The present study was focus on the interaction of alpha lactalbumin (α-LA) and Nano-berberine (Nano-Ber) in the absence and presence of beta lactoglobulin (β-LG) as binary and ternary systems by the exertion of different spectroscopic techniques, conductometry, β-galactosidase activity, and molecular simulation measurements. The outcomes of fluorescence quenching clarified the dominance of static quenching mechanism throughout the interaction of α-LA-Nano-Ber and (α-LA-β-LG) Nano-Ber complexes and also determined the KSV values to be (4.68±0.05) × 104 and (5.18±0.06) × 104 M−1, respectively. The measured thermodynamic parameters exhibited the participation of electrostatic forces in the binding interaction of α-LA and α-LA-β-LG complex with Nano-Ber. Moreover, the negative value of ΔG0 represents the spontaneous mode of this binding process in both systems. Synchronous fluorescence and resonance light scattering data demonstrated the impact of β-LG attendance on altering the interaction behavior of α-LA and Nano-Ber. The changes in the secondary structure of both proteins were affirmed by circular dichroism spectroscopy (CD) technique. According to the displayed β-galactosidase activity in the Michaelis-Menten plot of both binary and ternary systems, a greater Vmax refers to the stronger affinity of lactose and lactose hydrolysis that can be effective on the lactose intolerance regarding dairy products. Additionally, molecular dynamic simulations were employed to simulate the atomic-level interactions and explore the stability and conformational changes of protein-Nano Ber complexes over time. The results of these comprehensive biophysical investigations shed light on the binding characteristics, structural alterations, and dynamic behavior of Nano-Ber upon interaction with α-lactalbumin in the absence and presence of β-lactoglobulin as the binary and ternary systems. This study reveals the Nano-Ber role in the protein–protein interaction behavior and provides valuable insights into the potential applications of α-lactalbumin in the design of functional food formulations and drug delivery systems
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