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Purification, modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate
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Purification, modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate Mengliang Tao, Chaoyang Wang, Dankui Liao, Haibo Liu, Zhenxia Zhao, Zhongxing Zhao ∗ Guangxi Colleges and Universities Key Laboratory of New Technology and Application in Resource Chemical Engineering, School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China
a r t i c l e
i n f o
Article history: Received 12 September 2016 Received in revised form 20 December 2016 Accepted 22 December 2016 Available online xxx Keywords: Silkworm pupa protein Angiotensin I-converting enzyme Inhibitory peptide Stability Docking
a b s t r a c t Angiotensin I-converting enzyme (ACE) inhibitory peptide from silkworm pupa (Bombyx mori) was purified, modified, as well as inhibition mechanism by using molecular docking analysis. Silkworm pupa protein was hydrolyzed by neutral protease and the obtained hydrolysate was subjected to various types of chromatography to acquire peptide isolate. Then the molecular mass and amino acid sequence of the peptide was determined by MALDI-TOF/TOF MS. Subsequently, thermal and digestive stability of the peptide were explored through a high temperature processing and a simulated gastrointestinal digestion. Finally, the peptide was modified to smaller peptides and investigated their potentiate activities. Results showed that the peptide from silkworm pupa was determined to be Gly-Asn-Pro-Trp-Met (603.7 Da) with IC50 21.70 M. Stability testing showed that ACE inhibitory activities were not significantly changed at temperature from 40 to 80 ◦ C as well as during in vitro gastrointestinal digestion. The inhibitory activity of four modified peptides were Trp-Trp > Gly-Asn-Pro-Trp-Trp > Asn-Pro-Trp-Trp > Pro-Trp-Trp, and the IC50 of Trp-Trp was 10.76 M Docking simulation revealed that the inhibitory activity was closely related to the spatial structure of peptide and zinc ions. The purified peptide and four modified peptides may be beneficial as functional food or drug for treating hypertension. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Hypertension is a common cardiovascular disease worldwide, and can induce many complications such as prediabetes, atherosclerosis and heart stroke [1]. Recent research shows that this disease is mainly caused by the increasing concentration of angiotensin I-converting enzyme (ACE, EC 3.4.15.1) [2]. Herein, ACE inhibitors are the mainstay medications for treating high blood pressure. Many synthetic ACE inhibitors, including captopril, enalapril, lisinopril, can usually associated with unacceptable adverse effects such as dry cough, angioedema, taste disturbance and skin rash [3,4]. Currently, more and more bioactive peptides have been isolated from food protein sources which have significant ACE inhibitory activities and minimum side effects [5–7]. Quantitative structureactivity relationships between ACE structure and ACE inhibitory
∗ Corresponding author. E-mail address: [email protected] (Z. Zhao).
peptide have been reported that the peptide containing hydrophobic amino acids at C-terminal exhibited potent inhibitors [8–10]. Silkworm pupa protein is known as containing 18 known amino acids, which maintains high levels of hydrophobic amino acids like valine, methionine and phenylalanine [11,12]. Its hydrolysates by various enzymatic hydrolysis possess multifunctional property, for instance, ACE inhibitory activity [13,14], antioxidant activity [15], anti-obesity [16], antitumor activity [17], etc. Therefore, some ACE inhibitory peptides have been reported to be isolated from silkworm pupa protein, and their inhibition mechanisms have been also investigated by molecular docking [18,19]. However, there are few reports about the modification of peptides to enhance their activities. In this paper, a novel ACE inhibitory peptide derived from enzyme hydrolysate of silkworm pupa (Bombyx mori) protein was isolated and identified. The sequence of purified peptide was identified by matrix-assisted laser desorption ionization timeof-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Besides, the peptide was synthesized by using solid phase peptide method, and its thermal and digestive stability were also characterized through high temperature and simulated gastrointestinal
http://dx.doi.org/10.1016/j.procbio.2016.12.022 1359-5113/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: M. Tao, et al., Purification, modification and inhibition mechanism of angiotensin Iconverting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.12.022
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digestion process. Furthermore, the molecule docking mechanism of purified peptide with ACE was determined by Sybyl X-2.1.1. Finally, this peptide was modified and obtained five novel peptides with Trp at the C-terminal. The ACE inhibitory activity and action mechanism of modified peptides were also studied.
2. Materials and methods 2.1. Materials The silkworm pupa was purchased from Guangxi Jialian Silk Co., Ltd. (Yizhou, China). Neutral protease was provided by Pangbo Biological Engineering Co., Ltd. (Nanning, China). ACE from rabbit lung, hippuryl-l-histidyl-l-leucine (HHL) were offered from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Pepsin from porcine gastric mucosa was supplied by Coolaber Science & Technology Co., Ltd. (Beijing, China), and pancreatin from porcine pancreas was offered by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Sephadex G-15 was purchased from Pharmacia Fine Chemicals Co., Ltd. (Uppsala, Sweden). Methanol for HPLC analysis was supplied by Tianjin Shield Specialty Chemical Co., Ltd. (Tianjin, China). All starting materials were commercially available reagents of analytical grade and used without further purification.
Fig. 1. Flowchart of preparation of ACE inhibitory peptides from silkworm pupae protein. The purification procedures of the ACE-inhibitory peptide are laid out step by step. (I) Ultrafiltration, 5000 Da; (II) Ion-exchange chromatography, D201; (III) Gel filtration chromatography, Sephadex G-15; (IV) Reversed-phase high performance liquid chromatography, ZORBAX SB C18.
