Some 2S albumin from peanut seeds exhibits inhibitory activity against Aspergillus flavus

Plant Physiology and Biochemistry 66 (2013) 84e90

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Research article

Some 2S albumin from peanut seeds exhibits inhibitory activity against Aspergillus flavus Xiao Hua Duan 1, Rui Jiang 2, Yun Jie Wen 3, Jin Hua Bin* College of Life Sciences, South China Normal University, Guangdong Key Lab of Biotechnology for Plant Development, Guangzhou 510631, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 July 2012 Accepted 25 January 2013 Available online 21 February 2013

A crude 2S albumin fraction was separated from peanut (Arachis hypogaea L.) cotyledons. Untreated 2S albumin had little inhibitory activity against trypsin, spore germination, or hyphal growth of Aspergillus flavus. However, following treatment of 2S albumin with SDS, increased inhibitory activity was demonstrated. We further purified 2S albumin using Sephadex G-100 and DEAE cellulose (DE-32) chromatography. HPLC analysis showed that the partially pure 2S albumin consisted of two polypeptides, whereas SDS-PAGE analyzes exhibited six polypeptides. One of the polypeptides, 2S-1, was found to contain the same molecular weight and enzymatic properties as the peanut protease inhibitor (PI); however, the N-terminal amino acid sequence of 2S-1 differed from that of PI. An NCBI database search revealed that the 2S-1 polypeptide is homologous to the pathogenesis-related proteins from soybean, cowpea, chickpea, and Lupinus luteus. We hypothesize that the 2S-1 polypeptide might represent a novel antifungal protein. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: 2S albumin Activation of protein Antifungal peptide Aspergillus flavus Peanut seed Protein purification

1. Introduction Peanut (Arachis hypogaea L.) is a leguminous plant and its seeds are rich in storage proteins and oils. There are three kinds of storage proteins in peanut seeds: arachin, coarachin I, and coarachin II (2S albumin). Peanut 2S albumins were precipitated in 65e85% ammonium sulfate solution, and were first estimated to represent approximately 20% of the total soluble protein in the peanuts [38]. Subsequently, Shewry and Pandya described 2S albumin as a storage protein with additional biological functions, such as enzyme inhibitor, antifungal protein (AFP), calmodulin antagonist, sweet protein, and allergen [28]. Terras et al. showed that napins (2S albumin) from radish (Raphanus sativus L.) inhibit the growth of a number of plant pathogenic fungi, but the concentrations required for 50% growth inhibition were high [31]. The antifungal activities of napins were found to be enhanced in the presence of thionin [4].

* Corresponding author. E-mail address: [email protected] (J.H. Bin). 1 Present address: Jiangxi Subtropical Plant Resource Protection and Utilization Key Laboratory, College of Life Science, Jiangxi Normal University, Nanchang 330022, PR China. 2 Present address: Shunde No. 1 High School, Nanguo Road, Daliang Town, Shuande District, Foshan 528300, Guangdong Province, PR China. 3 Present address: Guangzhou Huayin Clinical Pathology Center, 2nd Floor, Life Science Building, Southern Medical University, 1023 Shatai Nan Road, Baiyun District, Guangzhou 510515, Guangdong Province, PR China. 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.01.015

Recent reports have demonstrated that 2S albumin has antifungal activity [1,2,20,25,31,32]. Peanut is a healthy food source due to its abundant proteins and essential amino acids, and is favored by many people. However, peanuts are easily contaminated by Aspergillus flavus and Aspergillus parasiticus, which produce aflatoxins that can induce liver cancer [15]. To date, only a few of the AFPs from peanut seeds had been isolated, such as hypogin [37], hypotin [35], and Ah PR10 [9], and their effect on A. flavus has not yet been demonstrated. In this study, we found that SDS-treated 2S albumin could inhibit the growth of A. flavus. Therefore, we purified 2S albumin from peanut seeds, characterized its properties, and found that one of its components might represent a novel class of AFP. 2. Results 2.1. Inhibitory activity of peanut 2S albumin against endopeptidase and the growth of A. flavus Crude 2S albumin treated with SDS strongly inhibited peanut cotyledon endopeptidase, whereas untreated 2S albumin demonstrated little inhibitory activity (Fig. 1). SDS is an anionic detergent, and although it can denature proteins, it did not inhibit the growth of A. flavus, which was demonstrated by the lack of an inhibition zone around discs containing 30e150 mg disc 1 of SDS (Fig. 2I). Similarly, natural crude 2S albumin did not inhibit the growth of

