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The structure and activity changes of phytohemagglutinin from red kidney bean (Phaseolus vulgaris) affected by ultra-high pressure treatments Yunjun Lu, Cencen Liu, MouMing Zhao, Chun Cui, and Jiaoyan Ren J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Sep 2015 Downloaded from http://pubs.acs.org on October 17, 2015

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Journal of Agricultural and Food Chemistry

23

ABSTRACT

24

Phytohaemagglutin (PHA) purified from red kidney beans (Phaseolus vulgaris)

25

by Affi-gel blue affinity chromatography, was subjected to ultra-high pressure (UHP)

26

treatment (150, 250, 350, 450 MPa). The purified PHA lost its haemagglutination

27

activity after 450 MPa treatment, and showed less pressure tolerance than crude PHA.

28

However, the saccharide specificity and α-glucosidase inhibition activity of the

29

purified PHA did not change much after UHP treatment. Electrophoresis staining by

30

perodic acid-Schif (PAS) manifested that the glycone structure of purified PHA

31

remained stable even after 450 MPa pressure treatment. However, electrophoresis

32

staining by Coomassie blue, as well as circular dichroism (CD) and differential

33

scanning calorimetry (DSC) assay, proved that the protein unit structure of purified

34

PHA unfolded when treated at 0-250 MPa, but re-aggregates at 250-450 MPa.

35

Therefore, the haemagglutination activity tends to be affected by protein unit structure,

36

while the stability of glycone structure contributed to the remaining of α-glucosidase

37

inhibition activity.

38

KEYWORDS

39

Ultra-high

40

pressure

treatment;

Phytohemagglutinin;

Haemagglutination; α-glucosidase inhibition

41 42 43

2

ACS Paragon Plus Environment

Red

kidney

bean;


44

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INTRODUCTION

45

Lectins represent a group of carbohydrate-binding proteins that agglutinate cells

46

or precipitate glycoconjugates.1 Phytohaemagglutinin (PHA), one of the three main

47

types of plant lectins, is commonly found in leguminous plants and can combine with

48

membrane receptors.2 Among various kinds of legumes, red kidney beans (Phaseolus

49

vulgaris) contains the highest concentration of PHA3, which is a tetrameric

50

combination

51

erythrocyte-specific (E).4 From previous researches, PHA has been found to

52

demonstrate distinct potential bioactive effect, such as antitumor5, 6, antifungal7,

53

antiviral8, mitogenic9,

54

Besides, PHA as a bioprobe could be applied to structural analysis of lectin

55

oligosaccharides through its characteristic of saccharide specificity.12

formed

of

10

two

subunits,

lymphocyte-specific

(L)

and

and HIV-1 reverse transcriptase inhibitory activities11.

56

Despite of these health-promoting benefits based on the pharmaceutical concern,

57

from the food safety point of view, PHA is also considered as an anti-nutritional

58

factor, which must be destroyed during food processing. The influence of PHA are

59

indicated by a loss of epithelial resistance which may lead to an acute gastroenteritis13,

60

and PHA would be fatal when ingested at high concentration14. Therefore, effective

61

processing approach must be taken to make PHA inactivation before PHA-containing

62

legumes are consumed as food.

63

Generally speaking, PHA had two remarkable characteristics. On one hand, it

64

binds specific monosaccharides or oligosaccharides reversibly, and facilitates

65

erythrocyte agglutinating or precipitating, which make it an anti-nutritional factor. On 3


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66

the other hand, PHA could inhibit the activity of α-glucosidase effectively 15, 16. As an

67

α-glucosidase inhibitor, it could postpone the transformation of polysaccharides and

68

disaccharides to glucose by invertible inhibition towards α-glucosidase of intestinal

69

brush border membrane. As a consequence, PHA might contribute to the regulation of

70

blood sugar level after meal, or increase the insulin secretion mildly and continuously.

71

From previous researches, α-glucosidase played an important role in the pathogenesis

72

of some serious diseases such as diabetes, human immunodeficiency virus (HIV)

73

infection

74

researches on α-glucosidase inhibitor had biological and practical significance.

