Trends in Pharmacological Sciences
Ligand-binding studies: old beliefs and new strategies
Section snippets
Equilibrium-binding experiments
Conventionally, two distinct types of equilibrium experiments are most commonly performed: the `saturation experiment' and the `competition experiment'. The latter might involve the same chemical species of the tracer (homologous displacement), or a different chemical species (heterologous displacement). Indeed, from a mathematical point of view, both saturation and homologous displacement curves contain exactly the same type of information1, 2and the choice between them is not a theoretical
Protocol optimization
Optimization of the experimental design can yield a dramatic improvement over conventional designs. To apply the theory of d-optimal design[9]to equilibrium-ligand-binding studies, a computer program has been developed, design[1], which is based on the parametric binding model of multiple ligands interacting with multiple independent binding sites. design can be used to optimize various protocols, including the simple saturation or homologous displacement curve[1], the complete self- and
Data analysis
If it was not for experimental error, many commonly used graphical techniques (e.g. Scatchard[11]and Eadie–Hofstee12, 13) would all yield exact results, at least in the simplest model for ligand–receptor interaction. Unfortunately, there are many popular misconceptions regarding the properties of graphical display of ligand-binding data in a variety of coordinate systems14, 15: (1) each method provides a slightly different view of the same data and, therefore, conclusions may be influenced by
Kinetic-binding experiments
Association and dissociation timecourses are mainly used in preliminary phases of the characterization of a receptor system, to optimize some of the conditions for subsequent use in equilibrium experiments and to demonstrate the reversibility of the ligand–receptor interaction. Equilibrium experiments are conventionally used to calculate affinities and capacities of a receptor system for a number of theoretical and practical reasons, whereas kinetic protocols are often considered as a `second
Open problems
The thermodynamic model of multiple independent binding sites is only one of the possible models that can be used to predict ligand–receptor interaction. For example, De Lean and co-workers[21]have published the so-called ternary-complex model, which was originally developed to explain the binding properties of a G protein-coupled receptor (GPCR) (β-adrenoceptor). The computer program equil[22]has been developed for simulation and analysis of arbitrary chemical systems in equilibrium and, thus,
Concluding remarks
Far from being an outdated technique, and despite its limitations, which are more practical than theoretical, ligand-binding studies will continue to prove to be a fundamental tool in many biological sciences such as pharmacology, physiology, protein chemistry and, of course, in the study of the theories and models of receptor mechanisms and functions.
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Assistant Professor of Pharmacology