A team of researchers from Kazan Federal University, Russia, led by Dr. Igor Sedov, reported an application of capillary differential scanning calorimetry technique for the study of the binding of albumin, a plasma transport protein, with different drug ligands.
The processes of binding of various biologically active ligands to proteins can be quantitatively characterized by the values of thermodynamic binding constants, which determine the ratio between the concentrations of free and bound ligand. Accurate determination of these quantities is not an easy task. Presently, a fairly sensitive and robust isothermal titration calorimetry method is most commonly used; however, it does not allow to study the processes with very high binding constants or separate binding steps.
The differentl scanning calorimetry (DSC) method, based on monitoring changes in the heat capacity of a heated sample, is widely used to study protein denaturation. A shift of the denaturation peak to higher temperatures in the presence of a ligand is a sign of protein-ligand binding. However, the relationship between the shift magnitude and the binding constant cannot be described by a simple mathematical formula. Therefore, only a few attempts were made to use DSC thermograms for the quantitative ligand binding studies. The authors of the paper developed a program that takes into account the denaturation and ligand binding equilibria and allows one to predict the DSC curve from the known binding constants and protein and ligand concentrations. Then the values of the constants can be optimized to provide the best fit of the calculated curves to the experimental ones.
The application of this approach to albumin is complicated by the mechanism of denaturation of this protein, which is usually considered to be two-stage. However, the authors showed that the two-state model of protein denaturation used in the program gives the results consistent with other experimental methods. Moreover, the analysis of DSC thermograms helps measure very high binding constants, as well as weak binding constants with the second molecule of a ligand, and the stoichiometry of a complex at the large excess of ligand, when simultaneous binding to several centers, including those with low affinity, occurs.
Albumin is the main transport protein in blood plasma and can bind to a wide variety of molecules. Albumin has a two-domain structure with at least two high-affinity and several low-affinity binding sites. The binding affinity of drugs to plasma proteins influences the fraction of the free drug in plasma, which, in turn, is one of the major factors governing drug permeability through physiological barriers. Only unbound drugs can cross these barriers. As an example, the studied strong albumin binders - naproxen and ibuprofen - have limited brain bioavailability due to low permeability through the blood-brain barrier. On the other hand, slowly metabolized drugs with weak plasma binding have increased clearance, which may result in lower efficacy. The binding constant values can give the idea about the fraction of an unbound drug in blood plasma, while the knowledge of sequential binding constants is important for the studies of competition between drugs and other substrates or between two different drugs for albumin binding centers. In the case when the strongest binding center is occupied by another molecule, the unbound fraction of the drug will be determined by its affinity to the other binding sites. The interest in albumin binding also arises from the prospective use of drug delivery systems based on albumin nanoparticles.
The DSC-based method has potential applications to a wide range of proteins and bioactive ligands and can be useful in drug development and protein science.
This work was supported by the Russian Science Foundation (grant 19-73-00209).