Isolation and Characterization of Angiotensin I-Converting Enzyme

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Bioactive Constituents, Metabolites, and Functions

Isolation and Characterization of Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from the Enzymatic Hydrolysate of Carapax Trionycis (the shell of the turtle Pelodiscus sinensis) Pengying Liao, Xiongdiao Lan, Dankui Liao, LiXia Sun, Liqin Zhou, Jianhua Sun, and Zhangfa Tong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01558 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 20, 2018

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Journal of Agricultural and Food Chemistry

Isolation and Characterization of Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptides from the Enzymatic Hydrolysate of Carapax Trionycis (the shell of the turtle Pelodiscus sinensis)

Pengying Liao, Xiongdiao Lan, Dankui Liao*, Lixia Sun*, Liqin Zhou, Jianhua Sun, Zhangfa Tong

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, Guangxi, P.R. China

* Corresponding authors. Tel: +86 771 327 2702 Fax: +86 771 323 3718 E-mail: [email protected]; [email protected]

Dankui Liao: School of Chemistry & Chemical Engineering, Guangxi University, 100 Daxue East Road, Nanning 530004, Guangxi, P.R. China.

Lixia Sun: School of Chemistry & Chemical Engineering, Guangxi University, 100 Daxue East Road, Nanning, 530004, Guangxi, P.R. China.

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ACS Paragon Plus Environment


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ABSTRACT

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Carapax Trionycis (the shell of the turtle Pelodiscus sinensis) was hydrolyzed by six different

3

commercial proteases. The hydrolysate prepared from papain showed stronger inhibitory activity

4

against angiotensin I-converting enzyme (ACE) than other extracts. Two noncompetitive ACE

5

inhibitory peptides were purified successively by ultrafiltration, gel filtration chromatography, ion

6

exchange column chromatography, and high-performance liquid chromatography (HPLC). The

7

amino acid sequences of them were identified as KRER and LHMFK, with IC50 values of 324.1 and

8

75.6 μM, respectively, confirming that Carapax Trionycis is a potential source of active peptides

9

possessing ACE inhibitory activities. Besides, both enzyme kinetics and isothermal titration

10

calorimetry (ITC) assay showed that LHMFK could form more stable complex with ACE than KRER,

11

which is in accordance with the better inhibitory activity of LHMFK.

12 13

KEYWORDS: Carapax Trionycis; ACE inhibitory peptides; isolation and characterization; enzyme

14

kinetics; isothermal titration calorimetry.

15 16 17 18 19 20 21 22 23 2


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INTRODUCTION

25

Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase containing a zinc ion in

26

the active site, plays an important role in the renin–angiotensin–aldosterone system (RAAS), which

27

is crucial in regulating body fluids and electrolyte balance. ACE can convert the decapeptide

28

angiotensin I (Ang I) to the octapeptide angiotensin II (Ang II) and inactivate the vasodilator

29

bradykinin, both of which can induce blood pressure enhancement.1 Thus, synthetic ACE inhibitors

30

(ACEI) such as perindopril, captopril, and enalapril are believed to be pivotal drugs in the treatment

31

of hypertension. However, adverse effects, such as coughing, allergic reactions, taste disturbances,

32

renal injury, and skin rashes, have always been associated with synthetic ACEI.2

33

The hypotensive effects of the ACEI peptides deriving from various sources including plant and

34

animals are usually weaker than synthetic ACEI, but they are milder and safer.3 Thus, the natural

35

peptides have been used in the development of new therapeutics or nutraceuticals. One important

36

method to obtain these active peptides is protein digestion using commercially available proteases.4

37

Various proteases have been used in acquiring oligopeptides owing to the different specific cleavage

38

sites of them, and the activities of the hydrolysates are the dominant factors in protease screening.5–7

39

The amino acid compositions of the peptides from the hydrolysates are totally dependent on the

40

substrate and the proteases, whereas the activity of the peptide is mainly influenced by amino acid

41

compositions and sequences.8 Numerous principles concerning the relationship between the

42

structures and the activities of the ACEI peptides have been summarized. The short sequences and

43

hydrophobic amino acid residues or charged amino acid residues at C-terminal are the common

44

characteristics of most known active peptides.2, 9

45

Soft-shelled turtle (SST) (Pelodiscus sinensis) is a type of edible aquatic animal possessing

46

antihypertensive effect in spontaneously hypertensive rats (SHR).10 The ACE inhibitory activity of 3



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the hydrolysate of SST treated by gastrointestinal enzymes (pepsin, trypsin, and chymotrypsin) has

48

been reported to be better than SST.11 Besides, three ACEI peptides have been purified from the

49

enzymatic hydrolysates of egg from SST.12–14 These results demonstrate that SST is a valuable source

50

of ACEI peptides. Carapax Trionycis, the shell of SST, is rich in amino acids and peptides.15, 16 Active

51

peptides associated with hepatic diseases have been isolated from the water extracts of Carapax

52

Trionycis.16–18 However, the potential ACE inhibition of this source has never been reported.

53

This work is focused on the isolation and characterization of ACEI peptides from the hydrolysate

54

of Carapax Trionycis. The extracts of Carapax Trionycis were hydrolyzed by six different proteases,

55

and the papain hydrolysates possessing the strongest ACE inhibitory activities were further purified

56

by series of chromatographic methods. After purification, matrix-assisted laser desorption/ionization

57

time-of-flight tandom mass spectrometry (MALDI-TOF/TOF) was used to identify the ACEI

58

peptides, whose inhibitory activities were further confirmed by synthetic authentic samples.

59

Moreover, the inhibitory mechanism of the ACEI peptides was proposed according to the results of

60

enzyme kinetics and ITC analysis.