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2.2. Purification of 2S albumin from peanut seeds To determine the functional protein responsible for the inhibition of endopeptidase and A. flavus growth, the crude 2S albumin fractions were applied to a Sephadex G-100 column. Fig. 3A shows the chromatographic profile obtained. The two active peaks were collected, pooled, and dialyzed, and were further purified on a DEAE-Cellulose column (Fig. 3B and C, respectively). Initially, there was no trypsin inhibitory activity observed for the proteins from natural peak 1, but their activity was greatly increased by SDS treatment. In contrast, the natural peak 2 proteins showed inhibitory activity. The active fractions from the DEAE-Cellulose column were collected, dialyzed, and dried at 4  C to obtain purified 2S albumin, which were named as partially pure 2S albumin. The purification procedure is shown in Table 1. 2.3. Characteristics of partially pure 2S albumin

Fig. 1. Inhibition of peanut crude 2S albumin against peanut cotyledon endopeptidase. :: natural 2S albumin, B: treated 2s albumin with SDS.

A. flavus (Fig. 2II). However, after the 2S albumin was treated with SDS, we observed inhibition with 30e100 mg protein disc 1, suggesting that the inhibitory activity of the 2S albumin was markedly increased (Fig. 2III).

HPLC was used to analyze 2S albumin that had been purified on two columns. The analysis revealed two major peaks and a little peak (the third peak and its retention time was about 4.5 min) in the profile (Fig. 4). As the third peak was so low and only two bands were present on the gel after analysis by polyacrylamide gel electrophoresis (PAGE, data not shown), it was considered a contaminating protein which indicates that the natural partially pure 2S albumin consisted of two different protein molecules. Six bands were noted after SDS-PAGE, one having high molecular weight and five having low molecular weight, and were about 67.0, 22.0, 20.8,

Fig. 2. Effect of SDS (I) and peanut crude 2S albumin (II, III) on the growth of A. flavus. I: A, PBS buffer; BeF, SDS contents were 30, 60, 70, 120, and 150 mg disc 1. II: A, PBS buffer; Be G natural 2S albumin. III: A, PBS buffer; BeG treated 2S albumin by SDS. The protein contents (BeG) were 10, 30, 50, 70, 90, and 100 mg disc 1 respectively in II or III.

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Fig. 3. Purification of 2S albumin from peanut seeds. The detailed experimental procedures are described in the Methods and materials section. A: profile on Sephadex G-100, B: profile of peak 1 proteins on a DEAE-Cellulose column, C: peak 2 proteins on a DEAE-Cellulose column. :: absorbance at OD 280, B: inhibitory activity of 2S albumin treated with SDS, ,: NaCl.

19.2, 17.8, and 7.1 kDa, respectively (Fig. 5). Thus, these results suggest that the partially pure 2S albumin of peanut does not comprise a pure monomeric protein, but two protein molecules and six polypeptides. The inhibitory activity of partially pure 2S albumin was not altered by incubation at 30e100  C for 10 min or at a pH of 2.0e11.0 (Fig. 6), suggesting that 2S albumin is thermostable and tolerant to a wide pH range. 2.4. Inhibitory activity of partially pure 2S albumin Similar to crude 2S albumin, partially pure 2S albumin treated with SDS strongly inhibited the endopeptidase activity of 5-day-old peanut seedlings, whereas natural, partially pure 2S albumin exhibited little inhibitory activity (data not shown). In addition, partially pure 2S albumin inhibited spore germination and hyphal growth of A. flavus (Fig. 7). Treated 2S albumin caused a prominent inhibition zone surrounding the discs (Fig. 7D), while a weaker inhibition zone was present around the discs containing natural 2S albumin (Fig. 7E), and there was no inhibition zone associated with

crude 2S albumin (Fig. 7C). These results strongly indicate that 2S albumin inhibits spore germination and hyphal growth of A. flavus. 2.5. Amino acid composition and N-terminal sequence of purified 2S-1 proteins The 2S-1 polypeptide, which exhibited the lowest molecular weight among the partially pure proteins (approximately 7.1 kDa; Fig. 5), was excised from an SDS-PAGE gel, and the polypeptides were subsequently extracted to analyze the amino acid composition and sequence of the N-terminus. Table 2 shows that similar to 2S albumin, the 2S-1 polypeptide is rich in glutamic acid (300.0 mg g 1), arginine (145.0 mg g 1), and aspartic acid

Table 1 Purification of 2S albumin from peanut seeds. Purification Yield (%) Purification steps Protein Total inhibitory Specific (mg) activity (U) inhibitory (N-fold) activity (U mg 1) Defatted powder Crude 2S Sephadex G-100 DEAECellulose

35,452

56,7232

1.6

1

10,500 253

39,900 14,168

3.8 56

2 35

7 2.5

48

6576

86

1.2

137

100

Inhibitory activity against trypsin was measured after the proteins were treated with SDS.

