Elucidating the Binding Mechanism of Kojic Acid with Human Hemoglobin by Molecular Docking and Multi-Spectroscopic Techniques.

Kojic acid (KA) is a natural secondary metabolite that is widely known for its skin-lightening properties and also used as food preservative. Here, we have explored the binding and interaction of KA with human hemoglobin (HHb), a multifunctional and the predominant protein in erythrocytes, using multi-spectroscopic techniques, enzymatic activities (esterase and peroxidase) and molecular docking method. The ultraviolet-visible absorption spectra of HHb showed hyperchromic effect at 275 nm upon addition of KA. The fluorescence experiments showed that KA quenches HHb fluorescence and alters the microenvironment around tryptophan residues. The fluorescence quenching mechanism is of static type and there is a single KA binding site on each HHb tetramer. KA binds spontaneously and interacts with HHb through ground state complex formation. The negative values of thermodynamic parameters ([Formula: see text]= -0.010 kcal mol K and [Formula: see text]= -8.35 kcal mol) indicated that van der Waals interactions and hydrogen bonds play an important role in stabilizing the HHb-KA complex. Circular dichroism studies revealed that KA induces changes in the secondary structure of HHb and decreases its α-helical content from 77.03 to 66.34%. The pseudo-esterase and peroxidase activities of HHb were significantly inhibited by KA in a concentration dependent manner. Molecular docking confirmed the formation of HHb-KA complex with binding free energy of -5.2 kcal mol and revealed the specific amino acid residues of HHb participating in binding to KA. The results of this study demonstrate that binding of KA to HHb induces structural alterations and impairs the function of this oxygen transporting protein.