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Graphitized Porous Carbon for Rapid Screening of ACE Inhibitory Peptide (GAMVVH) from Silkworm Pupae (Bombyx mori) Protein and Molecular Insight into Inhibition Mechanism Mengliang Tao, Huaju Sun, Long Liu, Xuan Luo, Guoyou Lin, Renbo Li, Zhenxia Zhao, and Zhongxing Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03195 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017
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Journal of Agricultural and Food Chemistry
Graphitized Porous Carbon for Rapid Screening of ACE Inhibitory Peptide (GMAVVH) from Silkworm Pupae (Bombyx mori) Protein and Molecular Insight into Inhibition Mechanism
Mengliang Tao, Huaju Sun, Long Liu, Xuan Luo, Guoyou Lin, Renbo Li, 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
*
Corresponding Authors Phone: +86-771-3233718; Fax: +86-771-3233718; E-mail:
[email protected] (Zhongxing Zhao) Phone: +86-771-3233718; Fax: +86-771-3233718; E-mail:
[email protected] (Zhenxia Zhao) 1
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ABSTRACT: A novel hydrophobic hexapeptide with high angiotensin converting
2
enzyme (ACE) inhibitory activity was screened from silkworm pupae protein (SPP)
3
hydrolysate using graphitized porous carbon and reverse-phase high performance
4
liquid chromatography methods. Graphitized porous carbon derived from dopamine
5
possessing high surface area and high graphitic carbon was used to rapidly screen and
6
enrich hydrophobic peptides from SPP hydrolysate. ACE inhibition pattern and ACE
7
inhibition mechanism of purified peptide were also systematically studied from
8
classic Lineweaver-Burk model and molecular docking/dynamic simulation. The
9
novel
hydrophobic
hexapeptide
was
identified
as
Gly-Ala-Met-Val-Val-His
10
(GAMVVH, IC50=19.39 ± 0.21 µM) with good thermal/anti-digestive stabilities.
11
Lineweaver-Burk plots revealed that GAMVVH behaved as competitive ACE
12
inhibitor. It formed hydrogen bonds with S1 and S2 pockets of ACE, and established
13
competitive coordination with Zn(II) of ACE. The synergy of hydrogen bonds with
14
active pockets and Zn(II) coordination would efficiently change 3D structure of ACE,
15
and thus inhibited bioactivity of ACE.
16
KEYWORDS: silkworm pupae protein, ACE inhibitory peptide, graphitized porous
17
carbon, adsorption, molecular docking and dynamics simulation
18
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■ INTRODUCTION
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In recent years, hypertension is one of the major public cardiovascular diseases that
21
seriously affect up to people’s health. As far as we known, angiotensin I-converting
22
enzyme (ACE, EC 3.4.15.1) having a structure of zinc-dependent dipeptidyl
23
carboxypeptidase cannot only contribute the generation of vasoconstrictor octapeptide,
24
but also inactive bradykinin.1 Thus, ACE has been thought to possess crucial function
25
on blood pressure regulation.2 Those who have ability to inhibit ACE activity can be
26
considered to have therapeutic effect of hypertension.
27
Currently, many artificial chemicals are available commercially with excellent ACE
28
inhibitor ability, such as captopril, enalapril and lisinopril.3 However, these drugs have
29
obvious negative influences, which can usually cause dry cough, angioedema, taste
30
disturbance and a skin rash.4 Therefore, searching for non-toxic ACE inhibitors from
31
natural sources has become attractive for treating hypertension. For example, some
32
natural ACE inhibitors isolated from edible sources, like milk protein,5 soy-whey,6
33
egg7,8 and fish9,10 were reported to have moderate ACE inhibitory effect.
34
In general, purifying target peptides with high ACE inhibitory activity from a new
35
edible source requires the complex procedure and long processing time. In most cases,
36
these active ingredients are extremely low levels in concentration of hydrolysates and
37
fermentation of natural sources. Extraction process is usually a complicating and
38
cumbersome work to isolate trace amount of bioactive peptides from natural sources.
39
It
40
chromatography, and reverse-phase high-performance liquid chromatography
41
(RP-HPLC).11 These methods have limited its practical application. Thus, rapid and
42
effective throughput screening method for isolating ACE inhibitory peptides from a
43
new natural source is a significant urgent and hot research topic.9
includes
continuous
ultrafiltration,
ion
exchange
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gel
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Adsorption separation using porous materials is an efficient protein coarse selection
45
method.12 Porous materials with large surface area, uniform pore sizes and tunable
46
functionality can form specific affinity and shape selectivity for enrichment of
47
bioactive peptides with unique bioactivities.13 Megias et al.14 reported to immobilize
48
ACE onto glyoxyl-agarose medium, and used this material to adsorb ACE inhibitor in
49
a very short time. G.B. Jiang prepared a Fe3O4@SiO2@graphene microsphere to
50
enrich bioactive peptides at low concentrations.13 Hippauf and co-workers15 employed
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microporous activated carbons to isolate ACE inhibitory peptides from lactalbumi
52
hydrolysate. These researches indicated that purification and capture of specific
53
bioactive molecules (i.e., peptides) with the aid of porous materials will exhibit a
54
multiplied efficiency.
