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International Journal of Food Science and Technology 2012, 47, 148154

Original article Purification and characterisation of a novel antioxidant peptide from porcine haemoglobin hydrolysate
Qian Sun,1 Yongkang Luo,1* Huixing Shen,2 Xue Li1 & Lei Yao1
1 College of Food Science & Nutritional Engineering, China Agricultural University; Beijing Higher Institution Engineering Research Center of Animal Product. Beijing 100083, China 2 College of Science, China Agricultural University, Beijing 100083, China (Received 26 February 2011; Accepted in revised form 14 September 2011)

Summary

Porcine haemoglobin, which is normally discarded as a by-product of meat industry, was hydrolysed using pepsin, AS1398 neutrase, trypsin, avorzyme, papain and alcalase respectively. The peptic hydrolysate exhibited the highest antioxidant activities than those of other hydrolysates, which was separated using ultraltration membranes, and consecutively using chromatographic methods including ion-exchange chromatography on SP Sephadex C-25 column, gel ltration chromatography on Sephadex G-25 column and reversed-phase high performance liquid chromatography. Finally, a novel antioxidant peptide from porcine haemoglobin (APPH) was puried, and its sequence was identied to be ARRLGHDFNPDVQAA (1666 Da) using mass spectrometry. APPH exhibited signicant higher lipid peroxidation inhibitory ability than that of a-tocopherol as positive control (P < 0.05), and eciently quenched hydroxyl radical (IC50 = 26.9 lm). APPH agrees with the 115129 residues of the b-chain from porcine haemoglobin. These results indicate that APPH would be a benecial ingredient for functional food and pharmaceuticals.
Antioxidant peptide, characterisation, lipid peroxidation, porcine haemoglobin hydrolysate, purication, radicals scavenging activity.

Keywords

Introduction

Reactive oxygen species (ROS) are thought to create oxidative stress, thereby causing various degenerative diseases, such as atherosclerosis, rheumatoid arthritis, diabetes mellitus and Alzheimers diseases (Athukorala et al., 2006; Manso et al., 2008; Mamelona et al., 2010). The chemical activity of hydroxyl radical is the strongest among ROS, and it easily induces severe damage in DNA leading to carcinogenesis, mutagenesis and cytotoxicity (Ardestani & Yazdanparast, 2007). In foods, lipid peroxidation (LPO) can lead to the development of undesirable o-avours and potentially toxic reaction products (Thiansilakul et al., 2007). To prevent foods from deterioration and to provide protection against serious diseases, it is very important to inhibit LPO occurring in foodstus and the living body. Antioxidants can protect the human body from ROS eects and retard the progress of many chronic diseases as well as discoloration and deterioration in foods (Je et al., 2004). Using synthetic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene and propyl gallate in
*Correspondent: Fax: +86 10 62737385; e-mails: luoyongkang@263.net or luoyongkang@cau.edu.cn

food products is under strict regulation because of their potential health hazards. Therefore, the search for natural antioxidants as alternatives to synthetic ones is of great interest among researchers. It has been reported that antioxidant peptides puried from many protein hydrolysates, such as canola (Cumby et al., 2008), smooth hound protein (Bougatef et al., 2009), marine species gelatin (Aleman et al., 2011) and pork hams (Park & Chin, 2011), possess antioxidant activities against free radicals and ROS. The slaughter of animals produces a considerable amount of by-products with high biological value, among which one of the most important is blood protein. The haemoglobin represents 80% of the protein content of blood, which has excellent nutritional value due to its physiologically well-balanced amino acid compositions (Piot et al., 1988). Hence, ecient recovery and utilisation of porcine haemoglobin are very important to reduce environmental problems and to maximise economic benets. An interesting approach for the eective application of haemoglobin would be enzymatic hydrolysis, which is widely used in food industry to convert to value-added products for upgrading the functional and nutritional properties of proteins. Recently, a study on the antioxidant activity of porcine

doi:10.1111/j.1365-2621.2011.02820.x
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haemoglobin hydrolysate (PHH) which produced using alcalase and avourzyme has been performed (Chang et al., 2007). However, the antioxidant peptides from haemoglobin were not further puried and identied. In this study, we investigated the antioxidant activity of PHH prepared using six dierent enzymes respectively. Furthermore, we adopted ultraltration (UF) and several chromatography techniques to isolate the most potent antioxidant peptide from PHH, and the amino acid sequences of the puried peptide were also determined.
Materials and methods

oxidation was evaluated using the ferric thiocyanate (FTC) method (Mitsuda et al., 1996). The colour development degree was measured at 500 nm (Model UV-2600A; UNICO, Shanghai, China). a-tocopherol was used as a positive control.
DPPH radical scavenging activity

