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PROCESSING, PRODUCTS, AND FOOD SAFETY |
Department of Animal Science, Iowa State University, Ames, 50011-3150
1 Corresponding author: duahn{at}iastate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: ovotransferrin • separation method • ethanol extraction • egg white
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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A metal-free ovotransferrin (apo-form) is colorless and easily destroyed by physical and chemical treatments, whereas iron-bound ovotransferrin (holo-form) shows a salmon-pink color and is resistant to proteolytic hydrolysis and thermal denaturation (Azari and Feeney, 1958). Because most chemical reagents and denaturing conditions decrease the affinity of iron to ovotransferrin, a particular spatial configuration of native ovotransferrin is needed for the formation of colored iron-bound ovo-transferrin (Fraenkel-Conrat, 1950). The binding of iron to ovotransferrin requires 1 molecule of CO2 as CO32– or HCO3– per atom of Fe3+ (Warner and Weber, 1953) to overcome the effect of citrate, which can bind iron (Masson and Heremans, 1968). Reiter et al. (1975), however, reported that bicarbonate rather than pH is a critical factor for bacteriostatic capability of lactoferrin.
To release Fe3+ from ferric transferrin, a simple anion, such as pyrophosphate, sulfate, and chloride, is required in vitro. The anion-induced Fe3+ release is closely related to the opening of a domain in either lobe (Baldwin and de Sousa, 1981; Cheuk et al., 1987; Bailey et al., 1997). At acidic pH, the N-lobe of ovotransferrin displays lower iron-binding capacity and faster release of Fe3+ than the C-lobe. In the absence of NaHCO3, the pH affects the efficiency of citrate-mediated release of Fe3+ from ovo-transferrin. Citrate-mediated Fe3+ release is more efficient at pH 6.8 than at pH 7.4 (Griffiths and Humpherys, 1977). Iron could be removed from iron-transport proteins at low pH (pH 4.5) in the presence of a large excess of citrate (Guo et al., 2003). Cunningham and Lineweaver (1965) reported that when holo-ovotransferrin solution was heated at pH 9, the protein was not altered significantly. However, ovotransferrin was denatured when the proteins were exposed to a pH below 4.2, but the influence of pH was reversible under certain experimental conditions (Phelps and Cann, 1956).
Ovotransferrin can be separated from egg white by aqueous and ethanol fractionation procedures (Bain and Deutsch, 1948; Warner and Weber, 1951), fractional precipitation with ammonium sulfate, or coagulating ovalbumin (Warner, 1954; Azari and Baugh, 1967). However, the drawback of these techniques such as ammonium sulfate precipitation at acidic conditions and ethanol precipitation for purification of ovotransferrin is that they denature ovotransferrin and the purity of the resulting products is relatively low (Vachier et al., 1995). To overcome these drawbacks from ethanol or ammonium sulfate precipitation, cation exchange chromatography such as carboxymethyl cellulose (Rhodes et al., 1958; Azari and Baugh, 1967), diethylaminoethyl (DEAE) cellulose anion exchange chromatography (Mandeles, 1960), and Q-Sepharose Fast Flow column (Vachier et al., 1995) were employed for purification of ovotransferrin from egg albumin. Guerin and Brule (1992) used a cation exchange (Duolite C464 and C476) chromatography for industrial-scale production of ovotransferrin from egg white devoid of lysozyme. Al-Mashikhi and Nakai (1987) used a single-step chromatographic method using an immobilized metal affinity chromatography (a copper-loaded Sepharose 6B column) to separate ovotransferrin from undiluted, blended egg white. Chung et al. (1991) used a bifunctional dye-ligand chromatography using DEAE Affi-Gel Blue as the first step to fractionate egg white proteins. The fraction containing ovotransferrin obtained from DEAE Affi-Gel Blue then was further purified using Fast Flow liquid chromatography. A 2-step chromatographic procedure involving gel permeation on a Superose-6 Prep grade column and anion-exchange chromatography on a Q-Superose Fast Flow was also employed (Awade et al., 1994).