55
Silkworm pupae is one of the substantial agricultural wastes in China and other
56
East Asia country. In this work, graphitized porous carbon (GPC) derived from
57
poly-dopamine (PDA) was used to select hydrophobic bioactive peptides from
58
silkworm pupae protein hydrolysate (SPP hydrolysate) for ACE inhibition. A novel
59
hydrophobic hexapeptide (GAMVVH) was successfully separated and further
60
identified by using MALDI-TOF-TOF. Its thermal stability and in-vitro anti-digestion
61
ability were investigated by using experimental methods. Moreover, ACE inhibition
62
pattern and ACE inhibition mechanism of the GAMVVH were also systematically
63
studied from classic Lineweaver-Burk model and molecular docking/dynamic
64
simulation. The studied results may guide others to high-throughput screen
65
anti-hypertensive candidates from many other potential bio-resources of agricultural
66
wastes.
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■ MATERIALS AND METHODS 4
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Materials. 3-Hydroxytyramine hydrochloride (dopamine, 98%) was purchased
70
from Aladdin industrial Co. Ltd (Shanghai, China). Hippuryl-His-Leu (HHL), and
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angiotensin I-converting enzyme (ACE) from rabbit lung acetone powder were
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purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Silkworm pupae
73
(Bombyx mori) was purchased from Guangxi Jialian Silk Co., Ltd. (Yizhou, China).
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Methanol for HPLC analysis was supplied by Thermo Fisher Scientific Co., Ltd.
75
(USA). The identified peptide of GAMWWH was synthesized (98% purity) by GL.
76
Biochem. Co., Ltd. All the other chemicals and reagents used in this study were of
77
analytical grade without further treatment.
78
Synthesis of GPC Material. Figure 1 shows a flowchart of GPC for rapid screen of
79
a novel ACE inhibitory hexapeptide from silkworm pupae protein. PDA spheres were
80
synthesized according to previous report.16 The procedure was as follows: deionized
81
water (100 mL) was mixed with 40 mL of absolute ethanol, and then ammonia
82
solution (0.4 mL) was injected. After mildly stirring for 30 min, 1.0 g of dopamine
83
hydrochloride was added into the above mixed solution. The mixed solution was
84
stirred gently for 20 h at room temperature. The obtained precipitate was filtered and
85
collected. After that, the product was washed and dried at 150 °C in a vacuum
86
overnight. After drying, PDA spheres were heated to 800 °C with a rate of 5 °C /min
87
and maintained at this temperature for 1.0 h under N2 atmosphere. Next, the
88
carbonized PDA was activated using KOH with a weight ratio of 1:4 (w/w), and then
89
heated at 700 °C for 2.0 h in N2. The porous carbonized PDA was washed with 1.0 M
90
HCl to remove the residual KOH, and designed as graphitized porous carbon (GPC)
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for use, seen in step I of Figure 1.
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Physical Characterization. The morphology of GPC was surveyed by a scanning 5
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electron microscope and a field-emission scanning electron microscope equipped with
95
an energy dispersive X-ray spectrometer (SEM-EDX, Hitachi S-3400N, Japan).
96
Powder X-ray diffraction (PXRD) measurement was performed on an energy
97
dispersive X-ray spectrometer (RIGAKU, Japan) with Cu Ka radiation (λ=1.5406 Å).
98
Specific surface area and pore size distribution were calculated based on the nitrogen
99
physical adsorption with a Micromeritics ASAP 2460. The element components of the
100
surface of GPC were analyzed by X-ray photoelectron spectroscopy (XPS) on a PHI
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5000C ESCA (PHI, USA) with Al Kα radiation (hυ = 1486.6 eV).
102
Assay of ACE inhibition activity. The inhibitory activity of ACE was performed
103
using spectrophotometry with some modifications.9 Firstly, ACE and HHL were
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dissolved in 100 mM sodium borate buffer (pH 8.3, containing 300 mM NaCl),
105
respectively. 40 µL of ACE solution and a certain concentration of inhibitor were
106
mixed to a total volume of 240 µL, and then incubated at 37 °C for 10 min. After that,
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10 µL of HHL was added to the above mixture and triggered the reaction. The
108
mixture was continually incubated at 37 °C for 60 min, and subsequently used 50 µL
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of 1.0 M HCl to terminate the incubation. The generated hippuric acid content of
110
mixture was determined through RP-HPLC (Agilent 1260, USA) with ZORBAX SB
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C18 column (4.6×150 mm, 5 µm particle size, Agilent, USA). The column was eluted
112
with 15% methanol in water (v/v) containing 0.1% trifluoroaceric acid (TFA) at a
113
flow rate of 1.0 mL/min, and monitored at 228 nm by a diode array detector (DAD).
