In recent years, thermophoresis has emerged as a promising tool for
quantifying biomolecular interactions. The underlying physical effect is still
not understood. To gain deeper insight, we investigate whether non-equilibrium
coefficients can be related to equilibrium properties. Therefore, we compare
thermophoretic data measured by thermal diffusion forced Rayleigh scattering
(TDFRS) (which is a non-equilibrium process) with thermodynamic data obtained
by isothermal titration calorimetry (ITC) (which is an equilibrium process). As
a reference system, we studied chelation reaction between
ethylenediaminetetraacetic acid (EDTA) and calcium chloride (CaCl2β) to
relate the thermophoretic behavior quantified by the Soret coefficient
STβ to the Gibb's free energy ΞG determined in the ITC
experiment using an expression proposed by Eastman [J. Am. Chem. Soc. 50, 283
(1928)]. Finally, we have studied the binding of the protein Bovine Carbonic
Anhydrase I (BCA I) to two different benzenesulfonamide derivatives:
4-fluorobenzenesulfonamide (4FBS) and pentafluorobenzenesulfonamide (PFBS). For
all three systems, we find that the Gibb' free energies calculated from
STβ agree with ΞG from the ITC experiment. In addition, we
also investigate the influence of fluorescent labeling, which allows
measurements in a thermophoretic microfluidic cell. Re-examination of the
fluorescently labeled system using ITC showed a strong influence of the dye on
the binding behavior