Recently, Ahlborn et al. (2006) separated ovotransferrin from egg white using Econo-Pac High S-Cation and High Q-Anion Exchange cartridges. Even though ion-exchange chromatography or immobilized metal affinity chromatography have been developed on a laboratory scale, the application of these techniques on a pilot-scale is difficult, because these methods are labor-intensive and very expensive (Awade, 1996).
The objective of this study was to develop a simple and rapid procedure for an economical, large-scale production of ovotransferrin from egg white.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Chicken eggs were purchased from a local store. Egg white (500 mL) was separated from the eggs and diluted with the same volume of distilled water. One liter of 2x-diluted egg white solution was blended for 2 min using an electric blender and was used as a starting material for ovotransferrin separation. The work was replicated 3 times.
Separation of Ovotransferrin from Egg White
Because iron-saturated, holo-ovotransferrin is more stable to chemicals such as ethanol than the apo-form, the iron-free ovotransferrin in egg white solution was converted to the iron-bound form using FeCl3 solution. The pH of 2x-diluted egg white solution was adjusted to pH 7.0, 8.0, or 9.0 first, and then NaHCO3 and NaCl were added to reach concentrations in the egg white solution to 50 mM and 0.15 M, respectively, to help iron binding, and then 0.25, 0.5, 1.0, 2.0, or 3.0 mL of 20 mM FeCl3.6H2O solution was added per 100 mL of 2x-diluted egg white solution. The egg white solution was stirred for 30 min at room temperature, and then appropriate amounts of 100% cold ethanol (to make 20, 33, 43, and 50%, final concentrations) were added to precipitate egg white proteins except for the holo-form of ovotransferrin. Holo-ovotransferrin was separated from the precipitated egg white proteins by centrifugation at 3,220 x g for 20 min. The precipitant was reextracted with the same concentration of ethanol and centrifuged at 3,220 x g for 20 min. The supernatants were pooled and filtered through a Whatman No.1 filter paper to remove floating materials. After filtering, cold ethanol (100%) was slowly added to aliquots of supernatant to determine the optimal ethanol concentration for precipitating iron-bound ovotransferrin in the supernatant. The precipitated holo-ovotransferrin was collected after centrifugation at 3,220 x g for 20 min, dissolved in 10 volumes of distilled water, and then the degree of iron saturation in each solution was estimated by measuring the absorbance at 468 nm.
Removal of Iron Using AG1-X2 Resin
AG1-X2 resin (chloride form, Bio-Rad, Richmond, CA) was used to remove iron from the iron-saturated ovotransferrin solution. To facilitate the release of the iron from holo-ovotransferrin, the pH of iron-saturated ovotransferrin solution was adjusted to pH 4.7 using 0.5 M citric acid based on the suggestion of Guo et al. (2003): iron bound to iron-transport proteins such as transferrin and lactoferrin can be removed by forming ferric citrate in the presence of a large excess of citrate. To know the suitable amount of AG1-X2 resin added to the final iron-bound ovotransferrin solution, 0.1, 0.2, 0.3, 0.6, or 0.9 g of AG1-X2 resin was added to 100 mL of iron-bound ovotransferrin solution containing around 6 mg/mL of ovotransferrin. The mixture was gently stirred for 1 h until the yellow color of ferric citrate completely disappeared. The mixture was filtered through a Whatman No.1 filter paper. After releasing iron, the residual iron in the ovotransferrin solution was estimated by the Ferrozine test (Carter, 1971), and the ovotransferrin was freeze-dried. Most of the citric acid added to adjust pH at the iron-removal step was removed as a ferric citrate form. However, the citrate remaining in solution after the iron removal step can affect the iron-binding capacity of ovotransferrin. So, citrate-free solution was prepared by dialyzing the solution against 20 mM phosphate buffer, pH 6.5, and the effect of residual citrate on the iron-binding capacity of ovotransferrin was determined.
