Exploration of the Binding Mechanism between Puerarin and β-Lactoglobulin Using Fluorescence Spectroscopy and Molecular Dynamics Simulation

The binding mechanism of puerarin (PUE) to β-lactoglobulin (β-lg) was investigated by fluorescence spectroscopy, molecular docking, and molecular dynamics simulation. The fluorescence spectroscopy results showed that PUE statically quenched the fluorescence of β-lg. At 25, 35, and 45 ℃, the binding constants were 7.24 × 10⁴, 1.34 × 10⁵, and 2.18 × 10⁵ L/mol, and the numbers of binding sites were 1.02, 1.18, and 1.15, respectively, indicating there was only one binding site or class of binding sites. Synchronous fluorescence and three-dimensional fluorescence spectroscopy indicated that the binding of PUE to β-lg resulted in an increase in the polarity of the microenvironment of β-lg, thereby weakening the hydrophobic force. Molecular docking results showed that PUE bound to the hydrophobic cavity of β-lg, forming hydrophobic interactions with six amino acid residues of β-lg and short hydrogen bonds with five amino acid residues. The molecular dynamics results showed that the root mean square deviation (RMSD), radius of gyration (R_g), and solvent accessible surface area (SASA) of the complex were (0.17 ± 0.02) nm, (1.47 ± 0.01) nm, and (88.94 ± 2.05) nm², and those of β-lg were (0.22 ± 0.03) nm, (1.48 ± 0.01) nm, and (90.09 ± 1.73) nm², respectively. The root mean square fluctuation (RMSF) of the complex was lower than that of β-lg, suggesting that the PUE/β-lg complex has better stability at the molecular level. This study is of reference significance for increasing the bioavailability of PUE.