A Review on the Ligand Binding Studies by Isothermal Titration Calorimetry

 A.A. Saboury

Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

(Received 20 December 2005, Accepted 1 February 2006)

     Thermodynamics of biomacromolecule ligand interaction is very important to understand the structure function relationship in proteins. One of the most powerful techniques useful to obtain additional information about the structure of proteins in biophysical chemistry field is isothermal titration calorimetry (ITC). An ITC experiment is a titration of a biomacromolecule solution by a solution containing a reactant (ligand) at constant temperature to obtain the exchanged heat of the reaction. The total concentration of ligand is the independent variable under experimental control. There are many reports on data analysis for ITC to find the number of binding sites (g), the equilibrium constant (K), the Gibbs free energy of binding process (DG), the enthalpy of binding (DH) and the entropy of binding (DS). Moreover, ITC gives information about the type of reaction, electrostatic and hydrophobic interactions, including determination of cooperativity characterization in binding process by calculating the Hill coefficient (n). A double reciprocal plot and a graphical fitting method are two simple methods used in the enzyme inhibition and metal binding to a protein. Determination of a binding isotherm needs more ITC experiments and more complex data analysis. Protein denaturation by ligand includes two processes of binding and denaturation so that ITC data analysis are more complex. However, the enthalpy of denaturation process obtained by ITC help to understand the fine structure of a protein.

Keywords: Isothermal titration calorimetry, Ligand binding, Metal binding, Enzyme inhibition, Protein denaturation, Enthalpy of binding, Equilibrium constant