Discrepancies in Thermodynamic Information Obtained from Calorimetry and Spectroscopy in Ligand Binding Reactions: Implications on Correct Analysis in Systems of Biological Importance

Abstract Thermodynamic signatures accompanying ligand binding interactions with proteins and nucleic acids have great potential in drug discovery and help in deriving guidelines for rational drug design. Frequent discrepancies have been observed between the results obtained from routinely used fluorescence spectroscopy and direct high sensitivity isothermal titration calorimetry (ITC). These discrepancies lead to incorrect data analysis even though experiments are done with extensive care. We analyze these discrepancies and discuss possible causes by taking eleven examples from literature where the data on binding processes has been obtained both by fluorescence spectroscopy and ITC. Further, a protocol has been suggested to obtain accurate thermodynamic signatures so that the information resulting from studies of biologically important ligand binding reactions is complete and leads to correct direction. Results from fluorescence quenching data on drug binding interactions have frequently been analyzed incorrectly, many times without even establishing the nature of the quenching process. This results in incorrect proposals for mechanism of binding of drugs with the target biological macromolecules. Relatively lesser problems have been observed when isothermal titration calorimetry has been employed. The analysis and suggested protocol have implications in deriving accurate thermodynamic signatures focused on rational drug design and hence in target oriented drug discovery.