP-ITC Unit measures heat evolved or absorbed in solution state samples as a result of mixing precise amounts of interacting molecules such as a protein and a ligand. A pair of identical coin shaped cells (the sample and reference cells) are enclosed in an adiabatic chamber. The sample and reference cells are accessible for filling and cleaning through the top of the unit. The sample cell is on the right as one faces the front of the unit. Access stems travel from the top exterior of the instrument to the cells. Both cells and the access stems must be totally filled with liquid during operation. This requires approximately 1.8 mL per cell even though the working volume of the cell is only 1.4 mL. A spinning syringe is utilized for injecting and mixing of the interacting species and spin rates are user selected. The normal operating range is 2°C to 80°C.
In the typical experiment, temperature differences between the reference cell and the sample cell are measured, calibrated to power units and displayed to the user as well as saved to disk. The data channel is referred to as the DP signal, or the differential power between the reference cell and the sample cell. This signal is sometimes referred to as the "feedback" power used to maintain temperature equilibrium. Calibration of this signal is obtained electrically by administering a known quantity of power through a resistive heater element located on the cell.
For the measurements, the syringe containing the "ligand" is titrated (injected) into the cell containing a solution of the "macromolecule". An injection that results in the evolution of heat (exothermic) within the sample cell causes a negative change in the DP power since the heat evolved chemically provides heat that the DP feedback is no longer required to provide. The opposite is true for endothermic reactions. Since the DP has units of power, the time integral of the peak yields a measurement of thermal energy, DH. This heat is released or absorbed in direct proportion to the amount of binding that occurs. When the macromolecule in the cell becomes saturated with added ligand, the heat signal diminishes until only the background heat of dilution is observed.
With the VP-ITC system the experiment is under computer control. The user inputs the experimental parameters (temperature, number of injections, injection volumes) and the computer carries out the experiment. Origin software is then used to analyze the ITC data using fitting models to calculate reaction stoichiometry (n), binding constant (Kb), enthalpy (DH) and entropy (DS).
From VP-ITC MicroCalorimeter User's Manual, MicroCal, LLC
Jelesarov, I. and Bosshard, H. R. (1999) Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition, J. Mol. Recognit,, 12:3-18.
Biocalorimetry: Applications of Calorimetry in the Biological Sciences, ed. John E. Ladbury and Babur Z. Chowdhry, 1998. John Wiley & Sons.
