Antioxidant activity of soybean peptides and Keap1 protein: A combined in vitro and in silico analysis

Antioxidant peptides can be obtained from diverse dietary protein sources with high safety.. The Kelch-like ECH-associated protein 1(Keap1) is a key protein in the cellular oxidative stress signaling pathway and it plays an important role in the field of antioxidant . Soybeans are a vital source for the production of bioactive peptides. In this study, firstly, we screened 468 antioxidant peptides from soybean protein with low molecular weight, high hydrophobicity and non-toxicity. The top three antioxidant peptides were isolated and extracted from the fractions using UHPLC-QQQ/MS analysis. The free radical scavenging rate demonstrated significant antioxidant activity of PHHADS (EC 50 = 4.26mmol/L). In addition , peptide PHHADS was demonstrated significantly protects HepG2 cells from oxidative stress-induced damage. Lastly, 200 ns molecular dynamics simulations were performed between the peptides and Keap1. The results revealed that PHHADS closely interacts with the active site of Keap1. Moreover, the interaction of the antioxidant peptides with Keap1 led to the disruption of the β-sheet structure of Keap1 in residues380-390380-390, transforming into an irregularly curved structure. This particular region in Keap1 is crucial for substrates binding, suggesting a potential inhibitory mechanism by the peptides. These findings contribute to the understanding of antioxidant peptide interactions and their potential application in developing functional foods for health benefits. • A database of 468 soybean antioxidant peptides was established by in silico hydrolysis. • The top three scoring antioxidant peptides were identified by UHPLC-QQQ/MS. • The peptide PHHADS is protective against oxidative damage in HepG2 cells. • Molecular docking reveals key residues for Keap1 protein binding to antioxidant peptides. • Molecular dynamics simulation reveals conformational changes of Keap1 protein upon binding of antioxidant peptides.