Effects of Disulfide Bond Reduction on the Conformation and Trypsin

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Effects of disulfide bond reduction on the conformation and trypsin/ chymotrypsin inhibitor activity of soybean Bowman-Birk inhibitor Hui He, Xingfei Li, Xiangzhen Kong, Caimeng Zhang, Yufei Hua, and Yeming Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05829 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Journal of Agricultural and Food Chemistry

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Effects of disulfide bond reduction on the conformation and

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trypsin/chymotrypsin inhibitor activity of soybean Bowman-Birk inhibitor

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Hui He, Xingfei Li, Xiangzhen Kong, Caimeng Zhang, Yufei Hua, Yeming Chen*

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State Key Laboratory of Food Science and Technology, School of Food Science and Technology,

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Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, PR China

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Corresponding Author:

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Yeming Chen

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Telephone/Fax: 86-510-85329091. E-mail: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT: Soybean seeds contain three groups (A, C, and D) of Bowman-Birk inhibitors

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(BBI). In this study, highly purified BBI-A (approximately 96%) was obtained from soybean whey

15

at the 0.1 g level by the complex coacervation method. BBI-A has seven disulfide bonds (SS) and

16

no sulfhydryl group and exhibits trypsin inhibitor activity (TIA) and chymotrypsin inhibitor

17

activity (CIA). The X-ray structure has shown that BBI-A has five exposed SS and two buried SS.

18

Due to steric hindrance, it was reasonable to consider that dithiothreitol first attacks the five

19

exposed SS and then the two buried SS, which was supported by the results that SS reduction with

20

dithiothreitol could be divided into quick and slow stages, and the critical point was close to .

ହ ଻

21

The effects of SS reduction on TIA and CIA could be divided into three stages: when one exposed

22

SS was reduced, both TIA and CIA decreased to approximately 60%; with increasing reduction of

23

exposed SS, CIA gradually decreased to 8%, and TIA gradually decreased to 26%; with further

24

reduction of buried SS, CIA gradually decreased to 2%, and TIA slightly decreased to 24%.

25

Far-UV CD spectra showed that the secondary structure of BBI-A was slightly changed, whereas

26

near-UV CD spectra showed that the conformation of BBI-A was substantially changed after the

27

five exposed SS were reduced, and further reduction of buried SS affected the conformation to

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some extent. The results from Tricine-SDS-PAGE and C8 column showed the same trend as

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near-UV CD spectra. BBI-A has a structural peculiarity in that two hydrophobic patches are

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exposed to the exterior (in contrast to typical soluble proteins), which was attributed to the seven

31

SS by some researchers. The results above supported the hypothesis that hydrophobic collapse of

32

the exposed hydrophobic patches into a regular hydrophobic core occurred after the reduction of

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SS in BBI-A.

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Keywords: Bowman-Birk inhibitor, disulfide bond, conformational change, bioactivity 2


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INTRODUCTION

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Bowman-Birk inhibitor (BBI) was initially considered an antinutritional component in

37

soybean.1 Then some studies showed that BBI appeared to be a universal cancer preventive agent.2

38

Recently, it was reported that BBI might exert a protective effect against proteolysis of lunasin by

39

digestive enzymes.3,4 BBI has seven disulfide bonds (SS) and no free sulfhydryl group (SH).5

40

During soymilk processing, BBI is extremely difficult to inactivate by heating compared to Kunitz

41

trypsin inhibitor (KTI).6,

42

chymotrypsin inhibitor activity (CIA) are mainly attributed to BBI in properly heated soymilk.6, 7

43

These findings reveal that BBI has a strong resistance to thermal treatment.

7

As a result, the residual trypsin inhibitor activity (TIA) and

44

It is believed by many researchers that the seven SS are responsible for the high heat stability.5−7

45

Wu and Sessa used sodium metabisulfite to reduce the SS in BBI, and it was found that only

46

approximately 16% of the original TIA remained.8 Sessa and Nelsen reported that steeping

47

soybean flour in 50 mM Na2S2O5 at 65 °C for 1 h could inactivate approximately 98% of the BBI

48

and 95% of the KTI.9 Jiao et al. used an NADP/thioredoxin system, dithiothreitol, and lipoic acid

49

to treat BBI, and approximately 29.4, 11.1, and 87.5% of the original TIA remained,

50

respectively.10

51

To date, five BBI isoforms (A, B, C-II, D-II, and E-I) have been found in soybean seed, and can

52

be largely classified into three groups (A, C, and D). 11, 12 This is because BBI-B is similar to

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BBI-A in its inhibitory specificity and amino acid composition,11, 12 and BBI-E-I is a variant of

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BBI-D-II, lacking nine amino acid residues at the N-terminal region.13 According to the UniProt

55

database, one reactive site (Lys16-Ser17) of BBI-A inhibits trypsin, and the other (Leu43-Ser44)

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inhibits chymotrypsin; two reactive sites (Arg24-Ser25 and Arg51-Ser52) of BBI-D-II inhibit 3



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trypsin; one reactive site (Ala22-Ser23) of BBI-C-II inhibits elastase, and the other (Arg49-Ser50)

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inhibits both trypsin and chymotrypsin. Clemente et al. examined the soybean BBI isoforms using

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a C4 column, and found that BBI-A and BBI-D, respectively, contributed 41 and 33% to the total

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BBI.14 Voss et al. examined the crystal structure of BBI-A at 0.28-nm resolution, and found that

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five SS (Cys14-Cys22, Cys41-Cys49, Cys12-Cys58, Cys32-Cys39, and Cys8-Cys62) were

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exposed to the exterior, and the other two (Cys9-Cys24 and Cys36-Cys51) were buried; in

