Macromolecule-ligand binding studied by the Hummel and Dreyer method: current state of the methodology.
The use of the Hummel and Dreyer method to measure binding parameters of ligand-macromolecule associations is reviewed. The possibility to determine the number of binding sites and their association constants, even in the case of low affinity, and to control the free ligand concentration as an independent variable are the main advantages of the method. The conditions of the validity are rapid equilibrium kinetics, independence between ligand binding and macromolecule association, and identical retention rates between free and bound macromolecules. Initially developed on soft gels, the method has been applied to high-performance chromatography and capillary zone electrophoresis. Technical progress such as increase in resolution, detection sensitivity, and automation have improved its utilization. The binding parameters given by the Hummel and Dreyer method are in general similar to those obtained by other techniques, in comparable experimental conditions (equilibrium dialysis, ultrafiltration, frontal elution, vacancy peak method, vacancy affinity capillary electrophoresis, retention analysis, affinity chromatography and affinity capillary electrophoresis, physical methods). The choice between these methods is directed by material availability and practical constraints. Separation by new types of chromatographic columns or by capillary zone electrophoresis would enable the study of the simultaneous binding of different drugs on the same macromolecule and their competition.
Berger
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G
Service de Bioenergetique, Departement de Biologie Joliot Curie, CEA Saclay, F-91191 Gif sur Yvette, France.
Girault
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G
D002853:Chromatography, Liquid; D019075:Electrophoresis, Capillary; D008024:Ligands; D011485:Protein Binding
D008024:Ligands