2.2. Measurement of ACE inhibitory activity The inhibitory activity of ACE was performed on reversed-phase high performance liquid chromatography (RP-HPLC) and assayed with a modified spectrophotometry [8]. HHL was dissolved in 100 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl and formed a concentration of 5.8 mM. Rabbit lung ACE was dissolved in the same buffer solution at a concentration of 10 mU/mL. 40 L of ACE solution and a certain concentration of sample solution were mixed with sodium borate buffer (460 L), which was incubated at 37 ◦ C for 10 min. After that, 40 L of HHL solution was added into above sodium borate buffer and kept the solution incubated for 60 min. The reaction was then terminated by adding 100 L of 1.0 M HCl, and then was filtered by 0.22 m membrane (Tianjin Jinteng Experiment Equipment Co., Ltd.). Finally, the product hippuric acid (HA) was detected with a ZORBAX SB C18 column (4.6 mm × 150 mm, particle size 5 m; Agilent, USA) and a Diode array detector (DAD) at 228 nm by RP-HPLC. The mobile phase was 0.1% trifluoroacetic methonal/water (3:17, v/v). The evaluation of ACE inhibition was based on the comparison between the concentrations of HA in the presence or absence of an inhibitor, and inhibition activity was calculated by using the following equation: I=
Ae − Af × 100% Ae − Ab
(1)
2.3.1. Ultrafiltration The lyophilized silkworm pupa protein hydrolysate was dissolved in ultrapure water, and then fractionated by ultrafiltration using Labscale System (Labscale TFF System, Millipore Co, Billerica, MA, USA) with a 5 kDa molecular-weight- cut-off membrane (Millipore Co, Billerica, MA, USA), seen in step I of Fig. 1. The SN1 and SN-2 were defined as large peptides with molecular weight more and less than 5 kDa, respectively, which were collected and lyophilized as the ACE inhibitory assay and for further purification. 2.3.2. Ion-exchange chromatography The fraction with the higher activity of the ACE inhibitory between SN-1 and SN-2 was further purified by ion-exchange chromatography. The lyophilized ultrafiltration sample was added in 10 mM phosphate buffer (pH 8.5) to give a final concentration of 10 mg/mL, and then 20 mL dissolved solution was loaded on a D201 anion exchange column (10 × 300 mm, HuiZhu resin Co., Ltd., Shanghai) previously equilibrated with 10 mM phosphate buffer (pH 8.5), seen in step II of Fig. 1. The column was eluted by a linear gradient of NaCl (0–1.0 M) at a flow rate of 1.0 mL/min within 300 min. The elution was monitored at 280 nm with a UV spectrophotometer (Model TU-19, Purkinje, Beijing, China). Two fractions, IE-1 and IE-2, were further collected, freeze dried and tested for ACE inhibitory activity.
2.3. Purification of ACE inhibitory peptide
2.3.3. Gel filtration chromatography The fraction with the maximum ACE inhibitory activity from the ion-exchange chromatography was further purified by gel filtration chromatography. The sample was first dissolved in ultrapure water, and then loaded onto a Sephadex G-15 column (20 × 500 mm) equilibrated with ultrapure water, seen in step III of Fig. 1. The absorbance was measured at 280 nm by a UV spectrophotometer. Three fractions, from GF-1 to GF-3, were pooled, concentrated and lyophilized for measurement of the ACE inhibitory activity.
The silkworm pupa protein and hydrolysate with neutral protease were prepared according to our previous reports [20,21]. Fig. 1 shows a flowchart of an entire preparation for ACE inhibitory peptides from silkworm pupae protein.
2.3.4. Reversed-phase high performance liquid chromatography, RP-HPLC The fraction showed the maximum ACE inhibitory activity was further separated on a ZORBAX SB C18 column by using RP-HPLC
Where I is the ACE inhibition activity, Ae , Af and Ab are the relative area of HA peak generated without ACE inhibitors, with ACE inhibitor component and without both of ACE and ACE inhibitors, mAU. Ratio of the IC50 (a total inhibitor concentration that reduces these activities by 50%) was estimated and used as a measure of isozyme selectivity.
Please cite this article in press as: M. Tao, et al., Purification, modification and inhibition mechanism of angiotensin Iconverting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.12.022
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(Agilent 1260, USA). The column was eluted by a linear gradient of acetonitrile (5–30%) containing 0.1% TFA at a flow rate of 0.50 mL/min within 60 min. The absorbance of the elution was monitored at 220 nm with DAD, seen in step IV of Fig. 1. Then ten fractions, from HP-1 to HP-10, were concentrated and freeze-dried. Subsequently, the freeze-dried sample represented the highest ACE inhibitory activity (HP-6) was subjected to a second round of RP-HPLC purification, eluting with 8% acetonitrile in water (v/v) containing 0.1% TFA with a flow rate of 0.50 mL/min. And four fractions, from HP-61 to HP-64, were collected and lyophilized to powder for further measurement of their ACE-inhibitory activities, respectively.
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The relative content and relative ACE inhibitory activity were calculated by the following equation: Crel =
Ct × 100% C0
(3)
Irel =
It × 100% I0
(4)
Where C0 and Ct are content of peptide before and after stabilities treatment, respectively, Crel is relative content of peptide. I0 and It are ACE inhibitory activities before and after stabilities treatment, respectively, Irel is relative ACE inhibitory activity.
2.4. Characterization of purified peptide Accurate relative molecular mass and amino acid sequence of the purified peptide with the highest ACE inhibitory activity were determined by 4800 plus MALDI-TOF/TOFTM Analyzer (Applied Biosystems, Beverly, MA, USA). The purified peptide was dissolved in ultrapure water, and then the purified peptide solution was mixed with matrix solution (␣-cyano-4-hydroxycinnamic acid solution, prepared with 50% acetonitrile containing 0.1% TFA). Spectra were acquired on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer with a 337 nm pulsed nitrogen laser (2-ns pulse duration, 3-Hz repetition rate). Peptide mass spectra were acquired in linear positive ion mode at a mass range of m/z 500–1500. Mass spectrometry/mass spectrometry data of the purified peptide were obtained by collision-induced dissociation (CID).
2.5. Stabilities of silkworm pupa protein derived ACE inhibitory peptide The identified peptide was synthesized by GL. Biochem Co., Ltd. (Shanghai, China) using conventional solid-phase chemistry. Then the peptide was dissolved in 100 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl and given a concentration of 1.0 mg/mL. The generated standard curve of identified peptide was as follows: y = 154.19x + 28.44
(2)
Where y is the chromatographic peak area of identified peptide, mAU, and x is the content of identified peptide, mg/mL. The analysis data showed a good linear relationship (R2 ≥ 0.9990) with the concentration ranges of 0.05–1.50 mg/mL. The identified peptide solutions (1.0 mg/mL) were incubated at 40, 60, and 80 ◦ C for 6 h, respectively. After being cooled to 30 ◦ C, the content and ACE inhibitory activity of the identified peptide were determined. Simulated gastric fluid was prepared by mixing 16.4 mL of 9.8 wt% HCl and 10.00 g of purified pepsin in 1000 mL of water. The pH of the solution was 1.0–1.4. Simulated intestinal fluid was prepared by dissolving 6.80 g of monobasic potassium phosphate (KH2 PO4 ) in 500 mL of water. The solution was brought to pH 6.8 with 0.1 M NaOH. After that, 10.00 g of pancreatin was added to the assay and then diluted to 1000 mL with water. The identified peptide solution was mixed with the simulated gastric at a ratio of 1:1 (v/v) and incubated at 37 ◦ C for 2 h. Afterwards, the solution was adjusted to pH of 6.5–7.0 with 0.5 M NaOH. Then simulated intestinal fluid was mixed with the above reacted solution with a ratio of 1:1 (v/v), incubated for 4 h at 37 ◦ C. After reaction, the solution was boiled to stop the enzyme reaction. The content and ACE inhibitory activity of the identified peptide were tested at different times of 0, 2.0 and 6.0 h.
