Study On The Design, Enzyme-resistant Activity And Inhibition Mechanism Of ACE Inhibitory Peptides

Objective: Hypertension is an important risk-factor to cardiovascular disease, sodeveloping new antihypertensive drugs which are safe, effective and without side-effectsbecomes the primary task to prevent and cure hypertension. ACE inhibitory peptides as a newfunctional food for antihypertensive effect is highlighted recently because of their high safetyand low side-effects. In this study, peanut protein (KIFLRLS) peptide was made as template,and a series of ACE inhibitory peptides were designed based on the net charge andhydrophobicity which are two key factors in structural development of peptides. The effect ofnet charge and hydrophobicity of peptides on ACE inhibitory peptides’ activities,enzyme-resistant activity was studied, as well as the interaction between ACE inhibitorypeptides and ACE.Methods: Sequence modification method was used to design analogues of parent peptideACEIP (net charge Q=+5, hydrophobic H=57%). The net charge and hydrophobicity ofanalogues are as followed, P1: Q=0, H=57%; P2: Q=0, H=42%; P3: Q=0, H=71%; P4: Q=+1,H=71%; P5: Q=+1, H=57%; P6: Q=+1, H=42%; P7: Q=+2, H=42%, P8: Q=+2, H=71%.Software was used to predict the physicochemical properties of the parent peptide andderivative peptides. The effect of peptides on cancer cells’ growth was studied by MTTmethod. Then the relationship between net charge and hydrophobicity of ACE inhibitorypeptides and their enzyme-resistant activity against pepsin and trypsin was researched. At lastthe interaction between ACE inhibitory peptides with ACE was investigated by fluorescencespectroscopy and molecular docking.Results: MTT results showed that the effect of ACE inhibitory peptides on HepG2cellsinhibitory growth was significantly and positively correlated with the net charge of peptides(P＜0.05). This indicated that HepG2cells were sensitive to the cationic peptides. The retentionrates of P3, P4and P8were all above70%after parent peptide and derivative peptides werehydrolyzed by pepsin (0.01mg/mL) for2h, while the retention rates of other peptides wereabout50%. The retention rates of P1, P5and ACEIP were all about40%after beinghydrolyzed by trypsin (0.01mg/mL) for2h, while the remaining peptides were above50%.Correlation analysis showed that enzyme-resistant activity of ACE inhibitory peptides wassignificantly and positively correlated with hydrophobicity of peptides(P＜0.05). However,the influence of net charge to enzyme-resistant activity of ACE inhibitory peptides is notsignificant. The retention rate of peptides with relatively high hydrophobicity were higher inthe same hydrolysis conditions. Though parent peptide and derivative peptides werehydrolyzed to some extent, their inhibitory activity remained, The anti-enzyme activity of P2,P7and P8increased after being hydrolyzed by pepsin. And in vitro test showed that peptideswith high hyrophobicity displayed higher activity than other peptides. The fluorescenceintensity of ACE was mainly quenched by P1, P2, P4, P6through collision with ACEmolecule or binding to ACE molecule. At low concentrations, P3, P5, P8and ACEIPquenched the fluorescence intensity by static binding to ACE molecule, while at highconcentrations, they collided with ACE molecule as well as static binding. P6and P7did not enter into the ACE molecule but only quenched the amino acids’ fluorescence in the proteinmolecular surface. By molecular docking, hydrogen bonds formed between P1, P2, P4,ACEIP and Zn2+active center and neighboring amino acids (His383/His387/Glu411) of theACE molecular, which showed good inhibitory activity.