Purification and Identification of Anti-Oxidant Soybean Peptides by Consecutive Chromatography and Electrospray Ionization-Mass Spectrometry

Introduction

Reactive oxygen species (ROS) or free radicals are highly reactive molecules, which are involved in a number of diseases such as diabetes, cardiovascular diseases, neurodegenerative disorders, and cancer. ¹ A variety of food-derived peptides have drawn increasing attention in recent years due to their anti-oxidant properties. Interestingly, proteins from routine food sources, including fish, eggs, and grains, are in fact ideal starting materials for generating anti-oxidant peptides. ² On the other hand, protein purification has been revolutionized at all scales in terms of speed and simplicity with the development of modern purification strategies. For example, the tripeptide Gly-Pro-Hyp with high anti-oxidative activity is obtained from Alaska green codfish skin gel hydrolysate using Sephadex G-25 gel chromatography, ion-exchange chromatography, and high performance liquid chromatography (HPLC) loaded with an octadecylsilyl (ODS) column. ³ The heptapeptide His-Gly-Pro-Leu-Gly-Pro-Leu capable of scavenging superoxide free radicals is purified by cation exchange chromatography, gel chromatography, and reversed-phase (RP) C18 HPLC from fish skin hydrolysate. ⁴

In this study, various peptides from soybean protein isolate (SPI) were obtained by enzymatic hydrolysis and then purified by ultrafiltration, ion-exchange chromatography, and gel filtration chromatography. The anti-oxidant activity of the peptides was evaluated by oxygen radical absorption capacity (ORAC), an assay based on hydrogen atom transfer reactions. ⁵ The sequence of a purified anti-oxidant peptide was identified using RP-HPLC connected to an electrospray ionization (ESI) mass spectrometer. The identified peptide was then synthesized and evaluated for anti-oxidant activity using hydrogen peroxide (H₂O₂)-induced PC12 cells.

Materials and Methods

SPI (protein content >90%) was hydrolyzed by Alcalase 2.4 L^® ([E]/[S]=0.8%, wt/wt; Novo Nordisk Co., Beijing, China) at the substrate protein concentration of 6% (wt/wt) for 4 hr at 55°C. The anti-oxidant activity of the obtained SPI hydrolysates (SPIH) was evaluated by an ORAC assay. ⁶ The hydrolysates were then subjected to a series of purification steps as follows (Fig. 1): (1) Ultrafiltration. The hydrolysates were separated to SPIH-I (>10 kD), SPIH-II (5–10 kD), SPIH-III (3–5 kDa), SPIH-IV (1–3 kDa), and SPIH-V (<1 kDa) using a membranes bioreactor system (ZJMP 10-002, Millipore, USA). (2) Ion-exchange chromatography. The fraction with the highest anti-oxidant activity after the ultra-filtration process was separated by an Amberlite IRC-50 (Fisher Scientific Co., Fair Lawn, NJ) cation-exchange column (2.6×60 cm). (3) Gel-filtration chromatography. The fraction with the highest anti-oxidant activity after ion-exchange chromatography was further separated with a Sephadex G-15 column (1.6×70 cm, Amersham Pharmacia Biotech, Piscataway, NJ). The sequences of anti-oxidant peptides were identified using an Agilent RP-HPLC system connected online to an ion trap instrument (liquid chromatography-electrospray ionization/multi-stage mass spectrometry [LC–ESI-MS/MS]; Bruker Daltonic, Bremen, Germany). The identified peptides were synthesized using the fluorenylmethoxycarbonyl (Fmoc) method and evaluated for anti-oxidant activity using hydrogen peroxide (H₂O₂)-induced rat adrenal pheochromocytoma (PC12) cells. Statistical analysis was performed using SPSS 15.0 (SPSS Inc., Chicago, IL).

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FIG. 1.

