Isothermal titration calorimetry binding properties of Cibacron Blue F3GA in complex with human serum albumin

Binding interactions between Cibacron Blue-F3GA (CB-F3GA) and human serum albumin (HSA, at physiologically ten-fold lower concentration) was studied by isothermal titration calorimetry (ITC) and in-silico docking computations. ITC experiments revealed two separate binding sites on HSA with different binding affinities for CB-F3GA. The high-affinity binding site (PBS-II) on HSA binds CB-F3GA at nanomolar scale (K-D1 = 118 +/- 107 nM) with favorable binding enthalpy (Delta H-1(o) = -6.47 +/- 0.44 kcal/mol) and entropy (-T Delta S-1(o) = -2.98 kcal/mol) energies. CB-F3GA binds to the low-affinity binding site (PBS-I) at mu M scale (K-D2 = 31.20 +/- 18.40 mu M) with favorable binding enthalpy (Delta H-1(o) = -5.03 +/- 3.86 x 10(-2) kcal/mol) and entropy (-T Delta S-1(o) = -1.12 kcal/mol) energies. ITC binding data strongly suggest that CB-F3GA binding to PBS-II site increases the formation of dimeric-HSA clusters (N-1 = 2.43 +/- 0.50), while binding to PBS-I leads to tetrameric-HSA clusters (N-2 = 4.61 +/- 0.90). These results suggest that a higher degree of HSA aggregation upon drug binding may be expected under physiological conditions, a notion that should be further investigated for the delivery and toxicity of drug-HSA interactions.