2.6. Molecular docking of the peptide with ACE The three-dimensional crystal structure of human ACElisinopril complex obtaining from the Protein Data Bank (PDB: 1O86) [22] was selected as working targets. The complete PDB file was downloaded and submitted to Sybyl X-2.1.1 software package (Tripos International, St. Louis, MO, USA) by only removing the inhibitor lisinopril and water molecular. The cofactors zinc and chloride atoms were retained in ACE model. Then the peptides structure were pre-analyzed and prepared for the docking runs using the biopolymer structure preparation tool with default settings. The protomol was created by extracting the original ligand with the help of MOLCADD program of the software. The crystal structure of peptides was subjected to energy minimization with Tripos force field using following the gradient termination of the Powell method for 10,000 iterations with a convergence criterion of 0.005 kcal/mol. This docking was simulated by using the Surflex-Dock program. The ligand (lisinopril) from the ACE-lisinopril complex was used as a reference molecule. The conformations were selected according to Consensus scores (CSCORE) and Total Score. The CSORES can range from 0 to 5, where 5 indicates that a ligand is judged good by all functions, and structures with CSORES of 3, 4 or 5 further consideration. The Total Score represents a binding affinity of conformation and the higher value of Total Score means a strong binding affinity. The top 20 conformations for each peptide had created and ranked based on Total Scores by Sybyl X-2.1.1 software and we chose the first conformation whose CSCORE ≥3 as the conformation of each peptide in ACE.
2.7. Statistical analysis All datas were expressed as the mean with standard deviation of triplicate determinations. Significance of differences between test samples was determined by the Student t-test. A p value of less than 0.05 was taken as significant.
3. Results and discussion To obtain ACE inhibitory peptide, we proposed to use neutral protease to hydrolyze silkworm pupa protein in this work. Next, the obtained hydrolysate was centrifuged, collected, and then lyophilized for further use. Followed by ultrafiltration, ionexchange chromatography, gel filtration chromatography and two steps of RP-HPLC, peptide with the highest ACE inhibitory activity was purified. Subsequently the sequence of peptide was identified by MALDI-TOF/TOF MS, then the peptide was synthesized, and its stabilities were studied.
Please cite this article in press as: M. Tao, et al., Purification, modification and inhibition mechanism of angiotensin Iconverting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.12.022
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Fig. 2. Purification of the fraction SN-2 and ACE inhibitory activity evaluation of its separated peptides. (a) Fraction IE-1 and IE-2 (87.1 g/mL); (b) fractions GF-1 to GF-3 (48.3 g/mL); (c) fractions HP series (37.2 g/mL); (d) fractions HP-6 series (29.8 g/mL).
Please cite this article in press as: M. Tao, et al., Purification, modification and inhibition mechanism of angiotensin Iconverting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.12.022
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Expressed as mean ± standard deviations of triplicates (n = 3). Values in the same column are significantly different at p < 0.05. a
3.1. Preparation of protein hydrolysate and its ACE inhibitory activity Table 1 showed the ACE inhibitory activities of the silkworm pupa protein, SN-1, and SN-2. As shown, The SN-2 (MW <5 kDa) exhibited 2.4 times higher value of ACE inhibitory activity than the SN-1 (MW >5 kDa). It was due to that SN-2 possessing smaller molecular mass was much easier to reach the active site of ACE [23,24]. Therefore, the SN-2 was chosen and purified for further studies.
39.04
b3 b1
60
b2
40
604.31
0.03 ± 0.41 29.36 ± 0.50 19.97 ± 0.23 47.63 ± 0.72
455.31
283.2 283.5 281.9 282.4
269.31
Silkworm pupae protein Protein hydrolysate SN-1 SN-2
b4
172.18
Inhibition ratea (%)
G N P W M
80
184.20
Concentration (g/mL)
b1 b2 b3 b4
58.12
Sample
100
Relative intensity (%)
Table 1 ACE inhibitory activity of silkworm pupae protein, protein hydrolysate, SN-1 and SN-2.
5
20 0 0
100
200
300
400
500
600
M ass (m /z) Fig. 3. Characterization of molecular mass and amino acid sequence of purified peptides. MS/MS spectrum of molecular ion m/z 604.31 Da.
3.2. Purification of ACE inhibitory peptide Fraction SN-2 was continuously divided into two fractions (IE1 and IE-2) by ion-exchange chromatography. Fig. 2(a) showed the ACE inhibitory activity of the fraction IE-1 and IE-2. Fraction IE-1 exhibited higher ACE inhibitory activity of 74.06 ± 1.83% than fraction IE-2. Thus, fraction IE-1 was further lyophilized and separated into three molecular weight (MW) groups by gel filtration chromatography, which were GF-1, GF-2 and GF-3. The ACE inhibitory activities of these fractions GF-1, GF-2 and GF3 are shown in Fig. 2(b). Fraction GF-2 shows the highest ACE inhibitory activity with a value of 69.28 ± 0.74%, followed by fraction GF-1 (50.11 ± 0.53%), and then fraction GF-3 (46.55 ± 1.23%). Furthermore, fraction GF-2 with highest ACE inhibitory activity was separated by RP-HPLC into ten major fractions (named from HP-1 to HP-10). Their ACE inhibitory activities were tested and shown in Fig. 2(c). Among which, fraction HP-6 exhibited the highest ACE inhibitory activity with a value of 76.27 ± 0.11%. Fractions obtained at the middle or end phase of HPLC exhibited a higher ACE inhibitory activity due to their lower molecular mass and greater amounts of hydrophobic amino acids [25]. Afterwards, HP6 was further separated by RP-HPLC and obtained four peaks; those were HP-61, HP-62, HP-63 and HP-64, respectively. As illustrated in Fig. 2(d), fraction HP-64 possessed the highest ACE inhi0bitory activity with a value of 77.60 ± 0.77%. 3.3. Characterization of ACE inhibitory peptide by MALDI-TOF/TOF MS The molecular mass and amino acid sequence of fraction HP64 were measured by MALDI-TOF/TOF MS and shown in Fig. 3. The sequence of the peptide was identified as Gly-Asn-Pro-TrpMet (GNPWM). The molecular mass of peptide was determined as 604.31 (M+H) + , which was consistent with its theoretical molecular mass (603.7 Da). This peptide from silkworm pupa protein is a novel ACE inhibitory peptide with the IC50 value of 21.70 M. The IC50 value was comparatively lower than some other peptides ASL (IC50 = 102.15 M) and APPPKK (IC50 = 73.81 M) obtained from the same source (silkworm pupa protein) [13,14]. The main reason was ascribed to the presence of aliphatic hydrophobic amino acids at Cterminal in the structure of GNPWM that could contribute to ACE inhibitory activity [26].
Fig. 4. Thermal stability (a) and digestion resistant (b) of the GNPWM peptide.
