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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
3 4
Hui He, Xingfei Li, Xiangzhen Kong, Caimeng Zhang, Yufei Hua, Yeming Chen*
5 6
State Key Laboratory of Food Science and Technology, School of Food Science and Technology,
7
Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, PR China
8 9
Corresponding Author:
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Yeming Chen
11
Telephone/Fax: 86-510-85329091. E-mail:
[email protected] 12
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ABSTRACT: Soybean seeds contain three groups (A, C, and D) of Bowman-Birk inhibitors
14
(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
28
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
30
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
33
SS in BBI-A.
34
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
53
BBI-A in its inhibitory specificity and amino acid composition,11, 12 and BBI-E-I is a variant of
54
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
61
five SS (Cys14-Cys22, Cys41-Cys49, Cys12-Cys58, Cys32-Cys39, and Cys8-Cys62) were
62
exposed to the exterior, and the other two (Cys9-Cys24 and Cys36-Cys51) were buried; in
63
addition, BBI-A did not exhibit a typical protein conformation with a hydrophobic core and a
64
hydrophilic surface exposed to the exterior, but contained two exposed hydrophobic patches.15
65
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
69
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
71
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
76
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,
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China). All other reagents were of analytical grade.
(BTpNA),
4-nitroanilide sodium
hydrochloride
dodecyl
sulfate
(BAPA),
(SDS),
N-benzoyl-L-tyrosine
dithiothreitol
(DTT),
and
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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
91
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.
96
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
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was collected and adjusted to pH 7.0 by 0.5 M KOH. By ultrafiltration (100 kDa cut off), the
103
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.
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Reduction of disulfide bond by DTT at different levels. DTT was added into BBI-A solution
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(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
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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%
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(w/v). The mixture was vortexed and kept at room temperature for 30 min. Then each sample was 6
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detected in triplicate at 324 nm against a sample blank and a reagent blank in a UV-2450 UV-VIS
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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
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BBI-A, and the sample blank was the above mixture without the DPS solution. The SH content
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was expressed as µmol SH/g protein by equation 1.
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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
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nm, respectively. ε is the molar extinction coefficient of 4-thiopyridone in the presence of 2% SDS
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(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,
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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
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water were added to a test tube and placed in a 37 °C water bath for 10 min. Five milliliters of
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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
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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)
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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.
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TIA (TIU/mg) = [(A410sb + A410rb – A410s)/0.01]* (D/C)
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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.
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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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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
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(Bio-Rad, USA).
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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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research by Clemente et al.,14 it was suggested that the two major peaks were the BBI-A and -B.
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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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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
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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
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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
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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
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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
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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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Clin. Nutr. 1998, 68, 1406–1412.
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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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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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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
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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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(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.
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(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.
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1999, 38, 10814−10822.
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(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)
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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
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proteins at low and neutral pH. Anal. Biochem. 1993, 213 (2), 362−369.
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(29) Kuipers, B. J. H.; Gruppen, H. Prediction of molar extinction coefficients of proteins and
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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.
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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.
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FIGURE CAPTIONS
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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
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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
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Figure 1.
455
23
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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
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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
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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
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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
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468 469 470
(A)
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(B)
Figure 6.
28
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TOC Graphic
472
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