Structure and ACE-Inhibitory Activity of Peptides Derived from Hen Egg White Lysozyme
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- Memarpoor-Yazdi, M., Asoodeh, A. & Chamani, J. Int J Pept Res Ther (2012) 18: 353. doi:10.1007/s10989-012-9311-2
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Angiotensin I-converting enzyme plays an important role in hypertension and therefore its inhibition is considered to be a useful procedure in the prevention of hypertension. Two novel ACE inhibitory peptides were purified and identified from the papain-trypsin hydrolysate of hen egg white lysozyme using reverse phase-high performance liquid chromatography. The sequences of identified peptides were NTDGSTDYGILQINSR (MW: 1,753.98 ± 0.5 Da) and VFGR (MW: 459.26 ± 0.5 Da), which were named F2 and F9 peptide, respectively. Analyses of the far-UV CD spectra of ACE in the absence and presence of the F2 peptide revealed ACE secondary structural changes. In the presence of the F2 peptide, a loss of helical content of ACE was observed, which can lead to decrease of the enzymatic activity. Lineweaver–Burk plots show that the identified peptides both act as non-competitive ACE inhibitors. These findings would be helpful on the understanding of interaction between ACE and its inhibitory peptides.
KeywordsRP-HPLCACE inhibitory activityInhibition patternCircular dichroism spectroscopy
Angiotensin I-converting enzyme
Reverse phase high-performance liquid chromatography
Hen egg white lysozyme
Matrix-assisted laser desorption/ionization time of flight
Much research has been carried out on bioactive peptides derived from food protein hydrolysates due to their potential nutraceuticals in relation to the development of functional foods (Hyun and Shin 2000). The peptides are not active within the origin protein but can be released and activated following enzymatic digestion (Phelan et al. 2009). Bioactive peptides exhibited many useful activities, such as immunoregulatory (Tsuruki et al. 2003), ACE inhibitory (FitzGerald et al. 2004), opioid (Pihlanto-Leppala 2000), antimicrobial (Mine and Kovacs-Nolan 2006), and antioxidant activities (Memarpoor-Yazdi et al. 2012; Hernandez-Ledesma et al. 2005). Amongst these, ACE inhibitory peptides have received more attraction due to having significant effect on prevention and treatment of hypertension. ACE (angiotensin I-converting enzyme; EC 22.214.171.124) is a dipeptidyl carboxypeptidase, which plays a critical role in human renin-angiotensin system. ACE catalyses the conversion of the inactive decapeptide (angiotensin I), by cleaving a dipeptide from the C-terminus, into a potent vasoconstriction octapeptide (angiotensin II). Additionally, ACE inactivates bradykinin, which has vasodilator activity in the kallikrein-kinin system (Asoodeh et al. 2012). Thus, inhibition of ACE leads to an overall antihypertensive effect and therefore, is considered to be a useful procedure in the prevention and treatment of hypertension and related diseases. The first report of natural ACE inhibitory peptides was found in sank venom (Ferreira et al. 1970). Some protein hydrolysates and their purified peptides have been reported with ACE inhibitory activity. ACE inhibitory peptides have been isolated from various protein sources, such as chicken egg (Yoshii et al. 2001), fish (Matsui et al. 1994), wheat germ (Matsui et al. 1999), casein (Muruyama and Suzuki 1982), whey (Pihlanto-Leppala et al. 1998), soybean (Shin et al. 1995), and garlic (Suersuna 1998). HEWL hydrolysates have been considered to produce antioxidant (Memarpoor-Yazdi et al. 2012; You et al. 2010), antimicrobial (Mine et al. 2004) and ACE inhibitory peptides (Asoodeh et al. 2012). Therefore, as in other studies, HEWL can be used for production of bioactive peptides with multifunctional activity.
In this study we have characterized new peptides with the highest ACE inhibitory activity from HEWL hydrolysate. The identified peptides were synthesised and their ability to inhibit of ACE activity were investigated. We have also analysed the conformational changes of ACE structure upon interaction with the inhibitory peptide using CD spectroscopy. The inhibition mode of the identified peptides was evaluated using Lineweaver–Burk plots, and finally the kinetic parameters (Km, Vmax, and Ki) were measured.