17

and cancer, showing enormous potential on illness treatment. Therefore,

75

Heating is the traditional processing technique to limit the toxicity of PHA, but

76

PHA is relatively resistant to thermal denaturation, so it takes a long processing time

77

to inactivate the PHA18, which would consume more energy, reduce the food quality

78

and cause the destroy of some useful bioactivity such as the α-glucosidase inhibition

79

activity. Ultra-high pressure treatment (UHP), a new processing technology, has the

80

superiority of less energy consumption, lower treatment temperature and more

81

nutrition maintenance. Ultra-high pressure technology was applied in more and more

82

food production, such as fruit juice, jam19 and peas20, since Hite21 had found that UHP

83

can be used to sterilize milk in 1899. The mechanism of UHP based on the changing

84

molecular volume through ultra-high pressure, which would destruct and restrict the

85

chemical bonds of protein molecules if the change is big enough. Therefore, UHP can

86

induce the changes of secondary and tertiary structures of the protein molecules,

87

which has been indicated by plenty of researches.22-25 Our previous work26 has 4



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investigated the effect of ultra-high pressure treatment towards red kidney bean

89

homogenates and found that UHP could impact the haemaggiutination activity and

90

change the structure of crude red kidney bean PHA.

91

In the present study, the effect of UHP treatment with different pressure levels

92

(150, 250, 350, 450 MPa) on the bioactivities of purified PHA including

93

haemagglutination activity, saccharide specificity activity and α-glucosidase

94

inhibition activity were studied. Besides, the underlying principles about how high

95

pressure changing these activities were also explored by the analysis of structural

96

conformation variations and the thermal behavior changes. The results from this work

97

might supply helpful information for using UHP processing techniques to enhance the

98

safety use of red kidney bean concerning PHA as its anti-nutritional, and also keep its

99

α-glucosidase inhibition activity as a beneficial factor.

100

MATERIALS AND METHODS

101

Materials

102

The raw material, dried red kidney beans, were obtained from Vanguard

103

Supermarket (Guangzhou, China). Red blood cells in buffer (Human group B,

104

glutaraldehyde treated) and the ɑ-glucosidase were purchased from Sigma-Aldrich (St.

105

Louis, MO, USA). The trypsin (2 × 103 U/g) was acquired from Sanye Biochemistry

106

Co. (Hangzhou, Zhejiang, China). Affi-gel blue gel and dialysis membrane (D36 mm,

107

6000-8000 Da) were purchased from Bio-Rad Laboratories (Shanghai) Co., Ltd.

108

(Shanghai,

109

Sigma-Aldrich (St. Louis, MO, USA). Molecular weight protein markers were

China).

The

p-N-phenylb-D-glucopyranoside

5


was

gained

from

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110

purchased from Dinguo Biotechnology Co., Ltd. (Shanghai, China). The

111

trishydroxymethylaminomethane,

112

methylene diacrylamide were purchased from GEN-VIEW Scientific Inc. (Florida,

113

USA). All other chemicals used in the present study were of analytical or higher

114

grade.

115

Extraction and Purification of PHA

β-mercaptoethanol,

acrylamide

and

116

The process of PHA extraction and purification were simply hint in Figure 1.

117

The beans were homogenized in a blender (National Co., Ltd., Osaka, Japan) after

118

soften by steeping in Tris-HCl buffer (10 mM, pH 7.4, 1:10, w/v) at 4 °C for 6 h.

119

Later, the homogenates were incubated at 4 °C for 36 h, and then centrifuged in a

120

CR22G high-speed centrifuge (Hitachi Co., Tokyo, Japan) at 8,000 g at 4 °C for 30

121

min after filtered through 4-layers of gauze. The supernatants were lyophilized by

122

ALPHA 1-2LD PLUS lyophilizer (Marin Christ, Germany) to gain the crude PHA.

123

The obtained lyophilized sample was then purified by the affinity

124

chromatography purification on an Affi-gel blue (Bio-Rad, USA) column according

125

to the method of Ye et al17 with some modifications. Briefly, the lyophilized samples

126

were dissolved in Tris-HCl buffer (10 mM, pH 7.4) to make a final concentration of

127

10 % (w/v) and then the sample solution was loaded on an Affi-gel blue gel column

128

(2.5 × 20 cm) which had been equilibrated with the same buffer. The unbound

129

proteins were all removed by washing the column with Tris-HCl buffer (10 mM, pH

130

7.4), and then the adsorbed proteins were eluted with 10 mM Tris-HCl buffer (pH 7.4)

131

containing 1.4 M NaCl. Later, the collected elution was dialyzed against double 6



132

distilled water for 36 h at 4 °C by changing water every 4 h. The dialyzed fractions

133

were lyophilized for further analysis.

