New insights into the pH dependence of anthocyanin-protein interactions by a case study of cyanidin-3-O-glucoside and bovine serum albumin

Anthocyanins are natural pigments that can undergo structural transformations depending on pH, and their interactions with proteins have received considerable attention in recent decades. This work intended to unravel the pH-dependence of anthocyanin-protein binding mechanisms by examining interactions between cyandin-3-O-glucoside (C3G) and bovine serum albumin (BSA) at different pH. Fluorescence quenching (FQ) and microscale thermophoresis (MST) demonstrated that their binding affinity was pH 7 > pH 5 > pH 3, with dissociation constant (Kd) at pH 7 of 43.1 mu M for FQ and 33.0 mu M for MST. The predominant C3G forms were determined by measuring UV-Vis absorption spectra with pH-jump experiments. Additionally, circular dichroism, Trp fluo-rescence, zeta potential and particle size measurements demonstrated the "molten globule" state of BSA at pH 3, and C3G had a negligible effect on BSA conformation. The binding mechanisms were investigated using mo-lecular dynamics simulation, which revealed the importance of electrostatic interaction. Flavylium cation rarely bound to BSA due to electrostatic repulsion at pH 3, whereas the uncharged forms of C3G at all pH values bound to BSA surface due to hydrophobic interactions and hydrogen bonds, but possibly in a weak and nonspecific manner. Anionic quinoidal bases, the most common C3G forms at pH 7, mostly bound to the positively charged pockets of BSA and formed several hydrogen bonds with surrounding amino acids, resulting in higher binding affinity. These findings provide insights into the interactions of proteins with different anthocyanin forms, which may guide the future research and applications in this field.