Fig. 4. HPLC analysis of partially pure 2S albumin.

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Fig. 7. Inhibition of purified 2S albumins against the spore germination and hypha growth of A. flavus. A: 100 ml sterile distilled water, B: 100 ml 50 mM TriseHCl buffer (pH 7.5), C: 50 mg crude 2S albumin, D: 50 mg partially pure 2S albumin treated with SDS, E: 50 mg natural partially pure 2S albumin. The inhibition zones are marked by the red arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. SDS-PAGE analysis of purified 2S albumin. A: crude 2S albumin, B: after Sephadex G-100 column purification, C: partially pure 2S albumin, D: molecular weight marker.

(118.0 mg g 1). However, unlike 2S albumin, methionine, tyrosine, and phenylalanine were undetectable in the 2S-1 polypeptide. The N-terminal amino acid sequence of the 2S-1 polypeptide was determined to be GVFTFEEESTSPVPPACLF. A sequence homology search using the NCBI database revealed that the 2S-1 polypeptide shares a significant homology with an allergen (Ara h8) and pathogenesis-related proteins in A. hypogaea (Table 3). 3. Discussion Most of the plant seeds are rich in nutrients and energy sources, such as proteins, starches, and oils (storage matter) [3]. These reserves are mobilized to supply the seedling with energy during

growth and germination. Many animals and microbes, including humans, mice, insects, worms, and fungi feed on plant seeds. Plants protect themselves using a variety of pre-formed and inducible defensive mechanisms. One of these mechanisms includes the use of AFPs [8,16,21]. AFPs have been identified in all plant tissues studied; even latex [11] and a fruit peel [13] and seeds have been proven to be a particularly rich source of proteins and peptides with such activity (e.g., defensins, ribosome inactive proteins, lectins, and thionin) [7,18,22,27,29,39]. The biological activity of several types of proteins, such as enzyme inhibitors and AFPs, has been studied in vitro [28]. Legume storage proteins play a role in defending seeds against bruchids [30]. Barley 2S albumin has been found to inhibit the activity of trypsin [34]. Moreover, the 2S albumin fraction from radish has been shown to possess antifungal properties in vitro [31]; however, none of the fungi tested were affected by 2S albumin at concentrations of up to 1000 mg ml 1, and the IC50 of 2S albumin was found to be higher than that of the defensin characterized in the same study. This result suggests that the inhibitory activity of radish 2S albumin is weak. Peanut seeds contain abundant 2S albumin protein. However, although 2S albumin is regarded as a storage protein, its function is not well understood. Previously, we found that partially pure 2S albumin exhibited no inhibitory activity against purified endopeptidase from peanut seeds [4]. In this study, we observed that both crude 2S albumin and partially pure 2S albumin could inhibit endopeptidase activity (Fig. 1) and the growth of A. flavus (Fig. 2)

Fig. 6. Effect of temperature (A) and pH (B) on the inhibitory activity of partially pure 2S albumin.

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Table 2 Amino acid composition of partially pure 2S albumin and the 2S-1 polypeptide (mg g 1). Amino acid