134

Ultra-High Pressure (UHP) Treatment

135

The lyophilized samples were dissolved in the phosphate buffer (10 mM, pH 7.4)

136

to obtain a concentration of 6 mg/mL (m/v) and divided into five equal aliquots. Each

137

sample was placed in double polyethylene sealable bags before vacuum sealing in a

138

DZ-280/2SD multi-functional vacuum packaging machine (Jinqiao Technology

139

Electronic Equipment Manufacturing Co., Ltd., Dongguan, Guangdong, China) and

140

made sure that no headspace was left. The vacuum sealed packages were subjected to

141

UHP-treatment in a high-pressure vessel (KEFA Hitech Food Machine Co., Ltd.,

142

Baotou, Neimenggu, China) at 25 °C with a filling volume of 5 L. The pressure

143

transmitting medium was oil. Untreated samples of purified PHA were used as control

144

(atmospheric pressure 0.1 MPa at 25 °C). The samples subjected to UHP treatment

145

were conducted in triplicate at 150, 250, 350 and 450 MPa respectively, holding for

146

15 min. The pressure increase and release rate were about 100 MPa/min and 200

147

MPa/min, respectively. All the samples with or without UHP treatments in the plastic

148

bag were lyophilized and preserved in a desiccator within 2 months for further

149

analyses.

150

Haemagglutination Assay

151

The suspension of red blood cells (type B) were washed with phosphate buffer

152

(10 mM, pH 7.4) 3 times before pipetting into a 96-well microtiter V-plate (Dinguo

153

Biotechnology Co., Ltd., Shanghai, China) for the haemagglutination assays. The 7


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lyophilized samples of purified PHA were dissolved in the phosphate buffer (10mM,

155

pH 7.4) containing 0.15 M NaCl to obtain a concentration of 6 mg/mL (m/v). Then

156

each sample (25 µL) was serially diluted two-fold, and added to each well with 25 µL

157

of blood. After incubated at 37 °C for about 40 min, the agglutination was determined

158

visually. The erythrocytes in the phosphate buffered saline (negative control) were

159

considered fully sedimented when a small dot was observed at the bottom of the well.

160

The haemagglutination titer was defined as the reciprocal of the highest dilution

161

exhibiting haemagglutination. This was assigned a value of one haemagglutination

162

unit.26, 27

163

Saccharides Specificity

164

The saccharide specificity of PHA was determined by the haemagglutination

165

assay after mixing different saccharides with the purified PHA. The monosaccharides

166

(glucose, galactose, xylose, ribose and rhamnose), disaccharides (sucrose, lactose and

167

maltose) and glycoconjugates (mannitol, glucuronic acid and galacturonic acid) were

168

dissolved separately in distilled water to obtain final concentration of 250 mM/L for

169

each saccharide. The solutions were pipetted into a 96-well microtiter V-plate, and

170

then the purified PHA was added at 25 °C and incubated about 10 min. After that, the

171

haemagglutination activity for each treated solution and the control (no PHA) was

172

tested as described above.

173

The α-Glucosidase Inhibition Assay

174

The α-glucosidase inhibition assay was modified based on the methods of Ye et

175

al.17 The ɑ-glucosidase was dissolved in MES-HCl buffer (10 mM, pH 6.4) to make 8



176

the final concentration of 0.05 U/L, and then it was added into each well in a 96-well

177

microtiter plate. Later, the purified PHA with or without UHP treatment were pitted

178

into the well respectively, and incubated with the α-glucosidase at 37°C for 10 min.

179

Then the substrate, p-N-phenylb-D-glucopyranoside (2 mM), was added to start the

180

reaction. After 15 min, 2 M NaOH (pH 10.0) was pitted to each well to terminate the

181

reaction. The absorbance of the solution was measured at 400 nm.

182

Averages of three replicates are presented. The α-glucosidase inhibition rate was

183

defined as the follow expression.

184

Inhibition Rate % =

185

Where A1 was the absorbance of the control with MES-HCl buffer instead of

−

186

purified PHA; A2 was the absorbance of the sample treated by purified PHA.

