Biochemical characterization of a novel antioxidant and angiotensin I-converting enzyme inhibitory peptide from Struthio camelus egg white protein hydrolysis

Abstract

A peptide from ostrich (Struthio camelus) egg white protein hydrolysate (OEWPH) was purified, characterized, and its antioxidant and enzyme inhibitory properties were evaluated. The OEWPH was prepared using pepsin and pancreatin, and then fractionated using reversed-phase high performance liquid chromatography. The antioxidant activity of the WG-9 peptide was investigated based on its scavenging capacity for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), superoxide (O₂^•-), hydroxyl (OH^•-), and lipid peroxidation inhibition. The angiotensin-converting enzyme (ACE) inhibitory activity and kinetic parameters of the peptide were determined using N-[3-(2-Furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG) as a substrate. Tandem mass spectrometry analysis of the purified peptide revealed a sequence of WESLSRLLG (MW: 1060 Da; WG-9). This peptide inhibited linoleic acid oxidation and acted as a DPPH (IC₅₀ = 15 ± 0.4 μg/mL), ABTS (IC₅₀ = 130 ± 4.5 μg/mL), superoxide (IC₅₀ = 160 ± 6.4 μg/mL), and hydroxyl (IC₅₀ = 150 ± 6.7 μg/mL) radical scavenger. The ACE-inhibitory activity and kinetic parameters of the WG-9 peptide were determined, showing an ACE inhibitory activity with IC₅₀ of 46.7 ± 1.4 μg/mL. The parameters of peptide/ACE interactions were investigated by molecule docking. Furthermore, viability assays showed that the identified peptide had no cytotoxicity against an HFLF-PI-5 cell line. In conclusion, the WG-9 peptide showed potent antioxidant and ACE-inhibitory activity.

Keywords: angiotensin I-converting enzyme; antioxidant peptide; molecular docking; ostrich egg white proteins.

Figure 1

(A) Semi-preparation of OEWPH on a C₈ column (10 mm × 250 mm). Nearly 15 mg OEWPH was dissolved in solvent A (water containing 0.1% TFA) and was injected on the column. By monitoring the absorbance at 214 nm, the elution was accomplished at a flow rate of 2 mL/min using a linear gradient of acetonitrile as solvent B (5–45% for 40 minutes). The major peak, which appeared after 26 minutes, was chosen to be identified by MALDI-TOF/TOF sequencing. The y-axis indicates absorbance units (mAU). Characterization of the antioxidant peptide: (B) MS/MS spectrum of the purified peptide (upper section) and analysis of MS/MS spectrum for purified peptide (lower section, the unit of X-axis is m/z). By manual calculation, the sequence of this peptide is displayed with the fragmentations observed in the spectrum. MALDI-TOF/TOF = matrix-assisted laser desorption/ionization-time of flight; MS/MS = tandem mass spectrometry; OEWPH = ostrich egg with protein hydrolysate.

Figure 2

(A) DPPH-, (B) hydroxyl-, (C) superoxide-, (D) ABTS-radical scavenging activities and (E) lipid peroxidation inhibition of the WG-9 peptide. GSH was used as a positive control for scavenging properties, while BHA was considered for the lipid peroxidation assay. The assay was carried out in triplicate. ABTS = 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt; BHA = butylated hydroxyanisole; DPPH = 1,1-diphenyl-2-picrylhydrazyl; GSH = glutathione.

Figure 3

(A) ACE inhibition curve at various concentrations of WG-9 peptide. (B) Lineweaver-Burk plots for ACE in the presence or absence of the peptide. The equations for the plot are as follows: y = 0.0003x + 0.0034 (R² = 0.996) for control (without peptide); y = 0.0004x + 0.0035 (R² = 0.999) at 46 μg/mL WG-9; and y = 0.00069x + 0.0034(R² = 0.995) at 90 μg/mL WG-9. (C) The secondary plot for the WG-9 peptide is the competitive inhibitor. ACE = angiotensin-converting enzyme.

Figure 4

(A) Docking results of WG-9 and the N-terminal region of tACE and (B) WG-9 and the C-terminal region of tACE. All interactions in the active site have been simplified. Hydrophobic, polar, and acidic residues of ACE are represented by green, violet, and red rings, respectively. Green arrows show hydrogen bands from donor atom to acceptor. ACE = angiotensin-converting enzyme.

Figure 5

(A) Time- and dose-dependent effects of the WG-9 peptide on viability of HFLF-PI-5 cells evaluated by MTT assay. The peptide had no significant effect on the treated cells after 24 hours (p < 0.05). (B) Hemolytic activity of the WG-9 peptide against human erythrocytes. The assay was carried out in triplicate. MTT = 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide.