Biology Reference
In-Depth Information
18.1 ISOTHERMAL TITRATION CALORIMETRY
18.1.1 Principles
Interaction of molecules, including protein-protein binding, is usually accompanied
by heat exchange. ITC is based on the measuring of this heat as a function of molar
ratio of interacting molecules. Subsequent fitting of raw data allows one to get most
thermodynamical parameters of interaction such as stoichiometry ( N ), enthalpy
(
S ) of binding, and association equilibrium constant ( K a ) from a sin-
gle 1-h-experiment ( Ladbury &Doyle, 2004 ). As heat exchange upon binding occurs
naturally, ITC does not require immobilization, as surface plasmon resonance does,
and/or modification of the reactants by addition of a fluorophore, for example. In
addition, ITC does not depend on the size or mass difference between the studied
interacting molecules, which enables ITC to be also used to study binding of low
molecular weight drugs and metal ions to proteins ( Tsvetkov et al., 2010 ). At last,
contrary to spectroscopic methods, ITC can be used with colored, turbid or even
not transparent solutions and suspensions. Continuous progress and improvements
in ITC instrumentation now allow using ITC routinely to characterize thermodynam-
ics of binding with association constants ranging from 10 3 to 10 8 M 1 . For all these
reasons, ITC is a very powerful method for the study of a wide range of biological
systems under near physiological conditions ( Ladbury & Doyle, 2004 ).
D
H ), entropy (
D
18.1.2 Experimental procedure
An ITC apparatus consists of a calorimetric cell in an isothermal jacket and a syringe
that is inserted in the cell ( Ladbury & Doyle, 2004; Pierce, Raman, & Nall, 1999 ).
To perform ITC experiments, the calorimetric cell is filled with protein solution at a
concentration close to the expected dissociation constant, and the titration syringe is
filled with about a 10-fold more concentrated solution of the second interactant (usu-
ally called ligand) in an identical buffer. During each successive injection of small
aliquots of ligand into the cell, the microcalorimeter registers heat exchange. The
released heat is proportional to the amount of complex formed after each injection
and decreases as the protein gets saturated by the ligand ( Freyer & Lewis, 2008 ).
During final injections, as there is no more binding of the ligand to the protein,
the measured heat corresponds to the heat of mixing of the two solutions, which
is often referred to as heat of dilution. The signal due to dilution can be significant
in the case of imbalance in compositions of buffers between the calorimetric cell and
the syringe, and/or a high intrinsic dilution heat of the ligand. This is why the sample
preparation is a crucial step in the experimental procedure (see Section 18.2.1 ). Areas
under each peak corresponding to heat exchanges are then plotted against molar ratio
of ligand over protein in order to obtain a thermogram or binding isotherm. To de-
termine the thermodynamic parameters, this binding isotherm should be fitted with a
theoretical binding isotherm curve. All ITC instruments are supplied with the
software that offers several standard models of interaction.
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