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

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-hydroxycou-marin (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 beta 2-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 x 104 M-1 and 5.73 x 104 M-1 at 298 K, respectively. This moderate form of protein-ligand asso-ciation could result in reversible binding to transport and release the ligand in the target tissue. Negative Delta G for both complexes suggested spontaneous binding. Positive Delta H and Delta S 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 Delta H and positive Delta S values. Changes in the microenvi-ronment of the binding site were observed through 3D fluorescence studies. Through Fo center dot rster 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 alpha-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.

Automated summary

This study investigated the molecular recognition of bovine hemoglobin with 7-hydroxycou-marin and 4-methyl-7-hydroxycoumarin. The results showed that the binding of these fluorescent compounds to hemoglobin is reversible and occurs through a static quenching mechanism. Hydrophobic forces and hydrogen bonding play important roles in the binding process.