187

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

188

The PHA constituents were subjected to SDS-PAGE analysis using a 12%

189

acrylamide separating gel and a 5% acrylamide stacking gel containing 0.1% SDS by

190

an electrophoresis apparatus (Bio-Rad Laboratories, California, USA). The

191

lyophilized purified PHA sample was dissolved in Tris-HCl buffer (60 mM, pH 8.8),

192

which contains 2% SDS, 5% 2-mercaptoethanol, 25% glycerol and 0.1%

193

bromophenol blue. And then the solution was heated in boiling water for 5 min,

194

centrifuged at 10,000 × g for 3 min, and then loaded to the electrophoresis gel. A

195

cocktail of protein standards were used as markers, containing rabbit phosphorylase b

196

(97.4 kDa), bovine serum albumin (66.2 kDa), rabbit actin (43.0 kDa), bovine

197

carbonic anhydrase (31.0 kDa), and trypsin inhibitor (20.1 kDa). Each sample (15 µL) 9


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198

and the protein standard cocktail (15 µL) were loaded onto a gel lane, respectively.

199

After electrophoresis, 0.25% Coomassie Brilliant Blue-R250 in 50% trichloroacetic

200

acid was applied to stain the gel for the protein chain and then the Perodic acid schif

201

(PAS) was used to stain for the saccharide chain. Finally, the gel was destained in

202

methanol/acetic acid/water (1:1:8, v/v/v).

203

Circular Dichroism (CD) Spectroscopy

204

CD measurements were performed on a Jasco J-810 spec-trometer (Jasco

205

International Co., Ltd., Tokyo, Japan) using nitrogen as protective gas and the

206

temperature was controlled by a constant thermostat during samples running. The

207

measurements were done using cuvette with 1 mm path length at 25°C. As for far-UV

208

region (250-190 nm) assay, the sample concentration 0.125 mg/mL was used for

209

measurements, while for near-UV region (300-250 nm) analysis, the sample

210

concentration tested was 2.5 mg/mL. The spectra were recorded using a scan speed of

211

100 nm/ min and the data were collected at 0.2 nm intervals. Each scan was set to

212

repeat five times in order to eliminate signal noise and finally averaged scan was

213

given by the instrument to be considered as the final result. The data obtained were

214

normalized by subtracting the baseline recorded for the blank. The phosphate buffer

215

without PHA was used as blank. Secondary structure estimates (α-helix, β-sheet and

216

β-turn) were analyzed by the method of K2D.

217

Differencial Scanning Calorimetry (DSC)

218

The thermal behaviour of PHA samples with or without pressure treatments was

219

measured by a TA Q100-DSC thermal analyzer (TA Instruments, New Castle, DE, 10



Page 10 of 33

220

USA). The indium standard was used to calibrate the calorimeter. The lyophilized

221

purified PHA samples (2.0 mg) were placed into coated aluminium pans. And then 20

222

µL of phosphate buffer (pH 7.4, 20 mmol/L) was added to the pans which was

223

hermetically sealed by then. The pans were incubated at 4 °C for at least 4 h before

224

the measurements. As for the measurements, the pans were heated in the calorimeter

225

from 25 to 120 °C at a heat rate of 5 °C/min and the nitrogen gas was used to control

226

temperature at the input rate of 30 mL/min. A pan without PHA sample was used as

227

control. From the thermograms, the denaturation temperature (Td) and denaturation

228

enthalpy (∆H) were acquired through the Universal Analysis 2000 software, Version

229

4.1D (TA Instruments-Waters LLC). All experiments were conducted in triplicate.

230

Statistical Analysis

231

Data processing was accomplished with one-way analysis of variance (ANOVA),

232

by the software of SPSS Version 19 for Windows (SPSS Inc.). Significant differences

233

between means (p < 0.05) were identified using the least significant difference (LSD)

234

test.

235

RESULTS AND DISCUSSION

236

Haemagglutination Assay

237

The hemoagglutinating activity of PHA was on account of its capacity to bind to 28, 29

238

red blood cells.

239

with their interactions with specific carbohydrate residues on the cell membrane

240

structure

241

Haemagglutination was indicated by the formation of a PHA-erythrocyte-PHA matrix.

and

The ability of PHA samples to haemagglutinate was associated

their

three

dimensional

structure of

11


themselves.

27,

30,

31

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242

The dense dots that sedimented to the bottom of the well were showed as negative in

243

Figure 2(A), which manifested no agglutinin activity and hence indicated the absence

244

of agglutinin. For comparison, the haemagglutination concentration were shown in

245

Figure 2(B), which represented the lowest concentration of PHA to induce the

246

agglutination of erythrocytes. After purified by Affi-gel blue affinity chromatography,

247

PHA demonstrated great increase on haemagglutination activity, as proved by the fact

248

that the haemagglutination concentration of the purified PHA (0.19 mg/mL) was

249

about 4 times lower than that of crude PHA (0.75 mg/mL). As also indicated by

250

Figure 2 (B), the variation trend of the purified PHA was almost the same as that of

251

the crude samples with the increase of pressure levels (150, 250, 350, and 450 MPa).

