Calorimetric determination of cooperative interactions in high affinity binding processes.
It is demonstrated that isothermal titration calorimetry can be used to determine cooperative interaction energetics even for extremely tight binding processes in which the binding affinity constants are beyond the limits of experimental determination. The approach is based on the capability of calorimetry to measure the apparent binding enthalpy at any degree of ligand saturation. When calorimetric measurements are performed under conditions of total association at partial saturation, the dependence of the apparent binding enthalpy on the degree of saturation is a function only of the cooperative binding interactions. The method developed in this paper allows an independent estimation of cooperative energetic parameters without the need to simultaneously estimate or precisely know the value of the association constants. Since total ligand association at partial saturation is achieved only at macromolecular concentrations much larger than the dissociation constants, the method is especially suited for high and very high affinity processes. Biological associations in this category include fundamental cellular processes like cell surface receptor binding or protein-DNA interactions.
Bains
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Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Freire
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D001665:Binding Sites; D002151:Calorimetry; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D008024:Ligands; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D008024:Ligands; D004247:DNA