114
ACE inhibition activity is calculated by using the following equation Eq. (1): =
− × 100% −
Eq. (1)
115
where I is the ACE inhibition activity, Ae is the relative area of HA peak generated
116
without ACE inhibitors, Af is the relative area of HA peak generated in the presence of
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inhibitor, Ab is the relative area of HA peak generated without ACE and inhibitor. The 6
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IC50 value defined as the concentration of inhibitor (mM) required to inhibit 50% of
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ACE activity, which is determined by regression analysis of ACE inhibition (%)
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versus peptide concentration.
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Purification of ACE inhibitory peptides. The preparation of the silkworm pupa
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protein hydrolysate (SPP hydrolysate) with alcalase was already described
123
elsewhere.11 The lyophilized SPP hydrolysate was firstly dissolved in ultrapure water,
124
and then10 mg of GPC was mixed with 10 mL SPP hydrolysate (1.0 mg/mL) and
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stirred at 30 °C for 10 min. After incubation with the hydrolysate, the GPC was
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recovered through centrifugation and washed three times with ultrapure water. After
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that, the peptides were eluted from the adsorbed GPC with absolute ethanol, seen in
128
step II of Figure 1.
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The effluent fraction having the highest ACE inhibitory activity was concentrated
130
and then separated on a ZORBAX SB C18 column by using RP-HPLC (Agilent 1260,
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USA) with a flow rate of 0.5 mL/min and a linear gradient acetonitrile (5~25%)
132
containing 0.1% TFA within 60 min at 25 °C. The effluent fraction was monitored at
133
220 nm with DAD. The fractions collected from HPLC were lyophilized for further
134
assay of ACE inhibitory activity. Subsequently, the lyophilized fraction with the
135
highest ACE inhibitory activity was purified for the second step HPLC, and eluted
136
with 18% acetonitrile in water (v/v) containing 0.1% TFA with a flow rate of 0.50
137
mL/min. These fractions were collected and lyophilized to powder for further
138
measurement of their ACE-inhibitory activities and sequence identification, seen in
139
step III of Figure 1.
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Characterization of purified ACE inhibitory peptide. Accurate relative molecular
141
mass and amino acid sequence of the purified peptide (peptide A) with the highest
142
ACE inhibitory activity in the second step HPLC were determined by 4800 plus 7
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MALDI-TOF/TOFTM Analyzer (Applied Biosystems, Beverly, MA, USA). Spectra
144
were acquired on a matrix-assisted laser desorption/ionization time-of-flight
145
(MALDI-TOF) mass spectrometer with a 337 nm pulsed nitrogen laser (2-ns pulse
146
duration, 3-Hz repetition rate). Mass spectrometry/mass spectrometry data of the
147
peptide A were obtained by collision-induced dissociation (CID), seen in step IV of
148
Figure 1. Finally, the toxicity of the identified peptide was simulated by toxic
149
prediction (http://www.imtech.res.in/raghava/toxinpred/) to analyze its toxicity.17
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Stabilities of the purified ACE inhibitory peptide. Peptide A was synthesized by
151
GL. Biochem Co., Ltd. (Shanghai, China) using conventional solid-phase chemistry.
152
Thermal stability of the peptide A was tested at temperature up to 90 °C. 1.63 mM
153
peptide A solution was incubated at various temperatures (40, 50, 70 and 90 °C) for 6
154
h, respectively, and then cooled to room temperature. Then the ACE inhibitory
155
activity was measured.
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Digestive stability of the peptide A was carried out through in vitro evaluation
157
method according to the method described Gawlik-Dziki with slight modification.18
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Simulated gastric fluid was prepared by mixing 16.4 mL of hydrochloric acid
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containing 9.8% (w/w) hydrogen chloride (HCl) and 800 mL of water and adding
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10.00 g of purified pepsin. Then, the solution was diluted with water to 1000 mL with
161
pH ≅1.4. Simulated intestinal fluid was prepared by dissolving 6.80 g of monobasic
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potassium phosphate (KH2PO4) in 500 mL of water, adjusting the solution to a pH of
163
6.8 with 0.1 M sodium hydroxide (NaOH), and adding 10.00 g of pancreatin with
164
water to 1000 mL. The peptide A solution was mixed with the simulated gastric fluid
165
at a ratio of 1:1 (v/v), and then incubated at 37 °C for 2 h. Afterwards, pH of the
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mixed solution was adjusted to 6.5~7.0 with 0.5 M of NaOH. Then, simulated
167
intestinal fluid was added into the reaction mixture with a ratio of 1:1 (v/v), and then 8
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was incubated at 37 °C for another 4 h. After that, the reaction was quenched by
169
boiling water for 10 min. The ACE inhibitory activity of the peptide A was monitored
170
each hour.
