Binding mechanism of Orange G to human serum albumin: Saturation transfer difference-NMR, spectroscopic and computational techniques

Abstract

Abstract Orange G (OG) was used as a model compound to investigate the binding mechanism between azo dye and human serum albumin (HSA) using a variety of methods. These included nuclear magnetic resonance (NMR), saturation transfer difference (STD)-NMR, steady-state fluorescence, UV–vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR), three-dimensional fluorescence, time-resolved fluorescence spectroscopy, and molecular modeling method under simulated physiological conditions. The data of NMR and STD-NMR indicated that OG was indeed bound to HSA and located in the hydrophobic pocket of HSA. The fluorescence quenching data showed that the binding of OG and HSA quenched the intrinsic fluorescence of HSA, and the dynamic quenching constants were acquired. Thermodynamics analysis and molecular modeling studies suggested that GO bound to the site I on HSA molecule, and indicated the presence of hydrophobic forces. The alterations of protein conformational structures were further examined by synchronous fluorescence, UV–vis absorption, CD, FT-IR, three-dimensional fluorescence, and time-resolved fluorescence spectroscopy.