Signal and binding. II. Converting physico-chemical responses to macromolecule-ligand interactions into thermodynamic binding isotherms

•In general, there is no linear relationship between observed physico-chemical signals and the total average degree of binding.•Signal and mass conservation relationships allow the construction of binding isotherms from physico-chemical titration curves.•Ligand Binding Density Function (LBDF) Method allows the construction of binding isotherms using the ligand signal.•The Empirical Function (EF) Method relates the observed signal to the total average degree of binding.

Physico-chemical titration techniques are the most commonly used methods in characterizing molecular interactions. These methods are mainly based on spectroscopic, calorimetric, hydrodynamic, etc., measurements. However, truly quantitative physico-chemical methods are absolutely based on the determination of the relationship between the measured signal and the total average degree of binding in order to obtain meaningful interaction parameters. The relationship between the observed physico-chemical signal of whatever nature and the degree of binding must be determined and not assumed, based on some ad hoc intuitive relationship/model, leading to determination of the true binding isotherm. The quantitative methods reviewed and discussed here allow an experimenter to rigorously determine the degree of binding and the free ligand concentration, i.e., they lead to the construction of the thermodynamic binding isotherm in a model-independent fashion from physico-chemical titration curves.

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