61

MATERIALS AND METHODS

62

Materials and Chemical Regents. Carapax Trionycis was purchased in Nanning, China, and

63

authenticated by Professor Songji Wei from Guangxi University of Chinese Medicine as the shell of

64

P. sinensis. ACE (from rabbit lung) and hippuryl-L-histidyl-L-leucine (HHL) were both purchased

65

from Sigma-Aldrich (St. Louis, MO, USA). Alcalase (300 000 U/g), neutral protease (400 000 U/g),

66

papain (300 000 U/g), and bromelain (20 000 U/g) were all provided by Pangbo Biological

67

Engineering Co., Ltd. (Nanning, China). Trypsin (300 000 U/g) and pepsin (10 000 U/g) were

68

purchased from Sinopharm Medicine Holding Co., Ltd. (Shanghai, China). Acetonitrile (HPLC grade)

69

was purchased from Fisher Scientific Inc. (Hudson, NH, USA). Other reagents were of analytical 4


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grade and purchased from Sinopharm Chemical Reagent Company (Shanghai, China).

71

Preparation of enzymatic hydrolysates and screening of the proteases. Carapax Trionycis was

72

crushed in a grinder and sieved through a 40 mesh. The powder (1 Kg) was then mixed with 10 L of

73

distilled water and heated at 60 °C for 2 h. The extract (WEP) was filtered and lyophilized as raw

74

material for enzymatic hydrolysis. The protein content of WEP was 77.02% ± 1.60% as determined

75

by Kjeldahl method, using 6.25 as protein conversion factor.19 WEP was dissolved in ultrapure water

76

at a protein concentration of 2% in a 500 mL glass reactor, and was digested by six different enzymes

77

(papain, neutral protease, trypsin, bromelain, alcalase, and pepsin) under their optimal conditions

78

(shown in Table 1), respectively. During enzymatic reactions, 0.1 M NaOH was added to maintain

79

pH at the optimal level, and the solution was continuously stirred for 7 h. Afterwards, the hydrolysate

80

was then placed in a boiling water bath for 10 min to inactivate the enzyme and the pH of the solution

81

was adjusted to 7.0. The hydrolysates prepared using six different proteases were centrifuged at 4 000

82

rpm for 30 min at 4 °C (Model 5810R, Eppendorf), and the supernatant was lyophilized to evaluate

83

the ACE inhibitory activities.

84

Determination of ACE inhibitory activities. ACE inhibitory activity was evaluated by

85

determining the amount of hippuric acid (HA) generated from HHL using a previously described

86

method with a few modifications.7 ACE was dissolved into the 100 mM borate buffer (pH 8.3)

87

containing 300 mM NaCl at the concentrations of 0.10 U/mL, and the substrate HHL was also

88

dissolved into the same buffer at the concentration of 5.40 mM. 20 μL of ACE solution was mixed

89

with 140 μL of sample/blank buffer for 10 min at 37 °C bath, and 40 μL of HHL solution was then

90

added and incubated for 15 min at 37 °C. At last, the reaction was terminated by adding 150 μL of 1

91

M HCl. 20 μL of the reaction solution was injected into HPLC (Agilent 1260) equipped with Zorbax

92

SB C18 column (4.6 mm × 150 mm, 5 μm, Agilent, USA) to detect the content of HA . The IC50 5



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value of the sample was defined as the concentration when 50% ACE was inhibited under the same

94

conditions.

95

Preparation of the papain hydrolysate and ultrafiltration. WEP (equivalent to 20 g of protein)

96

was mixed with 1 L of ultrapure water, and papain was added to begin the hydrolysis at an

97

enzyme/substrate ratio of 2:100 (w/w). The reaction was conducted at 37 °C and pH 7.0 for 7 h. The

98

hydrolysates (WEPH) were filtered through a 0.45 μm filtrate membrane (Shanghai Xingya Purifying

99

Materials Factory, China), and the filtrate was fractioned by ultrafiltration (UF) using a Labscale

100

system (Labscale TFF System, Millipore Co., Billerica, MA, USA) with molecular weight cut-off

101

(MWCO) membranes of 10 KDa (Pelllicon XL Biomax 10) to obtain the retentate (WEPH-I, Mw >

102

10 KDa) and the permeate (WEPH-II, Mw < 10 KDa). WEPH, WEPH-I and WEPH-II were

103

lyophilized and used for further study.

104

Purification of ACEI peptides. WEPH-II was loaded onto a Sephadex G-15 (Pharmacia Fine

105

Chemicals, Uppsala, Sweden) column (Φ 2 cm × 40 cm) pre-equilibrated with ultrapure water. After

106

the sample was loaded, the column was eluted with ultrapure water for 270 min at a flow rate of 0.5

107

mL/min, and the eluted solution was monitored at 280 nm. Each fraction was collected, reduced, and

108

lyophilized to investigate the ACE inhibitory activity. The fraction showing the strongest activity

109

against ACE was further purified using strong anion exchange chromatography HiPrep Q FF 16/10

110

(16 × 100 mm; GE Healthcare, Buckinghamshire, UK) coupled with an ÄKTA purifier system (GE

111

Healthcare) at 20 °C. The sample was dissolved into buffer A (10 mM pH 8.3 borate buffer) at 5

112

mg/mL, and 2 mL was loaded onto the column, which was equilibrated with 5 column volumes (CV)

113

of buffer A previously. The flow rate was set at 2 mL/min. The elution condition was as follows: 0–

114

85 min: 0% to 20% of buffer B (10 mM pH 8.3 borate buffer containing 1 M NaCl); 85–100 min:

115

20%–100% of buffer B; and 100–115 min: 100% of buffer B. The chromatograms were monitored at 6


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214 nm. The fractions of each major peak were collected, reduced, and lyophilized for the analysis

117

of ACE inhibitory activities.