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addition, BBI-A did not exhibit a typical protein conformation with a hydrophobic core and a

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hydrophilic surface exposed to the exterior, but contained two exposed hydrophobic patches.15

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Therefore, one hypothesis was proposed that the hydrophobic collapse of the two exposed

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hydrophobic patches into a regular hydrophobic core occurred after all seven SS were reduced.15,16

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To date, just one reference can be found on the effects of SS on soybean BBI conformation.8 In

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this reference, a decrease in β-sheet (from 61 to 53%) and an increase in β-turn structure (from 1

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to 5%) were detected in metabisulfite-treated BBI compared to native BBI, and it was suggested

70

that the magnitude of the change was not large. As a result, the authors proposed that BBI had a

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stable conformation even after SS were broken.8 However, the low residual TIA (16%) in

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metabisulfite-treated BBI likely contradicted their hypothesis.

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In this study, highly purified BBI-A (approximately 96%) was first obtained from soybean whey

74

at the 0.1 g level by the complex coacervation method. Then, dithiothreitol (DTT) was used to

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reduce SS in BBI-A at different levels, and oxidized DTT and excessive DTT were removed by a

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desalting column. Finally, the effects of stepwise reduction of disulfide bond on the conformation

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of BBI-A were examined by circular dichroism, Tricine-SDS-PAGE, and C8 column, and the

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relationship between conformational change and BBI’s TIA and CIA was also examined. 4


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MATERIALS AND METHODS

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Materials. Soybean Nannong 99-10, harvested in 2015, was purchased from Nanjing

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Agricultural University (Nanjing, China) and stored at 4 °C until use. Bovine α-chymotrypsin,

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Nα-benzoyl-DL-arginine

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p-nitroanilide

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4,4′-dithiodipyridine (DPS) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai,

85

China). All other reagents were of analytical grade.

(BTpNA),

4-nitroanilide sodium

hydrochloride

dodecyl

sulfate

(BAPA),

(SDS),

N-benzoyl-L-tyrosine

dithiothreitol

(DTT),

and

86

Preparation of highly purified BBI-A. BBI-A was prepared with the method reported by Li et

87

al.,17 with some modifications. Soybeans (100 g) were soaked in deionized (DI) water at 4 °C for

88

18 h. The soaked water was discarded, and pre-cooled (4 °C) DI water was added to a total mass

89

of 1000 g. This was ground in a Waring blender (18000 rpm, MJ-60BE01B, Midea) for 1 min, and

90

was filtered through four layers of gauze to obtain raw soymilk (about pH 6.6). The raw soymilk

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was adjusted to pH 4.5 by 2 M HCl, and separated into supernatant and precipitate by

92

centrifugation (10000g, 30 min; 4 °C). The supernatant was collected and adjusted to pH 8.0 by

93

1.5 M KOH, and treated by centrifugation (15000g, 30 min; 4 °C) to remove the insolubles. The

94

supernatant (soybean whey) was collected and adjusted back to pH 4.5 by 2 M HCl. The volume

95

of the soybean whey was recorded, and ammonium sulfate was added to reach 40% saturation.

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After centrifugation (12000g, 40 min; 4 °C), the precipitate was collected and dissolved into 50

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mL of DI water. The solution was adjusted to 7.0 by 0.5 M KOH and dialyzed (3.5 kDa cut off)

98

for 18 h (three times, six hours each time) at 4 °C. Its protein concentration was determined by the

99

bicinchoninic acid method and diluted to 0.3% (w/v) by DI water. Then it was mixed with

100

ι-carrageenan solution (0.3%, w/v; pH 7.0) at a protein/ι-carrageenan mass ratio of 4/1, and the pH 5



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was adjusted to 3.75 by 0.5 M HCl. After centrifugation (5000g, 20 min; 4 °C), the supernatant

102

was collected and adjusted to pH 7.0 by 0.5 M KOH. By ultrafiltration (100 kDa cut off), the

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filtrate was collected and dialyzed (3.5 kDa cut off) for 18 h (three times, six hours each time) at

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4 °C, and concentrated by polyethylene glycol before freeze-drying. Generally, approximately 0.1

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g of BBI-A powder could be obtained from 100 g of soybean.

106

Reduction of disulfide bond by DTT at different levels. DTT was added into BBI-A solution

107

(2 mg/mL) with DTT/SS molar ratios of 0, 1/4, 1/2, 1/1, 2/1, 4/1, and 8/1 and treated at 40 °C for

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1 h. This condition was selected to weaken the effects of thermal treatment on BBI-A as much as

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possible. Then oxidized DTT and unreacted DTT were removed by a desalting column. The

110

obtained samples were used as soon as possible, or stored at 4 °C before use. It was determined

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that more than 95% of the generated SH in totally reduced BBI-A remained after storing the

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sample at room temperature for 1.5 h or at 4 °C for 24 h. The SS reduction experiment was

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conducted two times, and the experiments below were also carried out two times, which showed

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similar results. To further examine the effects of DTT on SS in BBI-A, DTT was added into the

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BBI-A solution at a DTT/SS molar ratio of 1/1 and treated at 40 °C for 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,

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and 10 h. Then, oxidized DTT and unreacted DTT were removed by a desalting column before

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free sulfhydryl content determination.

