Application of Isothermal Titration Calorimetry in the Biological Sciences: Things Are Heating Up!
BioTechniques 885 During the last 15 years isothermal titration calorimetry (ITC) has come of age. There are now in excess of 2000 instruments sited in laboratories in more than 40 countries around the world. Research scientists in such diverse fields as biophysics, cell biology, pharmaceutical screening, and food research routinely investigate their systems of interest using ITC. Why is it that this methodology has sparked such enthusiasm and interest, and what use is the data obtained? The
... ta obtained? The dramatic advances in the field of structural biology in the last couple of decades fed a desire of biochemists to define molecular function and mechanism in ever increasing detail. Describing a biochemical process however, cannot be served by structure alone. A full understanding is only obtained with a quantification of the change of state of the system. In an equilibrium process, such as a biomolecular interaction, thermodynamic measurement provides quantification of the change in energy on going from the free to the bound state. The ITC instrument (for reviews, see References 1-5) uses the extremely accurate measurement of heat as a probe for an interaction as it occurs. Knowing the concentrations of the interacting moieties allows the calculation of the observed change in molar enthalpy of the interaction, ∆H obs . The term observed (denoted by the subscript obs) signifies that the quantity is not solely from the isolated events associated with forming a biomolecular interface (i.e., direct noncovalent bonds between the atoms of the interacting moieties), but also includes heat derived from perturbation of the solvent around the binding site, potential conformational changes occurring elsewhere in the interacting biomolecules (6), and direct formation of noncovalent bonds between other solutes such as ions or apolar compounds that may be incorporated as an ingredient of the bulk solvent. Since every biomolecular interaction has either an uptake or release of heat associated with it, the ITC is a universal detector of the occurrence of binding (at an appropriate temperature). The direct determination of the ∆H obs negates the indirect calculation of this parameter using a van't Hoff-based method, which can be problematic over extended temperature ranges due to the influence of the change in heat capacity (i.e., the ∆H changes with temperature; see Equation 3). Furthermore, since the two components of an interaction can be titrated, the measured heat gives a direct readout of the extent of interaction at any given concentration regime (see Figure 1 and Reference 7 for experimental tutorial). As a result, the concentrations of free and bound molecules and hence the observed binding or dissociation constant, (K B,obs or K D,obs , respectively; K B = 1/K D ) can be determined. Armed with the ∆H obs and the K B,obs , a full thermodynamic description of the interaction can be elucidated at a given experimental temperature (T) based on the following relationships: ∆G obs = -RTln K B,obs [Eq. 1] Figure 1. Schematic representations of isothermal titration calorimetry (ITC) instruments. (A) An ITC instrument prior to performing a titration. The sample cell and the reference cell as kept at the same temperature, which is typically 5°-10°C above the temperature maintained outside the jacket in which the cells are housed. The reference cell is always kept at the experimental temperature. One of the components of the interaction is placed in the syringe and the other in the cell. (B) A ITC instrument performing a titration. When an injection is made, the change in heat associated with binding (endothermic or exothermic) results in a change in temperature in the sample cell. A change in power (heat/s) is required to return the cells to identical temperatures (T) (i.e., ∆T = 0). This change in power is recorded as a series of injections is made. In the raw data presented in the inset, each injection is accompanied by an interaction where heat is given out (exothermic). As the course of injections is completed, the binding sites on the sample in the cell are gradually saturated, and the exothermic effect becomes reduced. For details, see References 1-5 and 7.