Interaction of rofecoxib with human serum albumin: Determination of binding constants and the binding site by spectroscopic methods

The interaction of rofecoxib with human serum albumin (HSA) under physiological condition was investigated by fluorescence, UV–vis absorbance and Fourier transfer infrared (FT-IR) spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by rofecoxib was the result of the formed complex of HSA–rofecoxib, and the site binding constants (Ka) were 4.840 × 104, 3.450 × 104, and 2.325 × 104 M−1 at 298, 304, and 310 K, respectively. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be −46.90 kJ mol−1 and −67.59 J mol−1 K−1 according to van’t Hoff equation. The spectroscopic measurements and the thermodynamic parameters suggested that van der Waals interaction and hydrogen bonds were the predominant intermolecular forces to stabilize the complex. The distance r = 5.1 nm between donor (Trp214) and accepter (rofecoxib) was obtained according to the Förster theory of non-radiative energy transfer. FT-IR spectra and UV–vis absorbance showed that the change of protein secondary structures resulted from the rofecoxib binding to several amino acids on the hydrophobic pocket of HSA. Furthermore, it is observed from the probe of competitive experiments that the binding location of rofecoxib with HSA could be the same as the warfarin site I of HSA, which was also revealed by fluorescence anisotropy.