Interaction Behavior between Spermidine and Pepsin Determined by Spectroscopic and Docking Methods

Pepsin (EC 3.4.23.1), the main digestive enzyme, is released as a zymogen by the chief cells in the stomach to degrade food proteins into peptides. Pepsin has a single polypeptide with 327 amino acid and mainly negative charge in pH stomach. Polyamine (Spermidine: NH2 (CH2)3NH-(CH2)4 NH2) are fully protonated at the physiological pH. Due to their cationic nature, they interact with negatively charged molecules including proteins that can induce structural changes. Information on protein stability is essential to study protein structure and interactions with ligands. On the other hand, the interactions of pepsin with other ligands be also very important for the treatment of some diseases related to pepsin. Therefore, the interaction of spermidine with pepsin was investigated intrinsic fluorescence spectroscopy, UV−Vis spectroscopy, and molecular docking. The complex formation between spermidine and pepsin was detected as a change in the absorbance at 280 nm, which is due to the aromatic amino acids (Trp residues) in pepsin. The intensity of this peak slightly increases after the addition of spermidine. On increasing the concentration of spermidine, the fluorescence intensity decreases. The decrease in the fluorescence intensity happens because of various types of molecular interactions such as ground‐state complex formation, excited state reaction, collisional quenching, molecular rearrangement, and energy transfer. The Stern−Volmer quenching constants (Ksv) for the pepsin−spermidine complex were obtained at three temperatures, revealing that spermidine quenched the intensity of pepsin through the static mode of the quenching mechanism. Thermodynamic parameters calculated at different temperatures revealed that negative value for the Gibbs free energy, so interaction was a spontaneous process. Docking results supported experimental findings and suggest aspermidine induced change in the structure of the active site by bindings with pepsin in locations of catalytic residues Asp32 and Asp215, which can stabilize the complex.