252

The visible haemagglutination activities and the haemagglutination titers of the

253

purified PHA under UHP treatments were shown in Figure 2(A). It was found that

254

high pressure treatment could cause obvious decrease of the hemoagglutinating

255

activity. The titer was 23 U at 0.1 MPa treatment but decreased to 22 U at 150 MPa.

256

Almost 50 % of the hemoagglutination activity of PHA were destroyed when the

257

treatment pressure was further increased to 250 - 350 MPa. Finally, the titer was

258

reduced to 20 U at 450 MPa. It indicated that the high pressure treatment led to the

259

structure changes in PHA, which may either effect the glycone chain or protein

260

configuration.

261

Saccharide Specificity

262

The specificity of PHA is usually defined by the saccharides (or other saccharide

263

analogues) that could specifically recognized by PHA. The saccharide specificity of 12



264

the purified PHA with or without UHP treatment was determined by the inhibition of

265

hemagglutinating activity (Figure 2C). It was found that untreated purified PHA (at

266

normal atmosphere 0.1 MPa) showed no specific inhibition on hemagglutination with

267

the addition of monosaccharides (glucose, galactose, xylose, ribose and rhamnose),

268

disaccharides (sucrose, lactose and maltose) or glycoconjugates (mannitol, glucuronic

269

acid and galacturonic acid). It appeared that untreated PHA samples did not exhibit

270

saccharide specificity towards the tested monosaccharides, disaccharides and

271

glycoconjugates. Just as R. Loris 32 also reported, it had not been clear so far that what

272

kind of saccharide could be specifically recognized by PHA from red kidney beans,

273

which belongs to the glycoprotein group termed “complex”. And the complex type of

274

legume lectins had a similar structure that the hydrogen bones binding with the

275

specific monosaccharide were removed and compensated by protein-carbohydrate

276

hydrogen bonds. Therefore, these complex specific groups of lectins were not

277

inhibited by any simple saccharide. As also shown in Figure 2(C), high pressure

278

treatments (150, 250, 350 and 450 MPa) did not change the saccharides specificity of

279

PHA. It seemed that PHA was quite tolerant towards high pressure treatment at even

280

450 MPa treatment.

281

The α-Glycosidase Inhibition

282

Carbohydrates that digested to monosaccharides could be absorbed through the

283

intestine. The inhibitors of α-glucosidase could be able to prevent the digestion of

284

carbohydrate and reduce the content of blood sugar. Therefore, they have been used

285

as drugs for diabetes mellitus type 2.33 In the present study, the ɑ-glucosidase 13


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286

inhibitory effects of the PHA samples were illustrated in Figure 2(D). The purified

287

PHA samples (at normal atmosphere 0.1 MPa) without high pressure treatments

288

exhibited strong ɑ-glucosidase inhibitory activity (39.9%). After 150 MPa UHP

289

treatment, the PHA samples showed slightly decreased inhibitory activity against

290

a-glucosidase, but it still remained to be 35.4%. Particularly, even after 450 MPa UHP

291

treatment, the PHA sample could also keep above 60 % of its original activity. The

292

PHA structure especially the saccharide chain structure characteristics contributed to

293

its α-glucosidase inhibitory activity, and minor changes that happened to the structure

294

might induce huge changes of its activity. Therefore, the remained α-glucosidase

295

inhibitory activity after UHP treatment further proved that the glycone units of PHA

296

processed high pressure tolerance.