Materials and chemicals

This method was according to the procedure described by Rajauria et al. (2010). Test samples which dissolved in 1.5 mL of water (1.5 mg mL)1) and were mixed with 1.5 mL of 1 mm DPPH ethanol solution. This mixture was shaken and kept at 25 C for 30 min, and then the absorbance of the mixture was measured at 517 nm against a blank.
Superoxide radical scavenging activity

Six independent batches of the porcine blood were taken from Shunxin agricultural Co. Ltd (Shun-Yi District, Beijing). Pepsin (pH 2.0, 37 C, from porcine stomach mucosa) used for protein hydrolysis with declared activities of 1:3000 U g)1 and a-tocopherol, were purchased from Amresco (Solon, OH, USA). Alcalase (pH 8.0, 55 C) and avorzyme (pH 6.5, 45 C) were from Novozymes (Novo Nordisk, Bagsvaerd, Denmark). The crude protease A.S.1398 (pH 7.0, 45 C) was from Donghua Qiangsheng Biotechnology Co., Ltd (Beijing, China) and Papain (pH 6.5, 37 C) from Javely Biological Products Co., Ltd (Nanning, China). Trypsin (pH 7.5, 45 C) was from Amresco. 2-Deoxy-D-ribose (D5899), linoleic acid (99%) (L1376) and 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH, D9132) were purchased from SigmaAldrich (St. Louis, MO, USA). The UF membrane reactor system was from XuBang membrane equipment Co., Ltd (Beijing, China). SP Sephadex C-25 and Sephadex G-25 were purchased from Pharmacia (Uppsala, Sweden). All reagents were of analytical grade.
Preparation of PHH by pepsin

This assay measured the inhibition of the auto-oxidation of pyrogallol using a slightly modied method of Marklund & Marklund (1974). A sample solution (0.3 mL, 5 mg mL)1) and 2.61 mL of 50 mm phosphate buer (pH 8.2) were added into freshly prepared 90 lL of 3 mm pyrogallol (dissolved in 10 mm HCl). After incubation for 4 min, the reaction mixture was added with 100 lL of 0.2 m ascorbic acid immediately. The absorbance was measured at 325 nm against a blank.
Hydroxyl radical scavenging activity

The haemoglobin was isolated from quarantine qualied porcine blood and transferred to the lab at 4 C immediately, and was lyophilised to dry powder then stored at 4 C until use. Five grams of dried haemoglobin were dissolved in 100 mL distilled water and digested by pepsin (enzyme to substrate ratio = 1.6%, w w) at 40.4 C for 60 min. During hydrolysis, pH was maintained at 1.6 by addition of 1 m HCl. After heating at 100 C for 10 min to inactivate the enzyme, the hydrolysate was centrifuged at 4200 g and 4 C for 10 min to remove insoluble substrate, was lyophilised to dry powder and then storage at )20 C.
Antioxidant activity Antioxidant activity in Linoleic acid FTC model system

This assay was measured using the deoxyribose method (Siddhuraju & Becker, 2007) with some modications. The reactions were performed in 0.1 m, phosphate buer (pH 7.4), containing 10 mm deoxyribose, 10 mm FeSO47H2O, 10 mm EDTA and samples. The reaction was activated by adding H2O2 to a concentration of 10 mm, and the reaction mixture was incubated for 1 h at 37 C in a water bath. After incubation, the colour was developed by adding 1 mL of 1% thiobarbituric acid and 1 mL of 2.8% ice-cold trichloroacetic acid and heated at 100 C for 10 min. After cooling, the absorbance was measured at 532 nm.
Purication and characterisation of antioxidant peptide Ultraltration (UF) system