2S-1

2S

Amino acid

2S-1

2S

Asp Thr Ser Glu Gly Ala Ile Val Pro

118.0 37.0 60.9 300.0 45.0 31.8 24.5 33.6 48.0

125.8 16.17 41.32 323.9 26.95 17.61 25.15 28.75 11.32

Met Cys Leu Tyr Phe Lys His Arg

Undetectable 9.0 52.7 Undetectable Undetectable 75.4 20.0 145.0

46.70 10.78 68.27 23.36 26.95 30.54 15.27 161.7

when treated with SDS. Further analysis revealed that the partially pure 2S albumin comprised two subunits (Fig. 4) consisting of six polypeptides with molecular weights of 67.0, 22.0, 20.8, 19.2, 17.8, and 7.1 kDa (Fig. 5). These results suggest that one of these polypeptides has an inhibitory function. Marcus et al. [19] isolated a new family of 4-kDa cysteine-rich antimicrobial peptides from germinating seedlings of Macadamia integrifolia, which were found to be produced from a large precursor protein related to the 7S globulins of other plants, suggesting that some vicilins play a role in defense during seed germination. Guo et al. [14] provided evidence of protein induction in corn kernels during imbibition or early stages of germination, and the induced proteins might be related to germination-associated resistance in the corn kernel, especially in susceptible kernels. Plant roots might also secrete a variety of proteins that function to repress the growth of root pathogen fungi [23,36]. The antifungal properties of 2S albumin homologs from passion fruit seeds include inducing plasma membrane permeabilization and ultrastructural alterations in yeast cells [2]. When we investigated the mechanism behind 2S albumin-mediated inhibition of A. flavus growth, we found that the hyphae and spores internalized the polypeptide, and plasma membrane permeability was disturbed (data not shown). During plant seed germination, a regulated biochemical process takes place, which results in the generation of multiple peptides or proteins with antifungal activities [12,36].

Sunflower 2S albumin maybe does not play a role in protecting the seeds from fungal pathogens [26], and thionin from the radish storage organ displays weak antifungal activity against Fusarium culmorum in vitro [33]. Similarly, in the present study, the antifungal activity of natural 2S albumin was observed to be weak, whereas treatment of 2S albumin with SDS strongly increased its antifungal activity. However, we found that the inhibitory activity of peanut 2S albumin against trypsin was greatly decreased by treatment with bmercaptoethanol (data not shown), suggesting that the inhibitory activity of 2S albumin could be related to the oxidation of thiol groups. Therefore, we hypothesize that the 2S albumin of peanut seeds might exhibit a function similar to that of vicilins and could activate AFPs by protein degradation (specific cutoff) or specific modifications during germination. This 2S albumin inhibitory activity could also be present during fungal aggression. The inhibitory activities of 2S albumin were not significantly altered at high temperatures and in acid or alkaline conditions (Fig. 6), suggesting that this protein is highly stable and is expected to generally be able to function during pathogen aggression, regardless of the environmental conditions. Antimicrobial peptides are usually enriched with a specific amino acid. For example, anionic peptides are rich in Asp and Glu, and cationic peptides are rich in Arg and Lys [6]. In this study, Glu, Arg, and Asp were also present in the partially pure 2S albumin and 2S-1 polypeptides (Table 2), thereby confirming the hypothesis of peanut seed 2S albumin representing a candidate antipathogenic agent. In a previous study, an antifungal peptide (hypogin) has been purified from peanut seeds, and its N-terminal sequence is KSPYYQKKTENPQAQRQLQSDDQEPAKLK [35]. Although this sequence differs from the N-terminal sequence of the 2S-1 polypeptide (Table 3), it resembles the sequence of the peanut allergen Ara h1. The molecular weights of hypogin and 2S-1 are 7.2 and 7.1 kDa, respectively; hence, they are different proteins. We analyzed the N-terminal sequence of a peanut protease inhibitor (PI, data not shown), which was also found to differ from that of the 2S-1 polypeptide, suggesting that they are two different proteins. A pathogenesis-related protein (Ah PR10) was previously identified from a 6-day-old peanut cDNA library [9]. The clone was

Table 3 N-terminal amino acid sequences of the 2S-1 polypeptide and other proteins (results of a BLAST Search).

Position and amino acid sequence of the

Proteins

2S-1 polypeptide from peanut seeds Ara h 8 allergen isoform [Arachis hypogaea] PR10 protein [Arachis hypogaea] Unnamed protein product [Glycine max] Protein PR-L6 PR protein 1 (PvPR1)

N-terminus 1 G 2 2 2 1 1 -

Cowpea PR protein 3 (CpPR3) Mal d 1.0902 [Malus x Domestica] Mal d 1.0903 [Malus x Domestica] Major allergen Mal d 1.09 [Malus x Domestica] Ara h 8 allergen [Arachis hypogaea]

2 2 2 2 -

Score

19 F 20 - - K 47.7 20 - - K 47.7 20 - - T 42.2 Y 16 41.4 19 - - T 41.4 Y 20 - - R 40.9 Y 20 - - R 40.5 20 - - R 40.5 20 - - R 40.5 17 - 40.1 -