297

Taken both hemoagglutinating activity and α-glucosidase inhibition activity into

298

consideration, the results revealed that UHP treatment could destroy the undesirable

299

hemoagglutinating activity of PHA, and at the same time keep the beneficial

300

α-glucosidase inhibition activity, making UHP-treated PHA a potential diabetes

301

treatment products. Besides, ɑ-glucosidase was related to HIV-1 envelope protein gp

302

120, a surface glycoprotein located on the viral coat, which was required to interact

303

with the human CD4 glycoprotein in order to initiate entry into the cells. Thus, the

304

glucosidase-inhibitory activity of purified PHA was also involved with anti-HIV

305

activity, which was supported from the findings of Ye et al. 17

306

SDS-PAGE Analysis

307

SDS-PAGE was conducted to explore the effects of UHP treatment on the 14



308

subunit changes of PHA samples. Figure 3(A) shows the electrophoretic patterns

309

stained with Coomassie Brilliant Blue-R250, which demonstrated the changes of

310

protein subunits in PHA. The band with a molecular weight of about 31 kDa

311

prominently presented in each sample, corresponding to the basic subunit of PHA as

312

previously reported.3 As a comparison, the sample that underwent 450 MPa pressure

313

treatment revealed two additional bands, one with low intensity located between 66.2

314

- 97.4 kDa and another located between 43.0 - 66.2 kDa. It indicated either the protein

315

subunits with high molecular weight disrupted or low molecular weight fractions

316

aggregated, as declared in our previous study. Similar phenomenon had been

317

observed on soy bean protein, with the dissociation of subunits after high pressure

318

treatment. 34

319

PAS staining used in electrophoresis is a very important technique to explore the

320

saccharide binding situation in glycoprotein molecules, which illustrates the

321

glycol-protein structure changes. In the present study, PAS staining of SDS-PAGE

322

profiles was performed and the gel was shown in Figure 3(B). It seems that no

323

obvious changes were observed for lanes running PHA samples with or without UHP

324

treatment (150, 250, 350 and 450 MPa). The results further confirmed that the

325

glycone units of PHA were more stable and less sensitive to the pressure treatment as

326

compared with the protein units of PHA.

327

Circular Dichroism (CD) Spectroscopy

328

The near UV-CD spectra of protein mainly reflected the conformation of

329

side-chains. The peak of tyrosine (Tyr) was mainly at about 275 nm, and the 15


Page 14 of 33

Page 15 of 33


330

migration of this peak was usually used as the indicator for structure change. A near

331

UV-CD spectra for PHA undergone different pressure treatments was shown in Figure

332

4. A red shift of 1-2 nm in the peak of tyrosine was found in purified PHA treated with

333

0-250 MPa. When treated with 350 MPa pressure, a peak indicating the interaction of

334

phenylalanine (Phe) and tyrosine (Tyr) was shown at 268 nm, and another peak

335

derived from the mutual effect of both tyrosine (Tyr) and tryptophan (Trp) residues

336

was found at 285 nm. As for 450 MPa treatment, a significant red shift of 3 nm in the

337

Tyr peak was observed as compared with that of the untreated PHA (0.1 MPa).

338

Compared with our preceding study35, PHA may have maximum mean at around 285

339

nm due to the side chains of Tyr and Trp residues, which was similar in the near

340

UV-CD spectra after UHP treatment.

341

The far UV-CD (190 - 250 nm) spectra represents the range of absorption peaks

342

of peptide bond, reflecting the conformation of protein backbone. The secondary

343

structure compositions of purified PHA samples with different UHP treatments were

344

shown in Table 1. Although the content of α-helix and β-sheet structures found in this

345

study seemed to be different with other kinds of legume lectins, the spectra were quite

346

similar with that found in our previous study35, which showed that the PHA purified

347

by two-step affinity chromatography was with 52.8% α-helix and 20.6% β-sheet.

348

From 0-250 MPa, the increase of pressure induced the expansion of protein

349

structure, indicated by the decrease of the ordered α-helix structure proportion and the

350

increase of β-sheet structure proportion. Also, the disordered structure ratio increased.

351

As the pressure further increased from 250 - 450 MPa, it forced the formation of 16



352

protein aggregation through non-covalent interaction, which caused the increase of

353

α-helix structure ratio and the decrease of disordered structure ratio. Similarly, the

354

high pressure treatment had same effects on the isolated soy protein, for example,

355

Puppo

356

the ordered protein structure was partly destroyed through ultra-high pressure

357

treatment.