Porcine haemoglobin hydrolysate was fractionated through three dierent UF membranes having a molecular weight cuto (MWCO) range of 10, 5 and 3 kDa respectively. All peptide fractions (PHH-I with MW distribution >10 kDa, PHH-II with MW distribution of 510 kDa, PHH-III with MW distribution of 35 kDa and PHH-IV with MW distribution <3 kDa) recovered were lyophilised in a freeze drier and stored at )20 C.
Ion-exchange chromatography and Gel ltration chromatography

The antioxidant activity was determined according to the method of Saha et al. (2004). The degree of

Among the UF fractions, PHH-IV was separated using SP Sephadex C-25 ion-exchange column (3.5 40 cm).

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The lyophilised PHH-IV (4 mg mL)1) was loaded onto a the column equilibrated with 20 mm ammonium acetate buer (pH 3.6) and eluted with a linear gradient of ammonium formate (01 m) in the same buer at a ow rate of 1 mL min)1, and 5 mL fractions were collected. Each fraction was monitored at 280 nm and the fraction eluted under the same elution peak were collected, lyophilised and tested for antioxidant activity. The pooled fraction (10 mg mL)1) with the highest antioxidant activity was consecutively puried on a Sephadex G-25 gel ltration column (2.6 100 cm) equilibrated with ammonium acetate buer (pH 3.6). Fractions (4 mL each) were collected at a ow rate of 0.8 mL min)1, and absorbance was measured at 280 nm to determine the elution prole of the sample. These two chromatographic runs were repeated 20 times. Fractions associated with each peak showing antioxidant activity were pooled and freeze-dried for next step.
Reversed-phase high pressure liquid chromatography (RP-HPLC)

Results and discussion

Enzymatic hydrolysis of porcine haemoglobin

The fraction exhibiting the highest antioxidant activity was further puried using RP-HPLC on a Daisogel C18 100A (20 250 mm; Daiso, Osaka, Japan) column with a linear gradient of acetonitrile (5% in 05 min, 545% in 540 min) containing 0.1% triuoroacetic acid (TFA) at a ow rate of 3.0 mL min)1. Elution peaks were detected at 220 nm, and active peak was concentrated using a rotary evaporator. For further purication, the most potent peak was loaded onto a Kromasil-5 C18 RPHPLC analytical column (4.6 250 mm; Akzo Nobel, Sweden) with a linear gradient of acetonitrile (5% in 05 min, 550% in 25 min) containing 0.1% TFA at a ow rate of 1.0 mL min)1. Elution peaks were detected at 220 nm, and the potent peaks were collected, evaluated for antioxidant activity and then lyophilised. The chromatographic run was repeated 35 times. The nal puried peptide was analysed for amino acid sequence.
Matrix-assisted laser desorption ionisation-time of ight (MALDI-TOF) mass spectrometry

Porcine haemoglobin was separately hydrolysed with six enzymes (Pepsin, AS1398 Neutrase, Trypsin, Flavorzyme Papain and Alcalase) at optimal conditions. The peptic hydrolysate exhibited higher DPPH radical scavenging ability (67.0 1.8%) and linoleic acid autoxidation inhibitory ability (86.1 2.1%) than other hydrolysates. The LPO inhibitory ability of PHH could be due to the properties that strong anity for oil and other nonpolar substances of porcine haemoglobin. Furthermore, pepsin preferably digests peptide bonds by cleaving after the N-terminal of aromatic amino acids such as Phe, Trp and Tyr. The phenyl groups of the residues at peptide ends were likely to scavenge the free radical to prevent DNA damages. Similar to our study, Je et al. (2007) also found that peptic hydrolysates from tuna backbone protein exerted the highest antioxidant activity among six hydrolysates prepared by dierent enzymes.
Purication and identication of antioxidant peptide

Molecular weight and amino acid sequence of the puried peptide was analysed using a MALDI-TOFTOF Bruker Ultraex II (Bruker, Germany). The peptide was mixed with matrix solution (50% acetonitrile and 0.1% TFA), and the mixture was spotted on the MALDI target plate. The spectrum was acquired in positive ion mode at 20 kV in linear mode.
Statistical analysis

