Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Isothermal Titration Calorimetry 121
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Isothermal Titration Calorimetry
Maria M. Lopez and George I. Makhatadze
1. Introduction
Thermodynamic characterization of the system provides us with important
information about the stability, strength, specificity, and stoichiometry of interacting
systems. The method of choice for the direct measurements of energetics
of protein-ligand interactions is the isothermal titration calorimetry (ITC)
technique. The use of ITC to measure the binding of a macromolecule (in general,
a protein) to its ligand (ion, peptide, another protein, DNA, RNA, and so
on) relies on the fact that such an interaction is accompanied by a heat effect.
The heat absorbed (endothermic) or released (exothermic) upon the interaction
Q is then used to obtain information about the binding constant, K a, and the
enthalpy of binding ∆H. The strength of ITC is that under proper experimental
conditions, from one single titration both the binding constant and the enthalpy
of binding can be obtained. Moreover, the temperature dependence of the
enthalpy of binding allows one to calculate another important thermodynamic
parameter: the heat capacity change of binding ∆C p. The ∆C p is calculated
from the slope of ∆H vs temperature, and it can be positive (hydrophobic interactions
are disrupted upon binding) or negative (hydrophobic interactions are
formed) (1).
The shape of the binding curve depends on the unitless parameter c. This
parameter is proportional to the equilibrium constant K a the total concentration
of protein in the cell [P t] , and the stoichiometry of the interaction n, as:
c = K a · [P t] · n (1)
High c values indicate very tight binding and low c values indicate low affinity
of the interactions. High-affinity binding constants have to be measured at low
protein concentration. However, one needs to keep in mind that the lower the
From: Methods in Molecular Biology, vol. 173:
Calcium-Binding Protein Protocols, Vol. 2: Methods and Techniques
Edited by: H. J. Vogel © Humana Press Inc., Totowa, NJ
121