Commercial human serum albumin (HSA) will be employed. Prior to the study, HSA will be dialyzed to rid some soluble additives and frozen-dried to form a solid state. It purity will be not less than 97%. Drug compounds of high purity grade (no less than 97%) such as phenytoin, and salicylic acid which preferentially binds with sub-domain IIA, and IIIA, respectively, with high affinity will be candidate ligands. The drug compound which binds both sub-domains with high affinity such as ceftriaxone is also employed. These ligands are subjected to change due to the compound availability, but the candidates must meet the requirements of preferentially binding to either sub-domain with considerably high affinity reported in the literature (e.g. Kratochwil et al, 2002). All other reagents are of analytical grade.

The DSC scanning of HSA solution will be undertaken to yield the characteristic heat flow-temperature profiles called thermograms. With various scanning rates, the thermal induced denaturation will be done between 25 and 90 degrees C. The dilution of solid HSA with appropriate solvents such as phosphate buffers may be done so as to suit for convenient sample handling, instrument loading with acceptable signal-to-noise DSC detection.

            Variety of the drug candidate will be equilibrated with HSA at concentration of 50 g/L in physiological pH (phosphate or other appropriate buffer) and dialyzed afterwards to make sure that only bound drug candidate is in the system under study. The solutions are subjected to differential scanning calorimetric study, DSC running against buffer. HSA at identical concentration without the drug candidate will be DSC run as a control group. Three replications will be performed. The thermograms will be analyzed using some thermodynamic theoretical concept to have the insight information of the drug-protein binding interactions. The DSC equilibrium thermodynamic analysis based on the theories proposed (e.g. Sanchez-Ruiz, 1992) will be done to examine the effect of protein binding on the protein stability including reversible unfolding and irreversible denaturation. In addition, the mixture solution will be frozen-dried. The solid form of HSA-drug candidate will be subjected to DSC run to examine the relaxation transitions of amorphous HAS as the drug interaction might contribute to glassy state stability of solid HSA (Ur’yash and Kokurina, 2011).

To validate the DSC method under study, the established methods of protein binding study will be done. The methods such as classical dialysis, 2-D electrophoresis, fluorescence quenching spectroscopy, and others will be employed. These methods will be modified from the literature (e.g. Faridbod et al, 2011; Keswani and Kishore, 2011; Xiong et al, 2011; Divsalar et al, 2009; and Sulkowska et al, 2008, etc.).