3.4. Stability and ACE inhibitory activity of synthetic peptides Fig. 4(a) gave the thermal stability of the GNPWM. Purified peptide possessing high thermal stability was a predominant of functional food preparation. From Fig. 4(a), the relative content of GNPWM showed a clear decrease from 77.14 to 65.83% with temperature. High temperatures could accelerate cleavage and rearrangements of disulfide and hydrophobic bonds of the GNPWM [27]. The relative ACE inhibitory activity of the GNPWM, however, showed a small increase from 102.2 to 107.4% as temperature increases. The increasing ACE inhibitory activity was probably caused some rearranged peptides presenting ACE inhibitory activity. Overall, the ACE inhibitory activity of the GNPWM showed a
Please cite this article in press as: M. Tao, et al., Purification, modification and inhibition mechanism of angiotensin Iconverting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.12.022
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Fig. 5. Predicted binding mode between ACE and peptide after the peptide was docked at the ACE active site. (a) GNPWM; (b) WW; (c) PWW; (d) GNPWW; (e) NPWW. The residues of ACE are shown as line, and the peptide is shown as stick. The Zn ion is shown as cyan sphere and the hydrogen bond is shown as yellow dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
small variation with temperature increasing under the test conditions of 40–80 ◦ C. Therefore, it can be concluded that thermal heating process exerted certain influence on the structure of the GNPWM, while had little impact on ACE inhibitory activity. In order to validate a certain digestive stability of the GNPWM, gastrointestinal enzyme was used to estimate digestion resistance with in vitro model. Fig. 4(b) showed the digestive stability of the GNPWM at approximately the temperature of the human body. It showed that the relative content of GNPWM slightly declined to 92.3 ± 0.4% after being digested with gastrointestinal enzyme, while the relative ACE inhibition activity of the GNPWM was essentially unchanged before and after digestion. Thus, the peptide displayed a good digestion resistance. 3.5. Modification of peptide Although the structure-activity relationship of ACE inhibitory peptides has not yet been fully elucidated, ACE prefers inhibitors containing hydrophobic amino acid residues at each of the three
C-terminal position [28], e.g., Pro, are more active if present at each of the three C-terminal positions. In addition, the presence of the positive charge of Lys and Arg as the C-terminal residue may contribute to the inhibitory potency [29]. So the C-terminal residue of GNPWM was replaced by 8 different hydrophobic amino acids and docked with ACE by Sybyl X-2.1.1. The result showed that the conformation of GNPWW had a best binding affinity with ACE, and then the peptide was modified by using hydrophobic amino acid Trp at C-terminal position. Moreover, to further investigate the inhibition mechanism, we changed the length of GNPWW by cutting off one amino acid from its N-terminal end one after another to get more peptides. Finally, the four modified peptides GNPWW, NPWW, PWW and WW were synthesized, and their IC50 were measured to be 19.74 M, 23.11 M, 83.69 M and 10.76 M, respectively. The IC50 value of peptide GNPWW had slightly decreased comparing with GNPWM. The peptide PWW with the highest IC50 means that it had the lowest ACE inhibitory activity, while the peptide WW with the lowest IC50 means that it had the highest ACE inhibitory activity.
Please cite this article in press as: M. Tao, et al., Purification, modification and inhibition mechanism of angiotensin Iconverting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.12.022
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This coincided with previous reports that Pro was an unfavorable N-terminal amino acid for binding to ACE [30]. 3.6. Molecular docking To investigate the inhibition mechanism of peptides further, the binding mode between ACE and peptides was predicted by Sybyl X-2.1.1. ACE has three main active site pocket, these are, S1 pocket contains Ala354, Glu384 and Tyr523 residues, S2 pocket contains Gln281, His353, Lys511, His513 and Tyr520 residues, and S1 includes Glu162 residue [31]. Meanwhile, Zn (II) at the ACE active site usually plays a significant role for ACE activity, which constitutes a tetrahedrally-coordinated Zn (II) with ACE by ACE residues His383, His387, Glu411 [32]. The molecular docking result revealed that hydrogen bonds were established between original GNPWM and three pockets, which were the S1 pocket (Ala354 and Glu384), the S2 pocket (Gln281, Lys511 and Tyr520) and the S1 pocket of ACE (Fig. 5a). Although GNPWM did not interact with Zn (II) directly, it could form the interaction with His383. As a result, it caused the distortion of the tetrahedrally-coordinated Zn (II) and therefore reducing ACE activity [13]. As seen, the WW has the highest inhibitory activity among the above peptides. WW peptide could only form hydrogen bonds with S1 pocket (His353) and S2 pocket (Ala354) (Fig. 5b). However, it could also form coordination with Zn (II), and generate hydrogen bond with His383. Thus, the consequences of the distorted tetrahedrally-coordinated Zn (II) resulted in a high inhibitory activity of the WW peptide. Among which, PWW possessed the lowest ACE inhibitory activity. PWW also had two hydrogen bonds with pockets. However, it was hardly to form coordination with Zn (II) and hydrogen bond with residues His383, His387 and Glu411 (Fig. 5c). Thus, its ACE inhibitory activity was much lower than the WW peptide. The modified peptides of GNPWW and NPWW had moderate ACE inhibitory activity in these five peptides. Compared to WW, GNPWW and NPWW could not form hydrogen bond with any of residues His383, His387 and Glu411 (Fig. 5d and e). Thus, it possessed lower ACE inhibitory activity, although more hydrogen bonds existed between modified peptides (GNPWW and NPWW) and ACE. 4. Conclusions In this work, GNPWM was successfully purified and identified from silkworm pupa protein. The peptide was synthesized with an IC50 value of 21.70 M. In addition, the GNPWM showed good stability to high processing temperatures and simulated gastrointestinal digestion. The molecule docking studies revealed that the GNPWM could effectively interact with the active site of ACE and therefore inhibit ACE activity. Four modified peptide (GNPWW, NPWW, PWW, WW) derived from GNPWM were also synthesized. Among these modified peptides, WW had the highest ACE inhibitory activity due to its formation of hydrogen bonds with His383 as well as the distortion of the tetrahedrally-coordinated Zn (II). As a result, high ACE inhibitory activity of the peptides is the result of synergistic effect of strong hydrogen bond between peptides with some critical ACE residues and coordination interactions with Zn (II). In conclusion, we obtained a novel WW peptide with excellent ACE inhibitory activity through selection of molecular docking method and peptide modification, and further work needs to be done to demonstrate the in vivo antihypertensive activity of peptides in order to provide an efficient utilization of silkworm pupa protein.
