Article pubs.acs.org/JAFC
Conjugation of Ovotransferrin with Catechin Shows Improved Antioxidant Activity Juan You,†,‡ Yongkang Luo,† and Jianping Wu*,‡ †
College of Food Science & Nutritional Engineering, China Agricultural University, Beijing 100083, People’s Republic of China Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada
‡
ABSTRACT: Ovotransferrin (OTF), representing 12−13% of the total egg white, is a member of transferrin family with antimicrobial and antioxidant activities. Catechin is a polyphenolic antioxidant found in green tea. The objective of the study was to conjugate ovotransferrin with catechin to improve the antioxidant activity of OTF. Conjugates were prepared either by the free radical method using hydrogen peroxide−ascorbic acid as the initiator or by the alkaline method at pH of 9.0. The oxygen-radical-scavenging effect was increased from 3.95 mol trolox equivalent (TE)/mol of ovotransferrin to 22.80 and 17.14 mol TE/mol sample, respectively, in radical and alkaline prepared conjugates, which indicated that conjugation with catechin is an effective way to improve antioxidant activity of the protein. Conjugation between ovotransferrin and catechin was analyzed by fluorescence analyses, ultra performance liquid chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatography coupled online to a tandem mass spectrometer. Catechin was covalently bound to lysine (residues 327) and glutamic acid (residues 186) in ovotransferrin. The ovotransferrin-catechin conjugate may have a potential application as a functional food and nutraceutical ingredient. KEYWORDS: ovotransferrin, catechin, conjugation, antioxidant activity, site of conjugation
1. INTRODUCTION Reactive oxygen species (ROS), a collective term used for free radicals or molecular species capable of generating free radicals, are thought to cause oxidative stress. Oxidative stress has been implicated in several human diseases like cancer, atherosclerosis, diabetics, neurological diseases such as Alzheimer’s disease, Parkinson’s disease, and so forth as well as in the aging process.1−3 In food materials, lipids or proteins may go through oxidation by a free radical mechanism, and the oxidation contributes to the subsequent development of unpleasant flavors, dark colors, poor texture and may also generate potentially toxic end products.4 Antioxidants can protect the human body from the damaging effects of oxidative stress and prevent food from deterioration by donating their own electrons to ROS and neutralizing the adverse effects of ROS.5 Although many synthetic antioxidants are proved to be successful in the management of ROS, their use is often limited because of toxic side-effects. Therefore, there is a need to develop new compounds with strong antioxidant activity. As an economical and nutritional food commodity, eggs are also a rich source of bioactive proteins with physiological benefits for human health.6 Ovotransferrin (OTF), the second major protein of egg white albumen, has antimicrobial activity as well as antioxidant activity.7 Recently, OTF was found to be a superoxide dismutase (SOD) mimic protein with a potent superoxide anion scavenging activity.8 The utilization of protein antioxidants in functional foods presents additional advantages © 2014 American Chemical Society
over other natural antioxidants because they also confer an additional nutritional value and other desired functional properties.9 Catechin, a polyphenol antioxidant found in tea, wine, fruits, and so forth, is well known to have a high affinity to bind protein.10,11 Binding phenolic compounds to proteins lead to significant changes in structure, physicochemical properties, and the activity of proteins.12 It was reported that phenolic compounds can interact with proteins via different covalent and noncovalent bonds (i.e., hydrogen bonding, π-bonding, hydrophobic, and ionic interactions).13,14 Most of reports on noncovalent bonds focused on the interactions between polyphenols and plasma proteins. 15 Recently, covalent conjugation has been studied for various proteins such as gelatin,16,17 bovine serum albumin,14,18 myofibrillar proteins,19 and milk proteins.20 However, despite the numerous studies, the antioxidant properties of the conjugates have some contradictory results.12 Arts et al.21 and Ryan and Petit22 showed that the interactions of protein and polyphenol significantly decreased the total antioxidant capacity comparing with polyphenol, whereas Dubeau et al.23 suggested milk protein can have dual effects on the tea antioxidant capacity. In Received: Revised: Accepted: Published: 2581
December 15, 2013 February 21, 2014 March 9, 2014 March 10, 2014 dx.doi.org/10.1021/jf405635q | J. Agric. Food Chem. 2014, 62, 2581−2587
Journal of Agricultural and Food Chemistry
Article