Materials and Methods
HEWL (hen egg white lysozyme) was purchased from Merck KGaA Co. [Darmstadt, Hesse, Germany]. ACE (angiotensin I converting enzyme) from rabbit lung, FAPGG (N-(3-[2-furylacryloyl-Phe-Gly–Gly])), HPLC grade acetonitrile, trypsin (from bovine pancreas), papain (from pawpaw sap) and TFA (trifluoroacetic acid) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Semi-preparative column was purchased from Macherey–Nagel GmbH Co. (St. Neumann Neander, Düren, Germany). Ultrafiltration membranes with a 3 kDa cut off were procured from Millipore (Bedford, MA, USA). All other used chemicals were of analytical grade.
Preparation of HEWL Hydrolysate and Peptide Isolation
HEWL (4.2 mg/mL) was dissolved in 50 mM Tris–HCl buffer (pH 7.5) and digested by a combination of trypsin and papain with a ratio of protein substrate to each enzyme (20:1 w/w) at 37 °C for 2 h. Each enzyme was dissolved (0.21 mg/mL) in the same buffer, separately. After the 2-h incubation at 37 °C, the enzymatic hydrolysis was stopped by heating in boiling water for 15 min. After the removal of insoluble materials by centrifugation (7,000×g, 10 min), the supernatant solution was transferred to fresh tubes for subsequent studies. To isolate low molecular weight peptides, the supernatant was passed through an ultra-membrane with a 3 kDa cut off. The resulting filtrate was fractionated using a C18 semi-preparative RP-HPLC column (10 × 250 mm, manufactured by Macherey–Nagel GmbH & Co. Düren, Germany). Elution was performed using solution A (0.1 % TFA in distilled water (v/v)) combined with a 5–50 % gradient of solution B (0.098 % TFA in acetonitrile) over a period of 45 min, at a flow rate of 2 mL/min. The absorbance of the elution peaks was monitored at 220 nm using a UV detector. Major peaks were collected, lyophilized and evaluated for their ACE inhibitory ability. The most active fractions were further purified using the same RP-HPLC conditions except that the elution was conducted using a 0.8 % per minute increasing gradient of solution B.
ACE Inhibition Assay
Determination of IC50 Values
Five different concentrations of inhibitory peptide were selected and evaluated for their %ACE inhibitory activity. The IC50 values (peptide concentration needs to inhibit 50 % of the ACE activity) were determined by plotting the ACE inhibition (%) against the different peptide concentrations. Experiments were carried out in triplicate.
Determination of Molecular Mass and Amino Acid Sequencing
The most active peptides, as novel ACE inhibitory peptides from HEWL, were characterized for identification for their molecular mass and amino acid sequence. The sample was desalted using ZipTips (Millipore, Billerica, MA, USA) and then analysed using MALDI TOF–TOF mass spectrometer by a 5800 Proteomics Analyzer (Applied Biosystems at Proteomics International Pty. Ltd., Nedlands, Western Australia). Determination of the amino acid sequence was carried out by using de novo sequencing method. The obtained MS/MS spectra was analysed using PEAKS Studio Version 4.5 SP2 (Bioinformatics Solutions Inc., Waterloo, ON, Canada).
The identified peptides were synthesized by Fmoc solid-phase using an Applied Biosystems Model 432A Synergy peptide synthesizer. The synthetic peptides were purified using RP-HPLC column on a C18 semi-preparative column (10 × 250 mm, manufactured by Macherey–Nagel GmbH & Co. Düren, Germany). The column was developed at a flow rate of 2 mL/min by a linear gradient of acetonitrile (5–45 % for 40 min) containing 0.1 % TFA.
