Investigation of Interactions between Naringenin and Bovine Serum Albumin by Native Mass Spectrometry

Serum albumin is a globular protein, which is the most abundant protein in human blood and plays a vital role in drug transportation, distribution and metabolism. Bovine serum albumin (BSA) and human serum albumin (HSA) have a high degree of homology with structural similarity of 76%. In addition, BSA is cheap and easy to obtain, which is often used as a model protein. Therefore, studying the interaction between the drug and BSA is helpful to understand the transportation and distribution of drug in the body, and is of great significance for elucidating the mechanism action of the drug. Naringenin is an important flavonoid compound that exists in various citrus fruits, tomatoes and grapes, which has been shown to have a variety of pharmacological effects, such as anti-cancer, antioxidant, anti-atherosclerosis, anti-thrombosis and vasodilation activities, etc. Although naringenin has a variety of pharmacological activities, its effect on plasma protein is still unclear. There are many methods for protein-ligand interaction analysis, but those methods have certain shortcomings, such as high sample consumption, long analysis time, the need to label and fix the protein or ligand before analysis, which may easily lead to deviations. Electrospray ionization (ESI) is used in native mass spectrometry to transfer the complete protein-ligand complex from the liquid phase to the gas phase. This process maintains weak interactions and retains the structure of biological macromolecules and biological function. Native mass spectrometry has the advantages of low sample consumption, no labeling, simplicity, rapidity, and sensitivity. It has been successfully applied to the study of the interaction between proteins and ligands in recent years. In this study, the interaction between BSA and naringenin was studied by native mass spectrometry, which could quickly obtain information about the interaction mode, stoichiometric ratio, equilibrium dissociation constant, and binding kinetics. The results showed that BSA and naringenin formed a reversible non-covalent complex with a stoichiometric ratio of 1∶1 and 1∶2. BSA and naringenin had a strong affinity (equilibrium dissociation constant Kd1=（7.82±0.03） μmol/L, Kd2=（9.63±0.02） μmol/L). The binding speed of BSA and naringenin was fast, which reached saturation after incubating for about 5 min. This method has the advantages of simplicity, rapidity, low sample consumption, no labeling, etc., and can provide reference for the study of the interaction between other drugs and serum albumin.