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 (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.