Iron-Binding Capacity
The quantity of ovotransferrin was determined using iron-binding capacity, which was measured by the modified method of Williams (1974). The calibration curve was obtained using commercial ovotransferrin (from chicken egg white, substantially iron-free, Sigma, St Louis, MO). To support its iron-binding capability, NaHCO3 and NaCl solution (50 mM and 0.15 M final concentration, respectively) were added to the apo-ovotransferrin solution. After pH adjustment to 8.0, 0.4 mL of 10 mM Fe-nitriloacetate in 50 mM Tris-HCl at pH 8.0 was added to 20 mL of prepared iron-free ovotransferrin, and then the solution was kept at room temperature for 1 h to develop color. Finally, iron-binding capacity of the solution was determined by measuring absorbance at 468 nm. To eliminate trace amounts of metal contaminants, water used in all the procedures was treated with Chelex (Chelex-100 sodium form, Sigma) according to the method reported by Willard et al. (1969). To determine the effect of pH on the iron-binding capacity of ovotransferrin, the pH of apo-ovotransferrin solution was adjusted to pH 4, 5, 6, 7, 8, or 9, and then the iron-binding capacity of each solution was measured by the same method as described above.
Ferrozine Test
After iron removal from the holo-ovotransferrin precipitation, the amount of residual iron in ovotransferrin was measured using the Ferrozine method (Carter, 1971). Disposable polyethylene vessels, which were essentially free of metal contamination, were used for the analysis. Aqueous solutions were prepared with deionized distilled water. Fresh ascorbic acid [1 mL, 0.1% (wt/vol) in 0.2 N HCl] was added to 1 mL of iron-free ovotransferrin solution, and then the mixture was stirred and allowed to stand at room temperature for 5 min. Trichloroacetic acid (11.3% solution, 1 mL) was added to the mixture and centrifuged at 2,500 x g for 10 min. Each standard iron solution under certain concentrations was prepared by serial dilution from 100 ppm Fe(NH4)2SO4·6H2O solution containing 50 ppm concentrated sulfuric acid with distilled water. Ferrozine color reagent (0.8 mL), which was prepared with 75 mg of ferroine (Sigma), 75 mg of neocuproin (Sigma), and 1 drop of 10 N HCl in 25 mL of distilled water, was added to 2 mL of sample and a standard iron solution, and then the mixture was stirred gently. Finally, the optical density of the sample was measured at 562 nm after standing for 5 min.
Yield of Ovotransferrin
To determine the yield of ovotransferrin, egg white was extracted twice using 43% ethanol. The holo-ovotransferrin from the first ethanol extraction was precipitated with 59% ethanol (final concentration), but the holo-ovotransferrin from the second extraction was precipitated with 64% ethanol, because 64% ethanol was the best for precipitating the second extract. The precipitate was dissolved with distilled water and then the iron bound to ovotransferrin was removed using AG1-X2 resin. After iron removal, the concentration of apo-ovotransferrin from each supernatant was determined by measuring iron-binding capacity. Also, after combining the first and second supernatants, the holo-ovotransferrin was precipitated with 59% ethanol to determine the total yield of apo-ovotransferrin. In calculating the yield of apo-ovotransferrin, 7 mg/mL of ovotransferrin in 2x-diluted egg white solution was used, because ovotransferrin comprises 12 to 13% of egg white proteins (Stadelman and Cotterill, 1986) and 11 to 11.5% of egg white is protein (Ahn et al., 1997).
SDS-PAGE
The most suitable amount of ethanol required for extraction and precipitation of holo-ovotransferrin was evaluated using SDS-PAGE. The SDS-PAGE was conducted under nonreducing conditions using Mini-Protean II cell (Bio-Rad). Ten percent SDS gel and Coomassie Brilliant Blue R-250 (Bio-Rad) staining were used. Broad-range SDS-PAGE molecular weight standards of 44 to 200 kDa (Bio-Rad) were used as a marker. Quantification of electrophoreograms and determination of molecular weight of protein bands were conducted with a Pharmacia Phast Imagine Gel Analyzer using AlphaEase FC software (Alpha Innotech Corp., San Leandrao, CA).
