Study of the binding mechanism between hydroxytyrosol and bovine serum albumin using multispectral and molecular docking

Abstract

Hydroxytyrosol (HT) is a naturally occurring amphiphilic plant polyphenol with tremendous potential as an antioxidant, antibacterial, anti-cancer, cardiovascular disease treatment, etc. However, the mechanisms of its interactions with proteins remain unclear. The interaction between HT and bovine serum albumin (BSA) in vitro was investigated through multispectral spectroscopy, molecular docking, particle size studies, and atomic force microscopy (AFM). Fluorescence spectra and molecular docking results indicated that HT was bound to the active site of BSA via hydrophobic forces through a spontaneous binding process (ΔG<0). A single HT binding site was observed on BSA, which was located in the hydrophobic cavity between Domain ⅡA and Domain ⅡB near the Trp213 residue, leading to the static quenching of endogenous BSA fluorescence. Synchronous fluorescence spectroscopy revealed that HT primarily quenched the fluorescence of Trp213 residues of the BSA. Three-dimensional fluorescence spectra confirmed the conformational alteration of BSA treated with HT, which was further supported by Ultraviolet–Visible spectra. Raman spectra showed that the α-helix content (from 36.420% to 59.968% at BSA:HT ratios of from 1:0 to 1:30, respectively) had a more obvious increase with higher HT concentrations. The solution particle sizes increased as the HT concentration increased. However, the AFM results suggested that HT had a negligible contribution toward changing the BSA microstructure. These results will provide reference materials for the wider application of HT and further research into protein-polyphenol interactions.