E

Sequence

Value identity

V F T F E E E S T S P V P P A C L - - H - - -

- - - - - -

-

- - H - - -

- - - - - -

-

I - - - - D - - - - - - A - - - - - -

- - - - T - A

- - - - - D Q T - - - - A 3 - - - - D - P - - Y -

-

- - - Y - S - - - V - -

-

- - Y - S -

-

- - - V - -

- - - Y - S - - - - V I - - - - - D - I - - T -

-

1e-04

89%

1e-04

89%

0.005

68%

0.009

81%

0.009

68%

0.011

72%

0.015

73%

0.015

73%

0.015

73%

0.021

81%

“-” Indicates that the amino acid is the same as in the 2S-1 polypeptide sequence at that position

X.H. Duan et al. / Plant Physiology and Biochemistry 66 (2013) 84e90

found to be expressed as a 20-kDa protein in Escherichia coli, and thus, is obviously different from the 2S-1 polypeptide (7.1 kDa). The 2S-1 polypeptide most closely resembles the PR10 proteins of A. hypogaea in its N-terminal sequence (89% identity, Table 3). Thus, the 2S-1 polypeptide might represent a novel AFP that participates in plant defense-related processes. Furthermore, Odintsova et al. reported that the 2S albumin of dandelion seeds represents a novel example of storage protein with defense functions [25]. Considering the above-mentioned findings as a whole, we believe that the 2S albumin of peanut seeds exhibits an inhibitory activity against A. flavus, and this antifungal role is present in the complex stored protein of the seed, and can be activated and released during seed germination. 4. Materials and methods 4.1. Purification of 2S albumin from peanut cotyledons Peanut seeds were ground into powder and immersed in hexane (20 ml g 1) to remove the lipids. The defatted powder (250 g) was resuspended by mixing with 20 mM phosphate buffer (pH 7.9, containing 100 mM NaCl and 10 mM b-mercaptoethanol) at 4  C for 4 h, and centrifuged at 8000 g for 20 min to remove undissolved materials. Ammonia sulfate was added to the supernatant for 3 h at 4  C to achieve 65% relative saturation. The undissolved materials were pelleted by centrifugation, and the supernatant was collected; ammonia sulfate was added to 85% relative saturation. After allowing the solution to stand for 3 h at 4  C, it was centrifuged and the pellet was resuspended in phosphate buffer and dialyzed against double distilled water using dialysis tubing with a molecular weight cutoff of 1000 Da at 4  C overnight (this dialysis tubing was also used in the other dialysis steps discussed in the following paragraphs). The protein solution was dried at 4  C and the obtained protein fraction was named as crude 2S albumin, and its inhibitory activity against peanut endopeptidase and A. flavus was tested. To further purify the crude fraction, natural 2S albumin was resuspended in phosphate buffer as described earlier and was centrifuged at 10,000 g at 4  C for 20 min. The supernatant was loaded onto a Sephadex G-100 gel filtration column (2.5  50 cm) followed by washing with phosphate buffer at a flow rate of 25 ml h 1. The elution profile was monitored by UV absorption at 280 nm. The fractions containing inhibitory proteinase activities were pooled and loaded onto a DEAE-cellulose ion exchange column (2.5  50 cm) and was subsequently eluted using a 0e0.4 M NaCl gradient in 25 mM TriseHCl buffer (pH 8.0). The flow rate was 25 ml h 1 and elution was monitored at 280 nm. The fractions with inhibitory proteinase activity were pooled and dialyzed against double distilled water at 4  C overnight. The dialyzed protein solution was frozen and dried to obtained purified proteins (were named as partially pure 2S albumin). The partially pure 2S albumin was subsequently used for characterization analyzes. 4.2. Determination of 2S albumin protein concentration The protein concentration was determined using the Bradford [5] method with bovine serum albumin as a reference protein.

