In recent years, thermophoresis has emerged as a promising tool for quantifying biomolecular interactions. The underlying microscopic physical effect is still not understood, but often attributed to changes in the hydration layer once the binding occurs. 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 the chelation reaction between ethylenediaminetetraacetic acid (EDTA) and calcium chloride (CaCl (Formula presented.)) to relate the thermophoretic behavior quantified by the Soret coefficient (Formula presented.) to the Gibb’s free energy (Formula presented.) determined in the ITC experiment using an expression proposed by Eastman. 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’s free energies calculated from (Formula presented.) agree with (Formula presented.) 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.
|Journal||International journal of molecular sciences|
|Publication status||Published - 2022 Nov|
Bibliographical noteFunding Information:
Most grateful we are for the support by the group of Jörg Fitter (RWTH Aachen) especially to Olessya Yukhnoevets, who taught us the fluorescent labeling of the protein. We thank Mona Sarter and Andreas Stadler for fruitful and helpful discussions. We are grateful to Peter Lang and Jan Dhont for inspiring ideas and their generous support of our work. SM acknowledges the support by the International Helmholtz Research School of Bio-physics and Soft Matter (BioSoft) and NL acknowledges the support by the Humboldt foundation.
© 2022 by the authors.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Computer Science Applications
- Physical and Theoretical Chemistry
- Organic Chemistry
- Inorganic Chemistry