In vitro investigation of domain specific interactions of phenothiazine dye with serum proteins by spectroscopic and molecular docking approaches

Abstract

In the present study the interaction of the chemotherapeutic agent, Azure A (AZA) with Human Serum Albumin (HSA) and Bovine Serum Albumin (BSA) was investigated by multi spectroscopic and molecular docking methods. The influence of inner filter effect (IFE) on the emission quenching of HSA/BSA at low concentration of AZA (absorption value <0.1) suggested the need to employ an IFE correction factor even for the low concentration regime. The emission titration experiments of HSA/BSA with AZA revealed the formation of AZA–HSA/BSA complexes. The binding parameters calculated from corrected emission intensities showed that AZA binds to HSA/BSA with moderately strong binding affinities. The negative free energy obtained for the binding of AZA with HSA/BSA indicated that the complexation process is spontaneous. The results from site maker competitive experiments with specific site markers revealed that the probable binding location of AZA is located near site I of HSA/BSA. An AutoDock based molecular docking approach was utilized to characterize the binding models of AZA–HSA/BSA complexes. The free energy calculations for the most stable conformer from molecular docking studies were utilized to examine the energy contributions and the role of various amino acid residues of HSA/BSA in AZA binding. The results of site-competitive replacement experiments with specific site markers and molecular docking simulation studies unambiguously helped us to conclude that AZA binds to site I of HSA/BSA. Constant wavelength synchronous emission, excitation–emission matrix (three-dimensional) emission, absorption and circular dichroism spectroscopic techniques have been exploited to unravel AZA induced tertiary and secondary conformational changes of HSA/BSA.