Identification of hepatoprotective peptides from porcine liver and its interaction with ethanol metabolizing enzymes in vitro

Alcoholic liver disease (ALD) is a worldwide disease caused by excessive ethanol consumption. In current study, hepatoprotective peptides were isolated from the porcine liver while their activities and interactions with ethanol metabolizing enzymes (alcohol dehydrogenase: ADH/acetaldehyde dehydrogenase: ALDH) were analyzed. The alcalase digested porcine liver hydrolysate (PLH) was confirmed to possess ADH/ALDH activation ability in a dose-dependent manner. The peptide sequences of PLH were identified by mass spectrometry. Molecular docking and in silico analysis were federatively used for screening. NTLPHPTAP had the lowest binding energy with ADH (−0.88 kcal/mol) and low toxicity. Fluorescence quenching data showed a dynamic fluorescence quenching constant (Kq) for NTLPHPTAP interacting with ADH/ALDH (ADH: 1.57 × 10−10 M−1 s−1; ALDH:1.961 × 10−10 M−1 s−1). Circular dichroism (CD) spectra and infrared spectrometer (FTIR) revealed that the enzyme secondary structures became flexible and disordered after interaction with NTLPHPTAP. Meanwhile, the ADH/ALDH was speculated to bind NTLPHPTAP via amido and carbonyl groups, respectively. These results indicated that the formation of ADH/ALDH-NTLPHPTAP complexes expanded the overall structure of the original enzymes and further increased the possibility of enzyme-substrate binding. In conclusion, the study proposed a method for the extraction of porcine liver hepatoprotective peptides and suggested further explanation for the interaction between NTLPHPTAP and ADH/ALDH in vitro.