171 172
The relative ACE inhibitory activity was calculated by the following equation Eq. (2): =
× 100%
Eq. (2)
173
where and are ACE inhibitory activities before and after stability treatment,
174
respectively, is relative ACE inhibitory activity.
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Kinetics of ACE Inhibition. To determine ACE inhibitory mechanism of peptide A,
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kinetics of the ACE inhibition was conducted according to the previously described
177
method, using Lineweaver-Burk plots.19 Various substrate (HHL) concentrations (0.47,
178
0.94, 1.41, and 1.88 mM) were incubated with ACE in the absence and presence of
179
peptide A (109.6 and 219.2 µM).20,21 Lineweaver-Burk plots were used to calculate
180
inhibition constant ( ), which were determined from the intercept of the
181
Lineweaver-Burk lines.
182
Molecular docking and molecular dynamics simulation. Molecular docking was
183
performed to investigate conformation between ACE active sites and inhibitor
184
(lisinopril or peptide A) using the flexible docking tool of Sybyl X-2.1.1 program
185
package (Tripos Inc., St. Louis, MO, USA).
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Firstly, the 3D structures of peptide A and lisinopril were generated and energy
187
minimized with the Tripos force field using the Powell conjugate gradient
188
optimization algorithm with a convergence criterion of 0.05 kcal/mol. Secondly, the
189
crystal structure of human ACE-lisinopril (1O86.pdb) complex was selected as
190
working target, which was obtained from the Protein Data Bank (http://www.rcsb.org).
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Before docking, water molecules and the inhibitor lisinopril were removed whereas 9
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the cofactor Zn(II) was retained in ACE model. Then the ACE structure was
193
pre-analyzed and prepared for the docking runs using the biopolymer structure
194
preparation tool with default settings as implemented in the Sybyl X-2.1.1 program
195
package. The Surflex-Dock program was used for docking. The binding affinity of the
196
ligand was predicted by the software in terms of Total Score, which was expressed as
197
log Kd (Kd was binding constant). Total Score is prospected the receptor-ligand
198
interaction. The higher the Total Score is, the more intensive the interaction. The
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conformation with the highest Total Score was chosen from the top 20 conformations
200
generated automatically for ongoing study.
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To enhance complementarity of the interaction between ACE and inhibitor
202
(lisinopril or peptide A), molecular dynamics simulation was performed with the
203
GROMACS software package (version 4.5.5) using the GROMACS-96 force field.22
204
First, the complex of ACE bound to inhibitors (peptide A and lisinopril) with the
205
highest Total Score among the top 20 conformations was imported into GROMACS
206
software for further molecular dynamics simulation. PRODRG server was used to
207
obtain topology for the inhibitors.23 Then, the enzyme-inhibitor complex was solvated
208
with the explicit SPC water embedded in a dodecahedron box with a length of 1 nm to
209
achieve water density 1.0. System was neutralized by addition of 12 Na+ counter ions
210
to replace water molecules, which was then subjected to a 1000-step energy
211
minimization with the steepest descent approach.
212
The molecular dynamics simulation was performed at 300 K and 1 bar for 15 ns
213
with a time step of 2 fs. The particle mesh Ewald (PME) algorithm24 was employed to
214
calculate electrostatic interactions with interpolation order of 4.0 and a grid spacing of
215
0.12. The cutoff for van der Waals interactions was determined as 1.4 nm. Images of
216
ACE residue and simulated complexes of ACE and inhibitors involving Zn(II) 10
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coordination were generated using Pymol, seen in step V of Figure 1.
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Statistical analysis. All assays of ACE inhibitory activity were conducted in
219
triplicates. Data were presented as mean ± standard deviation. Statistical analysis was
220
performed in MS Excel (Microsoft Windows 2003) by using Student’s t-test.
221
Significant difference in means between the samples was determined at a 5%
222
confidence level (p < 0.05).
223 224
■ RESULTS AND DISCUSSION
225
Physical characteristics. PDA sample was uniformly spherical in shape, and their
226
size could be controlled in the range from 400 to 600 nm (Figure S1). After
227
carbonization and activation, GPC also maintained the original morphology of PDA
228
spheres (Figure 2a). Element analysis in Figure 2a shows that GPC belongs to high
229
carbon material after being calcinated, whose C content reached 87.4 wt.%. During
230
activated process, large amount of oxygen groups was removed from PDA substrate,
231
resulting in the decrease concentration of O element in GPC sample.
232
Nitrogen adsorption isotherm of GPC is displayed in Figure 2b. As shown, the
233
isotherm of GPC belongs to type I isotherm according to the IUPAC classification,
234
demonstrating a typical microporous structure. Its BET surface area, micropore area,
235
and total pore volume were 1895.3 m2/g, 1763.2 m2/g, and 0.792 cm3/g, respectively.