118

The active fractions were further separated by semi-preparative RP-HPLC on a YMC-Pack ODS-

119

A column (10 × 250 mm, 5 μm, YMC Co., Japan) under the following chromatographic conditions:

120

flow rate, 1.8 mL/min; UV detector, λ 214 nm; mobile phase, 0.1% trifluoroacetic acid (TFA) in

121

acetonitrile (solvent A) and 0.1% TFA in water (solvent B), gradient elution from 0% to 50% A for

122

150 min. Two active fractions were further subjected repeatedly chromatographic runs by analytical

123

ODS-A column (4.6 × 250 mm, 5 μm (YMC); flow rate, 1 mL/min; detection and eluent, as above).

124

After removing the solvent under reduced pressure and freeze-drying, the samples were stored at

125

-20 °C before further analysis.

126

Characterization of ACEI peptides. The molecular mass and amino acid sequences of the

127

purified peptides were determined using a 4800 plus MALDI-TOF/TOF Analyzer (Applied

128

Biosystems, Beverly, MA, USA). The sample was mixed with matrix solution (a-cyano-4-

129

hydroxycinnamic acid solution) and dried on a conventional steel matrix-assisted laser

130

desorption/ionization target. The sample was ionized at 337 nm and operated in the positive ion

131

delayed extraction reflector mode. Spectra were recorded over the mass/charge (m/z) range of 0–1500

132

Da. Tandem mass spectrometry (MS) experiments were conducted by collision-induced dissociation,

133

and sequence information was obtained by tandem MS analysis.

134

Determination of inhibitory pattern of ACEI peptides. The ACE inhibitory kinetics of the active

135

peptides were investigated at different concentrations of HHL (0.25, 0.50, 1.00, and 2.00 mM), and

136

the ACE activities were measured in the absence and presence of ACEI peptides.13 The inhibitory

137

pattern was determined by the Lineweaver–Burk plot of the reciprocal of the production speed of HA

138

(y-axis) versus the reciprocal of the substrate concentration (x-axis). 7



139

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Simulated gastrointestinal digestion of ACEI peptides. To evaluate the stability of ACEI

140

peptides,

141

(http://web.expasy.org/peptide_cutter/) was used to evaluate the potential cleavage sites of the

142

gastrointestinal (GI) tract enzymes in the ACEI peptides. The pepsin (pH 1.3 and pH > 2.0), trypsin

143

and chymotrypsin (high and low specificity) were chosen to predict the peptides stability.

144 145

in

silico

analysis

was

performed.

The

program

Expasy

Peptide

Cutter

In silico prediction of toxicity of ACEI peptides. The ACEI peptides were assessed for potential toxicity in silico using ToxinPred, available at http://crdd.osdd.net/raghava//toxinpred/design.php.

146

Isothermal Titration Calorimetry (ITC) analysis of ACEI peptides. To measure the

147

thermodynamic parameters (enthalpy change ΔH, entropy change ΔS, the association constant Ka, the

148

dissociation constant Kd, and binding stoichiometry n) of the interactions between the active peptides

149

and ACE, ITC experiments were performed on the Nano ITC Low Volume (TA Instrument, USA) at

150

25 °C. All solutions were degassed thoroughly to avoid the bubble. Both the active peptides and ACE

151

were prepared in 50 mM N-[(2-amino-2-oxoethyl) amino] ethanesulfonic acid buffer solution (pH

152

7.0, containing 300 mM NaCl) (ACES). The reference cell was filled with the blank buffer solution,

153

and the sample cell was injected with 4.89 nM ACE solution (1.97 U/mg). The peptides concentration

154

in the syringe was 30.0 mM for KRER and 1.2 mM for LHMFK, respectively. While achieving the

155

baseline stability, the peptide solution was titrated into ACE via 20 individual injections, the volume

156

of each injection was 2.5 µL with a 300 s interval, and the stirring speed was fixed at 250 rpm. A plot

157

of the corrected heat rate (µJ/s) against time (s) was achieved after subtracting the dilution heat in the

158

blank experiment by using the Nano Analyze Software (TA Instrument, New Castle, DE 19720, USA).

159

The independent binding model was used to analyze the thermodynamic parameters of the binding

160

action.

161

Statistical analysis. All assays were conducted in triplicate. Data were presented as the mean ± 8


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standard deviation. Statistical analysis was performed using ANOVA one-way analysis by SPSS 19.0

163

Statistics software (IBM SPSS, Armonk, NY) with post-hoc least significant difference (LSD)

164

analysis. Significant difference was taken into account when p < 0.05.

165

RESULTS AND DISCUSSION

166

The screening of the proteases for Carapax Trionycis. Six proteases were used in the hydrolysis

167

of Carapax Trionycis protein. The IC50 values of the hydrolysates from papain, neutral protease,

168

trypsin, bromelain, alcalase, pepsin and the WEP were 0.99 ± 0.06 (a), 1.27 ± 0.08 (a), 1.30 ± 0.09

169

(a), 1.34 ± 0.11 (a), 1.35 ± 0.09 (a), 7.11 ± 0.57 (b), and 7.94 ± 0.38 (c) mg/mL respectively. The

170

papain hydrolysate possessed the lowest IC50 value, even if no significant differences were observed

171

among the neutral protease, trypsin, bromelain, and alcalase digestion (Data with the same letter

172

indicated that the differences were not significant) (p > 0.05). The results imply that Carapax

173

Trionycis protein was cleaved at different sites during hydrolysis by different enzymes, producing

174

different peptides with varying ACE inhibitory activities. The broad cleavage site of papain, which

175

originates from Carica papaya, shows a preference for Lys–Arg and Phe–X–COOH at the terminal

176

amino acid, whereas the basic amino acids at C-terminal is one of the preferred characteristics for

177

ACEI peptides.20, 21 The result demonstrated the effectiveness of papain for generating the ACE

178

inhibitory hydrolysates from Carapax Trionycis, which was in agreement with the screening results

179

from threadfin bream frames, bovine fibrinogen, and bovine brisket.21–23 Owing to the utilization as

180

a food-grade enzyme and a tenderizer in meat industry, papain was finally chosen for the protein

181

digestion of Carapax Trionycis.22, 23

182

Purification of ACEI peptides. MW is an important factor for ACEI peptides, and UF with

183

different MWCO membranes has always been used in the first step of purification to enrich the low-

184

molecular active fractions.5, 6, 12 WEPH were fractioned using an UF system with 10 KDa MWCO. 9



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The ACE inhibitory activities of the fractions WEPH, WEPH-I (MW > 10 KDa), and WEPH-II (MW

186

< 10 KDa) were assayed. The results showed that WEPH-II with an IC50 value of 0.72 ± 0.03 (a)

187

mg/mL presented significantly stronger inhibitory activity than WEPH and WEPH-I with IC50 values

188

of 0.99 ±0.06 (b) and 1.23 ±0.06 (c) mg/mL, respectively (p < 0.05).

