The interaction of biologicalmacromolecules, whether protein-DNA, antibody-antigen, hormone-receptor, etc., illustrates the complexity and diversity of molecular recognition. The importance of such interactions in the immune response, signal transduction cascades, and gene expression cannot be overstated. It is of great interest to determine the nature of the forces that stabilize the interaction. The thermodynamics of association are characterized by the stoichiometry of the interaction (n), the association constant (K(a)), the free energy (DeltaG(b)), enthalpy (DeltaH(b)), entropy (DeltaS(b)), and heat capacity of binding (DeltaC(p)). In combination with structural information, the energetics of binding can provide a complete dissection of the interaction and aid in identifying the most important regions of the interface and the energetic contributions. Various indirect methods (ELISA, RIA, surface plasmon resonance, etc.) are routinely used to characterize biologically important interactions. Here we describe the use of isothermal titration calorimetry (ITC) in the study of protein-protein interactions. ITC is the most quantitative means available for measuring the thermodynamic properties of a protein-protein interaction. ITC measures the binding equilibrium directly by determining the heat evolved on association of a ligand with its binding partner. In a single experiment, the values of the binding constant (K(a)), the stoichiometry (n), and the enthalpy of binding (DeltaH(b)) are determined. The free energy and entropy of binding are determined from the association constant. The temperature dependence of the DeltaH(b) parameter, measured by performing the titration at varying temperatures, describes the DeltaC(p) term. As a practical application of the method

@Mohsen Darabi... Any realistic solution is highly appreciated

There are two issues regarding those titrations:

1. The buffer-buffer assay dos not show a constant heat affect in the injections. Although the heats per injection seem to be smaller than in the other experiments with protein samples, it is strange. Do you really need 36% glycerol?

2. The protein B-buffer assay shows a profile similar to that of the protein B-protein A assay, in shape and size of heat effect per peak. This would mean that in the third assay you are basically observing protein B dilution (or dissociation), provided that after subtracting the buffer-buffer heat effects there is a remaining heat effect in each injection.

I would try:

1. Reducing the glycerol concentration (in all solutions).

2. Increasing concentrations.

3. Selecting another temperature.

4. Selecting another buffer with different ionization enthalpy.

5. Selecting another pH.