A comprehensive in vitro exploration into the interaction mechanism of coumarin derivatives with bovine hemoglobin: Spectroscopic and computational methods

Abstract

Biological functioning takes place through the interaction of biomacromolecules with each other or with other small molecules. The molecular recognition of a carrier protein, bovine hemoglobin (BHb), with 7-hydroxycoumarin (7-HC) and 4-methyl-7-hydroxycoumarin (4-Me-7-HC) was investigated using various biophysical and computational techniques. Fluorescence spectroscopy and molecular docking revealed the interaction of 7-HC and 4-Me-7-HC with β2-Trp37 fluorophore, quenching the intrinsic fluorescence of BHb. The mechanism of quenching was determined to be static. The binding constant (Kb) for BHb with 7-HC and 4-Me-7-HC was found to be 6.15 × 104 M−1 and 5.73 × 104 M−1 at 298 K, respectively. This moderate form of protein–ligand association could result in reversible binding to transport and release the ligand in the target tissue. Negative ΔG for both complexes suggested spontaneous binding. Positive ΔH and ΔS for BHb-7-HC indicated hydrophobic forces played a dominant role in binding. However, hydrogen bonding and hydrophobic forces dictated the binding process for BHb-4-Me-7-HC due to obtaining negative ΔH and positive ΔS values. Changes in the microenvironment of the binding site were observed through 3D fluorescence studies. Through Förster resonance energy transfer (FRET), a binding distance of less than 7 nm was measured between BHb and 7-HC/4-Me-7-HC. The interaction of coumarin derivatives with BHb resulted in a loss of α-helical content of the protein, as proven by circular dichroism (CD) and Fourier transform infrared (FTIR) measurements. Molecular dynamic simulations showed the binding of 7-HC supplemented the stability of BHb, whereas 4-Me-7-HC binding resulted in conformational changes in the structure of BHb.