chapter 2 - Bentham Science

Analytical Tools for Protein-Carbohydrate Interaction Studies Synthesis and Biological Applications of Glycoconjugates 257
ISOTHERMAL TITRATION MICROCALORIMETRY (ITC)
Isothermal Titration Calorimetry (ITC) is a well-established and powerful methodology for studying biomolecular
interactions. Dam and Brewer [22] have published a very comprehensive review on the use of ITC to study lectincarbohydrates
interactions whereas Lundquist and Toone [7] discussed the advantages and limitations of this technique.
Titration calorimetry measures the heat evolved during ligand binding as a function of titrant concentration. Usually the
lectin is placed in the calorimetric cell, the carbohydrate ligand is added in a series of 20 to 50 injections and the heat
absorbed or produced during the binding is recorded. The shape of the resulting titration curve is a function of the
number of lectin binding sites in the cell and of the binding constant. Analysis of the experimental data needs the use of
a binding model. This model is then used to fit the experimental binding curve, which hopefully gives access to binding
constant (Ka), free energy (G°), enthalpy (H°), entropy (S°) of binding and stoichiometry (n) of interaction for each
class of binding sites that are assumed in the model. From temperature dependence of enthalpy and entropy changes,
the change in heat capacity (Cp°) can be evaluated. Importantly, ITC is the only technique that allows the
simultaneous determination of the thermodynamic binding parameters (Ka, G°, H°, S°) and stoichiometry (n) in a
single experiment. Due to material availability, solubility limitations and sensitivity issues, ITC is practically effective
for studying systems exhibiting binding constants ranging roughly from 10 3 to 10 7 M -1 .
As previously discussed [7], ITC presents many advantages as compared to other methods. First ITC gives a
concentration binding constant rather than an IC50, which can be related directly to free energy change of the
interaction. It is also the only methods able to provide directly binding enthalpies in contrast to other methods that are
based on the temperature dependence of binding constant and use of Gibbs-Helmholtz relationship. Thus ITC is unique
in allowing direct measurement of the enthalpy of binding as a function of temperature. This in turn provides a direct
measure of the change in molar heat capacity accompanying binding. This molar heat capacity change is of primary
importance when studying the effect of solvent reorganization on the thermodynamic of ligand binding [23].
Main drawbacks concerns the large amount of needed material (typically milligram quantities of both lectin and
ligand), which is often a strong practical limitation. As a matter of fact, an adequate model describing the binding
process has to be elaborated and validated before using it for fitting experimental data in order to extract quantitative
thermodynamic information.
SURFACE PLASMON RESONANCE (SPR)
Surface plasmon resonance (SPR) biosensor instruments are sensitive to the common property of refractive index or
mass change that is induced by complex formation at the surface of a biospecific sensor [24-28]. This optical
method can provide both equilibrium and dynamic characterization of the interaction process [29, 30]. The method
is label-free since it is based on refractive index changes, and monitors the interactions in real time. Although SPR
was first observed in 1902 [31], its use for biosensors was only developed in the mid 1980s. Today the technique is
widely used for studying bio-interactions, in particular because of the commercial availability of several
instruments. Assessment of carbohydrate-lectin interactions by SPR has been reviewed in several articles [4, 7, 32].
A very complete and detailed article by Wilson et al. [33] focusing on the use of SPR for quantitative analysis of
small molecule- nucleic acid interactions provides fundamental and practical information of general interest.
The physical basis of SPR has been presented in several reviews [34-35]. SPR phenomenon is observed under
condition of total internal reflexion (incidence angle higher than critical angle, see Fig. 1) at an interface between a
dielectric medium of high refractive index (usually a prism) that is covered by a thin film (2.5 to few tens of nm) of an
adequate metal (aluminium, silver, gold), in contact with a dielectric medium of low refractive index (usually the liquid
buffer of interest).
Gold is usually preferred due to its inertness under biological conditions and to its easy and versatile
functionalization using Au-S bond formation by reaction with thiolated derivatives. In absence of a metallic film, the
fully reflected beam leaks an electrical field intensity (i.e. the evanescent field) into the medium of low refractive
index. No photons exit the reflecting surface but the intensity of the corresponding evanescent electrical field
decreases exponentially with distance from the interface, decaying over a distance of about ¼ wavelength beyond
the surface in the low refractive index medium (Fig. 2a).