Spectroscopic study on the interaction of celecoxib with human carbonic anhydrase II: Thermodynamic characterization of the binding process

Abstract

The effect of celecoxib, a sulfonamide drug, on the structure and function of human carbonic anhydrase II (hCA II) was investigated by various spectroscopic techniques such as UV–Vis, fluorescence and circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC), in 20 mM Tris, pH 7.75 at 27 °C. Kinetic results revealed that celecoxib inhibits the esterase activity of hCA II in a linear competitive manner with K i = 61.61 ± 3.05 nM. DSC data indicated that the thermal stability of the enzyme has a minor increment in the presence of celecoxib. Fluorescence measurements showed that the celecoxib acts as a quencher of the enzyme fluorescence and calculation of the protein surface hydrophobicity (PSH), using 1-anilinonaphthalene-8-sulfonate (ANS), revealed the decrement of its PSH upon interaction with the drug. Acrylamide quenching experiments indicated the less accessibility of the tryptophan residues to the acrylamide due to the presence of celecoxib. Stern–Volmer analysis of quenching data at different temperatures elucidated that the quenching of intrinsic fluorescence of hCA II is occurred through a static quenching mechanism. Analysis of the thermodynamic parameters of binding showed that hydrogen bonding and hydrophobic interactions play the major role in stabilization of the enzyme–drug complex. The Job’s plot confirmed the existence of one binding site for celecoxib in hCA II. The far- and near-UV CD experiments indicated that celecoxib causes a little increment in α-helicity content of hCA II whereas its flexibility is decreased somewhat upon celecoxib binding.