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Free sulfhydryl (SH) content determination. The free SH content was determined according

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to the method of Ruan et al.18 One hundred and twenty-five microliters of 4 mM DPS in 12 mM

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HCl solution was added into a solution of BBI-A (300 µL, 0.6 mg/mL) and then diluted with

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2.315% (w/v) SDS in 0.1 M phosphate buffer (pH 7.0) to give a final SDS concentration of 2%

122

(w/v). The mixture was vortexed and kept at room temperature for 30 min. Then each sample was 6


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123

detected in triplicate at 324 nm against a sample blank and a reagent blank in a UV-2450 UV-VIS

124

spectrophotometer (Shimadzu, Kyoto, Japan) until the absorbance reached the maximum value

125

(this time period was recorded). The reagent blank was the above mixture without the solution of

126

BBI-A, and the sample blank was the above mixture without the DPS solution. The SH content

127

was expressed as µmol SH/g protein by equation 1.

128

SH content = (A324 – A324r – A324s)/(ε×b×C)×1000000

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A324, A324r, and A324s are the absorbance of the sample, reagent blank, and sample blank at 324

130

nm, respectively. ε is the molar extinction coefficient of 4-thiopyridone in the presence of 2% SDS

131

(21200 M-1cm-1), and 4-thiopyridone is the product of reaction between SH and DPS. b is the

132

optical path (1 cm in this study). C is the protein concentration (g/L) in the sample.

(1)

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Trypsin inhibitor activity (TIA) assay. This assay was based on the method of Liu and

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Markakis,19 with some modifications. TIA was measured with 0.04% (w/v) BAPA as the substrate,

135

which was dissolved in 0.05 M Tris-HCl buffer (pH 8.2; 0.02 M CaCl2), and was freshly prepared

136

and pre-warmed in a 37 °C water bath before use. One milliliter of diluted sample and 1 mL of DI

137

water were added to a test tube and placed in a 37 °C water bath for 10 min. Five milliliters of

138

BAPA solution was added, followed by 2 mL of 0.012% (w/v) trypsin in 0.04 mM HCl solution.

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After exactly 10 min, the reaction was stopped by adding 1 mL of 30% acetic acid (v/v). After

140

mixing, the absorbance was measured using a spectrophotometer at 410 nm (WFZ UV-2100,

141

UNICO) against a reagent blank. The reagent blank was prepared simultaneously with the sample

142

by adding 1 mL of 30% acetic acid (v/v) to a test tube containing the diluted sample. DI water and

143

trypsin solution were added before adding the BAPA solution. A sample blank was prepared as

144

above, but 1 mL of the diluted sample was replaced with 1 mL of DI water. 7



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TIA was measured in trypsin inhibitor activity unit. One trypsin inhibitor activity unit (TIU)

146

was arbitrarily defined as a decrease of 0.01 absorbance unit at 410 nm per 10 mL of reaction

147

mixture under the conditions used. TIUs were calculated according to equation 2.

148

TIA (TIU/mg) = [(A410sb + A410rb – A410s)/0.01]* (D/C)

149

A410sb, A410rb, and A410s are the absorbance of the sample blank, reagent blank, and diluted

150

sample at 410 nm, respectively. D is the dilution ratio of the sample. C is the protein concentration

151

of the BBI-A solution. For accuracy, the samples were diluted to the point where 1 mL produced

152

40−60% trypsin inhibition. The experiment was performed in triplicate.

(2)

153

Chymotrypsin inhibitor activity (CIA) assay. CIA was determined using the method of Tan et

154

al.,20 with some modifications. One milliliter of diluted sample, one milliliter of DI water, and 4.2

155

mL of 0.05 M Tris-HCl buffer (pH 8.2; 0.01 M CaCl2) were pipetted into a test tube and placed in

156

a 37 °C water bath for 10 min. Two milliliters of 0.02% (w/v) α-chymotrypsin in 0.002 M HCl

157

solution was added, followed by 0.8 mL of 0.04% (w/v) BTpNA in acetone solution before

158

thorough mixing. After exactly 10 min, the reaction was terminated by adding 1 mL of 30% (w/v)

159

acetic acid. After mixing, the absorbance was measured at 385 nm by a spectrophotometer (WFZ

160

UV-2100, UNICO) against a reagent blank. The reagent blank was prepared simultaneously with

161

the sample by adding 1 mL of 30% acetic acid (v/v) to a test tube containing the diluted sample,

162

DI water, Tris-HCl buffer, and α-chymotrypsin solution before the BTpNA solution was added. A

163

sample blank was prepared as above, but with 1 mL of the diluted sample replaced with 1 mL of

164

DI water.

165

CIA was measured in chymotrypsin inhibitor activity unit. One chymotrypsin inhibitor activity

166

unit (CIU) was arbitrarily defined as a decrease of 0.01 absorbance unit at 385 nm per 10 mL of 8


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167

reaction mixture under the conditions used. CIUs were calculated according to equation 3.

168

CIA (CIU/mg) = [(A385sb + A385rb – A385s)/0.01]*(D/C)

169

A385sb, A385rb, and A385s are the absorbance of the sample blank, reagent blank, and diluted

170

sample at 385 nm, respectively. D is the dilution ratio of the sample. C is the protein concentration

171

of the BBI-A solution. For accuracy, the samples were diluted to the point where 1 mL produced

172

30−40% chymotrypsin inhibition. The experiment was performed in triplicate.

(3)

173

HPLC analysis. The HPLC equipment consisted of an Agilent 1100 series chromatograph

174

(Agilent Technologies, Santa Clara, CA, USA), equipped with a column oven (G1311C), a

175

quaternary pump (G1216A), and a UV detector (G1321B). An analytical C8 column

176

(OC20S05-1546WT, 5 µm particle size, 200 A, 150×4.6 mm id, YMC HPLC column) was used

177

for separation. Gradient elution was carried out at a flow rate of 1 mL/min using two solvents:

178

solvent A (0.1% (v/v) trifluoroacetic acid in water) and solvent B (0.085% (v/v) trifluoroacetic

179

acid in acetonitrile). The gradient was as follows: 95% A + 5% B from 0 to 5 min, 40% A + 60%

180

B from 5 to 60 min, 100% B from 60 to 65min, 95% A + 5% B from 65 to 70 min. The column

181

temperature was kept at 34 °C, and the detection was done at 214 nm. The injection volume of

182

BBI-A solution (2 mg/mL) was 20 µL.

