The Interaction Between Acacetin and Pepsin: A Studied by Multi-Spectral Method and Molecular Docking Simulation.

The binding interaction between acacetin and pepsin was explored by multispectral techniques combined with molecular docking. Fluorescence quenching revealed that acacetin quenched the intrinsic fluorescence of pepsin concentration-dependently, and the reduced KSV at higher temperatures verified a primary static quenching mechanism. The binding constant reached 106 L mol-1 with nearly one binding site, indicating specific intermolecular binding. Negative ΔG° reflected spontaneous binding, while negative values of ΔH° and ΔS° indicated that hydrogen bonding and van der Waals forces were the main driving forces. FRET calculations gave J = 9.8871 × 10-15 cm3·L·mol-1, E = 43.28%, R0 = 2.127 nm, and r = 2.225 nm, confirming close-range interaction. UV-visible, synchronous, 3D fluorescence, FT-IR, and circular dichroism spectra revealed that acacetin altered the microenvironment of aromatic residues and caused conformational alterations of pepsin, especially in β-sheet structure. Molecular docking indicated steady binding of acacetin near the pepsin active cleft through hydrogen bonds and non-covalent interactions. This work clarifies the molecular interaction mechanism, providing references for flavonoid-protein studies, ligand design and relevant food and pharmaceutical applications.