mixture was placed at room temperature in darkness for 17 h to generate radical before use. The ABTS•+ working solution was diluted with 5 mM phosphate buffered saline (pH 7.4) to an absorbance of 0.70 ± 0.02 at 734 nm. Radical scavenging activity of samples was measured by mixing 100 μL of the samples with 100 μL of diluted ABTS•+. The decline in absorbance was measured at 1 min interval for a total of 10 min. A standard curve was prepared by reacting 100 μL of a series of concentrations of Trolox (0, 2, 4, 8, 12, 16, and 20 μM) with 100 μL of diluted ABTS•+ solution. The degree of radical scavenging activity of the samples was calculated on the basis of the Trolox standard curve and was expressed as TEAC (μmol/mg sample). 2.3.3. Reducing Power Assay. The ability of samples to reduce iron(III) was determined by the method of Yildirim, Mavi, and Kara.26 An aliquot of 1 mL of sample (5 mg/mL) was mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% (w/v) potassium ferricyanide. The mixtures were incubated at 50 °C for 30 min. After incubation, 2.5 mL of 10% (w/v) trichloroacetic acid was added and the reaction mixtures were centrifuged for 10 min at 3000g. Finally, 2.5 mL of the supernatant solution was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% (w/v) ferric chloride solution. After a 10 min reaction time, the absorbance of the resultant solution was measured at 700 nm. Higher absorbance of the reaction mixture indicated higher reducing power. 2.4. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE was performed in a MiniPROTEAN tetra cell with a Power Pac Basic electrophoresis apparatus (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.) at a constant voltage of 200 V according to Offengenden et al.27 OTF and OTFcatechin conjugates solutions (2 mg/mL) were diluted in a ratio of 1:1 with Laemmli sample buffer containing 5% β-mercaptoethanol and loaded on 12% Mini-PROTEAN TGX precast gel (Bio-Rad Laboratories, Inc., Hercules, CA, U.S.A.). Loading volume of samples and protein marker “Precision Plus Standards Dual Xtra Protein” (BioRad Laboratories, Inc., Hercules, CA, U.S.A.) was 15 μL. The gel was stained with Coomassie brilliant blue R250 and scanned in an Alpha Innotech gel scanner (Alpha Innotech Corp., San Leandro, CA, U.S.A.) with Fluor Chem SP software. 2.5. Matrix-Assisted Laser Desorption/Ionization Time-ofFlight Mass Spectrometry (MALDI-TOF−MS). MALDI-TOF−MS was used to determine the molecular weights of OTF and OTFcatechin conjugates. Lyophilized OTF and OTF conjugates were dissolved in 20 μL of distilled H2O. A 0.8 μL aliquot of sample solution was spotted to an 800 μm Bruker’s anchor Chip MALDI target (Bruker Daltonics, Billerica, MA, U.S.A.). A 0.8 μL aliquot of matrix solution was then spotted on top of the sample and left to dry. 2,5-dihydroxybenzoic acid (DHB) was used as the matrix compound. Matrix solution was prepared in 50% acetonitrile at a final concentration of 3.5 mg/mL. MALDI MS was performed on an ultraflex Xtreme TM MALDI-TOF/TOF (Bruker Daltonics, Billerica, MA, U.S.A.) mass spectrometer in positive MS or lift mode. 2.6. Fluorescence. The fluorescence of samples was tested according to Roy et al.28 with slight modification. The steady-state fluorescence spectra of samples were recorded at an excitation wavelength of 280 nm in a SepctraMax M3 spectrometer (Molecular Devices, Inc., U.S.A.), with 10 nm excitation and 5 nm emission bandwidth, in a 96 well plate. Samples were dissolved in distilled water to make a protein concentration of 10 −5 M. 2.7. Ultra Performance Liquid Chromatography (UPLC). The hydrophilic/hydrophobic characteristics of OTF, catechin, and OTFcatechin conjugates were studied by reverse-phase chromatography using a UPLC (Waters, Miliford, MA, U.S.A.). In brief, samples were dissolved in double distilled water and filtered through a 0.2 μm nylon syringe filter prior to analyze with a Waters BEH 300 C18 column (2.1 × 100 mm). The column was eluted (0.5 mL/min) with a two solvent system: (A) 0.1% Trifluoroacetic acid (TFA) in water and (B) 0.1% TFA in acetonitrile, with a gradient from 0% solvent B to 75% solvent B over 30 min, and then decreased to 0% solvent B in 10 min. 2.8. LC−MS−MS. The identification of the sites of protein-catechin conjugates was performed by LC−MS−MS. Excised gel pieces from above SDS-PAGE containing the samples of interest were digested by
addition, the binding mechanism of polyphenols with proteins has not been clarified yet, nor has the conjugation site of amino acids of proteins been elucidated. Therefore, we studied the interaction of catechin and OTF in order to investigate the antioxidant activity of modified protein. Two different conjugating reactions between OTF and catechin were conducted. The identification of catechin was performed by liquid chromatography coupled online to a tandem mass spectrometer (LC−MS−MS). The possibility to graft antioxidant moieties like catechin into a proteic structure represents an interesting innovation that significantly improves the performance of the biomacromolecules, opening up new applications in the nutraceutical and functional food ingredient fields.
