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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 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 (1,2,3,4,5) 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, ΔHobs. 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 ΔHobs 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 ΔH 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, (KB,obs or KD,obs, respectively; KB = 1/KD) can be determined. Armed with the ΔHobs and the KB,obs, a full thermodynamic description of the interaction can be elucidated at a given experimental temperature (T) based on the following relationships:
from which the change in Gibbs free energy (ΔGobs) is obtained (where R is the gas constant), and
from which the change in entropy (ΔSobs) can be ascertained. A further thermodynamic characterization of an interaction, the change in constant pressure heat capacity (ΔCp) can be determined from measurement of the ΔH over a range of temperatures since
Figure 1.
Thus ITC has become the instrument of choice for thermodynamic analysis of biomolecular interactions, since it can provide the ΔH, ΔG, and the ΔS in one experiment. Furthermore, the experiment is rapid, typically taking less than an hour, there is no need for chemical modification or immobilization of any of the components, there is no size limitation on the molecules to be investigated, and the experiment can be conducted in turbid or colored solutions or in the presence of suspensions.
