Comparative Studies on the Interaction of Spermidine with Bovine Trypsin by Multispectroscopic and Docking Methods.

The effect of spermidine on the kinetics, conformation, and dynamics of native trypsin was studied by steady-state thermal stability, intrinsic fluorescence, circular dichroism (CD), ultraviolet-visible (UV-vis) spectroscopy, and kinetic techniques, as well as molecular docking, at the temperatures of 298 and 308 K. The Stern-Volmer quenching constants (Ksv) for the trypsin-spermidine complex were obtained at two temperatures, revealing that spermidine quenched the intensity of trypsin through the static mode of the quenching mechanism. The corresponding thermodynamic parameters, Gibbs free-energy, enthalpy, and entropy changes, showed that the binding process was spontaneous. These values and the molecular docking technique revealed that the hydrogen bonding and van der Waals forces played a major role in stabilizing the complex. CD, absorption, and fluorescence results also indicated that spermidine binding had a partial effect on trypsin structure. Spermidine could also influence the activity of trypsin. Upon spermidine binding, the Vmax value of the enzyme was increased and the kcat/Km values were enhanced slightly. The Tm of the trypsin-spermidine complex was enhanced probably due to the higher H-bond formation and lower surface hydrophobicity after spermidine modification, as confirmed by UV-vis spectroscopy and fluorescence spectra. UV absorption and CD studies also indicated that the binding of spermidine to trypsin had induced microenvironmental changes around the enzyme, leading to changes in its secondary structure.