Acridine Orange and Proflavin trapped in the biological nanocavity of β-Lactoglobulin: Comprehensive spectroscopic and docking analyses of the binding interactions

β-Lactoglobulin (βLG) is considered among the most extensively researched ligand-binding proteins in food science research. Acridine Orange and Proflavin, which are recognized as prospective candidates of photosensitizer in photodynamic therapy, exist in protonated forms under the physiological conditions i.e. as AOH + and PFH + respectively. This study reports spectral and docking analyses to elucidate the binding of AOH + and PFH + with βLG. AOH + perturbs the secondary structure of βLG more significantly compared to PFH + as evident from circular dichroism spectroscopic study. Furthermore, fluorescence spectroscopic study suggests that the binding interaction is hydrophobic in nature and at a particular temperature AOH + binds more strongly to βLG than PFH + . Ligand-induced steady-state fluorescence quenching of the tryptophanyl fluorescence of the protein shows an upward curvature for both AOH + and PFH + on Stern-Volmer analysis which is mainly attributed to the co-occurrence of static and dynamic quenching mechanisms.. Moreover, the phenomenon of energy transfer is found to take place from the protein to the ligands. The binding parameters as well as the thermodynamic parameters involved in the binding interactions are evaluated using fluorescence spectroscopic data. Molecular docking suggests that both the ligands enter the hydrophobic beta-barrel region of the protein. The pronounced effect of AOH + can be rationalized by the presence of the flanking methyl groups in AOH + making it more hydrophobic than PFH + .