236
The pore size distribution of sample calculated by DFT model revealed that the main
237
peak was observed at about 6.8 Å (inset of Figure 2b). The PXRD pattern of GPC is
238
depicted in Figure 2c. Two broad peaks appeared at 23º and 43º in pattern of GPC,
239
which corresponded to the (002) and (100) crystal planes of a typical graphitic
240
structure.
241
To further investigate the graphitic structure of the synthesized GPC, XPS 11
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measurement was carried out in this work. In Figure 2d, the GPC was mainly
243
composed of four peaks centered at around 284.7, 285.7, 287.0 and 289.0 eV, which
244
were assigned to Sp2C (graphitic carbon), Sp3C and C=N and C-O,25,26 respectively.
245
The relative intensity ratio of these two signals C Sp2/Sp3 was of ~ 2:1, revealing the
246
high content of graphitic carbon in GPC. It will give a more hydrophobic surface
247
property for GPC.
248
High surface area and hydrophobic surface feature of GPC will enable samples to
249
exhibit high enrichment capacity and high selectivity for hydrophobic peptides.
250
Therefore, adsorption approach is a green, facile and rapid process for rough
251
screening peptides from protein hydrolysate in comparison to traditional
252
high-performance liquid chromatography method.
253
Purification of ACE inhibitory peptides. The inhibitory activity for ACE in SPP
254
hydrolysate and the eluate fraction from GPC was determined. The measured value of
255
inhibitory activity indicated that the eluate fraction from GPC showed an increasing
256
from 20.39 ± 1.64% to 79.42 ± 2.47% at a concentration of 800 mg/L. That means
257
some peptides with high ACE inhibitory activity had been adsorbed by the GPC
258
sample. According to the hydrophobic character of the GPC, it would prefer to
259
adsorption of hydrophobic peptides by π-π interaction.27 From this, it can be known
260
that the selected hydrophobic peptides would possess high ACE inhibitory activity. It
261
is consistent with other observations that more hydrophobic peptides may contribute
262
to more ACE inhibitory activity than hydrophilic ones.28 This would provide further
263
evidence by using peptide sequences identification.
264
The eluted peptides from GPC sample were purified through RP-HPLC into nine
265
fractions (named from SH-1 to SH-9) (Figure 3a), and their ACE inhibitory activities
266
were tested and shown in Figure 3b. Among these fractions, the SH-8 fraction 12
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exhibited the highest ACE inhibitory activity with a value of 87.89 ± 3.65%, and thus
268
was further separated using the second RP-HPLC. From the retention time of these
269
peaks, the chosen SH-8 fraction was preliminarily judged to contain more
270
hydrophobic peptides. Further, SH-8 fraction was divided into four fractions (Figure
271
3c), among which fraction SH-84 exhibited the most potent ACE inhibitory activity
272
(84.35±3.42%, seen in Figure 3d).
273
Identification of ACE inhibitory peptide by MALDI-TOF/TOF MS. The molecular
274
mass and
amino acid
sequence
of fraction
SH-84
were measured
by
275
MALDI-TOF/TOF MS and shown in Figure 4. The sequence of fraction SH-84 was
276
identified as Gly-Ala-Met-Val-Val-His (GAMVVH). The molecular mass of peptide
277
was determined as 613.3 (M+H)+, which was consistent with its theoretical molecular
278
mass (612.8 Da). GAMVVH is composed of five typical hydrophobic amino acids
279
and one basic amino acid. Thus, GAMVVH can be thought as a relatively small and
280
hydrophobic hexapeptide. It can form a high affinity for the selective adsorption in
281
GPC in analogy to the lock-and-key mechanism of enzyme-substrate pairs.15 The
282
GAMVVH was synthesized by GL. Biochem Co., Ltd. (Shanghai, China) using
283
conventional solid-phase chemistry. Its ACE inhibitory activity was tested to IC50 =
284
~19.39 (±0.21) µM. And, its SVM score was about -0.8, and the negative value means
285
a nontoxic peptide.
286
Peptide stability of gastro-intestinal conditions. The relative ACE inhibitory
287
activities ( ) of GAMVVH at different temperatures are shown in Figure 5a in the
288
supporting information. As seen, of GAMVVH had no significantly decreased
289
when temperature increased up to 90 °C, indicating an excellent thermal stability for
290
practical application.
291
Another most crucial property of bioactive peptides is the resistance against 13
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digestive proteases, which enables itself to reach targeted organs retaining high ACE
293
inhibitory activity. In this work, digestive stability of GAMVVH was tested followed
294
by in vitro gastrointestinal tract. Figure 5b shows the variation of its relative ACE
295
inhibitory activity with sequential digestion by pepsin and then by pancreatin,
296
respectively. It can also be seen that of GAMVVH maintained 98.9% and 97.3%
297
of its initial ACE inhibitory activity after being digested with pepsin for 2 h and
298
further with pancreatin for another 4 h, respectively. As a result, sequential in vitro
299
digestion measurement clearly indicated that the peptide of GAMVVH exhibits a very
300
good anti-digestion performance.
