European Food Research and Technology

, 230:89

Detection of an angiotensin converting enzyme inhibitory peptide from peanut protein isolate and peanut polypeptides by western blot and dot blot hybridization

Authors

  • Li-na Liu
    • College of Food Science and TechnologyHuazhong Agricultural University
    • College of Food Science and EngineeringWuhan Polytechnic University
    • College of Food Science and TechnologyHuazhong Agricultural University
  • Dong-ping He
    • College of Food Science and EngineeringWuhan Polytechnic University
Original Paper

DOI: 10.1007/s00217-009-1136-7

Cite this article as:
Liu, L., Zhang, S. & He, D. Eur Food Res Technol (2009) 230: 89. doi:10.1007/s00217-009-1136-7

Abstract

The peptide P7, with an amino acid sequence of Cys-Val-Thr-Pro-Ala-Leu-Arg, inhibits angiotensin I-converting enzyme (ACE). In this paper, P7 was identified in peanut protein isolate (PPI) and peanut polypeptides (PPs) with a new method. The P7 peptide was synthesized and used in the preparation of an antiserum. Using the antiserum, P7 was specifically identified in PPI by western blot, and its level in PPI and PPs was assayed by dot blot hybridization. The results showed that two bands of P7 expression with molecular weight 18–25 and 25–35 kDa were seen in PPI by glucan gel chromatography. The positive reaction rate of P7 in PPs was higher than in PPI, consistent with the measured ACE inhibitory activity. The rate of P7 in sample no. 3 reached 39.29% of the positive control, using a dose of 20 μg/mL. This sample had an ACE inhibitory activity of 89.73%. Therefore, western blot and dot blot hybridization with prepared antibody against synthetic peptide was a very sensitive detection method for peptide.

Keywords

Peanut protein isolatePeanut polypeptidesAngiotensin I-converting enzyme inhibitory peptideWestern blotDot blot hybridization

Abbreviations

PPI

Peanut protein isolate

PPs

Peanut polypeptides

ACE

Angiotensin I-converting enzyme

P7

Synthetic peptide with an amino acid sequence of Cys-Val-Thr-Pro-Ala-Leu-Arg

Introduction

Angiotensin I-converting enzyme (ACE. dipeptidyl carboxpeptidase. EC 3.4.15.1) plays an important role in blood pressure regulation. ACE converts the inactive decapeptide angiotensin I into the potent vasoconstricting octapeptide angiotensin II, and also inactivates the vasodilator, bradykinin [1]. For this reason, ACE inhibitors are used in hypertension therapy. Many ACE inhibitors have been isolated as the enzymatic digestive products of food proteins such as zein, milk [2], wheat, blood [3], soybean [4], dried bonito [5], chicken muscle [6], and sardine muscle [7].

Peanuts (Arachis hypogeae L.) are an important oil-bearing crop in China, where their annual production of 14 million tons ranks first in global output [8]. Peanuts are also the third most important vegetable protein source in China. They contain 26–33% protein, and have good bioavailability and a relatively low content of anti-nutritional factors [9]. Peanut polypeptides (PPs), obtained by enzymatic hydrolysis of peanut proteins, have important physiological functions [10]. For instance, PPs possess in vitro ACE inhibitory activities, and improve the nonspecific and specific immunities of mice. Furthermore, PPs have recently been confirmed as having significant in vivo antioxidative activity [1115]. To date, however, there has been no report on the amino acid sequence of bioactivity peptides in PPs and the relationship between sequence and physiological function.

In this study, a new method was developed to detect the ACE inhibitory peptide in peanut protein isolate (PPI) and PPs. Peptide with specific sequences was synthesized and used in the preparation of an antiserum. The expression of the ACE inhibitory peptide in PPI was confirmed by western blot, and its level in PPI and PPs was assayed by dot blot hybridization.

Materials and methods

Plant materials

Samples of peanut protein flour were kindly supplied by Hongxin Peanut Protein Food Co. Ltd. (Dezhou, Shandong, China). The peanut protein flour was defatted with hexane for 48 h on a shaker. The resulting defatted peanut kernel flour was used as the starting material for the preparation of PPI and PPs.