358

Thermal Behavior

36

found the surface hydrophobicity of soy isolate protein was promoted and

359

Endothermic effects seen with DSC thermograms are mainly attributed to the

360

disruption of intra and intermolecular bonds. The denaturation peak temperature (Td)

361

can be used to monitor the thermal stability of protein, and the enthalpy value (∆H) is

362

correlated with the proportion of undenatured protein or the extent of ordered protein

363

structure.37 The denaturation peak temperature (Td) and the enthalpy value (∆H) of

364

PHA treated by different pressures were shown in Figure 5. When the treated pressure

365

was lower than 250 MPa, the Td of PHA (100.06 °C) increased with the raise of

366

pressure compared to that of the native PHA (97.34 °C). However, the Td of purified

367

PHA decreased slightly at 350 MPa treatment, and then went up to the highest level

368

(101.19 °C) at 450 MPa. In addition, a significant decrease in the enthalpy value (∆H)

369

from 2.413 to 1.319 J/g was seen with the enhancement of pressure from 0.1 to 250

370

MPa. While from 250 to 450 MPa, the ∆H increased from 1.319 to 2.458 J/g indicated

371

first a decrease then an increase tendency in the thermo stability of PHA, showing that

372

the higher pressure treatment may induce the expansion and aggregation in protein

373

structure of the PHA. In general, high pressure treatment could cause the expansion or 17


Page 16 of 33

Page 17 of 33


374

aggregation of PHA protein structure and exert effects on the thermal stability of PHA,

375

as indicated by CD spectroscopy.

376

CONCLUSIONS

377

The results from this work demonstrated that ultra-high pressure (UHP)

378

treatment had more obvious effects on haemagglutination activity of purified PHA

379

than crude PHA samples. Also, in terms of the three bioactivities of PHA (e.g.

380

haemagglutination activity, saccharide specificity and α-glucosidase inhibition

381

activity), we found that only haemagglutination activity was sensitive to UHP

382

treatment, while the saccharide specificity and the α-glucosidase inhibition activity

383

were barely influenced. The underlying mechanism was revealed by protein units and

384

glycone units structure analysis.

385

The results were shown simply in Figure 6. As for protein units analysis, the

386

electrophoretic analysis stained with Coomassie Brilliant Blue-R250 demonstrated the

387

disruption of high-molecular-weight protein subunits or the aggregation of

388

low-molecular-weight protein fractions for purified PHA treated at 450 MPa. As a

389

consequence, the haemagglutination activity of the purified PHA was almost

390

completely destroyed. Also, the data from both CD spectroscopy and DSC analysis

391

revealed the protein untis of purified PHA were very sensitive to pressure treatment,

392

resulting in the re-aggregation at 250-450 MPa. Therefore, the destroying of its

393

haemagglutination activity after high pressure treatment might be more likely to be

394

associated with the change of the PHA protein units. As a comparison, the glycone

395

untis of purified PHA was much stable towards UHP treatment as illustrated by PAS 18



396

staining electrophoresis. Accordingly, the α-glucosidase inhibition activity did not

397

change much after UHP treatment.

398

Therefore, the results from this work proved that UHP processing seemed to be

399

an effective way to protect the α-glucosidase inhibition activity as a beneficial factor

400

of PHA, while destroy its haemagglutination activity as an anti-nutritional factor. This

401

might supply useful information for further utilization of PHA from red kid bean.

402

ACKNOWLEDGEMENTS

403

The authors are grateful to the Guangdong Natural Science Funds for

404

Distinguished Young Scholars (No.S2013050013954), Program for New Century

405

Excellent Talents in University (NCET-13-0213), the Fundamental Research Funds

406

for the Central Universities (02-2015) and Key Laboratory of Aquatic Product

407

Processing, Ministry of Agriculture, P.R.China (NYJG201402).

408 409 410 411 412 413 414 415 416 417 19


Page 18 of 33

Page 19 of 33


418 419 420 421 422 423 424 425 426

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soy protein isolate and 7S and 11S globulins. Food Hydrocoll. 2001, 15, 263-269.

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studies of Fusarium solani lectin. IUBMB Life 2007, 59, 34-43.

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erythrocytes. J. Biol. Chem. 1970, 245, 2536-2545.

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Acta-Protein Struct. Molec. Enzym. 1998, 1383, 9-36.

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515

phytohaemagglutinin from red kidney bean (Phaseolus vulgaris) purified by different affinity

516

chromatography. Food Chem. 2008, 108, 394-401.

517

(36) Puppo, C.; Chapleau, N.; Speroni, F.; de Lamballerie-Anton, M.; Michel, F.; Añón, C.; Anton, M.,

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Physicochemical modifications of high-pressure-treated soybean protein isolates. J. Agric. Food. Chem.

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520

(37) Arntfield, S.; Murray, E., The influence of processing parameters on food protein functionality I.

