Abstract:

Morin is a natural flavonoid compound extracted from the bark of mulberry, orange, and other fruit trees. Serum albumin (SA) is the most abundant carrier protein in animal plasma, as well as the most common soluble protein in the circulatory system. The study of the binding behavior of Morin and the characteristics of the binding of Morin to SA would help in further elucidating its transport process and mechanism of action in vivo at the molecular level. Herein, the thermodynamics of the interaction between bovine serum albumin (BSA) and Morin was investigated by fluorescence, UV-Vis absorbance, CD, and molecular modeling under physiological conditions. The quenching constants (K_SV) decreased as the temperature increased, indicating that the fluorescence quenching of BSA by Morin was a static process. The static quenching mechanism was further supported by the measurement of the UV-vis spectra of the BSA-Morin system. Based on the van't Hoff equation, the ΔH^Ɵ, ΔS^Ɵ, and ΔG^Ɵ were calculated to be around −81.20 kJ·mol⁻¹, −181.01 J·mol⁻¹·K⁻¹, and −27.19 kJ·mol⁻¹, respectively. The negative ΔG^Ɵ value indicated that the interaction between Morin and BSA was a spontaneous process. The hydrogen bonds and van der Waals force played a predominant role in the binding process. Our data indicate that Morin binds solely with the BSA molecule. The apparent binding constant of the Morin-BSA system reached the order of 10⁴, which further confirmed the strong binding between Morin and BSA. This indicates that serum albumin can store and transport Morin molecules in the body, enabling them to reach the action site through blood circulation; thus, they can exert their physiological and biochemical effects. By using the fluorescence resonance energy transfer theory and the molecular simulation method, we found that Morin bound at Site Ⅱ in the hydrophobic cavity of the substructure domain IIIA of BSA, and the average distance between the two tryptophan residues and Morin was 3.09 nm. The synchronous fluorescence spectrum also revealed that Morin was far away from the two tryptophans of BSA, and therefore, cannot change the spatial structure near tryptophan. The CD spectra demonstrated that the α-helix content of BSA decreased from 59.5% to 53.9% after its interaction with Morin, while the disordered structure increased from 20.6% to 23.7%. The best-fitted docking poses reveal that Morin mainly contacted with the side-chains of surrounding hydrophobic amino acid residues. In addition, the generation of hydrogen bonds between hydroxyl groups on Morin molecules and the side-chains of R413 and K437 can be observed. These results provide basic knowledge for understanding the pharmacology of Morin, and useful guidance for designing, modifying, and screening flavonoid drug molecules.

Key words: Morin, Bovine serum albumin, Interaction, Thermodynamics