301
Kinetics of ACE Inhibition activity. Lineweaver-Burk plots were determined to
302
elucidate the ACE inhibition model for the GAMVVH and shown in Figure 6. The
303
generated plots show a coinciding intercept at the Y-axis (1/S) with increasing the
304
peptide concentration, exhibiting the competitive inhibition model of GAMVVH to
305
ACE. This inhibition model means that the inhibitory peptide acted through binding
306
to the active site of the ACE, and thus blocked the enzyme from interacting with
307
substrate.29 Besides, its inhibition constant ( ) was calculated to be appropriately
308
5.1×10-6 M. The lower value of is, the inhibitor binding to ACE-inhibitor forms
309
higher binding ability. In our case, the value of GAMVVH shows at least two
310
orders of magnitude smaller than some reported inhibition constant of protein
311
hydrolysates (6.0-8.9×10-4 M),30 indicating a more potential ACE inhibitory capacity.
312
Molecular docking and dynamics simulation. Molecular docking was performed to
313
further investigate and predict the inhibition mechanism of GAMVVH. For
314
comparison, a commercial compound (lisinopril) was also be simulated in this system.
315
First, the top 20 conformations were selected and ranked based on their calculated
316
Total Score (TS) by using Surflex-Dock program. The obtained conformation with 14
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highest TS (named No. 1) was selected as the research subject and displayed in Figure
318
S2. After that, we imported the selected conformation (No. 1) of the ACE/inhibitor
319
(GAMVVH and lisinopril) complexes into GROMACS software to adjust its
320
conformation for ligand binding by using molecular dynamics simulation.28 Backbone
321
root mean square deviations (RMSD) were used to the conformation changes of
322
ACE/inhibitor complexes during the molecular dynamics simulation. Figure S3 shows
323
RMSD as a function of simulation time for the complexes of ACE/inhibitor. Clearly,
324
the distance for both complexes of ACE/GAMVVH and ACE/lisinopril shows no
325
change during the course of about 15.0 ns molecular dynamics simulation. This
326
indicated that the system reached equilibrium state within a short time. Moreover, we
327
found that the radius of gyration (Rg) of ACE molecule can be used to detect the
328
structure change.31 Figure S4 depicts the Rg of ACE in complexes of ACE/GAMVVH
329
and ACE/lisinopril, and its average value throughout the simulation time was 23.0 Å
330
for ACE/GAMVVH, which was a little smaller than that of the ACE/lisinopril (23.1
331
Å). As seen, upon the binding of the inhibitors, their changes of Rg for
332
ACE/GAMVVH and ACE/lisinopril were no obvious variation. It shows no big
333
difference of ACE/GAMVVH and ACE/lisinopril complexes in this system,
334
suggesting a stability of the whole structure of complexes after inhibitors bonding to
335
ACE.
336
Then, the stable structures of the ACE/inhibitor complexes (molecular dynamics
337
simulation for 15 ns) were analyzed by Pymol to display the interaction of ACE and
338
inhibitors. The peptide of GAMVVH established hydrogen bonds with ACE residues
339
(Figure 7a) of Gln281 (1.7 Å), His353 (2.2 Å), Ala354 (1.9 Å), Asp453 (1.8/2.2 Å)
340
and Lys511 (2.0 Å), respectively. While lisinopril was found to form hydrogen bonds
341
with ACE residues of Glu162 (2.2 Å), Gln281 (2.3 Å), Ala354 (2.3 Å), Lys511 (2.4 Å) 15
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342
and Tyr520 (2.2 Å) (Figure 7b), which was similar to previous report. What the two
343
shares in common were the interaction occurring peptides with of S1(Ala354) and S2
344
(Lys511, Lys511and Gln281) pockets of ACE. Their slight differences were the way
345
that lisinopril could also interact with the third S1’ pocket (Glu162) of ACE, which
346
GAMVVH didn't. That would be the probable reason why the selected peptide
347
GAMVVH possessed relative weaker ACE inhibitory activity compared to lisinopril.
348
The knowledge of interaction with active pockets of ACE and inhibitors will be a
349
guide to the screening peptides with high ACE inhibitory activity.