Reagents and instruments

Chemicals and reagents were obtained from the following commercial sources: complete Freund’s adjuvant, incomplete Freund’s adjuvant, horseradish peroxidase (HRP) and goat anti-rabbit IgG (Sigma, United States); bovine serum albumin (BSA) from Tianyuan Bioscience Technology Co. (Wuhan, People’s Republic of China); mini-2 and mini-4 electrophoresis apparatus baths (BIO-RAD, Inc., USA); Waters 2690 High performance liquid chromatography (Waters, USA); Waters Platform ZMD 4000 Mass spectrometry (Waters, USA), and Biorad 680 Enzyme mark instrument from Queen & King Biochem. Co, LTD. (Shanghai, China).

Synthesis of ACE inhibitory peptides

An ACE inhibitory peptide (IC50, 29.3 μg/mL) with the amino acid sequence Cys-Val-Thr-Pro-Ala-Leu-Arg (P7) has been isolated from lactic acid bacteria fermentation of soybean meal aqueous extracts [16]. This ACE inhibitory peptide was synthesized by Beijing CWBio Co., LTD.

Preparation of PPI

Peanut protein flour (20 g) was diluted 1:8 with 160 mL of alkaline water (pH 9.0) for 1 h at 60 °C and centrifuged at 2,000×g for 20 min. The supernatant pH was adjusted to 4.5, approximately the isoelectric point of peanut protein, with 3 M citric acid. The protein precipitate after centrifugation was referred as the PPI.

Preparation of PPs

The PPI was dissolved in distilled water (1:10, w/v), and the solution was adjusted to the optimal pH and temperature of Alcalase (pH 8.0; 45 °C) before incubation for 2.5 h, at a 1:100 (w/v) ratio of enzyme solution to protein isolate. Samples were withdrawn from the proteolytic solution at degree of hydrolysis (DH) 8.16, 10.25, 12.08, 14.27, 16.89 and 19.53%, and the pH of the hydrolysate was immediately lowered to 4.5 with 3 M citric acid to inactivate the protease.

Analysis of DH and ACE inhibition activity

DH (%) was measured using the pH-stat method [17]. PPI (100 g) was diluted 1:10 (w/v) with pH 8.0 distilled water containing 1 g Alcalase at 45 °C. The pH was maintained with 3.0 M NaOH during hydrolysis. The percent DH was expressed as follows:
$$ {\text{DH}}(\% ) = {\frac{{C_{\text{NaOH}} { \times {V}}_{\text{NaOH}} }}{{\alpha \times M_{\text{P}} \times h_{\text{hot}} }}} \times 100 $$
where CNaOH is the concentration of alkali solution (mol/L), VNaOH is the consumption of alkali solution (mL), Mp is the weight of substrate protein (g), α is the average dissociation degree of amino groups and hhot is the total amount of peptides bonds (mmol/g protein).
ACE inhibitory activity was measured by the method of Cushman and Cheung, with some modifications [18, 19]. Briefly, a sample solution of 10 μL at 10 mg protein/mL was added to 50 μL of 6.5 mM Hip-His-Leu in 0.1 M sodium borate buffer (pH 8.3) with 500 mM NaCl, and preincubated at 37 °C for 6 min. The reaction was initiated by the addition of 5 μL of ACE solution (100 mU/mL), and the mixture was incubated at 37 °C for 30 min. The reaction was stopped by adding 25 μL of 1 M HC1, and cooled to room temperature, before analysis at 228 nm by HPLC for hippuric acid. The extent of inhibition was calculated as follows:
$$ {\text{ACE inhibitory activity }}\left( \% \right) \, = \, \left[ {\left( {A - B} \right)/ \, \left( {A - C} \right)} \right] \, \times 100 $$
where A represents the peak area of the control (buffer instead of test sample, mAU s), B is the peak area in the presence of ACE and sample, and C is the peak area of the reaction blank (HCl added before ACE, mAU s).

The IC50 value, defined as the concentration of peptide that inhibits 50% of the ACE activity, was determined by measuring the ACE inhibition and protein content of each sample after regression analysis. The protein contents were measured by the methods of Lowry, Roserbrough, Farr, and Randall [20]. All measurements were performed in triplicate.

