Graphitized Porous Carbon for Rapid Screening of Angiotensin

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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

ACS Paragon Plus Environment


1

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

2


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19

■ INTRODUCTION

20

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

3


chromatography,

gel


44

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

51

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.

67 68

■ 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

71

angiotensin I-converting enzyme (ACE) from rabbit lung acetone powder were

72

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).

74

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)

91

for use, seen in step I of Figure 1.

92 93

Physical Characterization. The morphology of GPC was surveyed by a scanning 5



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94

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

101

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

104

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,

107

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

109

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

111

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

117

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

119

ACE activity, which is determined by regression analysis of ACE inhibition (%)

120

versus peptide concentration.

121

Purification of ACE inhibitory peptides. The preparation of the silkworm pupa

122

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

125

stirred at 30 °C for 10 min. After incubation with the hydrolysate, the GPC was

126

recovered through centrifugation and washed three times with ultrapure water. After

127

that, the peptides were eluted from the adsorbed GPC with absolute ethanol, seen in

128

step II of Figure 1.

129

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,

131

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.

140

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



143

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

150

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.

156

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

158

Simulated gastric fluid was prepared by mixing 16.4 mL of hydrochloric acid

159

containing 9.8% (w/w) hydrogen chloride (HCl) and 800 mL of water and adding

160

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

162

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

166

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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168

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.

175

Kinetics of ACE Inhibition. To determine ACE inhibitory mechanism of peptide A,

176

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).

186

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).

191

Before docking, water molecules and the inhibitor lisinopril were removed whereas 9



192

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

199

conformation with the highest Total Score was chosen from the top 20 conformations

200

generated automatically for ongoing study.

201

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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217

coordination were generated using Pymol, seen in step V of Figure 1.

218

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



242

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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267

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



292

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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317

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



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


Page 16 of 33

Page 17 of 33


367

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



392

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

(1) Tsai, J. S.; Lin, T. C.; Chen, J. L.; Pan, B. S. The inhibitory effects of freshwater

398

clam (Corbicula fluminea, Muller) muscle protein hydrolysates on angiotensin I

399

converting enzyme. Process Biochem. 2006, 41, 2276-2281.

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(2) Ni, H.; Li, L.; Guo, S.; Li, H.; Jiang, R.; Hu, S. Isolation and Identification of an

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Angiotensin-I Converting Enzyme Inhibitory Peptide from Yeast (Saccharomyces

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cerevisiae). Curr. Anal. Chem. 2012, 8, 180-185.

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(3)Antonicelli, R.; Germano, G. What is new about stroke prevention?. Ital. Heart. J. 2003, 4, 958-964.

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to assay inhibitors of angiotensin I-converting enzyme. Food Chem. 2013, 141,

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3329-3334.

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(5) Li, X.; Li, Y.; Huang, X.; Zheng, J.; Zhang, F.; Kan, J. Identification and

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characterization of a novel angiotensin I-converting enzyme inhibitory peptide

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(ACEIP) from silkworm pupa. Food Sci Biotechnol. 2014, 23, 1017-1023.

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(6) Gu, Y.; Wu, J. LC-MS/MS coupled with QSAR modeling in characterising of

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angiotensin I-converting enzyme inhibitory peptides from soybean proteins. Food

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(7) Duan, X.; Wu, F.; Li, M.; Yang, N.; Wu, C.; Jin, Y.; Yang, J.; Jin, Z.; Xu, X.

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Naturally Occurring Angiotensin I-Converting Enzyme Inhibitory Peptide from a

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Fertilized Egg and Its Inhibitory Mechanism. J. Agric. Food Chem. 2014, 62, 18


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5500-5506.

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characterization of angiotensin I-converting enzyme inhibitory peptides of small red

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bean (Phaseolus vulgaris) hydrolysates. J. Funct. Foods. 2013, 5, 1116-1124.

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(9) Lan, X.; Liao, D.; Wu, S.; Wang, F.; Sun, J.; Tong, Z. Rapid purification and

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characterization of angiotensin converting enzyme inhibitory peptides from lizard fish

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protein hydrolysates with magnetic affinity separation. Food Chem. 2015, 182,

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136-142.

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(10) Wu, S.; Feng, X.; Lan, X.; Xu, Y.; Liao, D. Purification and identification of

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Angiotensin-I Converting Enzyme (ACE) inhibitory peptide from lizard fish (Saurida

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elongata) hydrolysate. J. Funct. Foods. 2015, 13, 295-299.

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(11) Tao, M.; Wang, C.; Liao, D.; Liu, H.; Zhao, Z.; Zhao, Z. Purification,

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modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory

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peptide from silkworm pupa (Bombyx mori) protein hydrolysate. Process Biochem.

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2017, 54, 172-179.

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(12) He, X.; Sun, H.; Zhu, M.; Yaseen, M.; Liao, D.; Cui, X.; Guan, H.; Tong, Z.;

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Zhao, Z. N-Doped porous graphitic carbon with multi-flaky shell hollow structure

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prepared using a green and 'useful' template of CaCO3 for VOC fast adsorption and

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small peptide enrichment. Chem. Commun. 2017, 53, 3442-3445.

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(13) Liu, Q.; Shi, J.; Cheng, M.; Li, G.; Cao, D.; Jiang, G. Preparation of

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graphene-encapsulated magnetic microspheres for protein/peptide enrichment and

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MALDI-TOF MS analysis. Chem. Commun. 2012, 48, 1874-1876.

