shown for other proteins [5], combining isothermal titration calo-
rimetry (ITC) and various nuclear magnetic resonance (NMR)
spectroscopy experiments, especially Carr-Purcell-Meiboom-Gill
(CPMG) relaxation dispersion and ZZ-exchange spectroscopy,
allowed the elucidation of both thermodynamic and kinetic data
of binding [3]. In this chapter we outline the application of these
latter experiments.
An ITC instrument works via a computer controlled thermal
feedback system. It consists of a reference cell and a sample cell with a
syringe that can titrate into the sample (Fig.1a). The sample, usually
the protein of interest, is placed in the sample cell, and the ligand, in
this case carbohydrate, is titrated in. As each injection occurs, a
protein-carbohydrate complex is formed, and heat is released (exo-
thermic) or absorbed (endothermic) upon the interaction. The feed-
back system then needs to increase or decrease the temperature to
keep the sample and reference cells at the same temperature. This
thermal power is then recorded as a series of peaks (Fig.1b), and the
area under each peak can be determined to find the heat of each
injection [6]. The heat of each injection can then be plotted against
protein-carbohydrate molar ratio and be fit to the appropriate non-
linear binding equation (single-site, multisite, sequential, or compet-
itive) in order to obtain the enthalpy (ΔH), stoichiometry (n)and
affinity (the association constant,Ka). From the affinity and enthalpy,Fig. 1Isothermal titration calorimetry—(a) Schematic of an isothermal titration calorimetry (ITC) instrument.
An ITC is composed of two cells (sample and reference) surrounded by a heat jacket, an injection syringe that
also stirs the sample, and a computer controlled thermostat. (b) Example of an ITC experiment. The additional
power needed to keep the sample cell at the same temperature as the reference cell is the monitored signal.
This is plotted as a function of time in the top graph as a series of downward peaks. The area under each peak
is plotted in the bottom graph as a function of molar ratio. This plot can be fitted with an appropriate model,
and the binding affinity (Ka), stoichiometry (n), and enthalpy (ΔH) can be obtained
88 Paul R. Gooley et al.