Analysis of P7 by HPLC

Synthetic peptides were analyzed by HPLC on a Kromasil C18 column (4.6 × 250 mm, 5 μm) in aseptic conditions, at a flow rate of 1 mL/min. Linear gradient elution conditions were used with acetonitrile as the organic modifier and trifluoroacetic acid (TFA) as the volatile buffer. Eluent A consisted of 0.1% TFA in acetonitrile; eluent B was 0.1% TFA in water (v/v). The chromatographic column was conditioned with 14% of eluent A and 86% of eluent B, after which 10 μL of peptide solution was applied and eluted with increasing eluent A concentrations of 0–25 min, 14–39% and 25–30 min, 39–100%. UV absorbance was monitored at 220 nm.

Detection of P7 by mass spectrometry

Synthetic P7 was analyzed by mass spectrometry in work conditions ionization mode: EI, electron energy 70 eV, interface temperature 250 °C, ion source temperature: 200 °C, gas flow: 1.5 L/min.

Preparation of antiserum against P7

Synthesis of P7 immunogen

The immunogen was synthesized with P7 coupled to keyhole limpet haemocyanin (KLH) carrier protein. KLH (20.4 mg) was dissolved in 3.5 mL phosphate buffered saline (PBS) solution. In a separate reaction, 10.35 mg P7, 17.24 mg 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), 6.91 mg N-hydroxysuccinimide (NHS) and 150 μL dimethyl pimelimidate·2 HCl (DMP) were dissolved in 1.5 mL PBS (pH 7.4) and shielded from light for 24 h. The reaction products were added to the KLH–PBS solution with stirring at 4 °C for 24 h, and dialysed against PBS with buffer changes three times a day for 72 h. The product was stored at −20 °C.

Preparation of antiserum against P7

Healthy white rabbits (n = 2) between 2 and 5 kg were subcutaneously injected with 0.5 mL antigen solution (1–2 mg/mL) emulsified with an equal volume of Freund’s complete adjuvant for the first immunization, and the antigen emulsified with an equal volume of Freund’s incomplete adjuvant for booster immunizations every 2 weeks. Antiserum titer was determined by indirect enzyme-linked immunosorbent assay (ELISA) 1 week after the third booster immunization [21]. At an acceptable titer, antiserum was harvested by cardiac puncture.

P7 in PPI by western blot

P7 in PPI was detected by western blotting. Electrophoresis was carried out as described by Laemmli [22] using a 12% gel at a contact current of 20 mA/gel for 2 h. Proteins were transferred to nitrocellulose filter membranes by wet electrophoretic transfer at 80 V for 2 h at 4 °C, and blots washed in 30 mL phosphate buffered saline with 0.1%(w/v) Tween 20 (PBST) for 2 h at room temperature. Antiserum (diluted 1:250 with 8 mL PBST) was added at 4 °C, and incubated overnight, before washing the membrane three times with PBST. Protein was visualized by horseradish peroxidase (HRP)-conjugated goat anti-rabbit second IgG (diluted 1:7,000 with 10 mL PBST) for 1 h, developed with 1-chlorine naphthol after washing the membrane with PBST and TBS buffer. The experiment repeated three times.

P7 in PPI and PPs by dot blot hybridization

Samples were spotted on NC membranes in four concentrations: 20, 10, 5, and 2.5 μg/mL, and the membrane washed in 30 mL PBST for 2 h. Antiserum (diluted 1:250 with 8 mL PBST) was added before incubation at room temperature for 2 h. The membrane was washed three times with TBS and visualized as described above. Using the positive control group as the standard, Image Q software was used to analyze the results. The experiment repeated three times.

Results

Analysis of ACE inhibitory activity of PPs

The ACE inhibitory activities of PPs with different DH are given in Fig. 1. The ACE inhibitory activity of PPs is higher at a relatively low DH than at high DH, and sample 3 was highest reaching 89.73% with a DH of 12.08%.
https://static-content.springer.com/image/art%3A10.1007%2Fs00217-009-1136-7/MediaObjects/217_2009_1136_Fig1_HTML.gif
Fig. 1

ACE inhibitory activity of PPs with different DH (unit %) 1 DH 8.16%, 2 DH 10.25%, 3 DH 12.08%, 4 DH 14.27%, 5 DH 15.89%, 6 DH 17.53%. Values are the means of three replicates ± SD