89

inhibitory activity of the protein against endopeptidase from peanut cotyledons and growth of A. flavus. 4.4. Proteinase inhibitor activity test We measured the partially pure 2S albumin-mediated inhibition of peanut seed endopeptidase according to the method described by Norioka et al. [24]. The endopeptidases used for these tests were purified from the cotyledons of peanut seeds that had been germinating for 5 days [10]. 4.5. Assays for antifungal activity The antifungal activity of the crude 2S albumin was assayed on sterile Petri dishes (diameter of 90 mm) containing 5 ml of Czapek medium. After the development of mycelial colonies, sterile filter paper discs (5-mm diameter) were placed at a distance of 1 cm from the edges of the colonies. The sterile discs were laid on the surface of the media, and 50 ml of crude 2S albumin solution (protein concentrations were 0.2e2.0 mg ml 1) was applied to the discs. The plate was then incubated at 25  C and the growth of the hyphae was monitored. The assay to determine the effect of SDS on A. flavus was performed as described earlier. We applied 50 ml of SDS solution (SDS contents were 0.6e3 mg ml 1) to the discs. Subsequently, the plates were incubated at 25  C and the growth of the hyphae was monitored; the control was 50 ml 50 mM TriseHCl buffer (pH 7.5). The partially pure 2S albumin was assayed for its ability to inhibit spore germination and growth of A. flavus. We dispersed 0.5 ml of spore solution (107 spore ml 1) onto the plates using a dauber and laid sterile filter paper discs (7 mm of diameter) onto the surface of the media. We subsequently applied 50 mg of partially pure 2S albumin treated with or without SDS and the crude 2S albumin to the discs, respectively. Next, the plates were incubated at 25  C and the growth of hyphae was monitored. A 100 ml sterile distilled water disc or 100 ml 50 mM TriseHCl buffer (pH 7.5) disc was used as control. 4.6. HPLC analysis An SGE-C18 column (Bio-Rad Company, Hercules, CA, USA; 0.45  25 cm) was used for HPLC analysis of the partially pure 2S albumin. The column was equilibrated with 25 mM TriseHCl buffer (pH 8.0) before sample injection. The sample was eluted with a 0e 100% watereacetonitrile solution at a flow rate of 1 ml min 1. The retention time was recorded, with the acetonitrile content measuring about 20%. Elution was monitored by UV absorption at 280 nm. 4.7. Molecular weight of the partially pure protein The molecular weights of partially pure 2S albumin and its components were determined using 12.5% acrylamide SDS-PAGE, according to the method of Laemmli [17]. The reference proteins for this analysis were phosphorylase (97.4 kDa), albumin (66.2 kDa), ovalbumin (43.0 kDa), carbonic anhydrase (31.0 kDa), soybean trypsin inhibitor (20.1 kDa), and lysozyme (14.4 kDa).

4.3. SDS treatment The crude 2S albumin was resuspended in 50 mM TriseHCl buffer (pH 7.5, 2.5% SDS, 20 mM CaCl2) at 4  C overnight. SDS was subsequently removed from the protein using an SDS-OutÔ Precipitation Kit (99.9% of SDS was removed from the treated 2S albumin), and 2S albumin (treated) was used to determine the

4.8. Effects of temperature and pH on the inhibitory activity of 2S albumin The partially pure 2S albumin was resuspended in 50 mM Trise HCl buffer (40 mg ml 1, pH 7.5, 20 mM CaCl2) for 90 min. Then, this protein solution was kept in a water bath at 30e100  C for 30 min,

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and subsequently, the inhibitory activity of 2S albumin was determined [24] against soybean trypsin. The partially pure 2S albumin was resuspended in 50 mM Trise HCl buffer (40 mg ml 1, pH 7.5, 20 mM CaCl2) for 90 min. The pH was adjusted to 2e11 using 1% HCl or NaOH solution, and following a 3 h incubation at room temperature, the solution was dialyzed overnight in 50 mM TriseHCl buffer (pH 7.5, 20 mM CaCl2) at 4  C. The dialysis solution was centrifuged and the supernatant was used for detecting the inhibitory activity of 2S albumin [24] against soybean trypsin.

[12]

[13] [14]

[15] [16]

4.9. Amino acid analysis and N-terminal sequence of 2S albumin [17]

The partially pure 2S albumin was subjected to separation by SDS-PAGE using a 12.5% acrylamide gel. Following electrophoresis, the polypeptide of interest (2S-1) was excised from the gel using a lancet and ground with a mortar and pestle. Phosphate buffer was subsequently added and the solution was centrifuged. The supernatant containing the proteins of interest was filtered by ultrafiltration for several minutes to concentrate the proteins and remove glycine which originated from the electrophoresis buffer. This purified protein (2S-1) was used to determine the amino acid sequence of the N-terminus of protein (Edman degradation method) and was tested for its amino acid composition (ninhydrin staining method). The partially pure 2S albumin was also was tested for its amino acid composition. The samples were hydrolysis with 6 N HCl at 110  C for 24 h and a nitrogen sequence analysis was carried out using ion-exchange chromatography in the analyzer L8800 (Hitachi Company, Japan). Furthermore, N-terminal sequence analysis was performed on an automatic protein sequencer (Applied Biosystems, Procise 491).

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Acknowledgments [26]

This work was supported by the Guangdong Natural Science Foundation (31531, 320404) and the Chinese Science Foundation (30671338, 30971841). References

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