In-depth analysis of dynamic binding of sardine-derived functional tripeptide with serum albumin in diverse buffers.

Food-derived angiotensin-converting enzyme inhibitory peptides (ACEIPs) are commonly utilized in functional food development. In-depth research on their interactions with serum albumin can enhance their bioavailability and in vivo functionality from functional food. This study systematically investigated the impact of the ACEIP Gly-Arg-Pro (GRP) derived from sardine muscle protein on the conformation and interaction mechanisms of bovine serum albumin (BSA) under different physiological buffer environments using multispectral techniques, molecular docking theory and isothermal titration calorimetry. GRP demonstrated potent angiotensin-converting enzyme inhibitory activity and had good biosafety in cytotoxicity assays. Steady-state and time-resolved fluorescence spectroscopy indicated the formation of a moderately bound BSA-GRP complex in various buffer environments. The existence of the BSA-GRP complex was further confirmed by ultraviolet-visible spectroscopy. Three-dimensional fluorescence spectroscopy, synchronous fluorescence spectroscopy and circular dichroism spectroscopy demonstrated that adding GRP to BSA affected its microenvironment and structural conformation, with more significant changes observed in the HEPES buffer system due to its distinct physicochemical properties. The thermodynamic parameters indicated that the interaction was mainly driven by hydrophobic forces, making it a spontaneous process regarding Gibbs free energy change. Molecular docking theory showed that GRP is preferentially bound to site II of subdomain IIIA. This work established a research system for BSA-GRP interactions and obtained relevant parameters, which will benefit the subsequent development of nutritional supplements and functional foods. © 2025 Society of Chemical Industry.