189

WEPH-II was subsequently applied to Sephadex G-15 column chromatography, and three primary

190

fractions (A, B, and C) were collected and lyophilized. Fraction A exhibited the strongest ACE

191

inhibitory activity with IC50 value of 0.46 ± 0.06 mg/mL (a), whereas fractions B and C showed

192

activities with IC50 values of 0.68 ± 0.07 (b) and 0.61 ± 0.05 (b) mg/mL, respectively (p < 0.05).

193

Fraction A, which was consecutively separated using anion exchange column chromatography

194

(Hiprep Q FF 16/10), was divided into five main fractions (A1–A5) (Figure 1). The A2 fraction

195

showed the lowest IC50 value of 0.17 ±0.04 mg/mL, compared with the fractions A1 and A3–A5 with

196

IC50 values of 0.34 ±0.05, 0.64 ±0.10, 0.50 ±0.07 and 0.94 ±0.12 mg/mL, respectively (p < 0.05).

197

The A2 fraction was loaded onto semi-preparative HPLC with ODS-A column for further

198

purification. Fractions A21–A28 were collected respectively, and the ACE inhibitory activities of

199

fractions A21 and A22 were both higher than 85% at the concentration of 0.30 mg/mL, and the IC50

200

value of fraction A22 (0.05 ± 0.01 mg/mL) was the lowest among them (the IC50 values of fractions

201

A21, and A23–A28 were 0.19 ±0.02, 0.24 ±0.05, 0.27 ±0.04, 0.28 ±0.04, 0.59 ±0.08, 0.64 ±0.06,

202

and 0.48 ± 0.09 mg/mL, respectively) (p < 0.05) (Figure 2). Both of A21 and A22 were further

203

purified on analytical HPLC with ODS-A column and fractions A21-1 and A22-1 were obtained

204

whose IC50 values were determined as 0.19 ±0.00 and 0.05 ±0.00 mg/mL. The ACEI peptides from

205

papain hydrolysates of Carapax Trionycis were purified 5-fold for fraction A21-1 and 20-fold for

206

fraction A22-1 by using a five-step purification procedure. Consecutive chromatography methods

207

including ion-exchange chromatography, gel-filtration chromatography, and HPLC have been usually 10


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208

adopted to purify the ACEI peptides, and the purification fold ranged between 15–30 folds, like 15-

209

fold for tilapia hydrolysates, 19-fold for hydrolysates of marine sponge Stylotella aurantium, and 24-

210

fold for hydrolysate of duck skin.6, 24–26

211

Identification of ACEI peptides by MALDI-TOF/TOF MS. Peptide sequences of fractions A21-

212

1 and A22-1 were identified by MALDI-TOF/TOF Analyzer (Figure 3). The molecular weight of

213

fraction A21-1 was 587.4 Da (588.3652 [M + H]+), and the amino acid sequence was identified as

214

KRER with IC50 value of 324.1 ± 2.5 μM. The molecular weight of fraction A22-1 was 674.3 Da

215

(675.3256 [M + H]+), and the amino acid sequence was identified as LHMFK with IC50 value of 75.6

216

±1.5 μM. Peptides with the same sequences were synthesized (98% purity) by Shanghai GL Biochem.

217

Ltd., China, and their ACE inhibitory activities were investigated. The IC50 values of synthetic KRER

218

and LHMFK were 328.1 ±3.5 and 72.9 ±1.3 μM, respectively. Some ACE inhibitory peptides derived

219

from Pelodiscus sinensis egg have been reported. The peptides were IVR, IVRDPNGMGAW,

220

DPNGMGAW AKLPSW and ILTKQDPYHGPF with the IC50 values of 0.8 μM, 4.4 μM, 14.4 μM,

221

15.3 μM, and 231.7 μM, respectively, which differ greatly from the two novel peptides KRER and

222

LHMFK.12–14 This may be due to the differences in protein sources and digestive enzymes. Moreover,

223

the IC50 values of two novel peptides were within 0.3–1500.0 μM, which is the rang of IC50 values of

224

ACE inhibitory peptides derived from foodstuffs.24 Thus, KRER and LHMFK have the prospects for

225

application in preventing hypertension and for therapeutic purposes.

226

To our knowledge, these two peptides have never been reported to be ACEI peptides before in the

227

literature by searching in different peptides databases, including AHTPDB, BIOPEP and EROP-

228

Moscow (last search on March 26th, 2018).

