Interactive multiple binding of oleic acid, warfarin and ibuprofen with human serum albumin revealed by thermal and fluorescence studies.

Human serum albumin binds a wide variety of drugs with different structure and affinity to two main binding sites, drug site 1 (DS1) and drug site 2 (DS2), which partially or totally overlap with fatty acid (FA) sites. Although multiple binding sites are available for endogenous compounds, FAs are the primary physiological ligands of albumin and their competition in the occupancy of DS1 and DS2 affects the binding of exogenous molecules, with a possible impact on drug delivery. In this work, we have investigated the simultaneous binding of oleic acid, warfarin and ibuprofen to albumin using differential scanning calorimetry and fluorescence to evaluate the impact on the conformational stability of the protein. The two drugs are widely used for their anticoagulant (warfarin) and anti-inflammatory (ibuprofen) properties, and can be also considered as site markers to probe DS1 and DS2, respectively. Oleic acid is one of the most important fatty acids from a physiological point of view for its role as a source of energy for cells, and also it binds albumin with the highest association constant. When complexed with oleic acid the calorimetric profile of albumin shows a biphasic trend whose line shape depends on the ligand concentration. The binding capacity of either warfarin or ibuprofen to albumin is modulated by oleate molecules in a concentration-dependent mode being synergic cooperative (warfarin) or competitive-like (ibuprofen). The overall results provide insights on the dynamics of albumin/ligands complex, which in turn may have important pharmacokinetic and pharmacodynamic implications.