Ligand-binding studies: old beliefs and new strategies

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Abstract

Ligand-binding studies remain a very popular technique among many experimentalists. As far as equilibrium experiments are concerned, saturation and displacement curves are commonly performed for simplicity, convenience or for the sake of tradition. However, alternative protocols, such as `mixed'-type protocols or multiligand experiments, are also possible. Indeed, there are cases where kinetic experiments, usually considered a `second-choice' experiment, might have a superior resolving power compared to equilibrium ones. A combination of equilibrium and kinetic experiments might be a powerful solution to overcome limits and shortcomings of each specific technique and is discussed in this issue by G. Enrico Rovati. Thus, a careful choice of the design, a protocol optimization and a computerized analysis of the data can yield a dramatic improvement in the precision of the parameter estimation over more conventional approaches.

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.

References (27)

  • G.E. Rovati et al.

    Anal. Biochem.

    (1988)
  • J.C. Kermode

    Trends Pharmacol. Sci.

    (1995)
  • G.E. Rovati et al.

    Anal. Biochem.

    (1990)
  • R.B. Rothman

    Neuropeptides

    (1983)
  • H.A. Feldman et al.

    Anal. Biochem.

    (1972)
  • G.S. Eadie

    J. Biol. Chem.

    (1942)
  • A.K. Thakur et al.

    Anal. Biochem.

    (1980)
  • P.J. Munson et al.

    Anal. Biochem.

    (1980)
  • A. De Lean et al.

    J. Biol. Chem.

    (1980)
  • R.F. Goldstein et al.

    Anal. Biochem.

    (1990)
  • R.J. Lefkowitz et al.

    Trends Pharmacol. Sci.

    (1993)
  • P.J. Munson

    J. Recept. Res.

    (1983)
  • Rovati, G. E., Rabin, D. and Munson, P. J. (1991) in Horizon in Endocrinology (Vol. II) (Maggi, M. and Geenen, E. V.,...
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