Isothermal titration calorimetry

Isothermal Titration Calorimetry (ITC) is a biophysical technique used in the study of molecular interactions. It directly measures the heat released or absorbed during a molecular binding event, providing a detailed understanding of the interaction in terms of thermodynamics. ITC is widely used in the fields of biochemistry, pharmacology, and molecular biology to quantify the binding affinity, stoichiometry, enthalpy (ΔH), and entropy (ΔS) changes associated with the interactions between molecules such as proteins, DNA, RNA, lipids, and small molecule ligands.

Principle

The principle of ITC involves the measurement of heat change that occurs when two molecules interact in a solution to form a complex. The technique is performed using an isothermal titration calorimeter, which consists of two cells: a sample cell containing one of the molecules of interest and a reference cell filled with a solvent, typically water. The other molecule is then titrated into the sample cell in small increments, and the heat change associated with each titration step is measured. The reference cell serves to compensate for baseline drifts and provides a comparison for the heat measurements.

Procedure

The procedure of an ITC experiment involves several steps: 1. Preparation of the sample and titrant solutions, ensuring they are of compatible pH and ionic strength. 2. Calibration of the calorimeter to ensure accurate heat measurements. 3. Loading the sample into the sample cell and the titrant into the syringe. 4. Incremental titration of the titrant into the sample cell, with continuous stirring to ensure rapid mixing and uniform distribution of heat. 5. Measurement of the heat change after each titration step, compared to the reference cell.

Data Analysis

The data obtained from an ITC experiment are plotted as heat change per injection versus the molar ratio of the titrant to the sample. The resulting curve can be analyzed to determine the binding parameters of the interaction, including: - Binding affinity (Kd), which indicates how tightly the two molecules interact. - Stoichiometry (n), which reveals the number of binding sites involved in the interaction. - Enthalpy (ΔH) and entropy (ΔS) changes, providing insights into the forces driving the binding process.

Applications

ITC has a wide range of applications in the study of molecular interactions, including: - Determining the binding specificity and affinity of enzyme-substrate or receptor-ligand interactions. - Investigating the effects of mutations on the binding affinity and thermodynamics of protein-protein interactions. - Characterizing the binding of small molecules to proteins, which is of particular interest in drug discovery and development. - Studying the thermodynamics of nucleic acid interactions, including DNA-DNA, DNA-RNA, and protein-DNA/RNA complexes.

Advantages and Limitations

Advantages of ITC include its ability to provide a complete thermodynamic profile of the interaction without the need for any labeling or immobilization of the molecules. It is also versatile, allowing for the study of a wide range of molecular interactions under near-physiological conditions.

However, ITC has some limitations, such as the requirement for relatively large amounts of sample and the difficulty in analyzing interactions with very high or very low binding affinities. Additionally, the interpretation of ITC data can be complex, especially for interactions involving multiple binding sites or conformational changes.