Studies on the binding of rhaponticin with human serum albumin by molecular spectroscopy, modeling and equilibrium dialysis

Abstract

Rhaponticin (RHA) possesses a variety of pharmacological activities including potent antitumor, antitumor-promoting, antithrombotic, antioxidant and vasorelaxant effects. In the solvation effect, RHA exhibited bathochromic shift in emission spectra with the increasing solvent polarity. The binding between RHA and HSA was investigated by molecular spectroscopy combining with modeling and equilibrium dialysis. Fluorescence data showed that the quenching of HSA by RHA was result of forming the complex of RHA-HSA. According to Stern-Volmer equation, the binding parameters between RHA and HSA were determined. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be -2.75kJmol(-1) and 1.58Jmol(-1)K(-1), indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding. The conformational investigation revealed the α-helical structure was decreased and the polypeptides of HSA were slightly folded upon the addition of RHA. The effect of common ions on the binding between RHA and HSA was also investigated. Furthermore, the result of warfarin displacement site indicated that RHA could bind to the site I of HSA, which was in agreement with the molecular modeling. When excitation wavelength was set at 260 or 355nm, RHA exhibited a fluorescence peak at 390nm, based on which, a simple and rapid fluorimetric method was developed and validated to determine RHA in the equilibrium dialysis. Calibration curves of RHA were linear over the concentration range of 1.1-15.0μM with the detection limits of 0.03μM. Examination of protein binding ability showed that RHA with 8.0μM concentrations in HSA achieved the percent of bound 82.3±2.5%.