P7 HPLC and mass spectrometry detection of synthetic peptide

The characteristic main peak of the P7 synthetic peptide had a relative retention time in HPLC fingerprinting of 9.638 min (Fig. 2). The purity of P7 based on the peak areas was higher than 98.70%.
https://static-content.springer.com/image/art%3A10.1007%2Fs00217-009-1136-7/MediaObjects/217_2009_1136_Fig2_HTML.gif
Fig. 2

P7 HPLC detection of synthetic peptides Chromatographic Column, Kromasil C18 column (4.6 × 250 mm, 5 μm); injection volume, 10 μL; temperature, 25 °C; detection wavelength: 220 nm; mobile phase: eluent A consisted of 0.1% TFA in acetonitrile; eluent B was 0.1% TFA in water (v/v); flow rate: 1 mL/min; linear gradient elution conditions: 0 min 14% A, 86% B; 25 min 39% A, 61% B; 30 min 100% A, 0% B

Mass spectrometry was used to identify the exact molecular weight of the target compound as 759.40 Da. It carried a positive charge and was composed of P7 and hydrogen ion conjugates, denoted [P7 + H]+. The difference between the theoretical molecular weight of 758.94 Da and the experimental molecular weight of 758.40 Da was less than 0.1% after removal of the hydrogen ion, so the synthetic peptides were the same as native P7, and had an amino acid sequence of Cys-Val-Ser-Thr-Pro-Ala-Leu-Arg by amino acid sequence analysis (Fig. 3).
https://static-content.springer.com/image/art%3A10.1007%2Fs00217-009-1136-7/MediaObjects/217_2009_1136_Fig3_HTML.gif
Fig. 3

P7 Mass spectrometry detection of synthetic peptides mass spectrometry, Waters Platform ZMD 4000; ionization mode, EI; electron energy, 70 eV; interface temperature 250 °C, ion source temperature: 200 °C, gas flow: 1.5 L/min

Analysis of ACE inhibitory activity of P7

P7 has a strong inhibitory action against ACE. The highest inhibition rate was 98.75% in a dose range of 10–500 μg/mL. Inhibition was dose dependent, the correlative coefficient was r = 0.9901 and the IC50 was 26.84 μg/mL.

Expression of P7 in PPI

The expression of P7 in PPI was examined by western blot (Fig. 4). Proteins in PPI were specifically bound by antiserum against P7, giving two bands of molecular weight 18–25 and 25–35 kDa. This demonstrates the presence of P7 in PPI.
https://static-content.springer.com/image/art%3A10.1007%2Fs00217-009-1136-7/MediaObjects/217_2009_1136_Fig4_HTML.gif
Fig. 4

Expression of P7 in PPI 1, Marker (18–113 kDa); 2–5 PPI parallel samples

Level of P7 in PPI and PPs

Using the antiserum against P7, PPI and six PPs processed to different DH were analyzed for the level of ACE inhibitory peptide P7 by dot blot hybridization. Using the P7 positive control group as standard, Image Q software was used to determine the P7 positive reaction rate of the samples. As shown in Fig. 5 and Table 1, the P7 positive control groups were strongly positive and no positive reaction was seen in the bovine serum albumin (BSA) negative controls. PPI and PPs sample spotted at different concentrations showed positive reactivity with the antiserum, indicating that the antiserum bound specifically to P7 in dot blot. This confirmed the expression of P7 in both PPI and PPs.
https://static-content.springer.com/image/art%3A10.1007%2Fs00217-009-1136-7/MediaObjects/217_2009_1136_Fig5_HTML.gif
Fig. 5

Dot blot hybridization analysis of PPs with antiserum against P7 16 PPs at different DH (1 8.16%, 2 10.25%, 3 12.08%, 4 14.27%, 5 15.89%, 6 17.53%); 7 PPI, 8 P7 positive control, 9 BSA negative control

Table 1

Positive reaction rate of P7 in samples with different DH compared with positive control (unit %)

Varieties (μg/ml)

Positive reaction rate of P7 [percent of positive control (%)]

