Quantitative thermodynamic analyses of spectroscopic titration curves

Elucidation of ligand - macromolecule interactions requires detailed knowledge of energetics of the formed complexes. Spectroscopic methods are most commonly used in characterizing molecular interactions in solution. The methods do not require large quantities of material and most importantly, do not perturb the studied reactions. However, spectroscopic methods absolutely require the determination of the relationship between the observed signal and the degree of binding in order to obtain meaningful interaction parameters. In other words, the meaningful, thermodynamic interaction parameters can be only determined if the relationship between the observed signal and the degree of binding is determined and not assumed, based on an ad hoc model of the relationship. The approaches discussed here allow an experimenter to quantitatively determine the degree of binding and the free ligand concentration, i.e., they enable to construct thermodynamic binding isotherms in a model-independent fashion.