Probing the binding of 8-Acetyl-7-hydroxycoumarin to human serum albumin by spectroscopic methods and molecular modeling

Interaction of 8-Acetyl-7-hydroxycoumarin with human serum albumin (HSA) at pH 7.40 has been investigated at 291, 301 and 310 K, respectively, employing the steady fluorescence, circular dichroism (CD) and molecular modeling methods. The quenching mechanism and binding constants were determined by the fluorescence quenching experiments. Thermodynamic data showed that 8-Acetyl-7-hydroxycoumarin was included in the hydrophobic cavity of HSA via hydrophobic interactions. The result of CD indicated that the binding of 8-Acetyl-7-hydroxycoumarin to HSA causes a slight conformational change of the protein. Furthermore, upon binding with HSA, the fluorescence spectra of the 8-Acetyl-7-hydroxycoumarin exhibits appreciable hypsochromic shift associated with an enhancement in the fluorescence intensity. The binding constant (K) and the standard free energy change (ΔG0) have been also calculated according to the fluorescence data of the ligand, which is in good agreement with the values determined by fluorescence quenching data of HSA. Computational mapping of the possible binding sites of 8-Acetyl-7-hydroxycoumarin revealed that the molecule was bound in the large hydrophobic cavity of subdomain IIA mainly by the hydrophobic interaction and also by the hydrogen bonding interactions between 8-Acetyl-7-hydroxycoumarin and the residues His 242, Arg 222, and Arg 218.