229

Structure-activity correlations of ACEI peptides. One of the hydrophobicity trends of the 20

230

amino acids was as follows: F > L = I > W = Y > V > M > P > C > A > G > T > S > K > Q > N > H > 11



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E > D > R.9 Most of the ACEI peptides from nature are composed of 2–20 amino acid residues and

232

possess hydrophobic amino acids residues at the C-terminal, however, the active peptides in this work

233

both possessed hydrophilic amino acids residues at the C-terminal. The positive charge of the amino

234

acids R and K are the relative factors contributing ACE inhibitory activities.27

235

Table 2 shows part of the purified ACEI peptides possessing similar C-terminals to KRER and

236

LHMFK. Log P values of the peptides, the logarithm (base 10) of the oil-water partition coefficient

237

(P), which can be calculated in silico by ACE/ChemSketch (Freeware 2017.2.1), have also been listed

238

out. Obviously, for the ACEI peptides purified in this work, Log P is correlated with the inhibitory

239

activity. LHMFK (1.45) assessing the hydrophobic character showed much stronger inhibitory

240

activity against ACE than KRER (-4.37) possessing the hydrophilic character. But the Log P value is

241

not always proportional to ACE inhibitory activity, e.g. the IC50 value of APER (-2.72), ER (-2.38)

242

and PER (-2.66) shown in Table 2 are all higher than KRER, and the ACE inhibitory activity of FK

243

(0.28) is stronger than VFK (1.36). Besides, the IC50 values of LHMFK, FK, and VFK were in a wide

244

range despite possessing the same dipeptidyl sequence, and the length of the peptides may be one of

245

the main reasons for the differences in ACE inhibitory activities of them.

246

Determination of inhibitory pattern of ACEI peptides. The inhibition patterns of KRER and

247

LHMFK against ACE were estimated by Lineweaver–Burk plot analysis. Both peptides exhibited

248

noncompetitive inhibition pattern, as shown in Figure 4. A great many of ACEI peptides purified

249

from food origin acted in noncompetitive manner. The peptide AKLPSW from SST yolk exhibited a

250

noncompetitive pattern towards ACE, while the other two active peptides IVRDPNGMGAW and

251

IVR from egg white exhibited a competitive pattern.12–14

252

The Vmax values decreased in a dose-dependent pattern for KRER and LHMFK, whereas less

253

change was observed in the Km value, as shown in Table S1 of Supporting Information. According to 12


Page 13 of 32


254

the Michaelis–Menten model, a noncompetitive inhibitor is marked by the Vmax value but exerts a

255

minimum effect on Km. Noncompetitive inhibitors interact with the non-catalytic site of the enzyme

256

and combine with the enzyme to produce an enzyme–inhibitor complex regardless of whether the

257

substrate is bound.28 The Ki value of KRER (1.11 mM) is about 9 times of LHMFK (0.12 mM),

258

indicating the stronger affinity of LHMFK to ACE, which is in line with the better inhibitory activity

259

of LHMFK.

260

Simulated GI tract digestion of ACEI peptides. Some of the ACEI peptides fail to show the

261

hypotensive activity after oral administration in vivo, and the stability against GI enzymes needs

262

evaluation. The stability of ACEI peptides during a simulated GI digestion were predicted by using

263

Peptide Cutter, a useful and efficient tool for predicting potential cleavage sites of the proteases in

264

given peptides in silico22, 28. The peptide KRER would be cleaved by trypsin into the di-peptide ER,

265

while the peptide LHMFK would be cleaved into tetra-peptide LHMF by chymotrypsin (high

266

specificity), and di-peptides HM and FK by chymotrypsin (low specificity) and pepsin.

267

As shown in Table 2, the IC50 values of ER and FK are 667.0 μM and 265.4 μM, both of which are

268

larger than the peptides KRER and LHMFK, respectively. The degradation of the peptides may lead

269

to the decreased ACE inhibitory activities, but the in vivo studies of the stability of the peptides in

270

animal model are required to certificate the judgement. The resistance of the peptides against the

271

digestive proteases is fatal for the development as food ingredients, for the weakened activity in vivo

272

may interfere the bioavailability of the peptides.

273

Determination of toxicity of ACEI peptides using in silico method. In recent decades, peptides

274

have been developed as potential therapeutic molecules against many diseases, while toxicity has

275

become one of the bottlenecks in the development of peptide-based functional food/drugs. ToxinPred

276

has been proved to be a useful in silico method to predict toxicity of peptides/proteins.29 KRER, 13



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LHMFK and their possible GI digestive products ER, LHMF and FK were analyzed to estimate their

278

potential toxicity by using ToxinPred. The support vector machine (SVM) score of them were -0.71,

279

-0.82, -0.80, -0.81, and -0.80, respectively, where the negative value reflects nontoxic.30 The results

280

suggested that the two peptides had the potential to be used as a potent natural ingredient for the

281

manufacture of functional foods or pharmaceuticals against for hypertension treatment.

282

Binding of the active peptides to ACE characterized by ITC. ITC method has been thoroughly

283

used to study the binding mechanism between some inhibitors and ACE.31, 32 The ITC diagrams of

284

the active peptides KRER and LHMFK binding to ACE were shown in Figure 5. Both of the reaction

285

equilibrium was achieved at the 10th injection. The fitting curve of the integrated injection heat versus

286

the injection number is like a typical “S” curve and has been fitted to the independent binding model

287

which means that there existed a set of identical peptide binding sites on ACE (Other binding models

288

including multiple sites, dimer dissociation, and cooperative have also been explored, but the

289

independent model has the best fit).33

290

The upward ITC titration peaks indicated that the interaction between the active peptides and ACE

291

was an endothermic reaction. The thermodynamic parameters from the ITC experiments were all

292

summarized in Table 3. The ΔG value of KRER are mainly contributed by -TΔS, indicating that the

293

reaction was mainly driven by entropy. For the active peptide LHMFK, the ΔH and -TΔS values are

294

almost the same, indicating that the reaction was both enthalpically and entropically driven. The

295

positive entropy change (ΔS) can be explained by a strong hydrophobic effect, which means that the

296

interactions between apolar groups from inhibitors and ACE requiring the dehydration.34 KRER

297

possessing strong hydrophilic character cannot interact with the active site of ACE fluently. For

298

forming the stable complex with ACE, the interfacial water is transferred to the bulk solvent, and the

299

entropy change is obtained. However, the hydrophobic groups of KRER is hard to interact with ACE, 14


Page 15 of 32


300

proved by the phenomenon that the corrected heat value was too little to detect at the low

301

concentration of KRER in the syringe (data not shown). Only by increasing the concentration of

302

KRER, the typical ITC profile could be obtained. LHMFK, possessing some typical characters of

303

ACEI peptides, may interact with ACE by hydrophobic effect, electrostatic interactions and some

304

other actions derived from conformation change which need further exploring.