183

Tricine-SDS-PAGE. This was conducted according to the method of Schägger, 21 with some

184

modifications. Solutions (0.5 mL, 2 mg/mL) of BBI-A reduced at different levels were mixed with

185

0.5 mL of Tricine-SDS-PAGE sample buffer. Then, each sample (10 µL) was loaded into the

186

sample well, and electrophoresed at a constant voltage of 30 mV until all samples entered into the

187

stacking gel and then at a constant voltage of 100 mV until the end. The gel was stained using

188

Coomassie Brilliant Blue G-250, and band intensities were analyzed by Image Lab Software 9



189

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(Bio-Rad, USA).

190

Measurement of circular dichroism (CD). CD spectra were obtained using a MOS-450

191

spectropolarimeter (BioLogic Science Instrument, France). The Far-UV CD spectroscopic

192

measurement was done according to the method of Wu and Sessa,8 which was performed in a

193

1-mm quartz cuvette with a protein concentration of 0.5 mg/mL in DI water. The sample was

194

scanned at 25 °C from 190 to 250 nm, and the spectra were an average of nine scans. The

195

following parameters were used: step resolution, 1 nm; acquisition duration, 2 s; bandwidth, 0.5

196

nm; sensitivity, 100 mdeg. The recorded spectra were corrected by subtraction of the spectra of DI

197

water. For near-UV spectra, the sample (0.5 mg/mL) was scanned at 25 °C from 250 to 320 nm,

198

which was performed in a 1-cm quartz cuvette. The spectra were also an average of nine scans,

199

and the following parameters were used: step resolution, 1 nm; acquisition duration, 2 s;

200

bandwidth, 0.5 nm; sensitivity, 100 mdeg. The recorded spectra were corrected by subtraction of

201

the spectrum of DI water.

202

RESULTS AND DISCUSSION

203

Purity of Bowman-Birk inhibitor (BBI). BBI was prepared from soybean whey at the 0.1 g

204

level by the complex coacervation method, and its TIA and CIA were determined to be 2395

205

TIU/mg and 817 CIU/mg, respectively. It was found that the TIA/CIA ratio of BBI-A was

206

obviously larger than those in two other research works.14,22 This might be due to the different

207

substrates and detection wavelengths used for the CIA assay, i.e., N-benzoyl-L-tyrosine

208

p-nitroanilide (detection at 385 nm) in this study and N-benzoyl-L-tyrosine ethyl ester (detection

209

at 256 nm) in the other two studies.14,22 BBI solution was treated by a C8 column (Figure 1) and

210

showed two major peaks at 26.78 and 27.52 min. According to one unpublished data set and 10


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211

research by Clemente et al.,14 it was suggested that the two major peaks were the BBI-A and -B.

212

BBI-A has a molecular weight of 7860 Da (UniProt database), so its theoretical value of

213

cysteine content was calculated as approximately 1780 µmol/g BBI-A. The free and total SH

214

contents of the BBI sample were, respectively, determined to be 16 and 1732 µmol/g.11 It was

215

considered that the free SH (16 µmol/g) should originate from the non-BBI proteins. As a result,

216

the purity of BBI-A and -B was calculated as approximately 96%, in agreement with the results in

217

Figure 1. BBI-B is similar to BBI-A in its inhibitory specificity and amino acid composition,11, 12

218

so the obtained BBI sample could be deemed as BBI-A (group A of BBI) in this study.

219

Effects of DTT on reduction of SS in BBI-A. DTT was added into BBI-A solution at DTT/SS

220

molar ratios of 1/4, 1/2, 1/1, 2/1, 4/1, and 8/1. After incubation in a 40 °C water bath for 1 h, these

221

solutions were cooled to room temperature. The oxidized DTT and unreacted DTT were removed

222

by a desalting column. Figure 2A shows that SS were quickly reduced by DTT from DTT/SS

223

molar ratio of 1/4 (approximately 10%) to 2/1 (approximately 62%), but the reduction rate of SS

224

decreased afterwards. Then, the effects of reduction time on SS were examined at DTT/SS molar

225

ratio of 1/1 in a 40 °C water bath. It was found that SS was reduced at the same rate from 1 to 5 h,

226

which decreased afterwards. At 5 h, approximately 78% of SS was reduced. The X-ray structure

227

of BBI-A showed that five SS were exposed to the exterior, whereas the other two were buried

228

(steric hindrance).15 In other words, approximately 71% of SS in BBI-A was exposed. The values

229

of 62 and 78% are close to 71%, so it was reasonable to consider that DTT first attacks the

230

exposed SS and then the buried SS, and the two buried SS should be gradually exposed during the

231

reduction of the exposed SS.

