New insights into the binding mechanism between osthole and β-lactoglobulin: Spectroscopic, chemometrics and docking studies.

Osthole, a type of coumarin derivative, owns many biological functions, but the poor water solubility and low bioavailability limit its usage in food and pharmaceutical fields. β-Lactoglobulin (BLG) is a major protein in bovine milk whey which is usually used as a carrier of poorly soluble bioactive components. Researching the interaction between osthole and BLG can help us in understanding their binding mechanism and design an osthole delivery system using BLG as carrier protein to further promote the application of osthole in functional food and drug. Hence, this study was devoted to explore the binding properties of osthole with BLG and the effect on the protein structure by employing multispectroscopic approaches, chemometrics and molecular simulation studies. The results of the concentration profiles and pure spectra of the components resolved from highly overlapping spectral signals by the multivariate curve resolution-alternating least squares (MCR - ALS) algorithm verified the formation of an osthole-BLG complex. Moreover, osthole was found to strong quench the intrinsic fluorescence of BLG and the quenching mechanism was determined to be a static procedure. The binding constant of osthole with BLG at 298 K was (4.06 ± 0.03) × 104 L mol-1. Hydrophobic forces and hydrogen bonds played major roles in the binding process. The analysis of synchronous fluorescence, circular dichroism and Fourier transform infrared spectra found that the presence of osthole caused BLG structure compact and the change in the polarity and hydrophobicity of the microenvironment around Tyr residues of the protein. Modeling docking predicted that osthole bound to the hydrophobic cavity of BLG through stable hydrogen bonds primarily with the amino acid residues Lys75 and Thr76.