305

The dissociation constant Kd values can reflect the binding affinity of the active peptides and

306

ACE.31 The Kd value of KRER is about 8 times of LHMFK, which means that LHMFK could form

307

more stable complex with ACE. This is well accordance with the Ki value derived from enzyme

308

kinetics study, also showing the stronger affinity of LHMFK to ACE.

309

In summary, this is the first investigation of the ACE inhibitory activities of the papain hydrolysates

310

from Carapax Trionycis. Two novel noncompetitive inhibitory peptides KRER and LHMFK were

311

purified successively by sequential chromatographic separation. Both enzyme kinetics and ITC assay

312

confirmed that LHMFK could bind more tightly to ACE. Combining enzyme kinetics and ITC

313

experiments in present work to study the interactions mechanism between the inhibitor and the

314

enzyme open up a new way for elucidating the in vitro inhibitory mechanism. More comprehensive

315

and thorough research on the interaction mechanism is in progress and will be reported. In silico study

316

predicted that the peptides in this work were not stable against the digestive enzymes. However, in

317

silico study is not enough to prove the stability and effectiveness of the peptides in vivo, and the

318

further study investigating the bioavailability of the peptides after ingestion in animal models is

319

required. The results obtained in this work demonstrated the potential of Carapax Trionycis for

320

generation of ACEI peptides, and have broaden up its application in nutraceuticals against

321

hypertention and other related diseases.

322

Supporting information 15



323

Kinetics Constants of ACE catalyzed reaction at different concentrations of the active peptides.

324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 16


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REFERENCES

347

(1) Orio, L. P.; Boschin, G.; Recca, T.; Morelli, C. F.; Ragona, L.; Francescato, P.; Arnoldi, A.;

348

Speranza, G. New ACE-inhibitory peptides from Hemp seed (Cannabis sativa L.) proteins. J. Agric.

349

Food Chem. 2017, 65, 10482–10488.

350

(2) Vercruysse, L.; Camp, J. V.; Smagghe, G. ACE inhibitory peptides derived from enzymatic

351

hydrolysates of animal muscle protein: A review. J. Agric. Food Chem. 2005, 53, 8106–8115.

352

(3) Lee, S. Y.; Hur, S. J. Antihypertensive peptides from animal products, marine organisms, and

353

plants. Food Chem. 2017, 228, 506–517.

354

(4) Di Bernardini, R.; Harnedy, P.; Bolton, D.; Kerry, J.; O’Neill, E.; Mullen, A. M.; Hayes, M.

355

Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products.

356

Food Chem. 2011, 124, 1296–1307.

357

(5) Salampessy, J.; Reddy, N.; Kailasapathy, K.; Phillips, M. Functional and potential therapeutic

358

ACE-inhibitory peptides derived from bromelain hydrolysis of trevally proteins. J. Funct. Foods

359

2015, 14, 716–725.

360

(6) Toopcham, T.; Roytrakul, S.; Yongsawatdigul, J. Characterization and identification of

361

angiotensin I-converting enzyme (ACE) inhibitory peptides derived from tilapia using Virgibacillus

362

halodenitrificans SK1-3-7 proteinases. J. Funct. Foods 2015, 14, 435–444.

363

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

364

characterization of angiotensin converting enzyme inhibitory peptides from lizard fish protein

365

hydrolysates with magnetic affinity separation. Food Chem. 2015, 182, 136–142.

366

(8) Wiriyaphan, C.; Chitsomboon, B.; Yongsawadigul, J. Antioxidant activity of protein hydrolysates

367

derived from threadfin bream surimi byproducts. Food Chem. 2012, 132, 104–111.

368

(9) Hayes, M.; Stanton,C.; Slattery, H.; O’Sullivan, O.; Hill, C.; Fitzgerald, G. F.; Ross, R. P. Casein 17



Page 18 of 32

369

fermentate of Lactobacillus animalis DPC6134 contains a range of novel propeptide angiotensin-

370

converting enzyme inhibitors. Appl. Environ. Microbiol. 2007, 73, 4658–4667.

371

(10) Feng, H.; Matsuki, N.; Saitot, H. Attenuation of the increase in blood pressure and vascular

372

reactivity by chronic oral administration of Soft-shelled Turtle powder in spontaneously hypertensive

373

rats. Phytother. Res. 1990, 4, 57–61.

374

(11) Chiu, L. H.; Hsu, G. S. W.; Lu, Y. F. Antihypertensive capacity of defatted soft-shelled turtle

375

powder after hydrolysis by gastrointestinal enzymes. J. Food Biochem. 2006, 30, 589–603.

376

(12) Rawendra, R. D. S.; Aisha Chang, C. I.; Aulanni’am; Chen, H. H.; Huang, T. C.; Hsu, J. L. A

377

novel angiotensin converting enzyme inhibitory peptide derived from proteolytic digest of Chinese

378

soft-shelled turtle egg white proteins. J. Proteomics 2013, 94, 359–369.

379

(13) Rawendra, R. D. S.; Aisha; Chen, S. H.; Chang, C. I.; Shih, W. L.; Huang, T. C.; Liao, M. H.;

380

Hsu, J. L. Isolation and characterization of a novel angiotensin-converting enzyme-inhibitory

381

tripeptide from enzymatic hydrolysis of soft-shelled turtle (Pelodiscus sinensis) egg white: In vitro,

382

in vivo, and in silico study. J. Agric. Food Chem. 2014, 62, 12178–12185.

383

(14) Pujiastuti, D. Y.; Shih, Y. H.; Chen, W. L.; Sukoso; Hsu, J. L. Screening of angiotensin-I

384

converting enzyme inhibitory peptides derived from soft-shelled turtle yolk using two orthogonal

385

bioassay-guided fractionations. J. Funct. Foods 2017, 28, 36–47.