232

Effects of stepwise reduction of SS on TIA and CIA of BBI-A. To avoid the effects of a long 11



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233

time on BBI-A, the samples obtained from the DTT/SS experiment were used for the following

234

sections. Figure 3 shows that the effects of stepwise reduction of SS on TIA and CIA of BBI-A

235

can be divided into three stages. When approximately 10% of SS was reduced, TIA and CIA

236

quickly decreased to 59 and 61%, respectively. Then TIA gradually decreased to 26%, and CIA

237

gradually decreased to 8% when 62% of SS were reduced. Finally, TIA slowly decreased to 24%,

238

and CIA decreased to 2% after all the SS were reduced. Together with the results in Figure 2, it

239

can be suggested that the five exposed SS were key to the TIA and CIA of BBI-A, and the two

240

buried SS affected the CIA to some extent. Furthermore, the value of 10% is close to

ଵ ଻

(14%), so

241

it was considered that the reduction of one exposed SS in BBI-A could obviously affect the TIA

242

and CIA. In addition, the decrease in CIA easily occurred compared to TIA as more SS were

243

reduced. Ponne et al. reported that CIA appeared to be more heat sensitive than TIA as the total

244

inactivation level of BBI was higher.23 Odani and Ikenaka proteolytically separated trypsin- and

245

chymotrypsin-reactive subdomains of BBI-A, and the former retained 80% of its activity, and the

246

latter retained only 20%.24 The X-ray structure of BBI-A showed the presence of more

247

interactions in the trypsin-reactive subdomain than in the chymotrypsin-reactive subdomain.15

248

These

249

chymotrypsin-reactive subdomain in BBI-A.

revealed

that

the

trypsin-reactive

subdomain

was

more

stable

than

the

250

Effects of stepwise reduction of SS on the conformation of BBI-A. Figure 4A shows that the

251

far-UV CD spectra of nonreduced BBI-A solution were similar to those reported by Wu and

252

Sessa.8 At a DTT/SS molar ratio of 1/4, the far-UV CD spectra were obviously changed compared

253

to the nonreduced BBI-A, which might explain the quick decrease in TIA and CIA in the first

254

stage (Figure 3). From DTT/SS molar ratio of 1/4 to 8/1, the far-UV CD spectra slowly changed 12


Page 13 of 29


255

and showed a blue shift until all the SS were reduced, which was also in agreement with the

256

results reported by Wu and Sessa.8

257

In the BBI-A molecule, there are two Tyr residues (Tyr45 and Tyr59) and two Phe residues

258

(Phe50 and Phe57), which are exposed to the exterior. In addition, five SS are also exposed to the

259

exterior, while BBI-A does not have Trp residues. It is known that the spectra in the region of

260

260-320 nm arise from aromatic amino acids and SS. Figure 4B shows a minimum at 278 nm and

261

broad absorption bands between 250 and 260 nm. The former one is from the Lb transition of two

262

exposed Tyr, and the latter one is attributed to the five exposed SS and two exposed Phe.25 By

263

reducing SS, a blue shift in the minimum from 278 to 272 nm with a considerable loss in the

264

signal occurred, and the broad absorption bands between 250 and 260 nm obviously decreased.

265

These results revealed that the tertiary structure of BBI-A gradually changed with DTT/SS molar

266

ratio from 0 to 2/1 and slowly changed afterwards. In addition, the results above also meant that

267

the two Tyr and two Phe were buried after the reduction of SS, indicating that the five exposed SS

268

were important for the conformation of BBI-A, and the two buried SS also affected it to some

269

extent. This should be correlated with the structural peculiarity of BBI-A, i.e., two hydrophobic

270

patches are exposed to the exterior,15 which can explain the formation of BBI dimer to some

271

extent.5,26 The two Phe and two Tyr above were located in the two hydrophobic patches. Therefore,

272

it was reasonable to consider that hydrophobic collapse of the two exposed hydrophobic patches

273

into a regular hydrophobic core occurred after the reduction of SS.15,16 This also could be used to

274

explain the residual TIA and CIA after the reduction of SS. According to the conformation of

275

BBI-A, trypsin reactive site (KSNPP) is more hydrophilic than chymotrypsin reactive site

276

(LSYPA). As a result, it is highly possible that the trypsin reactive site tends to be exposed to the 13



Page 14 of 29

277

exterior after the reduction of SS, whereas the chymotrypsin reactive site tends to be buried.

278

However, it should be noted that the interaction between the trypsin reactive site and trypsin was

279

weakened due to the conformational change of BBI-A.

280

Effects of stepwise reduction of SS on BBI-A band on Tricine-SDS-PAGE gel. Figure 5

281

shows that there were one intense band and one weak band with apparent molecular weights of

282

approximately 10 kDa, in agreement with the results in Figure 1. Unexpectedly, the intensity of

283

the BBI-A band decreased from DTT/SS molar ratio of 0 to 1/1 and then increased (Figure 5B). It

284

was reported that the intensity of the Coomassie Brilliant Blue G-250 stained protein band was

285

approximately proportional to the number of basic amino acid residues (lysine and arginine) of

286

proteins.27, 28 BBI-A has five Lys (Lys6, Lys16, Lys37, Lys63, and Lys69) and two Arg (Arg23

287

and Arg28), and the X-ray structure showed that only Arg28 was buried due to its electrostatic

288

interaction with Asp26 and His33.15 In addition, there are eight Asp (Asp1, Asp2, Asp10, Asp26,

289

Asp53, Asp56, Asp67, and Asp68) and four Glu (Glu3, Glu60, Glu66, and Glu70) in BBI-A.

290

Therefore, it can be suggested that the positively and negatively charged residues above were

291

gradually buried via electrostatic interaction with increasing reduction of the five exposed SS, and

292

then were partially exposed to the exterior with further reduction of the two buried SS; the

293

exposed basic amino acid residues of nonreduced BBI-A were more than those of the totally

294

reduced one. In addition, the partially reduced BBI-A (from DTT/SS molar ratio of 0 to 1/1) had

295

the same electrophoretic mobility as nonreduced BBI-A, whereas partially and totally reduced

296

BBI-A (from DTT/SS molar ratio of 2/1 to 8/1; approximately 12 kDa) showed smaller

297

electrophoretic mobility than nonreduced BBI-A. These results showed that the conformation of

298

BBI-A gradually changed from DTT/SS molar ratio of 0 to 1/1 and substantially changed after 2/1, 14


Page 15 of 29


299

indicating that the five exposed SS were key for maintaining the native conformation of BBI-A.