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Acknowledgments This work was financially supported by National Natural Science Foundation of China (No. 31401629, 31360020, 21376090, 21676059 and 21666004), Scientific Research Foundation of Guangxi University (No. XGZ 130080 and 2016JJA120072) and GuangXi University (Grant No. XGZ130963). References [1] H.I. Kim, Y. Song, W. Kim, J.E. Lee, Association of adherence to the seventh report of the Joint National Committee guidelines with hypertension in Korean men and women, Nutr. Res. 33 (2013) 789–795. [2] J.S. Tsai, T.C. Lin, J.L. Chen, B.S. Pan, The inhibitory effects of freshwater clam (Corbicula fluminea, Muller) muscle protein hydrolysates on angiotensin I converting enzyme, Process Biochem. 41 (2006) 2276–2281. [3] J. Chen, Y. Wang, R. Ye, Y. Wu, W. Xia, Comparison of analytical methods to assay inhibitors of angiotensin I-converting enzyme, Food Chem. 141 (2013) 3329–3334. [4] N.K. Sweitzer, What is an angiotensin converting enzyme inhibitor? Circulation 108 (2003) e16–e18. 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Purification, modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate Mengliang Tao, Chaoyang Wang, Dankui Liao, Haibo Liu, Zhenxia Zhao, Zhongxing Zhao ∗ Guangxi Colleges and Universities Key Laboratory of New Technology and Application in Resource Chemical Engineering, School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China
a r t i c l e
i n f o
Article history: Received 12 September 2016 Received in revised form 20 December 2016 Accepted 22 December 2016 Available online xxx Keywords: Silkworm pupa protein Angiotensin I-converting enzyme Inhibitory peptide Stability Docking
a b s t r a c t Angiotensin I-converting enzyme (ACE) inhibitory peptide from silkworm pupa (Bombyx mori) was purified, modified, as well as inhibition mechanism by using molecular docking analysis. Silkworm pupa protein was hydrolyzed by neutral protease and the obtained hydrolysate was subjected to various types of chromatography to acquire peptide isolate. Then the molecular mass and amino acid sequence of the peptide was determined by MALDI-TOF/TOF MS. Subsequently, thermal and digestive stability of the peptide were explored through a high temperature processing and a simulated gastrointestinal digestion. Finally, the peptide was modified to smaller peptides and investigated their potentiate activities. Results showed that the peptide from silkworm pupa was determined to be Gly-Asn-Pro-Trp-Met (603.7 Da) with IC50 21.70 M. Stability testing showed that ACE inhibitory activities were not significantly changed at temperature from 40 to 80 ◦ C as well as during in vitro gastrointestinal digestion. The inhibitory activity of four modified peptides were Trp-Trp > Gly-Asn-Pro-Trp-Trp > Asn-Pro-Trp-Trp > Pro-Trp-Trp, and the IC50 of Trp-Trp was 10.76 M Docking simulation revealed that the inhibitory activity was closely related to the spatial structure of peptide and zinc ions. The purified peptide and four modified peptides may be beneficial as functional food or drug for treating hypertension. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Hypertension is a common cardiovascular disease worldwide, and can induce many complications such as prediabetes, atherosclerosis and heart stroke [1]. Recent research shows that this disease is mainly caused by the increasing concentration of angiotensin I-converting enzyme (ACE, EC 3.4.15.1) [2]. Herein, ACE inhibitors are the mainstay medications for treating high blood pressure. Many synthetic ACE inhibitors, including captopril, enalapril, lisinopril, can usually associated with unacceptable adverse effects such as dry cough, angioedema, taste disturbance and skin rash [3,4]. Currently, more and more bioactive peptides have been isolated from food protein sources which have significant ACE inhibitory activities and minimum side effects [5–7]. Quantitative structureactivity relationships between ACE structure and ACE inhibitory
∗ Corresponding author. E-mail address: [email protected] (Z. Zhao).
peptide have been reported that the peptide containing hydrophobic amino acids at C-terminal exhibited potent inhibitors [8–10]. Silkworm pupa protein is known as containing 18 known amino acids, which maintains high levels of hydrophobic amino acids like valine, methionine and phenylalanine [11,12]. Its hydrolysates by various enzymatic hydrolysis possess multifunctional property, for instance, ACE inhibitory activity [13,14], antioxidant activity [15], anti-obesity [16], antitumor activity [17], etc. Therefore, some ACE inhibitory peptides have been reported to be isolated from silkworm pupa protein, and their inhibition mechanisms have been also investigated by molecular docking [18,19]. However, there are few reports about the modification of peptides to enhance their activities. In this paper, a novel ACE inhibitory peptide derived from enzyme hydrolysate of silkworm pupa (Bombyx mori) protein was isolated and identified. The sequence of purified peptide was identified by matrix-assisted laser desorption ionization timeof-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Besides, the peptide was synthesized by using solid phase peptide method, and its thermal and digestive stability were also characterized through high temperature and simulated gastrointestinal
http://dx.doi.org/10.1016/j.procbio.2016.12.022 1359-5113/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: M. Tao, et al., Purification, modification and inhibition mechanism of angiotensin Iconverting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.12.022
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digestion process. Furthermore, the molecule docking mechanism of purified peptide with ACE was determined by Sybyl X-2.1.1. Finally, this peptide was modified and obtained five novel peptides with Trp at the C-terminal. The ACE inhibitory activity and action mechanism of modified peptides were also studied.
2. Materials and methods 2.1. Materials The silkworm pupa was purchased from Guangxi Jialian Silk Co., Ltd. (Yizhou, China). Neutral protease was provided by Pangbo Biological Engineering Co., Ltd. (Nanning, China). ACE from rabbit lung, hippuryl-l-histidyl-l-leucine (HHL) were offered from SigmaAldrich Chemical Co. (St. Louis, MO, USA). Pepsin from porcine gastric mucosa was supplied by Coolaber Science & Technology Co., Ltd. (Beijing, China), and pancreatin from porcine pancreas was offered by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Sephadex G-15 was purchased from Pharmacia Fine Chemicals Co., Ltd. (Uppsala, Sweden). Methanol for HPLC analysis was supplied by Tianjin Shield Specialty Chemical Co., Ltd. (Tianjin, China). All starting materials were commercially available reagents of analytical grade and used without further purification.
Fig. 1. Flowchart of preparation of ACE inhibitory peptides from silkworm pupae protein. The purification procedures of the ACE-inhibitory peptide are laid out step by step. (I) Ultrafiltration, 5000 Da; (II) Ion-exchange chromatography, D201; (III) Gel filtration chromatography, Sephadex G-15; (IV) Reversed-phase high performance liquid chromatography, ZORBAX SB C18.
