Probing the interaction of doxycycline to trypsin and bovine hemoglobin by using multi-spectral techniques and molecular docking

Abstract

The interactions of doxycycline (DC) with digestive enzyme (trypsin) and intracellular protein (bovine hemoglobin, BHb) were investigated by using multi-spectral techniques and molecular docking. Fluorescence studies suggested that DC quenched trypsin fluorescence in a static quenching and BHb fluorescence in a combined quenching (dynamic and static quenching) with binding constants of 0.22 and 1.45×104L⋅mol−1 at 298K, respectively. The thermodynamic parameters demonstrated that the main forces of BHb-DC and trypsin-DC systems were hydrophobic forces, hydrogen bonds and van der Waals forces in the binding processes. Based on the results of FRET, the binding distances between DC and the inner tryptophan residues of trypsin and BHb were calculated to be 4.09 and 3.86nm, respectively. Furthermore, the results of circular dichroism spectra (CD) indicated the secondary structures of trypsin and BHb were partially changed by DC with the α-helix percentage of trypsin-DC system increased (6.3%–7.3%) and that of BHb-DC system also increased (38.5%–46.2%), the β-sheet percentage of trypsin-DC system decreased from 43.3% to 41.0%. UV–vis and three-dimensional fluorescence spectra results showed these binding interactions could cause conformational and some micro-environmental changes of trypsin and BHb. With the results of molecular docking, for trypsin, DC can bind to the active sites residues HIS 57 and SER 195 in the pocket of trypsin, thereby increasing the activity of trypsin, for BHb, DC binds into the hydrophobic cavity of BHb via multiple interactions. The work contributes to understand the interactions of DC with trypsin/BHb at molecular level.