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Approaches to Determining Protein-Ligand Binding Constants

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The non-covalent binding of small molecules, ligands to proteins, is playing a crucial role in biopharmaceutical research.  This interaction would alter the stereochemistry of a protein molecule (a drug candidate) and also could modify its molecular recognition ability and ultimately its bioactivity.  Therefore, it is important to develop a suitable method which is able to quantitatively determinate the binding strengths of the molecule.  A variety of different approaches have been developed to quantify molecular interactions.  Methods applied to drug-protein binding studies in the pharmaceutical and biomedical sciences include equilibrium dialysis, ultra-filtration, ultra-centrifugation, gel filtration, calorimetry, microdialysis, spectroscopic, HPLC, and capillary electrophoresis-based methods.

The methods that are traditionally used for measuring the binding strengths (e.g., fluorescence polarization or radioassays) require labeling of the analytes that cause problems related ether with complicated synthesis, generates radioactive waste, or produces incorrect results.

Normally, interaction between a protein and a ligand shows the dependence of pH, reflecting the linkage between the binding of the ligand and the binding of protons.  This linkage is quantitated as a change in the ligand binding constant with pH, or as a change in the proton affinity (i.e., pKa) of an ionizable group in the protein upon ligand binding.  In determining proton linkage, one typically measures the affinity constant for the binding of the ligand at a number of pH values and calculates the pKa of the protein in the free and liganded states.  However, this is problematic if the ligand binding constant is too large to be readily measured or if the pKa shift is small.

A common, truly label-free solution for measurement is Isothermal Titration Calorimetry (ITC).

ITC is a thermo-dynamic technique that allows the study of the interactions of two species.  When these two species interact, heat is either generated or absorbed.  By measuring these interaction heats, binding constants (K), reaction stoichiometry (n) and thermodynamic parameters including enthalpy (∆ H) and entropy (∆ S), can be accurately determined.  In addition, varying the temperature of the experiment allows the determination of the heat capacity (∆ C_p) for the reaction.

Titration experiments are typically fast (approximately 1 hour) yielding accurate values of K (in the range of 10² to 10⁸ M^-1), n, ∆ H and ∆ S.  No labeling or immobilization is required.  Also, ITC is not limited by the ligand or protein size.  It is relatively artifacts-free and is not affected by the optical properties of the samples.  ITC allows researchers to study almost any kind of interaction, including solutes with immobilized enzymes, tissue samples, or other solid materials in suspension.  The only major disadvantage of ITC is that it requires relatively high concentrations of samples.