2. METHODS AND MATERIALS 2.1. Chemicals and Reagents. Ovotransferrin was provided by Neova Technologies Inc. (Abbotsford BC, Canada), and the purity was >89%. (+)-Catechin was purchased from Sigma (Oakville, ON, Canada), and the purity was >98%, HPLC grade. Asorbic acid, 6hydroxy-2,5,7,8-tetramethylchromate-2-carboxylicacid (Trolox), ethylenediaminetetraacetic acid (EDTA) were also purchased from Sigma (Oakville, ON, Canada). Fluorescein disodium salt and 2,20-azobis(2methylpropionamide) dihydrochloride (AAPH) were obtained from Fisher Scientific (Ottawa, ON, Canada). All other chemicals and reagents used in this study were obtained from Sigma and were of the analytical grade. 2.2. Conjugation of OTF and Catechin. Conjugates were prepared either by free radical16 or alkaline method.14 To prepare conjugates by the free radical method, in a 100 mL glass flask, 0.5 g of ovotransferrin was dispersed in 50 mL of distilled H2O, then 1.0 mL of 5.0 M H2O2 (5.0 mmol) and 0.25 g of ascorbic acid (1.4 mmol) were added, and the mixture was maintained at 25 °C under atmospheric air. After 2 h, 0.35 mmol of catechin were added to solution to react for 24 h; unreacted catechin was removed by dialysis (dialysis tube with 1000 molecular weight cutoffs) at 4 °C for 48 h with Milli-Q water with eight changes. Conjugate was also prepared by the alkaline method. In the presence of alkaline, catechin is oxidized to its respective quinone. The reaction of this quinone with the nucleophilic side chains of proteins such as lysine, cysteine, methionine and tryptophan leads to a protein-phenol derivative.14 The alkaline conjugate was prepared in a 100 mL glass flask; 0.5 g of ovotransferrin was dispersed in 50 mL of distilled H2O, the pH was adjusted to 9.0, and the mixture was maintained at 25 °C under atmospheric air. After 2 h, 0.35 mmol of catechin were added to solution for 24 h with continuous stirring; unreacted catechin was removed by dialysis as above. The resulting solutions were frozen and freeze-dried. 2.3. Determination of Antioxidant Activities. 2.3.1. Oxygen Radical Absorbance Capacity (ORAC) Assay. The ORAC assay using fluorescein as the fluorescent probe was performed according to Huang et al.24 The reaction was carried out in 96-well black microplates (96F nontreated, Nunc, Roskilde, Denmark). Samples at different concentrations were mixed with 75 mM phosphate buffer (pH 7.4; 80 μL) and 200 nM fluorescein (50 μL) and incubated at 37 °C for 15 min. Then, 80 mM AAPH solution (50 μL) was added to each well using a dispenser. The fluorescence was recorded every min for 100 min by a microplate reader (Fluoroskan Ascent; Thermo Electron Corp., Vantaa, Finland). The excitation and emission wavelengths were 485 and 538 nm, respectively. Phosphate buffer was used instead of sample solution as blank. The area under curve was calculated by integrating the relative fluorescence curve. The ORAC value was calculated by dividing the slope of sample regression curve by the slope of Trolox regression curve. Final ORAC values were expressed as Trolox equivalents (TE) per mol. All the tests were performed at least in triplicates. 2.3.2. ABTS•+ Radical Scavenging Assay. ABTS was performed according to You et al.25 ABTS•+ was produced by mixing 7 mM ABTS•+ stock solution with 2.45 mM potassium persulfate. Then the 2582
dx.doi.org/10.1021/jf405635q | J. Agric. Food Chem. 2014, 62, 2581−2587
Journal of Agricultural and Food Chemistry
Article
trypsin following the protocol of Offengenden et al.27 OTF-catechin conjugates were analyzed by a Waters (Micro mass) Q-TOF Premier (Milford, MA, U.S.A.) according to Gu and Wu.29 Mass was detected through quadrupole time-of-flight (Q-TOF) analyzer operated in a positive ion MS/MS mode. A MS/MS full-scan was performed for each sample. The peptides’ sequences were identified from respective monoisotopic mass. Instrumental control and data analysis were performed using Mass Lynx software (Micromass U.K. Ltd.). Peaks Viewer 4.5 (Bioinformatics Solutions Inc., Waterloo, ON, Canada) in combination with manual de novo sequencing was used to process the MS/MS data. 2.9. Statistical Analysis. All sample determinations were performed in triplicate and the results were expressed as mean value ± standard deviation (SD) in this study. The fluorescence decay curves and data figures were prepared using GraphPad Prism Version 5.02 (GraphPad Software, Inc., CA, U.S.A.). Analysis of variance (ANOVA) with Tukey’s post hoc test was used to determine statistical differences. Differences were considered significant with p value