Initially, the peptic hydrolysate was separated using three kinds of UF membranes (10-, 5- and 3-kDa MWCO membranes), and four kinds of permeates (PHH-I, PHH-II, PHH-III and PHH-IV) were obtained. PHH-IV group had a higher LPO inhibition ability than the other fractions. The high inhibitory eect by PHH-IV was found to be 93.80 0.97% (5 mg mL)1). Furthermore, PHH-IV exhibited signicant higher radical scavenging eects than those of other groups (P < 0.05) (Table 1). Therefore, PHH-IV was employed for the purication and identication of the antioxidant peptide. Porcine haemoglobin hydrolysate-IV was further loaded onto a SP Sephadex C-25 column, and three absorbed portions were eluted with a linear gradient of ammonium formate (Fig. 1a). Fraction C possessed the highest hydroxyl radical scavenging activity among all
Table 1 Free radical scavenging activities (%) of fractionated porcine haemoglobin hydrolysate (PHH)a DPPH radical 69.9 54.2 66.7 83.4 1.7b 2.0c 3.1b 3.8a Hydroxyl radical 38.0 22.3 31.6 55.8 4.6b 5.7c 2.8bc 2.2a Superoxide radical 11.9 14.8 18.1 27.8 1.1c 0.8c 1.3b 1.6a

Fractions PHH-I PHH-II PHH-III PHH-IV

a

The antioxidant activity assays were carried out in triplicates, and data were expressed as means with standard deviations. A signicant dierence was considered at P < 0.05 (SAS Institute Inc., Cary, NC, USA; 1999).

Values are means of three determinations standard deviation. Means in the same columns with different letters are signicantly different (P < 0.05).

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Figure 1 Purication proles of antioxidant peptides from porcine haemoglobin. (a) Ionexchange chromatography on SP Sephadex C-25 chromatography. (b) Gel ltration chromatography on Sephadex G25 column. Upper panels represent the antioxidant activity measured using hydroxyl radical scavenging assay.

Figure 2 Reversed-phase high performance liquid chromatography pattern on a Daisogel C18 100A column of fraction CII obtained using gel chromatography. Upper panels represent the antioxidant activity measured using hydroxyl radical scavenging assay.

fractions. Following activity analysis, the lyophilised active fraction C was subjected on a Sephadex G-25 column equilibrated with ammonium acetate buer (20 mm, pH 3.6) (Fig. 1b). The potent fraction CII was further separated using RP-HPLC on a Daisogel C18 100A (20 250 mm) column, and more than 20 peaks were detected using this chromatography (Fig. 2). There were eight peaks exhibited potent antioxidant activities, and the CII-4 fraction possessed the strongest activity. To obtain a puried peptide, we rechromatographed CII-4 fraction on a Kromasil-5 C18 (4.6 250 mm) RP-HPLC analytical column (Fig. 3). Finally, we obtained a puried antioxidant peptide

from porcine haemoglobin (APPH). This puried peptide can eectively quench in hydroxyl radical (IC50 = 26.9 lm). Then, the molecular mass and amino sequence of APPH was determined using MALDITOF mass spectroscopy. As shown in Fig. 4, APPH was composed of 15 amino acid residues, which was determined to be Ala-Arg-Arg-Leu-Gly-His-Asp-PheAsn-Pro-Asp-Val-Gln-Ala-Ala (MW = 1666 Da). This amino acid sequence agrees with the 115129 residues of the b-chain from porcine haemoglobin. The molecular mass of the APPH determined using MALDITOF spectroscopy was in excellent agreement with theoretical mass calculated from the sequence. This

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Figure 3 Reversed-phase high performance liquid chromatography pattern on a Kromasil-5 C18 column of subfraction CII-4 obtained using Daisogel C18 100A RP-HPLC. Elution proles (lower panel) and antioxidant activities of the fractions (upper panel) are measured using hydroxyl radical scavenging assay.

