Signal and binding. I. Physico-chemical response to macromolecule-ligand interactions

- Physico-chemical titration curves are governed by conservation rules independent of the nature of the signal.
- In general, an equilibrium single physico-chemical titration curve is not a thermodynamic binding isotherm.
- An equilibrium single physico-chemical titration curve does not provide information about the maximum complex stoichiometry.
- A physico-chemical titration curve is a plot of two parametric equations; for the signal and the total ligand concentration.
- Fluorescence intensity can be modulated by a collisional quencher, which increases the resolution of the binding experiments.

Obtaining a detailed knowledge about energetics of ligand-macromolecule interactions is a prerequisite for elucidation of the nature, behavior, and activities of the formed complexes. The most commonly used methods in characterizing molecular interactions are physico-chemical techniques based mainly on spectroscopic, calorimetric, hydrodynamic, etc., measurements. The major advantage of the physico-chemical methods is that they do not require large quantities of material and, if performed carefully, do not perturb examined reactions. Applications of several different physico-chemical approaches, commonly encountered in analyses of biochemical interactions, are here reviewed and discussed, using examples of simple binding reactions. It is stressed that without determination of the relationship between the measured signal and the total average degree of binding, the performed analysis of a single physico-chemical titration curve may provide only fitting parameters, instead of meaningful interaction parameters, already for the binding systems with only two ligand molecules. Some possible pitfalls in the analyses of single titration curves are discussed.

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