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(14)Megias, C.; Pedroche, J.; Del Mar Yust, M.; Alaiz, M.; Giron-Calle, J.; Millan, F.;

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Vioque, J. Immobilization of angiotensin-converting enzyme on glyoxyl-agarose. J.

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Agric. Food Chem. 2006, 54, 4641-4645. 19



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Towards a continuous adsorption process for the enrichment of ACE-inhibiting

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peptides from food protein hydrolysates. Carbon 2016, 107, 116-123.

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(16) Zhu, M.; Zhou, K.; Sun, X.; Zhao, Z.; Tong, Z.; Zhao, Z. Hydrophobic N-doped

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porous biocarbon from dopamine for high selective adsorption of p-Xylene under

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humid conditions. Chem. Eng. J. 2017, 317, 660-672.

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(17) Gawlik-Dziki, U. Changes in the antioxidant activities of vegetables as a

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consequence of interactions between active compounds. J. Funct. Foods. 2012, 4,

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872-882.

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(18) Wu, J. P.; Aluko, R. E.; Muir, A. D. Improved method for direct

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high-performance

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enzyme-catalyzed reactions. J. Chromatogr. A. 2002, 950, 125-130.

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(19) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms

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for highly efficient, load-balanced, and scalable molecular simulation. J. Chem.

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Theory Comput. 2008, 4, 435-447.

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(20) Schuttelkopf, A. W.; van Aalten, D. PRODRG: a tool for high-throughput

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crystallography of protein-ligand complexes. Acta Crystallogr., Sect. D: Biol.

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Crystallogr. 2004, 60, 1355-1363.

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(21) Eslami, H.; Mojahedi, F.; Moghadasi, J. Molecular dynamics simulation with

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weak coupling to heat and material baths. J. Chem. Phys. 2010, 133, (0841058).

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(22) Ai, K.; Liu, Y.; Ruan, C.; Lu, L.; Lu, G. M. Sp2 C-Dominant N-Doped Carbon

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Sub-micrometer Spheres with a Tunable Size: A Versatile Platform for Highly

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Efficient Oxygen-Reduction Catalysts. Adv. Mater. 2013, 25, 998-1003.

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(23) Wei, Y.; Wu, Y.; Chang, Q.; Xie, M.; Wang, X.; Mo, J.; He, X.; Zhao, Z.; Zhao,

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Z. Ultrasonic-assisted modification of a novel silkworm-excrement-based porous

liquid

chromatography

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of

20


angiotensin-converting

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carbon with various Lewis acid metal ions for the sustained release of the pesticide

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thiamethoxam. RSC Adv. 2017, 7, 30020-30031.

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(24) Hippauf, F.; Lunow, D.; Borchardt, L.; Henle, T.; Kaskel, S. Extraction of

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ACE-inhibiting dipeptides from protein hydrolysates using porous carbon materials.

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Carbon 2014, 77, 191-198.

472

(25) Xie, C.; Choung, S.; Cao, G.; Lee, K. W.; Choi, Y. J. In silico investigation of

473

action mechanism of four novel angiotensin-I converting enzyme inhibitory peptides

474

modified with Trp. J. Funct. Foods. 2015, 17, 632-639.

475

(26) Rui, X.; Boye, J. I.; Simpson, B. K.; Prasher, S. O. Purification and

476

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

479

Enzyme Inhibition and Antihypertensive Effects of Hemp Seed (Cannabis sativa L.)

480

Protein Hydrolysates. J. Am. Oil Chem. Soc. 2011, 88, 1767-1774.

481

(28) Zhou, M.; Du, K.; Ji, P.; Feng, W. Molecular mechanism of the interactions

482

between inhibitory tripeptides and angiotensin-converting enzyme. Biophys. Chem.

483

2012, 168, 60-66.

484

(29) Pan, D.; Cao, J.; Guo, H.; Zhao, B. Studies on purification and the molecular

485

mechanism of a novel ACE inhibitory peptide from whey protein hydrolysate. Food

486

Chem. 2012, 130, 121-126.

487 488

Funding

489

This work was financially supported by National Natural Science Foundation of

490

China (No. 31401629, 21666004, 21676059 and 21606054), Natural Science

491

Foundation of Guangxi Zhuang Autonomous Region, China (No. 2016JJA120072),

492

Scientific Research Foundation of Guangxi University (No. XGZ130963) and 21



493

Innovation and Entrepreneurship Training Program of Guangxi Zhuang Autonomous

494

Region (No. 201610593169 and 201710593185).

495 496

Notes

497

The authors declare no competing financial interest.

498 499

22


Page 22 of 33

Page 23 of 33


500

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



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.

24


Page 24 of 33

Page 25 of 33


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

25



Figure graphics

Figure 1

26


Page 26 of 33

Page 27 of 33


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

27


288

290


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


SH82

SH83

SH84

9


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 (%)

Page 29 of 33

40 20 0 0

100

200

300

400

500

Mass (m/z)

Figure 4

29


600


(b) 100

80

80 Irel (%)

Irel (%)

(a) 100

60

60

40

40

20

20

0

Page 30 of 33

40

70 50 Temperature (°C)

pepsin

pancreatin

0

90

0

1

Figure 5

30


2

3 Time (h)

4

5

6


0 µM 0.15

1 09.6 µM

219.2 µM

-1

1/V0/(L·min·mmol )

Page 31 of 33

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

31



Figure 7

32


Page 32 of 33

Page 33 of 33


Table of Contents Graphic

33


Graphitized Porous Carbon for Rapid Screening of Angiotensin

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