350
Besides of hydrogen bonds, Zn(II) at the ACE active site usually plays a significant
351
role for ACE inhibitory activity, which constitutes a tetrahedrally-coordinated Zn(II)
352
with ACE by ACE residues His383, His387 and Glu411.32 The coordination
353
interactions between tetrahedrally-coordinated Zn(II) in the ACE and inhibitors were
354
also calculated using molecular dynamics simulation and shown by Pymol. It can be
355
found that Zn(II) was bound to ACE residues His383(NE2), His387(NE2) and
356
Glu411(OE1) with an approximate equidistance of 2.0 Å, which built a tetrahedron
357
with Zn(II) in its exact center (Figure S5). The result indicated a stable structure of
358
ACE molecule before contacting with inhibitors. The output images of the complexes
359
show coordination and distances between ACE/inhibitors and Zn(II) (Figure 7c-d,
360
Table 1). It was displayed that the Zn(II) tetrahedron in complexes of ACE/inhibitors
361
suffered distortion. By comparison, the bonds between Zn(II) and Glu411 oxygen
362
(2.1/2.2 Å for GAMVVH, and 2.3 Å for lisinopril) as well as His383 oxygen (2.0 Å
363
for both simulated inhibitors) remained in the new generated structure. The bond
364
towards His387, however, was replaced by a specific binding of other ACE residues
365
and oxygen groups in the inhibitors. The break of coordination Zn(II)-His387 in ACE
366
would distort Zn tetrahedral geometry, and thus would inhibit ACE activity. 16
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In this case, the relatively significant difference is the distance of Zn(II) with the
368
inhibitors in two compounds. Clearly, the distance between GAMVVH and Zn(II)
369
(2.5 Å) was about 0.4 Å longer than that between lisinopril and Zn(II) (2.1 Å). It
370
revealed that GAMVVH formed weaker interaction than lisinopril with Zn(II) sites.
371
The results may prove again that GAMVVH exhibited lower ACE inhibitory activity
372
compared with lisinopril. Thus, the coordination of Zn(II) with inhibitor and
373
hydrogen-bond interaction of ACE active pockets with inhibitor were the key index of
374
performance in ACE inhibitory activity Thereinto, molecular docking and dynamics
375
simulation can be the effective tools for determining and identifying high activity of
376
ACE inhibitor from various nature sources.11 Taken as a whole, a novel hydrophobic
377
hexapeptide (GAMVVH) with high inhibitory activity of ACE was rapidly selected
378
from SPP hydrolysate using graphitized porous carbon, and ACE inhibition
379
mechanism revealed that synergistic effect of hydrogen bonds and Zn-coordination
380
with ACE pockets was crucial to inhibitory activity of hexapeptide. The studied
381
results may guide others to high-throughput screen anti-hypertensive candidates from
382
many other potential bio-resources of agricultural wastes.
383 384
■ ACKNOWLEDGMENTS
385
We appreciate the helpful suggestion from Dr. Wei hu and Dr. Bingfeng Wang of State
386
Key Laboratory for Conservation and Vtilization of Subtropical Agro-bioresources.
387 388
■ Supporting Information
389
SEM image and element contents of PDA; The molecular docking of inhibitors
390
(GAMVVH and Lisinopril) binding to ACE; RMSD as a function of simulation time
391
for the complexes of ACE; Radius of gyration (Rg) of the complexes during 17
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molecular dynamics simulation; Distance between Zn (II) and ACE residues (His383,
393
His387, Glu411) without inhibitor. This material is available free of charge via the
394
Internet at http://pubs.acs.org
395 396
■ References
397
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Angiotensin-I Converting Enzyme Inhibitory Peptide from Yeast (Saccharomyces
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Carbon 2014, 77, 191-198.
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modified with Trp. J. Funct. Foods. 2015, 17, 632-639.
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(26) Rui, X.; Boye, J. I.; Simpson, B. K.; Prasher, S. O. Purification and
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characterization of angiotensin I-converting enzyme inhibitory peptides of small red
477
bean (Phaseolus vulgaris) hydrolysates. J. Funct. Foods. 2013, 5, 1116-1124.
478
(27) Girgih, A. T.; Udenigwe, C. C.; Li, H.; Adebiyi, A. P.; Aluko, R. E. Kinetics of
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Enzyme Inhibition and Antihypertensive Effects of Hemp Seed (Cannabis sativa L.)
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Protein Hydrolysates. J. Am. Oil Chem. Soc. 2011, 88, 1767-1774.
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(28) Zhou, M.; Du, K.; Ji, P.; Feng, W. Molecular mechanism of the interactions
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between inhibitory tripeptides and angiotensin-converting enzyme. Biophys. Chem.
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(29) Pan, D.; Cao, J.; Guo, H.; Zhao, B. Studies on purification and the molecular
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mechanism of a novel ACE inhibitory peptide from whey protein hydrolysate. Food
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Chem. 2012, 130, 121-126.
487 488
Funding
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This work was financially supported by National Natural Science Foundation of
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China (No. 31401629, 21666004, 21676059 and 21606054), Natural Science
491
Foundation of Guangxi Zhuang Autonomous Region, China (No. 2016JJA120072),
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Scientific Research Foundation of Guangxi University (No. XGZ130963) and 21
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Innovation and Entrepreneurship Training Program of Guangxi Zhuang Autonomous
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Region (No. 201610593169 and 201710593185).