2.2. Measurement of ACE inhibitory activity The inhibitory activity of ACE was performed on reversed-phase high performance liquid chromatography (RP-HPLC) and assayed with a modified spectrophotometry [8]. HHL was dissolved in 100 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl and formed a concentration of 5.8 mM. Rabbit lung ACE was dissolved in the same buffer solution at a concentration of 10 mU/mL. 40 L of ACE solution and a certain concentration of sample solution were mixed with sodium borate buffer (460 L), which was incubated at 37 ◦ C for 10 min. After that, 40 L of HHL solution was added into above sodium borate buffer and kept the solution incubated for 60 min. The reaction was then terminated by adding 100 L of 1.0 M HCl, and then was filtered by 0.22 m membrane (Tianjin Jinteng Experiment Equipment Co., Ltd.). Finally, the product hippuric acid (HA) was detected with a ZORBAX SB C18 column (4.6 mm × 150 mm, particle size 5 m; Agilent, USA) and a Diode array detector (DAD) at 228 nm by RP-HPLC. The mobile phase was 0.1% trifluoroacetic methonal/water (3:17, v/v). The evaluation of ACE inhibition was based on the comparison between the concentrations of HA in the presence or absence of an inhibitor, and inhibition activity was calculated by using the following equation: I=
Ae − Af × 100% Ae − Ab
(1)
2.3.1. Ultrafiltration The lyophilized silkworm pupa protein hydrolysate was dissolved in ultrapure water, and then fractionated by ultrafiltration using Labscale System (Labscale TFF System, Millipore Co, Billerica, MA, USA) with a 5 kDa molecular-weight- cut-off membrane (Millipore Co, Billerica, MA, USA), seen in step I of Fig. 1. The SN1 and SN-2 were defined as large peptides with molecular weight more and less than 5 kDa, respectively, which were collected and lyophilized as the ACE inhibitory assay and for further purification. 2.3.2. Ion-exchange chromatography The fraction with the higher activity of the ACE inhibitory between SN-1 and SN-2 was further purified by ion-exchange chromatography. The lyophilized ultrafiltration sample was added in 10 mM phosphate buffer (pH 8.5) to give a final concentration of 10 mg/mL, and then 20 mL dissolved solution was loaded on a D201 anion exchange column (10 × 300 mm, HuiZhu resin Co., Ltd., Shanghai) previously equilibrated with 10 mM phosphate buffer (pH 8.5), seen in step II of Fig. 1. The column was eluted by a linear gradient of NaCl (0–1.0 M) at a flow rate of 1.0 mL/min within 300 min. The elution was monitored at 280 nm with a UV spectrophotometer (Model TU-19, Purkinje, Beijing, China). Two fractions, IE-1 and IE-2, were further collected, freeze dried and tested for ACE inhibitory activity.
2.3. Purification of ACE inhibitory peptide
2.3.3. Gel filtration chromatography The fraction with the maximum ACE inhibitory activity from the ion-exchange chromatography was further purified by gel filtration chromatography. The sample was first dissolved in ultrapure water, and then loaded onto a Sephadex G-15 column (20 × 500 mm) equilibrated with ultrapure water, seen in step III of Fig. 1. The absorbance was measured at 280 nm by a UV spectrophotometer. Three fractions, from GF-1 to GF-3, were pooled, concentrated and lyophilized for measurement of the ACE inhibitory activity.
The silkworm pupa protein and hydrolysate with neutral protease were prepared according to our previous reports [20,21]. Fig. 1 shows a flowchart of an entire preparation for ACE inhibitory peptides from silkworm pupae protein.
2.3.4. Reversed-phase high performance liquid chromatography, RP-HPLC The fraction showed the maximum ACE inhibitory activity was further separated on a ZORBAX SB C18 column by using RP-HPLC
Where I is the ACE inhibition activity, Ae , Af and Ab are the relative area of HA peak generated without ACE inhibitors, with ACE inhibitor component and without both of ACE and ACE inhibitors, mAU. Ratio of the IC50 (a total inhibitor concentration that reduces these activities by 50%) was estimated and used as a measure of isozyme selectivity.
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(Agilent 1260, USA). The column was eluted by a linear gradient of acetonitrile (5–30%) containing 0.1% TFA at a flow rate of 0.50 mL/min within 60 min. The absorbance of the elution was monitored at 220 nm with DAD, seen in step IV of Fig. 1. Then ten fractions, from HP-1 to HP-10, were concentrated and freeze-dried. Subsequently, the freeze-dried sample represented the highest ACE inhibitory activity (HP-6) was subjected to a second round of RP-HPLC purification, eluting with 8% acetonitrile in water (v/v) containing 0.1% TFA with a flow rate of 0.50 mL/min. And four fractions, from HP-61 to HP-64, were collected and lyophilized to powder for further measurement of their ACE-inhibitory activities, respectively.
3
The relative content and relative ACE inhibitory activity were calculated by the following equation: Crel =
Ct × 100% C0
(3)
Irel =
It × 100% I0
(4)
Where C0 and Ct are content of peptide before and after stabilities treatment, respectively, Crel is relative content of peptide. I0 and It are ACE inhibitory activities before and after stabilities treatment, respectively, Irel is relative ACE inhibitory activity.
2.4. Characterization of purified peptide Accurate relative molecular mass and amino acid sequence of the purified peptide with the highest ACE inhibitory activity were determined by 4800 plus MALDI-TOF/TOFTM Analyzer (Applied Biosystems, Beverly, MA, USA). The purified peptide was dissolved in ultrapure water, and then the purified peptide solution was mixed with matrix solution (␣-cyano-4-hydroxycinnamic acid solution, prepared with 50% acetonitrile containing 0.1% TFA). Spectra were acquired on a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer with a 337 nm pulsed nitrogen laser (2-ns pulse duration, 3-Hz repetition rate). Peptide mass spectra were acquired in linear positive ion mode at a mass range of m/z 500–1500. Mass spectrometry/mass spectrometry data of the purified peptide were obtained by collision-induced dissociation (CID).
2.5. Stabilities of silkworm pupa protein derived ACE inhibitory peptide The identified peptide was synthesized by GL. Biochem Co., Ltd. (Shanghai, China) using conventional solid-phase chemistry. Then the peptide was dissolved in 100 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl and given a concentration of 1.0 mg/mL. The generated standard curve of identified peptide was as follows: y = 154.19x + 28.44
(2)
Where y is the chromatographic peak area of identified peptide, mAU, and x is the content of identified peptide, mg/mL. The analysis data showed a good linear relationship (R2 ≥ 0.9990) with the concentration ranges of 0.05–1.50 mg/mL. The identified peptide solutions (1.0 mg/mL) were incubated at 40, 60, and 80 ◦ C for 6 h, respectively. After being cooled to 30 ◦ C, the content and ACE inhibitory activity of the identified peptide were determined. Simulated gastric fluid was prepared by mixing 16.4 mL of 9.8 wt% HCl and 10.00 g of purified pepsin in 1000 mL of water. The pH of the solution was 1.0–1.4. Simulated intestinal fluid was prepared by dissolving 6.80 g of monobasic potassium phosphate (KH2 PO4 ) in 500 mL of water. The solution was brought to pH 6.8 with 0.1 M NaOH. After that, 10.00 g of pancreatin was added to the assay and then diluted to 1000 mL with water. The identified peptide solution was mixed with the simulated gastric at a ratio of 1:1 (v/v) and incubated at 37 ◦ C for 2 h. Afterwards, the solution was adjusted to pH of 6.5–7.0 with 0.5 M NaOH. Then simulated intestinal fluid was mixed with the above reacted solution with a ratio of 1:1 (v/v), incubated for 4 h at 37 ◦ C. After reaction, the solution was boiled to stop the enzyme reaction. The content and ACE inhibitory activity of the identified peptide were tested at different times of 0, 2.0 and 6.0 h.