Determination of IC50 and Inhibition Pattern of the Synthetic Peptides
ACE inhibitory effects of the synthetic peptides were evaluated and expressed as IC50 values using the method described previously. Determination of the inhibition pattern on ACE activity was performed based on the method described by Asoodeh et al. (2012). Fifty microlitres of different concentrations of the FAPGG substrate (0.6, 1.2, 1.8 and 2.4 mM) were used to characterize the inhibitory mechanism of the identified peptides. The enzyme activities were measured in the absence and presence of different concentrations (0.01 and 0.02 mg/mL) of inhibitory peptides. The kinetic of ACE inhibition in the presence of the inhibitory peptides was determined by Lineweaver–Burk plots. The kinetic parameters of Vmax, Km, as well as Ki (inhibitor constant) were obtained from Lineweaver–Burk and secondary plots, respectively (Palmer 2001).
Circular Dichroism Spectroscopy
Circular dichroism (CD) spectroscopy is an excellent method to analyse the conformation of proteins, peptides and their interactions in solution (Greenfield 1996). In this study, CD spectroscopy was used to analyse the ACE conformation in the absence and presence of the F2 peptide. Far-ultraviolet CD spectra were obtained with Jasco-815 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a Jasco 2-syringes titrator under constant nitrogen flush at room temperature. The instrument was con-trolled by Jasco’s Spectra Manager™ software. The instrument was calibrated by ammonium d-10-Camphorsulfonate (Takakuwa et al. 1985). A quartz cuvette having path length of 0.5-mm was used.
To explore changes in secondary structure of ACE, far-UV CD spectra were obtained over a wavelength range of 195–250 nm in the absence and presence of the F2 peptide at the same conditions. CD spectra were recorded with a time constant of 2 s, a 1-nm bandwidth, and a scanning rate of 50 nm min−1. They were signal-averaged over at least five scans, and baseline corrected by subtracting the buffer spectrum (Greenfield 1996). ACE (0.14 mg/mL) was dissolved in 50 mM Tris–HCl, pH 7.5 containing 0.03 M NaCl and 1 mM ZnCl2. The F2 peptide was dissolved in ACE buffer at a concentration of 0.02 mg/mL. The spectra for ACE were obtained at 0.07 mg/mL and the spectra of the peptide were obtained at 0.01 mg/mL. The spectra of ACE in the presence of the F2 peptide were obtained at approx. 7:1 ACE: peptide mass ratio. The spectra for ACE used as control were obtained at 0.07 mg/mL.
The data were expressed as molar residue ellipticity [θ], which is defined as [θ] = 100 θobs/cl, where θobs is the observed ellipticity in degrees, c is the concentration in residue mol cm−3, and l is the length of the light path in cm. To evaluate the content of secondary structure elements software packages as SELCON3 (Matsuo et al. 2012), CDSSTR (Sreerama and Woody 2000) and CONTIN (Sreerama and Woody 2000) were used.
Results and Discussion
Fractionation and ACE Inhibition Assay
The F7 fraction was the most active fraction obtained from the HEWL hydrolysate prepared as herein described. This has been previously reported by Asoodeh et al. (2012). However, two other fractions (F2 and F9) were also found to show important ACE inhibitory activity. Therefore, in this work we have focused on their characterization.
Molecular Mass and Amino Acid Sequencing
Determination of ACE Inhibition Pattern
The kinetic parameters for the F2 and F9 peptides were measured and compared to the control
F2 peptide (0.01 mg/mL)
F2 peptide (0.02 mg/mL)
F9 (0.01 mg/mL)
F9 (0.02 mg/mL)
Vmax or Vmax′a (mM/min)−1
HEWL hydrolysates are known to contain bioactive peptides such as ACE inhibitory, antioxidant and antimicrobial peptides. In this study, the most active peptides were F2 (NTDGSTDYGILQINSR) and F9 (VFGR) and identified as new ACE inhibitory peptides. The purified peptides both non-competitive inhibited ACE. The results of CD spectroscopy showed that there are significant changes in ACE conformation upon the interaction with F2 peptide as denoted by an alpha-helix content decrease and a coil content increase. These changes are indicative of a mild denaturation with loss of enzymatic activity. Two identified peptides can serve as lead compounds in the preparation of functional foods and antihypertensive drugs. However, more detailed researches as an indication of their ACE inhibitory activity in vivo are required.
This work was financially supported by Mashhad-Branch, Islamic Azad University and Ferdowsi University of Mashhad, Mashhad, Iran. The authors gratefully acknowledge Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran for beneficial help.