495 496
Notes
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The authors declare no competing financial interest.
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Figure captions
501
Figure 1
Flowchart of GPC for rapid screen of a novel ACE inhibitory hexapeptide from silkworm pupae protein. The flowchart is laid out step by step. (I) Synthesis of GPC Material; (II) Elute peptides from GPC; (III) Reversed-phase high performance liquid chromatography; (IV) Peptide identification; (V) Molecular simulation.
Figure 2
Physical characteristics of GPC (a) SEM image; (b) Nitrogen adsorption isotherm and DFT pore size distribution; (c) PXRD; (d) High-resolution C 1s XPS spectrum.
Figure 3
Chromatograms of (a) the first RP-HPLC and (c) the second RP-HPLC; ACE inhibitory activities of (b) nine fractions from first RP-HPLC (sample concentration of 300 mg/L) and (d) the four fractions from second RP-HPLC (sample concentration of 80 mg/L).
Figure 4
Characterization of molecular mass and amino acid sequence of GAMVVH.
Figure 5
Thermal stability (a) and digestive resistibility on pepsin and pancreatin (b) of GAMVVH. Values are presented as mean ± standard deviations from three replications.
Figure 6
Lineweaver-Burk plots of ACE inhibition by GAMVVH peptide.
Figure 7
Predicted binding mode between ACE and inhibitors ((a) GAMVVH and (b) lisinopril) after being docked with the ACE active sites; Predicted binding mode between Zn (II) and ACE residues (His383, His387, Glu411) with (c) GAMVVH and (d) Lisinopril after molecular dynamic simulation. The GAMVVH/lisinopril and Zn atom were shown 23
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as stick and cyan sphere, respectively. The hydrogen bond, coordination bond and distance of Zn (II) and inhibitor were shown as yellow dashed lines, red dashed lines and light blue dashed lines, respectively.
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Table
Table 1. Zinc Interactions With ACE/inhibitors After Using Molecular Dynamics Simulation Zn-O(N) distance (Å) Residues GAMVVH
Lisinopril
His383 (NE2)
2.0
2.0
Glu384 (OE1)
2.2
2.2
Glu384 (OE2)
2.1
2.2
Glu411 (OE1)
2.2
2.3
Glu411 (OE2)
2.1
---
GAMVVH (OBE)
2.5
---
Lisinopril (O3)
---
2.1
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Figure graphics
Figure 1
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500
(b) dV/dW (cm3 g-1 A )
3
-1
Amount of adsorbed N2 (cm ⋅g STP)
600
-1
400 300 200 100
6.8 Å
4
8
0 0.0
0.2
12 16 20 24 28 Pore width (Å)
0.4
0.6
0.8
1.0
Relative pressure (P/Po)
500
(d)
(c)
Raw Simulated 2 Sp C 3 Sp C&C-N C=N C-O
400
Intensity (a.u.)
(002)
350
(100)
Relative Intensity (a.u.)
450
300 250 200 150 10
20
30
40
50
60
70
282
80
2θ (degree)
284
286 Binding energy (eV)
Figure 2
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288
290
Journal of Agricultural and Food Chemistry
SH-1 ~ SH-7
SH-8
500
100
SH-9
mAU
90
400
75
mAU
60
300
45 32
33 34 35 Time (min)
36
200 100 0 10
20
50
mAU
57.15
54.35 39.45
40 23.86
18.49
20
60
0
SH82
800 600 SH81
400
65.36 60
11.38
2
1
3
4 5 6 SH - Series
7
8
100
(c)
1000
30 40 Time (min)
76.77
80
ACE inhibitory activity (%)
0
87.89
(b)
Relative inhibitory activity (%)
(a)
600
Page 28 of 33
SH84 SH83
200
(d)
84.35
80 55.81
60
39.75
40 20
19.97
0 0
3
6 Time (min)
9
12
0
SH81
Figure 3
28
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SH82
SH83
SH84
9
Journal of Agricultural and Food Chemistry
39.04
b1 b2 b3 b4 b5
GAMV VH
b4
b3 260.38
58.12
129.31
b1
60
b2
b5 547.36
80
359.14
100
Relative intensity (%)
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40 20 0 0
100
200
300
400
500
Mass (m/z)
Figure 4
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600
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(b) 100
80
80 Irel (%)
Irel (%)
(a) 100
60
60
40
40
20
20
0
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40
70 50 Temperature (°C)
pepsin
pancreatin
0
90
0
1
Figure 5
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2
3 Time (h)
4
5
6
Journal of Agricultural and Food Chemistry
0 µM 0.15
1 09.6 µM
219.2 µM
-1
1/V0/(L·min·mmol )
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0.10
0.05
0.00 -1.0
-0.5
0.0
0 .5
1 .0
1 .5
2 .0
1/[S] (L /m m ol)
Figure 6
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Figure 7
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Table of Contents Graphic
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