300

This was also in good agreement with the results above.

301

Effects of stepwise reduction of SS on the chromatographic profile of BBI-A. In this study,

302

the chromatographic profiles of BBI-A solutions were obtained at 214 nm. It was reported that the

303

absorbance at 214 nm was affected not only by peptide bonds and amino acid residues of

304

proteins,29 but also by conformations of proteins.30 Therefore, it was considered that the

305

chromatographic profiles obtained at 214 nm could also be used to examine the conformational

306

change of BBI-A. As stated above, the amino acid residues contributed to the absorbance at 214

307

nm, and it was clarified that Pro (not at the N terminus), Tyr, Phe, His, and Trp residues had much

308

higher molar extinction coefficients than the other amino acid residues.29 BBI-A has two Tyr

309

(Tyr45 and Tyr59), two Phe (Phe50 and Phe57), six Pro (Pro7, Pro19, Pro20, Pro46, Pro61, and

310

Pro64), and one His (His33), and only His33 is buried. Among them, two Tyr, two Phe, and four

311

Pro are located in the two exposed hydrophobic patches (right-hand side: Met27, Leu29, Ile40,

312

Tyr45, Phe50, Val52, and Pro46; left-hand side: Pro7, Pro19, Pro20, Phe57, and Tyr59).15

313

Figure 6A shows that nonreduced BBI-A possessed two peaks around 27 min, and the area of

314

the two peaks gradually decreased from DTT/SS molar ratio of 0 to 1/1, and disappeared at 2/1. At

315

a DTT/SS molar ratio of 4/1, two peaks appeared around 36 min, which obviously increased at a

316

DTT/SS molar ratio of 8/1. Therefore, it was suggested that the two exposed hydrophobic patches

317

were gradually buried during the reduction of the five exposed SS and were exposed to the

318

exterior to some extent after further reduction of the two buried SS. This meant that BBI-A had the

319

lowest molar extinction coefficient when the five exposed SS were reduced, and the nonreduced

320

BBI-A had the largest molar extinction coefficient. These results also revealed that the 15



Page 16 of 29

321

conformation of BBI-A gradually changed from DTT/SS molar ratio of 0 to 1/1 and substantially

322

changed after 2/1.

323

BBI-A has seven SS. Among them, five are exposed to the exterior, whereas the other two are

324

buried. Due to steric hindrance, DTT could not attack the two buried SS at the beginning.

325

According to the results in Figure 2, the five exposed SS might be attacked by DTT equally. It was

326

found that the conformation of BBI-A could be changed to some extent after reduction of one

327

exposed SS (Figures 3 and 4A). With increasing reduction of SS, the conformation of BBI-A was

328

continually changed, and substantially changed after all exposed SS were reduced. Moreover, it

329

should be noted that the two buried SS should be gradually exposed to the exterior during the

330

reduction of the five exposed SS. As a result, the two buried SS were also reduced by DTT

331

afterwards, and the conformation of BBI-A continually changed to some extent. With the

332

conformational change of BBI-A, TIA and CIA of BBI-A changed correspondingly. Overall, this

333

study supplied much evidence supporting the hypothesis that hydrophobic collapse of the exposed

334

hydrophobic patches into a regular hydrophobic core occurred after the reduction of SS in BBI-A.

335

Finally, this study recommends that nonreducing Tricine-SDS-PAGE should be used to detect BBI

336

due to its high quality.

337 338

AUTHOR INFORMATION

339

Corresponding Author

340

Yeming Chen

341

Phone/Fax: 86-0510-85329091

342

E-mail: [email protected] 16


Page 17 of 29


343

Funding

344

This study was supported by the National Great Project of Scientific and Technical Supporting

345

Programs of China (No. 2012BAD34B04-1), and Hi-tech Research and Development Program of

346

China (No. 2013AA102204-3).

347

Notes

348

The authors declare no competing financial interest.

349

REFERANCES

350

(1) Liener, I. E. Legume toxins in relation to protein digestibility-a review. J. Food Sci. 1976, 41,

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1076–1081.

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(2) Kennedy, A. R. The Bowman-Birk inhibitor from soybeans as an anticarcinogenic agent. Am. J.

353

Clin. Nutr. 1998, 68, 1406–1412.

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(3) Cruz-Huerta, E.; Samuel Fernández-Tomé, S.; Arques, M. C.; Amigo, L.; Recio, I.; Clemente,

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A.; Hernández-Ledesma, B. The protective role of the Bowman-Birk protease inhibitor in soybean

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lunasin digestion: the effect of released peptides on colon cancer growth. Food & Funct. 2015, 6,

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2626–2635.

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(4) Park, J. H.; Jeong, H. J.; de Lumen, B. O. In vitro digestibility of the cancer-preventive soy

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peptides lunasin and BBI. J. Agric. Food Chem. 2007, 55 (26), 10703–10706.

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(5) Birk, Y. The Bowman-Birk inhibitor. Trypsin- and chymotrypsin-inhibitor from soybeans. Int.

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J. Pept. Protein Res. 1985, 2, 11113–11131.