1

2

3

4

5

6

7

20

33.18 ± 2.3

32.87 ± 1.6

39.29 ± 0.4

28.72 ± 2.4

24.32 ± 1.8

28.01 ± 0.5

19.25 ± 0.6

10

18.23 ± 1.2

7.95 ± 1.4

14.52 ± 2.6

12.11 ± 2.0

8.61 ± 1.2

7.73 ± 1.5

11.73 ± 1.5

5

4.97 ± 0.5

4.37 ± 0.3

6.90 ± 0.8

5.34 ± 0.7

3.73 ± 0.6

2.98 ± 1.0

5.36 ± 0.3

2.5

4.16 ± 0.7

2.84 ± 0.4

5.88 ± 1.3

5.50 ± 1.0

3.47 ± 0.9

2.60 ± 0.2

3.87 ± 0.3

Values are the means of three replicates ± SD

1–6 PPs at different DH (1, 8.16%; 2, 10.25%; 3, 12.08%; 4, 14.27%; 5, 15.89%; 6, 17.53%); 7 PPI

Hydrolysis increased the reactivity with P7 antibodies and the ACE inhibitory activity of hydrolysates, so both were higher in PPs than in PPI. Over the course of hydrolysis, P7 cross-reactivity was elevated then decreased, with the presence of P7 in sample 3 reaching 39.26% of the positive control with 20 μg/mL, with a DH of 12.08%. With greater levels of hydrolysis, P7 reactivity was reduced, consistent with the ACE inhibitory activity (Fig. 1). Sample 3, which had the highest P7 cross-reactivity, also had the highest positive reaction rate of ACE inhibitory activity at 89.73%, suggesting that the increase in PPs ACE inhibitory activity resulted from the interaction of P7 with other ACE inhibitory peptides in PPs, in the DH range of 16.89–19.53%.

Discussion

Purification of compounds and determination of the relationship between sequence and physiological function is a common method of research on polypeptides. The separation and purification of polypeptides is difficult for bioactive peptides. Generally, the purification of polypeptides proceeds by hydrolysis of proteins by protease, adsorption of the hydrolysate to activated octadecylsilyl (ODS) columns, solvent extraction of the target peptides, purification of biologically active peptides by SP-Sephadex G-25 columns using RP-HPLC, and identification of peptides by SDS-PAGE and mass spectrometry. Polypeptides are easily inactivated during the process of purification because of lengthy procedures, complicated technology and the complexity of the process. The molecular weights of many polypeptides after enzymatic hydrolysis are close to each other, creating another obstacle to their purification.

The study of the ACE inhibitory activity of PPs has been an active area of research on biologically active peptides. Zhang [23] conducted a detailed study of ACE inhibitory peptide purification in PPs. Four components were obtained by DEAE-Sephadex G-25 and DEAE-Sephadex G-15 column chromatography. These had an IC50 against ACE of 0.94, 0.47, 0.25 and 0.086 mg/mL. Tricine-SDS-PAGE electrophoresis determined the molecular weight of each component, showing that small peptides less than 1,500 Da had the highest ACE-inhibiting activity. HPLC and mass spectrometry showed that the components were a mixture of several polypeptides with molecular weight from 300 to 700 Da. The components with the highest ACE inhibitory activity were purified by semi-preparative reversed phase HPLC, and the final product was a mixture of peptides of similar molecular weights, with a markedly decreased ACE inhibitory activity.

In this paper, we developed a method for detecting the ACE inhibitory peptides in PPs. The ACE inhibitory peptide with amino acid sequence Cys-Val-Thr-Pro-Ala-Leu-Arg was isolated by lactic acid bacteria fermentation of soybean meal aqueous extract. Without a means of detection, the question of whether the same polypeptides exist in other plants is still unanswered. Soybean and peanut are species in the same family, and share high homology in many amino acid sequences, and many antigenic determinants show similarity. Therefore, the western blot and dot blot hybridization methods developed in this work not only provide a detection method for research and application of peanut ACE inhibitory peptides, but they also allow the screening and identification of ACE inhibitory peptides in other plants. The results showed that western blot and dot blot hybridization with prepared antibody against synthetic peptide was a very sensitive detection method for peptide. P7 can be detected by western blot and dot blot hybridization in PPI and PPs, with a higher positive reaction rate of P7 in PPs than in PPI. The rate of P7 corresponded to the ACE inhibitory activity of PPs, suggesting that P7 can significantly contributed to research on peanut ACE inhibitory peptides.

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© Springer-Verlag 2009