Identification of novel antioxidant and anti-inflammatory peptides from bovine hemoglobin by computer simulation of enzymolysis, molecular docking and molecular dynamics

Due to the structural diversity and complex mechanisms of action of bioactive peptides, screening for specific functional peptides is often challenging. To efficiently screen bioactive peptides with antioxidant and anti-inflammatory effects from bovine hemoglobin, we employed bioinformatics methods to perform virtual enzymatic hydrolysis using online tools and predicted the bioactivity, toxicity, and sensitization scores of the resulting peptides. Molecular docking and molecular dynamics simulations with Keap1 and TLR4 were subsequently conducted to screen for antioxidant and anti-inflammatory peptides. Finally, peptides ARRF and ARNF were synthesized using the Fmoc solid-phase method. The oxidative stress and inflammation model in RAW264.7 cells was induced using lipopolysaccharide (LPS), followed by treatment with peptides ARRF and ARNF to verify their antioxidant and anti-inflammatory activities. The results demonstrated that 529 bovine hemoglobin oligopeptides were produced following virtual enzymatic hydrolysis, of which nine were identified as eligible based on predictions of biological activity, toxicity, solubility, and sensitization. Molecular docking results indicated that the oligopeptides ARNF, QADF, and ARRF exhibited favorable interactions with Keap1, while ARNF, RRF, and ARRF showed strong interactions with TLR4. The primary active sites binding to the Keap1 receptor included Val465, Thr560, and Gly464. The main active sites binding to the TLR4 receptor were Asn309, Asn305, and Glu286. Hydrogen bonding, electrostatic interactions, and hydrophobic interactions were identified as the primary modes of interaction between the oligopeptides and the Keap1 and TLR4 receptors. Molecular dynamics simulations further confirmed that the selected bovine hemoglobin peptides could stably bind to Keap1 and TLR4 receptors. Cell experiments demonstrated that ARRF and ARNF effectively ameliorated LPS-induced oxidative stress and inflammation in RAW264.7 cells.

Conclusion: Compared to traditional methods, this study promptly screens bovine hemoglobin antioxidant and anti-inflammatory peptides, offering a novel approach for rapidly identifying food-derived bioactive peptides.