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(6) Xu, Z.; Chen, Y.; Zhang, C.; Kong, X.; Hua, Y. The heat-induced protein aggregate correlated

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with trypsin inhibitor inactivation in soymilk processing. J. Agric. Food Chem. 2012, 60,

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8012−8019. 17



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(7) Chen, Y.; Xu, Z.; Zhang, C.; Kong, X.; Hua, Y. Heat-induced inactivation mechanisms of

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Kunitz trypsin inhibitor and Bowman-Birk inhibitor in soymilk processing. Food Chem. 2014, 154,

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108−116.

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(8) Wu, Y. V.; Sessa, D. J. Conformation of Bowman-Birk inhibitor. J. Agric. Food Chem. 1994,

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42, 2136−2138.

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(9) Sessa, D. J.; Nelsen, T. C. Chemical inactivation of soybean protease inhibitors. J. Am. Oil

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Chem. Soc. 1991, 68 (7), 463−470.

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(10) Jiao, J.; Yee, B. C.; Kobrehel, K.; Buchanan, B. B. Effect of Thioredoxin-linked reduction on

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the activity and stability of the Kunitz and Bowman-Birk soybean trypsin inhibitor proteins. J.

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Agric. Food Chem. 1992, 40, 2333−2330.

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(11) Odani, S.; Ikenaka, T. Studies on soybean trypsin inhibitors. X. Isolation and partial

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characterization of four soybean double-headed proteinase inhibitors. J. Biochem. 1977, 82,

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1513−1522.

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(12) Deshimaru, M.; Yoshimi, S.; Shioi, S.; Terada, S. Multigene family for Bowman-Birk type

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proteinase inhibitors of wild soja and soybean: the presence of two BBI-A genes and pseudogenes.

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Biosci. Biotech. Biochem. 2004, 68, 1279−1286.

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(13) Odani, S.; Ikenaka, T. Studies on soybean trypsin inhibitors. XII. Linear sequences of two

382

soybean double-headed trypsin inhibitors. J. Biochem. 1978, 83, 737−745.

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(14) Clemente, A.; Moreno, F. J.; Marin-Manzano, M. C.; Jimenez, E.; Domoney, C. The cytotoxic

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effect of Bowman-Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29

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human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases. Mol.

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Nutr. Food Res. 2010, 54, 396−405. 18


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(15) Voss, R. H.; Ermler, U.; Essen, L. O.; Wenzl, G.; Kim, Y. M.; Flecker, P. Crystal structure of

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the bifunctional soybean Bowman-Birk inhibitor at 0.28-nm resolution. Eur. J. Biochem. 1996,

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242, 122−131.

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(16) Lins, L.; Brasseur, R. The hydrophobic effect in protein folding. FASEB J. 1995, 9, 535−540.

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(17) Li, X.; Hua, Y.; Chen, Y.; Kong, X.; Zhang, C. The selective complex behavior between

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soybean whey proteins and ι-carrageenan and isolation of the major proteins of the soybean whey.

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Food Hydrocolloid. 2016, 56, 207−217.

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(18) Ruan, Q.; Chen, Y.; Kong, X.; Hua, Y. Comparative studies on sulfhydryl determination of

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soy protein using two aromatic disulfide reagents and two fluorescent reagents. J. Agric. Food

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Chem. 2013, 61, 2661−2668.

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(19) Liu, K. S.; Markakis, P. An improved colorimetric method for determining antitryptic activity

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in soybean products. Cereal Chem. 1989, 66 (5), 415–422.

399

(20) Tan, N. H.; Zubaidah, H. A.; Rahim. H. T. K.; Wong, K. C. Chymotrypsin inhibitor activity in

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winged beans (Psophocarpus tetragonolobus). J. Agric. Food Chem. 1984, 32 (1), 163–166.

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(21) Schägger, H. Tricine-SDS-PAGE. Nat. Protoc. 2006, 1, 16–22.

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(22) Arques, M. C.; Marin-Manzano, M. C.; da Rocha L. C. B.; Hernandez-Ledesma, B.; Recio, I.;

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Clemente, A. Quantitative determination of active Bowman-Birk isoinhibitors, IBB1 and IBBD2,

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in commercial soymilks. Food Chem. 2014, 155, 24−30.

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(23) Ponne, C. T.; Meijer, M. M. T.; Bartels, P. V. Effect of radio frequency energy on the activity

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of Bowman-Birk trypsin/chymotrypsin inhibitor. J. Agric. Food Chem. 1994, 42, 2583–2588.

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(24) Odani, S.; Ikenaka, T. Studies on soybean trypsin inhibitors. XIII. Preparation and

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characterization of active fragments from Bowman-Birk proteinase inhibitor. J. Biochem. 1978, 83, 19



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747−753.

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(25) Sreerama, N.; Manning, M. C.; Powers, M. E.; Zhang, J. X.; Goldenberg, D. P.; Woody, R.

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W. Tyrosine, phenylalanine, and disulfide contributions to the circular dichroism of proteins:

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circular dichroism spectra of wild-type and mutant bovine pancreatic trypsin inhibitor. Biochem.

413

1999, 38, 10814−10822.

414

(26) Bawadi, H. A.; Antunes, T. M.; Shin, F.; Losso, J. N. In vitro inhibition of the activation of

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pro-matrix metalloproteinase 1 (Pro-MMP-1) and pro-matrix metalloproteinase 9 (Pro-MMP-9)

416

by rice and soybean Bowman-Birk inhibitors. J. Agric. Food Chem. 2004, 52, 4730−4736.

417

(27) Tal, M.; Silberstein, A.; Nusser, E. Why does coomassie brilliant blue R interact differently

418

with different proteins. J. Biol. Chem. 1980, 260, 9976−9980.

419

(28) Chial, H. J.; Splittgerber, A. G. A comparison of the binding of Coomassie Brilliant Blue to

420

proteins at low and neutral pH. Anal. Biochem. 1993, 213 (2), 362−369.

