Probing midazolam interaction with human serum albumin

The features of midazolam (MDZ) induced structural perturbation of human serum albumin (HSA) has been studied in detail by Fourier transformation infrared spectra (FT-IR),and circular dichroism (CD).The CD spectra was deconvoluted to quantify the changes in α helix, β sheets, β turns and random coils of the protein. UV-Vis absorption studies suggested the static type quenching procedure for this binding interaction. The mechanism of MDZ-HSA binding was elucidated by steady state and synchronous fluorescence spectroscopy. The binding parameters for the reaction have been calculated according to Stern-Volmer equation at different temperatures. The plots indicated that the binding of HSA to MDZ is characterized by one high affinity binding site with the association constants of the order of 10. Employing protein unfolding pathway and site specific marker, site II in domain III of HSA has been assigned as the primary binding site for MDZ. Binding mode was expounded by thermodynamic parameters: enthalpy change (∆H) and entropy change (∆S), which imply that hydrogen bonding and hydrophobic interaction play a major role in stabilizing the complex. The molecular distance between donor (HSA) and acceptor (MDZ) was estimated according to Fluorescence resonance energy transfer (FRET).