386

(15) Liao, P. Y.; Zhou, L. Q.; Liao, D. K.; Sun, L. X.; Sun, J. H.; Wei, J. H. Determination of the

387

amino acids in the raw and processed products of Turtle shell by HPLC combining pre-column

388

derivatization. Zhongyaocai 2016, 39, 802–805.

389

(16) Wang, P.; Zhang, Y.; An, Y.; Xu, K.; Xu, X.; Fu, C.; Lin, J. Protection of a new heptapeptide

390

from Carapax trionycis against carbon tetrachloride-induced acute liver injury in mice. Chem. Pharm.

391

Bull. 2013, 61, 1130–1135. 18


Page 19 of 32


392

(17) Tang, Y.; Hu, C.; Liu, Y. Effect of bioactive peptide of Carapax Trionycis on TGF- β1 -induced

393

intracellular events in hepatic stellate cells. J. Ethnopharmacol. 2013, 148, 69–73.

394

(18) Hu, C. L.; Peng, X. Z.; Tang, Y. P.; Liu, Y. W.; Hu, Z. L.; Gao, J. R. Purification, characterization,

395

in vitro anti-hepatic fibrosis activity of bioactive peptides derived from Carapax Trionycis

396

hydrolysates. J. Chin. Pharm. Sci. 2017, 26, 605–610.

397

(19) AOAC. Official Methods of Analysis, 17th ed.; Association of Official Analytical Chemists:

398

Gaithersberg, Maryland, 2000.

399

(20) Zarei, M.; Forghani, B.; Ebrahimpour, A.; Abdul-Hamid, A.; Anwar, F.; Saari, N. In vitro and in

400

vivo antihypertensive activity of palm kernel cake protein hydrolysates: sequencing and

401

characterization of potent bioactive. Ind. Crops Prod. 2015, 76, 112–120.

402

(21) Gajanan, P. G.; Elavarasan, K. Bioactive and functional properties of protein hydrolysates from

403

fish frame processing waste using plant proteases. Environ. Sci. Pollut. Res. 2016, 23, 24901–24911.

404

(22) Lafarga, T.; Rai, D. K.; O’Connor, P.; Hayes, M. A bovine fibrinogen-enriched fraction as a

405

source of peptides with in vitro renin and angiotensin-I-converting enzyme inhibitory

406

activities. J. Agric. Food Chem. 2015, 63: 8676–8684.

407

(23) Di Bernardini, R.; Maria Mullen, A.; Bolton, D.; Kerry, J.; O'Neill, E.; Hayes, M. Assessment

408

of the angiotensin-I-converting enzyme (ACE-I) inhibitory and antioxidant activities of hydrolysates

409

of bovine brisket sarcoplasmic proteins produced by papain and characterisation of associated

410

bioactive peptidic fractions. Meat Sci., 2012, 90, 226–235.

411

(24) Puchalska, P.; Alegre, M. L. M.; López, M. C. G. Isolation and characterization of peptides with

412

antihypertensive activity in foodstuffs. Crit. Rev. Food Sci. Nutr. 2015, 55, 521–551.

413

(25) Ko, S. C.; Jang, J.; Ye, B. R.; Kim, M. S.; Choi, I. W.; Park, W. S.; Heo, S. J.; Jung, W. K.;

414

Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory 19



Page 20 of 32

415

peptides from hydrolysates of marine sponge Stylotella aurantium. Process Biochem. 2017, 54, 180–

416

187.

417

(26) Lee, S. J.; Kim, Y. S.; Kim, S. E.; Kim, E. K.; Hwang, J. W.; Park, T. K.; Kim, B. K.; Moon, S.

418

H.; Jeon, B. T.; Jeon, Y. J.; Ahn, C. B.; Je, J. Y.; Park, P. J. Purification and characterization of a

419

novel angiotensin I-converting enzyme inhibitory peptide derived from an enzymatic hydrolysate of

420

duck skin byproducts. J. Agric. Food Chem. 2012, 60, 10035–10040.

421

(27) Li, Y.; Yu, J. Research progress in structure-activity relationship of bioactive peptides, J. Med.

422

Food 2015, 18, 147–156.

423

(28) Lin, K.; Zhang, L. W.; Han, X.; Cheng, D. Y. Novel angiotensin I-converting enzyme inhibitory

424

peptides from protease hydrolysates of Qula casein : Quantitative structure-activity relationship

425

modeling and molecular docking study. J. Funct. Foods 2017, 32, 266–277.

426

(29) Lafarga, T.; Wilm, M.; Wynne, K.; Hayes, M. Bioactive hydrolysates from bovine blood

427

globulins: Generation, characterisation, and in silico prediction of toxicity and allergenicity. J. Funct.

428

Foods. 2016, 24: 142–155.

429

(30) Lin, K.; Zhang, L. W.; Han, X.; Meng, Z. X.; Zhang, J. M.; Wu, Y. F.; Cheng, D. Y.

430

Quantitative structure-activity relationship modeling coupled with molecular docking analysis in

431

screening of angiotensin I-converting enzyme inhibitory peptides from Qula casein hydrolysates

432

obtained by two-enzyme combination hydrolysis. J. Agric. Food Chem. 2018, 66: 3221–3228.

433

(31) Xie, J. L.; Chen, X. J.; Wu, J. J.; Zhang, Y. Y.; Zhou, Y.; Zhang, L. J.; Tang, Y. J.; Wei, D. Z.

434

Antihypertensive effects, molecular docking Study, and isothermal titration calorimetry assay of

435

angiotensin I-converting enzyme inhibitory peptides from Chlorella vulgaris. J. Agric. Food Chem.

436

2018, 66, 1359–1368.

