eBooks & Guides

6 Isothermal titration calorimetry (ITC) is considered the most quantitative technique available for measuring the thermodynamic properties of interactions. How it works ITC is a calorimetric method which measures binding-induced heat changes. For this, a sample in-solution is placed into a sample cell, and water is placed into a reference cell. Both cells are kept at exactly the same temperature. Then, titrating amounts of the binding partner are added into the sample cell while stirring. The energy required to keep the cells at identical temperatures is measured, meaning that the heat released or absorbed due to a binding event results in a measurable signal. This signal becomes smaller with increasing occupancy of the target molecule. From the data, one can calculate K d , stoichiometry, and binding thermodynamics (ΔH, ΔS). Strengths This technique relies upon the accurate measurement of heat changes that result from the interaction of molecules in solution. There is no need to label or immobilize either binding partner as the absorption or production of heat is an intrinsic property of virtually all biochemical reactions. In addition, ITC can be used to measure affinity and stoichiometry, and is the only technique in this list to enable direct determination of thermodynamics ( ΔH, ΔS). Weaknesses ITC requires large quantities of sample. Any miniaturization of the instrument would require a massive improvement in sensitivity, which is technically challenging since the heat changes are so small. Also, since one always works at concentrations above the K d , the amount of target in the sample cell has to be quite high. In addition, the concentration of the ligand in the syringe used to introduce one of the binding partners must be extremely high, especially when investigating rather weak interactions (in the higher μM range). This is often difficult to achieve for many protein preparations, or simply may exceed the solubility of the ligand because the volume per injection is small (1-5 μL). Moreover, the buffers in the sample cell and syringe have to be identical, which is typically achieved by dialysis overnight. Discrepancies between the buffers results in additional signals which preclude a precise analysis of the data. Another constraint of ITC is the low throughput. One titration typically takes 30 to 60 minutes, so the throughput is limited to ~20 to 40 K d s per day (with the automated version taking up to 24 hours). Because no single-use consumables are used, the instrument needs trained personnel to take care of regular washing and maintenance of the sample cells and syringes. Replacement of broken components can be costly and time- consuming. Conclusion ITC is o en considered the "gold standard" for label-free protein binding analysis. In contrast to SPR, it is truly label-free, since it does not require any modification of the interaction partners. However, it's not suited for high-throughput screening because of the large amounts of sample required and long titration times. Isothermal Titration Calorimetry

• Cover