Figure 4 Identication of molecular mass and amino acid sequence of antioxidant peptide from porcine haemoglobin using a MALDITOF-TOF mass spectrometer. Sequencing of active peptide was acquired over the m z range 502500.

identied peptide is a novel peptide with antioxidant activity that had never been reported.
Antioxidant activities of puried peptide (APPH)

The antioxidant activity of APPH was further investigated using LPO inhibition assay. We adopted a-tocopherol, a widely used natural antioxidant, as the positive control to evaluate the LPO inhibitory ability of APPH. As shown in Fig. 5, APPH eectively inhibited

LPO in linoleic acid emulsion system up to the 48 h, and the activity was signicant higher than that of a-tocopherol (P < 0.05). Cheng et al. (2003) reported that phenolic compounds aorded their protective actions in LPO by scavenging the lipid derived radicals (R, RO or ROO) to stop the chain reactions in a heterogeneous lipid phase. Furthermore, Tong et al. (2000) revealed that high molecular weight fraction of whey protein was able to inhibit LPO via scavenging of free radicals. Therefore, the LPO

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90 80

Antioxidant activity (%)

70 60 50 40 30 20 10 0

-tocopherol APPH

25 M

50 M

100 M

Uchida & Kawakishi, 1992). The hydrogen donors such as Gly and Asp are able to quench unpaired electrons or radicals by supporting protons. The two Asp residues located around the central in the APPH sequence would be very important to the antioxidant activity of APPH. Hsu et al. (2009) reported that the two puried peptide from tuna cooking juice hydrolysates exhibited strong radical scavenging activity. The two peptides and APPH sequence contained His-Asp sequence. Therefore, it can be expected that His-Asp sequence may be play a vital role in the observed radical scavenging activity of APPH. As a whole, the presence of specic amino acids and their specic positioning in the sequence could have been attributed to antioxidant activity of the puried peptide.
Conclusions

Figure 5 Effects of antioxidant peptide from porcine haemoglobin (APPH) on antioxidant activity using linoleic acid oxidation system. The antioxidant activity was estimated as the rate of inhibition of peroxide production at 48 h. a-tocopherol was used as positive control. The concentration of APPH and a-tocopherol was indicated on x-axis.

inhibitory ability of peptide or protein is dependent on molecular size and chemical properties such as hydrophobicity and electron transferring ability of amino acid residues in the sequence. In the sequence of APPH, hydrophobic amino acid residue such as Ala, Leu and aromatic amino acid composed approximately 46.7% of the puried peptide sequence, which exert scavenging eect of free radicals. The hydrophobic property of the APPH may have played an important role, exerting high anity to linoleic acid. In addition, as hydrophobicity of antioxidants is important for accessibility to hydrophobic targets (Chen et al., 1996), it is presumed that the presence of hydrophobic amino acids in puried peptides may contribute to LPO inhibitory activity by increasing solubility of peptides in lipid and thereby facilitating better interaction with radical species. Mendis et al. (2005) reported that hydrophobic amino acids might increase the anity and reactivity to the cell membrane in the living cells. In the APPH sequence, the Ala, Val, Leu, Pro with non-polar aliphatic groups have high reactivity to linoleic acid, and His, Phe with aromatic residues can make ROS stable through direct electron transfer. Antioxidant peptides that were isolated from soybean hydrolysate (Chen et al., 1995), egg yolk hydrolysate (Park et al., 2001) and tuna cooking juice hydrolysates (Hsu et al., 2009) have also been attributed to His due to the proton-donation ability of its imidazole group. It has also been reported that His and Pro play an important role in the antioxidant activity of peptides from soybean protein hydrolysates (Chen et al., 1996). In addition, presence of Asp seems to play a vital role irrespective of its position as observed in several antioxidant peptide sequences (Rajapakse et al., 2005;

In this study, a novel antioxidative peptide (Ala-ArgArg-Leu-Gly-His-Asp-Phe-Asn-Pro-Asp-Val-Gln-AlaAla) that could eciently inhibited LPO and was a potent free radical scavenger was puried using UF membranes and consecutive chromatographic methods from PHH. In addition, further detailed studies on APPH with regard to antioxidant activities in vivo and stability of this peptide when incorporated in foods, are needed.
Acknowledgments

This work was supported nancially by National Science Technology Ministry of China (award nr 2008GA741002 and nr 2006BAD05A16) and Ministry of Agriculture of China (award nr 2011-G8).
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