Identification And Prediction Of Bovine Hemoglobin Derived ACE-inhibitory Peptides And Its Inhibitory Molecular Mechanism

Hypertension is one of the major risk factors for cardiovascular diseases whichinclude coronary heart disease, peripheral artery disease and stroke. The globalprevalence of raised blood pressure in adults aged18years and over was around22%in2014. ACE plays a crucial role in regulation of blood pressure and cardiovascularfunction. Currently, inhibition of angiotensin I-converting enzyme has been reported as afirst-line therapy for hypertension. However, the chemical synthetical and modificatoryACE inhibitors are reported plethora of side effects, such as renal impairment, dry cough,skin rashes, hyperkalaemia and angioedema. Therefore, developing novel, safe, and mildACE inhibitors from natural food-derived proteins has attracted more and more attention.As the main byproducts of beef processing industry, bovine hemoglobin was not utilizedeffectively even it has been reported as potential protein resource of ACE-inhibitorypeptides. Nowadays, the traditional way (hydrolyzation-isolation-purification) toprepare ACE-inhibitory peptide has been well developed but costly and time consuming.Hence, the computer simulation technology has been applied to discover the potentialACE-inhibitory peptides and to investigate the ACE-inhibitory molecular mechanism.In this study, the potential ACE-inhibitory peptides from bovine hemoglobin wereobtained by the traditional way and by the computer-added modeling, and then theirACE-inhibitory activity was determined. The effects of peptide degradation on theirACE-inhibitory activity and the synergistically inhibitory effects of these degradedpeptides were also investigated. Finally, the ACE-inhibitory molecular mechanisms ofthese peptides were elucidated by molecular docking.Firstly, seven active peptides were identified from the purified fraction of bovinehemoglobin hydrolysate, obtained by in vitro simulated gastrointestinal digestion andidentified by HPLC-QTrap. Among the peptides, Phe-Leu and Thr-Tyr possess strongACE-inhibitory activities with the IC50value of290.66and96.43μM, respectively. The IC50values of the rest dipeptides were all above1000μM. The results of thestructure-activity relationship analysis indicated that the amino acid residues of bothsides of the active peptides play an important role in their ACE inhibition. The aromatic,heterocyclic, hydrophobic amino acid residues in C-terminus and/or hydrophobic aminoacid residues in N-terminus of the peptides could enhance their ACE-inhibitory activity.The docking results indicated the stability of peptide-ACE system of Phe-Leu andThr-Tyr was maintained by noncovalent interaction such as the hydrogen bond and thehydrophobic interaction.Following, computer-aided modeling technology was applied in peptide discoveryto enhance the obtainable efficiency of ACE-inhibitory peptides derived from bovinehemoglobin. A bovine hemoglobin virtual hydrolysis di-to penta-peptide database wasgenerated by Peptide Cutter software (Pepsin pH1.3) and a pharmacophore model basedon the chemical structure and spatial characteristics of twenty-one reportedACE-inhibitory peptides was established to predict the ACE-inhibtory activity ofpeptides in the database. The IC50values of the obtained Ala-Ser-His-Leu andTry-Thr-Gln-Arg-Phe were3.13mM and42.82μM, respectively. The docking resultssuggested all of these peptides accord with the non-competitively inhibitory mode.With the aim to enhance the prediction accuracy, a back propagation neural networkprediction model based on the structural, spatial, thermodynamic and electronicproperties of twenty-four reported ACE-inhibitory peptides was set up to forecast theinhibitory activity of the pentapeptides in the bovine hemoglobin virtual hydrolysispentapeptide database, obtained by Peptide Cutter software (Pepsin pH1.3, Pepsin pH>2and Trypsin). The Try-Thr-Gln-Arg-Phe was also obtained in this model. The flexibledocking technology was applied to investigate the interactions betweenTry-Thr-Gln-Arg-Phe and ACE for the further study on this pentapeptide’sACE-inhibitory mechanism. It is found that the coordination between the carbonyloxygen in the hydrophilic side chain of glutamine of the peptide and the Zn2+might playsa crucial role in ACE inhibition.Afterwards, we investigated the ACE-inhibitory activity and the synergistic effect ofthe degradated peptides from Try-Thr-Gln-Arg-Phe, and found that both the Thr-Gln-Arg and Try-Thr are the key parts of the peptide to exert its ACE-inhibitory effects. Inaddition, there are synergistic effects between Thr-Gln and Gln-Arg, and betweenTry-Thr-Gln and Arg-Phe. The results of molecular docking indicated that the positivessynergistic effect is inclined to occur in the group of two different kinds of peptides, ofwhich one is in correspond with the feature of competitive inhibition mode and the otherone is in correspond with the feature of noncompetitive inhibition mode.This research will contribute to further clarifying the ACE-inhibitory mechanisms ofbioactive peptides, which is of practical and theoretical significance.