2.6. Molecular docking of the peptide with ACE The three-dimensional crystal structure of human ACElisinopril complex obtaining from the Protein Data Bank (PDB: 1O86) [22] was selected as working targets. The complete PDB file was downloaded and submitted to Sybyl X-2.1.1 software package (Tripos International, St. Louis, MO, USA) by only removing the inhibitor lisinopril and water molecular. The cofactors zinc and chloride atoms were retained in ACE model. Then the peptides structure were pre-analyzed and prepared for the docking runs using the biopolymer structure preparation tool with default settings. The protomol was created by extracting the original ligand with the help of MOLCADD program of the software. The crystal structure of peptides was subjected to energy minimization with Tripos force field using following the gradient termination of the Powell method for 10,000 iterations with a convergence criterion of 0.005 kcal/mol. This docking was simulated by using the Surflex-Dock program. The ligand (lisinopril) from the ACE-lisinopril complex was used as a reference molecule. The conformations were selected according to Consensus scores (CSCORE) and Total Score. The CSORES can range from 0 to 5, where 5 indicates that a ligand is judged good by all functions, and structures with CSORES of 3, 4 or 5 further consideration. The Total Score represents a binding affinity of conformation and the higher value of Total Score means a strong binding affinity. The top 20 conformations for each peptide had created and ranked based on Total Scores by Sybyl X-2.1.1 software and we chose the first conformation whose CSCORE ≥3 as the conformation of each peptide in ACE.
2.7. Statistical analysis All datas were expressed as the mean with standard deviation of triplicate determinations. Significance of differences between test samples was determined by the Student t-test. A p value of less than 0.05 was taken as significant.
3. Results and discussion To obtain ACE inhibitory peptide, we proposed to use neutral protease to hydrolyze silkworm pupa protein in this work. Next, the obtained hydrolysate was centrifuged, collected, and then lyophilized for further use. Followed by ultrafiltration, ionexchange chromatography, gel filtration chromatography and two steps of RP-HPLC, peptide with the highest ACE inhibitory activity was purified. Subsequently the sequence of peptide was identified by MALDI-TOF/TOF MS, then the peptide was synthesized, and its stabilities were studied.
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Fig. 2. Purification of the fraction SN-2 and ACE inhibitory activity evaluation of its separated peptides. (a) Fraction IE-1 and IE-2 (87.1 g/mL); (b) fractions GF-1 to GF-3 (48.3 g/mL); (c) fractions HP series (37.2 g/mL); (d) fractions HP-6 series (29.8 g/mL).
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Expressed as mean ± standard deviations of triplicates (n = 3). Values in the same column are significantly different at p < 0.05. a
3.1. Preparation of protein hydrolysate and its ACE inhibitory activity Table 1 showed the ACE inhibitory activities of the silkworm pupa protein, SN-1, and SN-2. As shown, The SN-2 (MW <5 kDa) exhibited 2.4 times higher value of ACE inhibitory activity than the SN-1 (MW >5 kDa). It was due to that SN-2 possessing smaller molecular mass was much easier to reach the active site of ACE [23,24]. Therefore, the SN-2 was chosen and purified for further studies.
39.04
b3 b1
60
b2
40
604.31
0.03 ± 0.41 29.36 ± 0.50 19.97 ± 0.23 47.63 ± 0.72
455.31
283.2 283.5 281.9 282.4
269.31
Silkworm pupae protein Protein hydrolysate SN-1 SN-2
b4
172.18
Inhibition ratea (%)
G N P W M
80
184.20
Concentration (g/mL)
b1 b2 b3 b4
58.12
Sample
100
Relative intensity (%)
Table 1 ACE inhibitory activity of silkworm pupae protein, protein hydrolysate, SN-1 and SN-2.
5
20 0 0
100
200
300
400
500
600
M ass (m /z) Fig. 3. Characterization of molecular mass and amino acid sequence of purified peptides. MS/MS spectrum of molecular ion m/z 604.31 Da.
3.2. Purification of ACE inhibitory peptide Fraction SN-2 was continuously divided into two fractions (IE1 and IE-2) by ion-exchange chromatography. Fig. 2(a) showed the ACE inhibitory activity of the fraction IE-1 and IE-2. Fraction IE-1 exhibited higher ACE inhibitory activity of 74.06 ± 1.83% than fraction IE-2. Thus, fraction IE-1 was further lyophilized and separated into three molecular weight (MW) groups by gel filtration chromatography, which were GF-1, GF-2 and GF-3. The ACE inhibitory activities of these fractions GF-1, GF-2 and GF3 are shown in Fig. 2(b). Fraction GF-2 shows the highest ACE inhibitory activity with a value of 69.28 ± 0.74%, followed by fraction GF-1 (50.11 ± 0.53%), and then fraction GF-3 (46.55 ± 1.23%). Furthermore, fraction GF-2 with highest ACE inhibitory activity was separated by RP-HPLC into ten major fractions (named from HP-1 to HP-10). Their ACE inhibitory activities were tested and shown in Fig. 2(c). Among which, fraction HP-6 exhibited the highest ACE inhibitory activity with a value of 76.27 ± 0.11%. Fractions obtained at the middle or end phase of HPLC exhibited a higher ACE inhibitory activity due to their lower molecular mass and greater amounts of hydrophobic amino acids [25]. Afterwards, HP6 was further separated by RP-HPLC and obtained four peaks; those were HP-61, HP-62, HP-63 and HP-64, respectively. As illustrated in Fig. 2(d), fraction HP-64 possessed the highest ACE inhi0bitory activity with a value of 77.60 ± 0.77%. 3.3. Characterization of ACE inhibitory peptide by MALDI-TOF/TOF MS The molecular mass and amino acid sequence of fraction HP64 were measured by MALDI-TOF/TOF MS and shown in Fig. 3. The sequence of the peptide was identified as Gly-Asn-Pro-TrpMet (GNPWM). The molecular mass of peptide was determined as 604.31 (M+H) + , which was consistent with its theoretical molecular mass (603.7 Da). This peptide from silkworm pupa protein is a novel ACE inhibitory peptide with the IC50 value of 21.70 M. The IC50 value was comparatively lower than some other peptides ASL (IC50 = 102.15 M) and APPPKK (IC50 = 73.81 M) obtained from the same source (silkworm pupa protein) [13,14]. The main reason was ascribed to the presence of aliphatic hydrophobic amino acids at Cterminal in the structure of GNPWM that could contribute to ACE inhibitory activity [26].
