The interaction between macromolecules such as proteins or macromolecular systems with proteins, ligands, ions, lipids or detergents is of great importance for many biological functions. In contrast to optical spectroscopy, thermoanalytical techniques are especially suitable for the investigation of nanoparticulate systems. Based on a given model, microcalorimetric titrations can be carried out to determine binding enthalpies (ΔH) together with binding affinities and stoichiometries, provided the enthalpy change of the system under consideration is not equal to zero. For equilibria, the binding enthalpy (ΔS) can be calculated according to
ln Kth = (ΔH –TΔS) / RT ,
where Kth is the thermodynamic association constant, which is identical with the reciprocal dissociation constant. As a major advantage, the resulting enthalpic and enthropic contributions to the binding affinity can be obtained and interpreted in more detailed molecular and structural terms than if only the association, respectively dissociation constant is known.
In the case of a one-step binding process (stoichiometric coefficient equal 1) between a protein P and a ligand L, titration calorimetry under conditions of constant ionic strength and temperature allows to determine an apparent association constant Kapp according to
Kapp = Kth / Kγ = cPL / (cP cL) ,
if the standard concentration is expressed in molar terms and where Kγ is the term related to activity coefficients and c denotes concentrations (for details cf.). Kapp represents a suitable parameter to compare affinities of equilibria determined under identical experimental conditions.
The available experimental setup consists of a quasi adiabatic MCS ITC instrument of MicroCal, equipped with a 1.3 ml cell. Association constants can be determined in the range of 107 to close to 103 M-1, corresponding to dissociation constants of 100nM and 1 mM.
A typical application related to a membrane system is shown in the figure, where 15 µM Na,K-ATPase (in form of membrane discs) is titrated in the absence of Mg2+ with 0.5 mM TrisATP (15 identical additions) in 10 mM imidazole/HCl, 0.25 mM CDTA and 120 mM choline chloride pH 7.5, containing 25% glycerol (w/w) at 25°C. The evaluation provides a stoichiometric coefficient of 0.7, a Kapp value of 5 x 105 M-1 and a ΔH value of -59 kJ/mol.
Selected publications on calorimetry:
 E. Grell, E. Lewitzki, A. Schacht and M. Stolz, Nucleotide/Protein Interaction, J.Therm. Anal. Calor. 77, 471-481 (2004)
[2 ] E. Grell, E. Schick and E. Lewitzki, Membrane receptor calorimetry, Thermochim. Acta 380, 245-254 (2001)
 E. Grell, A. Geoffroy, M. Stolz, E. Lewitzki and M.von Raumer, Membrane proteins in thin films, J. Therm. Anal. Calor. 89, 723-727 (2007)
Dr. Ernst Grell
Tel.: +49 (0) 69 6303 2400
Fax: +49 (0) 69 6303 2402