Characterization of non-covalent binding of 6-hydroxyflavone and 5,7-dihydroxyflavone with bovine hemoglobin: Multi-spectroscopic and molecular docking analyses

Flavonoids are biologically imperative compounds used as anti-oxidants, anti-cancer, anti-bacterial agents etc. The current work reports comprehensive binding studies of two important Flavonoids, 6-hydroxyflavone and 5,7-dihydroxyflavone (chrysin) with bovine hemoglobin (BHb) at 298K and 308K, in aqueous medium using UV-vis spectroscopy, steady state fluorescence, circular dichroism (CD) measurements, Fourier Transform infrared spectroscopy (FT-IR) and molecular docking studies. Both 6-hydroxyflavone and chrysin can quench the intrinsic fluorescence intensity of BHb via static quenching mechanism. The values of binding constant (K_b) for BHb-chrysin complex (3.177±0.992×10⁴M^-1, at 298K) was found to be greater than that of BHb-6-hydroxyflavone complex (2.874±0.863×10⁴M^-1, at 298K) and the K_b values decreased with the rise in temperature. The thermodynamic parameters indicated that hydrophobic forces and H-bonding play crucial role in BHb-6-hydroxyflavone complexation whereas electrostatic interaction plays the major role in the binding of BHb and chrysin. The binding distances from donor BHb to the acceptor ligands (6-hydroxyflavone and chrysin) were estimated using the Föster's theory and the possibility of non-radiative energy transfer from BHb to 6-hydroxyflavone/chrysin was observed. The ligands, 6-hydroxyflavone and chrysin induced conformational change around Trp residues in BHb as confirmed by synchronous and 3D fluorescence results. CD and FT-IR studies indicated that the % α-helicity of BHb was enhanced due to 6-hydroxyflavone/chrysin binding. Both the Flavonoids showed remarkable inhibitory effect towards BHb glycation. Hydrophobic probe (8-anilino-1-naphthalenesulfonic acid, ANS) displacement and molecular docking studies revealed that the ligands bind within the hydrophobic pocket of BHb.