Flow chart of separation and purification of anti-oxidant peptides from soybean protein isolate hydrolysates (SPIH). LC-ESI-MS/MS, liquid chromatography-electrospray ionization/multi-stage mass spectrometry.

Results and Discussion

The SPIH showed anti-oxidant activity with an ORAC value of 1910.48±52.52 μmol Trolox equivalent/gram peptide. After ultrafiltration, the strongest ORAC activity (2419.21±59.60 μmol Trolox equivalent/gram peptide) was observed in SPIH-V fraction (<1 kD). This fraction was further separated by cation-exchange chromatography, and two sub-fractions (A1 and A2) were obtained. The neutral and weak acidic peptide fraction A1 had an ORAC value of 2721.22±54.25 μmol Trolox equivalent per gram of peptide, whereas the alkaline peptide fraction had a stronger anti-oxidant capacity (ORAC=2974.79±50.10 μmol Trolox equivalent per gram of peptide). This is in agreement with a previous report that the peptides isolated from soybean beta-conglycinin hydrolysate with high anti-oxidant activities, such as VNPHDHQN, LVNPHDHQN, LLPHH, LLPHHADADY and LNSGDALRVPSGTTYY, also contain alkaline amino acid residues. ⁷

The most active fraction from cation-exchange chromatography, SPIH-V-A2, was further subjected to size-exclusion chromatography on a Sephadex G-15 column. Fraction 2 from Sephadex G-15 chromatography, i.e., SPIH-V-A2-B2, was found to have a very high ORAC value (4083.01±68.39 μmol Trolox equivalent/gram of peptide). Therefore, this fraction was analyzed using RP-HPLC coupled with an ESI⁺ mass spectrometer for peptide sequence identification. The MS/MS spectrum of a single charged ion with m/z at 353.4 was found in SPIH-V-A2-B2-C, and therefore the molecular mass of the anti-oxidant peptide was determined to be 352.4 Daltons. After further manual calculation and data search, the sequence of the peptide was identified to be Ser-Phe-Val. Interestingly, the anti-oxidant activity of the peptide fractions rich in di-, tri-, and tetra-peptides from an in vitro digest of zein is shown to be higher than non-hydrolyzed zein. ⁸ Together, our results suggest that short peptides with 2–10 amino acids may have greater anti-oxidant activity than parent proteins or hydrolysates with larger polypeptides. ⁹

To further test the bioactivity of Ser-Phe-Val, the tripeptide was chemically synthesized, and its protection capacity against radical-mediated cellular injuries was evaluated using PC12 cells. After exposing the cells to H₂O₂-induced oxidation, cytotoxicity was determined by MTT assay, and the synthetic tripeptides (0.01 mM) were found to exhibit dose-dependent protection against H₂O₂-induced injuries in PC12 cells.

The amino acid composition and sequence of a peptide are without doubt very important for their anti-oxidant activity, and hydrophobic amino acids can enhance the activities of anti-oxidant peptides. ^10,11 In the sequence of the most active tripeptide (Ser-Phe-Val) purified from SPIH, two amino acids (Phe and Val) are hydrophobic and thus are expected to favor its effect on oxidation inhibition. Phe can restrain peroxidation chain reaction triggered by free radicals because its side chain is at the plane of the structure of benzene conjugate. ¹² The nonpolar aliphatic amino acid Val may also play an important role in the anti-oxidant activity of the purified tripeptide. ¹³

Conclusion

Anti-oxidant peptides were isolated and purified from soybean protein isolate hydrolysates by ultrafiltration, cation-exchange chromatography, and gel-filtration chromatography. We found that the low-molecular-weight peptides (molecular weight <1 kD) exhibited stronger anti-oxidant activity than the parent proteins and that the alkaline peptide fraction was more active than its neutral or weak acidic counterparts. The peptide Ser-Phe-Val was identified from the hydrolysates as a powerful anti-oxidant, and this tripeptide was also chemically synthesized to confirm its protection against H₂O₂-induced damage in PC12 cells.