Binding\nInteraction of Coumarin Derivative Daphnetin\nwith Ovalbumin: Molecular Insights into the Complexation Process and\nEffects of Metal Ions and pH in the Binding and Antifibrillation Studies

This study investigates the interaction between daphnetin\nand ovalbumin\n(OVA) as well as its potential to inhibit OVA fibrillation using both\nspectroscopic and computational analysis. A moderate binding affinity\nof 1 × 10<sup>4</sup> M<sup>–1</sup> was observed between\nOVA and daphnetin, with a static quenched mechanism identified during\nthe fluorescence quenching processes. Metal ions’ (Cu<sup>2+</sup> and Zn<sup>2+</sup>) presence led to an increase in the binding\naffinities of daphnetin toward OVA, mirroring a similar trend observed\nwith the pH variation. Synchronous and 3D fluorescence studies indicated\nan increase in the polarity of the microenvironment surrounding the\nTrp residues during binding. Interestingly, circular dichroism and\nFourier transform infrared studies showed a significant change in\nthe secondary structure of OVA upon binding with daphnetin. The efficacy\nof daphnetin in inhibiting protein fibrillation was confirmed through\nthioflavin T and Congo Red binding assays along with fluorescence\nmicroscopic imaging analysis. The thermodynamic assessment showed\npositive Δ<i>H</i>° [+(29.34 ± 1.526) kJ\nmol<sup>–1</sup>] and Δ<i>S</i>° [+(181.726\n± 5.465) J mol<sup>–1</sup>] values, indicating the presence\nof the hydrophobic forces, while negative Δ<i>G</i>° signifies spontaneous binding interactions. These experimental\nfindings were further correlated with computational analysis, revealing\ndaphnetin dynamics within the binding site of OVA.