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

Abstract

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 (KD1 = 118 ± 107 nM) with favorable binding enthalpy (ΔHo1 = − 6.47 ± 0.44 kcal/mol) and entropy (−TΔSo1 = −2.98 kcal/mol) energies. CB-F3GA binds to the low-affinity binding site (PBS-I) at μM scale (KD2 = 31.20 ± 18.40 μM) with favorable binding enthalpy (ΔHo1 = − 5.03 ± 3.86 × 10−2 kcal/mol) and entropy (−TΔSo1 = −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 (N1 = 2.43 ± 0.50), while binding to PBS-I leads to tetrameric-HSA clusters (N2 = 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.