Multi-spectroscopic and computational studies of esculetin/esculin-ovalbumin interactions at molecular level: Effects of metal ions and pH on binding, inhibition of amyloid aggregation.

This study examines the interaction between ovalbumin (OVA) and two coumarin derivatives, esculin and esculetin, using spectroscopic and computational methods. Fluorescence quenching confirmed a static mechanism, with binding constants (Kb) in the range of 1.776-3.340 × 104 M-1 for esculetin and 1.198-3.870 × 104 M-1 for esculin across 291-308 K. The binding affinity increased in the presence of Cu2+ and Zn2+ and was enhanced under acidic conditions. Thermodynamic analysis revealed that OVA-esculetin interaction is driven by hydrophobic forces and hydrogen bonding (ΔH = +31.18 kJ mol-1, ΔS = +188.06 J mol-1 K-1, ΔG = - 24.86 kJ mol-1), whereas OVA-esculin binding is dominated by hydrogen bonding and van der Waals interactions (ΔH = -53.09 kJ mol-1, ΔS = -93.21 J mol-1 K-1, ΔG = - 25.31 kJ mol-1). Spectroscopic analyses (synchronous, 3D fluorescence, CD, and FT-IR) indicated conformational and microenvironmental changes in OVA upon ligand binding. Thermal studies showed that esculetin enhanced protein stability (Tm = 72.01 °C), while esculin reduced it (Tm = 60.14 °C). ThT and Congo Red assays, supported by fluorescence microscopy, confirmed inhibition of OVA fibrillation. Molecular docking and MD simulations validated the experimental data, showing stable binding of esculin and esculetin within the OVA binding cavity.