437

(32) Andujar-Sanchez, M.; Ca ́mara-Artigas, A.; Jara-Perez, V. A calorimetric study of the binding 20


Page 21 of 32


438

of lisinopril, enalaprilat and captopril to angiotensin-converting enzyme. Biophys. Chem. 2004, 111,

439

183–189.

440

(33) Guo, J.; Catchmark, J. M.; Mohamed, M. N. A.; Benesi, A. J.; Tien, M.; Kao, T.; Watts, H. D.;

441

Kubicki, J. D. Identification and characterization of a cellulose binding heptapeptide revealed by

442

phage display. Biomacromolecules 2013, 14, 1795–1805.

443

(34) Tellez-Sanz, R.; Garcia-Fuentes, L.; Baron, C. Calorimetric analysis of lisinopril binding to

444

angiotensin I-converting enzyme. FEBS Letters 1998, 423, 75–80.

445

(35) Escudero, E.; Sentandreu, M. A.; Arihara, K.; Toldrá, F. Angiotensin I-Converting enzyme

446

inhibitory peptides generated from in vitro gastrointestinal digestion of pork meat. J. Agric. Food

447

Chem. 2010, 58, 2895–2901.

448

(36) Sagardia, I.; Roa-Ureta, R. H.; Bald, C. A new QSAR model, for angiotensin I-converting

449

enzyme inhibitory oligopeptides. Food Chem. 2013, 136, 1370–1376.

450

(37) Kodera, T.; Nio, N. Identification of an Angiotensin I-converting enzyme inhibitory peptides

451

from protein hydrolysates by a soybean protease and the antihypertensive effects of hydrolysates in

452

spontaneously hypertensive model rats, J. Food Sci. 2006, 71, 164–173.

453

(38) Pihlanto, A.; Mäkinen, S. Antihypertensive properties of plant protein derived peptides. In

454

Bioactive food peptides in health and disease. Hernandez-Ledesma B., Hsieh C. C. Eds.; Intechopen

455

Limited: London: England, 2013; 149.

456

(39) Wu, J.; Aluko, R. E.; Nakai, S. Structural requirements of angiotensin I-converting enzyme

457

inhibitory peptides: quantitative structure-activity relationship study of Di- and tripeptides. J. Agric.

458

Food Chem. 2006, 54, 732–738.

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Funding

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This work was supported by National Natural Science Foundation of China (51372043), Natural

463

Science Foundation of Guangxi Province (2017GXNSFDA198052, 2017GXNSFBA198215,

464

2017GXNSFAA198289) and Project of Guangxi Department of Education (KY2016YB206,

465

KY2016YB035).

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Figure captions: Figure 1. Chromatography of fraction A on Hiprep Q FF 16/10 and the ACE inhibitory activities of the fractions A1–A5 (upper panel). Different letters (a–d) above the bar represents the significant differences (p < 0.05). Figure 2. Chromatography of the fraction A2 by using semi-prep HPLC on ODS-A column and the ACE inhibitory activities of fractions A21–A28 (upper panel). Different letters (a–d) above the bar represents the significant differences (p < 0.05). Figure 3. Characterization of molecular mass and amino acid sequences of the purified peptides from Carapax Trionycis hydrolysate: (a) MS/MS spectrum of molecular ion m/z 587.4 Da of fraction A211 and (b) m/z 674.3 Da of fraction A22-1. Figure 4. Lineweaver–Burk plot for the determination of the inhibitory pattern of the active peptides (a) KRER and (b) LHMFK purified from Carapax Trionycis hydrolysate. The ACE activities were measured in the absence (control) or presence of the purified peptides. ●, absence of the active peptides; ▲, 256 μM for KRER and 148 μM for LHMFK; ■, 1022 μM for KRER and 296 μM for LHMFK. 1/V and 1/S represent the reciprocals of velocity and substrate, respectively. Figure 5. ITC diagram of ACEI peptides binding to ACE. (a) KRER; (b) LHMFK. The upper panel shows the corrected heat data of calorimetric titration of the ACEI peptides binding to ACE, and the lower panel shows the integrated injection heat from the upper panel. The integrated heat data were fit to an independent model.

23



Table 1. Optimum Conditions of Enzymatic Hydrolysis for Various Enzymes enzyme pH 7.0 7.0 8.0 6.0 9.5 2.0

papain neutral protease trypsin bromelain alcalase pepsin

24


optimum conditions temperature (°C) 37.0 50.0 37.0 45.0 45.0 37.0

Page 24 of 32

Page 25 of 32


Table 2. IC50 Values of ACEI Peptides from Various Sources sources antarctic krill (Euphausia superba)24 this study bromelain digest of trevally proteins5 pepsin and pancreatin digest of pork meat35 skeletal muscle gated chloride channel35 soybean treated by protease from Bacillus subtilis36 soybean protein hydrolysate by protease D337 this study trypsin digest of sweet-potato tuber defensin38 milk39

sequences of the ACEI peptides VFER KRER APER ER PER PGTAVFK YVVFK LHMFK FK VFK

25


IC50 (μM)

Log P

152.8 324.1 530.2 667.0 > 1000.0 26.5 44.0 75.6 265.4 1029.0

-0.19 -4.37 -2.72 -2.38 -2.66 -0.31 2.76 1.45 0.28 1.36


peptides KRER LHMFK

Page 26 of 32

Table 3. Thermodynamic Parametres for ACEI Peptides Binding to ACE Ka (1/µM) Kd (µM) -TΔS (KJ/mol) ΔH (KJ/mol) ΔG (KJ/mol) 0.05 19.36 -24.34 -2.55 -26.89 0.41 2.42 -15.88 -15.63 -31.51

26


Page 27 of 32


Figure 1.

27



Figure 2.

28


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Page 29 of 32


Figure 3.

29



Figure 4.

30


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Page 31 of 32


Figure 5.

31



Table of Contents Graphic (TOC)

32


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Isolation and Characterization of Angiotensin I-Converting Enzyme

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