Fig. 4. Thermal stability (a) and digestion resistant (b) of the GNPWM peptide.
3.4. Stability and ACE inhibitory activity of synthetic peptides Fig. 4(a) gave the thermal stability of the GNPWM. Purified peptide possessing high thermal stability was a predominant of functional food preparation. From Fig. 4(a), the relative content of GNPWM showed a clear decrease from 77.14 to 65.83% with temperature. High temperatures could accelerate cleavage and rearrangements of disulfide and hydrophobic bonds of the GNPWM [27]. The relative ACE inhibitory activity of the GNPWM, however, showed a small increase from 102.2 to 107.4% as temperature increases. The increasing ACE inhibitory activity was probably caused some rearranged peptides presenting ACE inhibitory activity. Overall, the ACE inhibitory activity of the GNPWM showed a
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Fig. 5. Predicted binding mode between ACE and peptide after the peptide was docked at the ACE active site. (a) GNPWM; (b) WW; (c) PWW; (d) GNPWW; (e) NPWW. The residues of ACE are shown as line, and the peptide is shown as stick. The Zn ion is shown as cyan sphere and the hydrogen bond is shown as yellow dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
small variation with temperature increasing under the test conditions of 40–80 ◦ C. Therefore, it can be concluded that thermal heating process exerted certain influence on the structure of the GNPWM, while had little impact on ACE inhibitory activity. In order to validate a certain digestive stability of the GNPWM, gastrointestinal enzyme was used to estimate digestion resistance with in vitro model. Fig. 4(b) showed the digestive stability of the GNPWM at approximately the temperature of the human body. It showed that the relative content of GNPWM slightly declined to 92.3 ± 0.4% after being digested with gastrointestinal enzyme, while the relative ACE inhibition activity of the GNPWM was essentially unchanged before and after digestion. Thus, the peptide displayed a good digestion resistance. 3.5. Modification of peptide Although the structure-activity relationship of ACE inhibitory peptides has not yet been fully elucidated, ACE prefers inhibitors containing hydrophobic amino acid residues at each of the three
C-terminal position [28], e.g., Pro, are more active if present at each of the three C-terminal positions. In addition, the presence of the positive charge of Lys and Arg as the C-terminal residue may contribute to the inhibitory potency [29]. So the C-terminal residue of GNPWM was replaced by 8 different hydrophobic amino acids and docked with ACE by Sybyl X-2.1.1. The result showed that the conformation of GNPWW had a best binding affinity with ACE, and then the peptide was modified by using hydrophobic amino acid Trp at C-terminal position. Moreover, to further investigate the inhibition mechanism, we changed the length of GNPWW by cutting off one amino acid from its N-terminal end one after another to get more peptides. Finally, the four modified peptides GNPWW, NPWW, PWW and WW were synthesized, and their IC50 were measured to be 19.74 M, 23.11 M, 83.69 M and 10.76 M, respectively. The IC50 value of peptide GNPWW had slightly decreased comparing with GNPWM. The peptide PWW with the highest IC50 means that it had the lowest ACE inhibitory activity, while the peptide WW with the lowest IC50 means that it had the highest ACE inhibitory activity.
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This coincided with previous reports that Pro was an unfavorable N-terminal amino acid for binding to ACE [30]. 3.6. Molecular docking To investigate the inhibition mechanism of peptides further, the binding mode between ACE and peptides was predicted by Sybyl X-2.1.1. ACE has three main active site pocket, these are, S1 pocket contains Ala354, Glu384 and Tyr523 residues, S2 pocket contains Gln281, His353, Lys511, His513 and Tyr520 residues, and S1 includes Glu162 residue [31]. Meanwhile, Zn (II) at the ACE active site usually plays a significant role for ACE activity, which constitutes a tetrahedrally-coordinated Zn (II) with ACE by ACE residues His383, His387, Glu411 [32]. The molecular docking result revealed that hydrogen bonds were established between original GNPWM and three pockets, which were the S1 pocket (Ala354 and Glu384), the S2 pocket (Gln281, Lys511 and Tyr520) and the S1 pocket of ACE (Fig. 5a). Although GNPWM did not interact with Zn (II) directly, it could form the interaction with His383. As a result, it caused the distortion of the tetrahedrally-coordinated Zn (II) and therefore reducing ACE activity [13]. As seen, the WW has the highest inhibitory activity among the above peptides. WW peptide could only form hydrogen bonds with S1 pocket (His353) and S2 pocket (Ala354) (Fig. 5b). However, it could also form coordination with Zn (II), and generate hydrogen bond with His383. Thus, the consequences of the distorted tetrahedrally-coordinated Zn (II) resulted in a high inhibitory activity of the WW peptide. Among which, PWW possessed the lowest ACE inhibitory activity. PWW also had two hydrogen bonds with pockets. However, it was hardly to form coordination with Zn (II) and hydrogen bond with residues His383, His387 and Glu411 (Fig. 5c). Thus, its ACE inhibitory activity was much lower than the WW peptide. The modified peptides of GNPWW and NPWW had moderate ACE inhibitory activity in these five peptides. Compared to WW, GNPWW and NPWW could not form hydrogen bond with any of residues His383, His387 and Glu411 (Fig. 5d and e). Thus, it possessed lower ACE inhibitory activity, although more hydrogen bonds existed between modified peptides (GNPWW and NPWW) and ACE. 4. Conclusions In this work, GNPWM was successfully purified and identified from silkworm pupa protein. The peptide was synthesized with an IC50 value of 21.70 M. In addition, the GNPWM showed good stability to high processing temperatures and simulated gastrointestinal digestion. The molecule docking studies revealed that the GNPWM could effectively interact with the active site of ACE and therefore inhibit ACE activity. Four modified peptide (GNPWW, NPWW, PWW, WW) derived from GNPWM were also synthesized. Among these modified peptides, WW had the highest ACE inhibitory activity due to its formation of hydrogen bonds with His383 as well as the distortion of the tetrahedrally-coordinated Zn (II). As a result, high ACE inhibitory activity of the peptides is the result of synergistic effect of strong hydrogen bond between peptides with some critical ACE residues and coordination interactions with Zn (II). In conclusion, we obtained a novel WW peptide with excellent ACE inhibitory activity through selection of molecular docking method and peptide modification, and further work needs to be done to demonstrate the in vivo antihypertensive activity of peptides in order to provide an efficient utilization of silkworm pupa protein.
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