421

(29) Kuipers, B. J. H.; Gruppen, H. Prediction of molar extinction coefficients of proteins and

422

peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative

423

reverse phase high-performance liquid chromatography-mass spectrometry analysis. J. Agric.

424

Food Chem. 2007, 55, 5445−5451.

425

(30) Rosenheck, K.; Doty, P. Far ultraviolet absorption spectra of polypeptide and protein

426

solutions and their dependence on conformation. P. Natl. Acad. Sci. USA 1961, 47, 1775−1785.

20


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427

FIGURE CAPTIONS

428

Figure 1. Fractionation of BBI-A solution (2 mg/mL) by C8 column. The absorbance is given in

429

milliabsorbance units (mAu).

430

Figure 2. (A) The effects of DTT/SS molar ratio (0, 1/4, 1/2, 1/1, 2/1, 4/1, and 8/1) on SS in

431

BBI-A. BBI-A was reduced by DTT in a 40 °C water bath for 1 h. (B) The effects of reduction

432

time (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h) on SS in BBI-A. The DTT/SS molar ratio was 1/1, and it

433

was reduced in a 40 °C water bath. The oxidized DTT and unreacted DTT were removed by a

434

desalting column.

435

Figure 3. The effects of stepwise reduction of SS on TIA and CIA of BBI-A. SS reduction was

436

conducted at DTT/SS molar ratios of 0, 1/4, 1/2, 1/1, 2/1, 4/1, and 8/1 in a 40 °C water bath for 1

437

h. The oxidized DTT and unreacted DTT were removed by a desalting column. The two dashed

438

lines divided the effects of stepwise reduction of SS on TIA and CIA into three stages.

439

Figure 4. (A) The far-UV CD spectra of nonreduced and reduced BBI-A solutions (0.5 mg/mL).

440

(B) The near-UV CD spectra of nonreduced and reduced BBI-A solutions (0.5 mg/mL). SS

441

reduction was conducted at DTT/SS molar ratios of 0, 1/4, 1/2, 1/1, 2/1, 4/1, and 8/1 in a 40 °C

442

water bath for 1 h. The oxidized DTT and unreacted DTT were removed by a desalting column.

443

Figure 5. (A) Tricine-SDS-PAGE profiles of nonreduced and reduced BBI-A. (B) Band intensity

444

of BBI-A in Figure 5A. SS reduction was conducted at DTT/SS molar ratios of 0, 1/4, 1/2, 1/1, 2/1,

445

4/1, and 8/1 in a 40 °C water bath for 1 h. The oxidized DTT and unreacted DTT were removed by

446

a desalting column.

447

Figure 6. (A) The C8 column chromatographic profiles of nonreduced and partially reduced

448

BBI-A solutions (2 mg/mL; from DTT/SS molar ratio of 0 to 1/1). (B) The C8 column 21



Page 22 of 29

449

chromatographic profiles of partially and totally reduced BBI-A solutions (2 mg/mL; from

450

DTT/SS molar ratio of 2/1 to 8/1). SS reduction was conducted at DTT/SS molar ratios of 0, 1/4,

451

1/2, 1/1, 2/1, 4/1, and 8/1 in a 40 °C water bath for 1 h. The oxidized DTT and unreacted DTT

452

were removed by a desalting column.

22


Page 23 of 29


453 454

Figure 1.

455

23



100 Reduced disulfide bond/original disulfide bond * 100 (%)

Reduced disulfide bond/original disulfide bond * 100 (%)

100 90 80 70 60 50 40 30 20 10

90 80 70 60 50 40 30 20 10 0

0 0

1

2

3

4

5

6

DTT/disulfide bond in BBI (mol/mol)

456

7

8

0

1

2

3

4

5

6

7

8

9

10

Reduction time (h)

(A)

457 458

Page 24 of 29

(B)

Figure 2.

24


Page 25 of 29


Residual TIA/original TIA * 100 (%) or Residual CIA/original CIA * 100 (%)

100 90

TIA

80

CIA

70 60 50 40 30 20 10 0 0

460

20

40

60

80

100

Percentage of reduced disulfide bond (%)

459 Figure 3.

25



5

Page 26 of 29

2

0 190

200

210

220

230

240

1

250

-10 -15 -20

BBI-0

-25

BBI-1/4

-30

BBI-1/2

-35

BBI-1 BBI-2

-40

BBI-4 -45 Wavelength (nm)

461

0 250

260

270

280

290

300

310

320

-1 BBI-0

-2

BBI-1/4 -3

BBI-1/2 BBI-1

-4

BBI-2 BBI-4

-5 Wavelength (nm)

(A)

462 463

BBI-8

[θ] (deg cm2 dmo1-1)

[θ] (deg cm2 dmo1-1)

-5

BBI-8

(B)

Figure 4.

464

26


Page 27 of 29


kDa 75 50

120000000

25 20 15

Totally reduced BBI-A

10

Band intensity of BBI-A

100000000

37

80000000 60000000 40000000 20000000

Partially reduced BBI-A

Nonreduced BBI-A

0 0

465

DTT/SS (mol/mol)

1/4

1/2

1/1

2/1

4/1

8/1

1/2

1/1

2/1

4/1

8/1

DTT/disulfide bond in BBI-A (mol/mol)

(A)

466 467

0

1/4

(B)

Figure 5.

27



468 469 470

(A)

Page 28 of 29

(B)

Figure 6.

28


Page 29 of 29


471

TOC Graphic

472

29


Effects of Disulfide Bond Reduction on the Conformation and Trypsin

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