Analysis of the Binding Mechanism of Bioactive Coumarins with Ovalbumin: Further Investigation into the Inhibitory Effects toward Protein Fibrillation

Abstract

Two bioactive coumarin derivatives, 7-hydroxycoumarin (7-HC) and 4-methyl-7-hydroxycoumarin (4-Me-7-HC), were used to investigate the molecular interactions with the main food allergen from egg white, ovalbumin (OVA), along with their inhibitory effects on OVA aggregation. The mechanism of binding interaction was elucidated by multispectroscopic and computational methods. Circular dichroism (CD) and Fourier transform infrared (FT-IR) experiments confirmed the ligand-induced conformational changes of OVA. The fluorescence spectroscopic experiments demonstrated that the quenching mechanism was an unusual static quenching method with binding constants (Kb) in the range of 104 M–1, indicating a moderate nature of binding between OVA and coumarins. Thioflavin T (ThT) and Congo red (CR) binding assays confirmed the production of OVA fibrils, and the aggregation was shown to be inhibited by the coumarin derivatives in vitro. Thermodynamic parameters for OVA-7-HC/4-Me-7-HC interactions showed positive ΔH and ΔS values, indicating hydrophobic forces predominated in the binding processes and a negative ΔG value, implying the spontaneity of the complex formation, which was then further confirmed by computational studies.