Characterization of the binding of flavanone hesperetin with chicken egg lysozyme using spectroscopic techniques: Effect of pH on the binding

The interaction between hesperetin and hen egg lysozyme has been investigated using UV-vis, steady-state fluorescence, synchronous fluorescence, three dimensional (3D) fluorescence, circular dichroism (CD) and molecular docking studies. The structural characteristics of hesperetin are monitored at different pH conditions (5.4, 6.4, 7.0, 7.4, 8.0, 8.4 and 9.0) and its binding affinity towards lysozyme is also determined using fluorescence study. It has been found that binding constant increased from pH 5.4 (0.154 × 10 M) to pH 7.0 (1.943 × 10 M), followed by a decreasing trend from pH 7.0 to 9.0 (1.293 × 10 M). The number of binding sites for hesperetin in lysozyme is estimated to be 1.394 ± 0.128 and negative δG (-23.345 ± 2.231 kJ mol) indicates the spontaneous nature of binding. Spectrofluorimetric analyses rationalized that the static quenching mechanism is present in the binding and the reason behind the upward curvature nature of Stern-Volmer plots are also discussed. The binding distance between lysozyme and hesperetin has been estimated according to Förster’s theory and a possibility of non-radiative energy transfer from lysozyme to hesperetin is observed. Synchronous and 3D fluorescence studies indicate that hesperetin binding induced apparent structural perturbation in lysozyme and it increased α-helicity of lysozyme by ~ 4 % (CD results). The interaction of hesperetin with lysozyme decreased the enzymatic activity of lysozyme and revealed the affinity of hesperetin towards the active site of lysozyme. Molecular docking study reveals that hesperetin prefers to bind in the close proximity of Trp 62 and Trp 63 and it is vital for catalytic activity of lysozyme.