521

Differential scanning calorimetry as an indicator of protein denaturation. Can. I. Food Sci. Tech. J.

522

1981, 14, 289-294.

523 524

FIGURES CAPTIONS

525

Figure 1. The flow chart for the extraction, purification and UHP treatment of PHA

526

from red kidney bean (Phaseolus vulgaris).

527

Figure 2. The effects of UHP treatment on different activities of PHA. (A) The visible 24



Page 24 of 33

528

haemagglutination titers of the purified PHA. The erythrocytes were considered no

529

agglutination when a small dot was observed at the bottom of the well. The

530

concentration of purified PHA was 1.5 mg/mL at the haemagglutination titer of 20.The

531

negative group was treated with PBS instead of PHA. (B) The haemagglutination

532

concentration of crude and purified PHA, representing the lowest concentration of

533

PHA to induce the agglutination of erythrocytes. (C) The saccharide-binding

534

specificity of purified PHA. Line 1-11 represents for glucose, galactose, xylose, ribose,

535

rhamnose, mannitol, glucuronic acid, galacturonic acid, sucrose, lactose and maltose,

536

respectively. The final concentration of each saccharide was 250 mmol/L. The signal

537

“-” means no agglutination activity, while “+” represents the sample has agglutination

538

activity, which reveals the PHA does not inhibit by the saccharide. (D) The

539

α-glucosidase inhibitory activity of purified PHA. The reaction was conducted in 0.05

540

U/L of ɑ-glucosidase and 8 mg/mL of purified PHA at 37 °C for 15 min.

541

Figure 3. SDS-PAGE pattern of purified PHA samples treated by ultra-high pressure.

542

(A) Electrophoresis conducted with Coomassie Brilliant Blue-R250 staining; (B)

543

Electrophoresis conducted with PAS staining. Lane 1-5 represent purified PHA

544

samples by 0.1 MPa, 150 MPa, 250 MPa, 350 MPa and 450 MPa of high pressure

545

treatments. M represents for protein molecule marker.

546

Figure 4. Near UV-CD spectra of purified PHA samples by UHP treatment.

547

Figure 5. Changes in thermal properties of UHP treatment PHA. Different letters

548

within a column are significantly different (P < 0.05).

549

Figure

6.

The

assumed

structure-bioactivity

(haemagglutination

25


activity,

Page 25 of 33


550

saccharide-binding specificity and α-glucosidase inhibitory activity) relationship of

551

purified PHA as affected by UHP treatment.

Figure 1

Extraction Homogenization

Extraction

Red Kidney Beans

Centrifugation

Oil

Lyophilization

PHA

Crude PHA

Purification

Affinity chromatography

Dialysis

Ultra-high pressure treatment Purified PHA

26



(A)

(C)

(B)

(D)

Figure 2 27


Page 26 of 33

Page 27 of 33


Figure 3 B

A

28



Page 28 of 33

Figure 4

3

2

CD (medg)

1 275 nm

285 nm

0

450 MPa 350 MPa 250 MPa 150 MPa 0.1 MPa

-1

-2

240

250

260

270

280

290

Wavelengh (nm)

29


300

310

Page 29 of 33


Figure 5 Td 104

∆H a

a

2.5

2.0

b 102

a

c b

100

c

1.0

d 98

1.5

0.5

e

0.0

-0.5

96 0.1

150

250

350

Pressure (MPa)

30


450

∆H (J/g)

Td(°C)

c


Page 30 of 33

Figure 6 α-helix

PHA-E/L monomer

β-sheet

Glycone

Ø

UHP 0-250 MPa

Protein Unit Change Protein Chain Expansion Glycone Unit Stable

α-helix

Disorder structure

Glycone

UHP 250-450 MPa

Ø

Haemagglutination Activity Lost

Saccharide Specificity Unchanged

Protein Chain Folded

Glycone units

α-helix

Disorder structure

β-sheet

31


The α-glycohydrolase inhibition activity Remained

Page 31 of 33


Table 1. Secondary structure compositions of purified phytohemagglutinin samples treated by ultra-high pressure. Pressure (MPa)

α- Helix (%)

β- Sheet (%)

Other (%)

0.1

49.95

11.13

38.92

150

42.92

13.58

43.50

250

33.10

16.86

50.04

350

44.17

14.22

41.61

450

47.14

12.48

40.38

32



Table of Contents Graphic

33


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Structure and Activity Changes of ... - ACS Publications

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