Statistical Analysis
All analyses were replicated 3 times, and data were analyzed using the JMP software (version 5.1.1, SAS Institute., Cary, NC). Differences in the mean values were compared by Tukey’s honestly significant difference procedure, and mean values and standard deviations were reported (Kuehl, 2000).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The yield of ovotransferrin was calculated by multiplying the concentration and volume of ovotransferrin and was significantly different depending upon the amount of FeCl3 added. Addition of 2x and 3x the irons required for saturating ovotransferrin produced higher absorbance at 468 nm than those of lower amounts of iron. There was no significant difference in absorbance values at 468 nm between adding 2x and 3x amounts of irons required to saturate ovotransferrin, indicating that all the apo-ovotransferrin in egg white solution was saturated by 2x iron treatment (Figure 2
).
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Addition of ethanol to diluted egg white solution with-out adding FeCl3 denatured apo-ovotransferrin, which co-precipitated with the other proteins present in egg white. Also, addition of 2x iron required for ovotransferrin saturation was the most appropriate for preventing loss of ovotransferrin during ethanol extraction.
pH Effect on Ovotransferrin Separation
The pH of egg white solution had a significant effect on the recovery of the apo-ovotransferrin during ethanol extraction (Table 1). Among the different pH conditions, pH 9.0 produced the highest recovery rate for ovotransferrin, which was around 96%. Even though iron saturation stabilized ovotransferrin by converting the apo-form to the holo-form, high pH (pH 9.0) stabilized holo-ovotransferrin. Also, the effect of pH conditions on the iron-binding capacity of apo-ovotransferrin indicated that iron binds well with ovotransferrin at pH >6.0, but the iron-binding capacity of ovotransferrin rapidly decreased at <pH 6.0 (Figure 3). Griffiths and Humpherys (1977) reported that the N-lobe of ovotransferrin displayed lower iron-binding stability and more accelerated release of Fe3+ than the C-lobe at acidic pH. Also, pH influenced the efficiency of citrate-mediated release of Fe3+ from ovotransferrin in the absence of NaHCO3. Warner and Weber (1951) reported that even though ovotransferrin can be separated from egg white at any pH using ethanol, the intensity of salmon-pink color of iron-bound ovotransferrin solution decreased at <pH 5.5. Williams (1975) reported that iron was bound preferentially to the N-terminal site at pH 8.0, whereas iron binding occurs preferentially at the C-terminal site in acid pH conditions (Williams et al., 1978).
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When 33% of ethanol was added to egg white solution, about 1/2 of the total ovalbumin remained in the supernatant. However, only a small amount of ovotransferrin precipitated at 43% ethanol solution, and most of the ovalbumin precipitated at 43% ethanol (Figure 4). The ovotransferrin separated at 50% ethanol had higher purity than 43% ethanol, but a larger amount of ovotransferrin was precipitated. This suggested that 43% of ethanol was the most suitable condition to produce ovotransferrin possessing high purity and yield from 2x-diluted egg white solution.
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). When 2.5 mL of ethanol was added to 5 mL of supernatant (corresponding to 62% ethanol), the amount of residual ovotransferrin in the supernatant was the smallest. When ethanol added was less than 2.5 mL, some proteins possessing less than 45 kDa of molecular weight appeared in the supernatant. For the production of ovotransferrin both with high purity and yield, therefore, adding 2 mL of ethanol to 5 mL of supernatant obtained from the 43% ethanol extraction (59% ethanol, final) is the most appropriate condition for precipitating iron-bound ovotransferrin.
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When 0.6 or 0.9 g of AG1-X2 resin was added to a 100-mL iron-bound ovotransferrin solution, residual iron content was 1.8 and 1.6 ppm, respectively (Figure 6). The concentrations of residual iron decreased to <0.5 ppm after second addition of 0.6 or 0.9 g of AG1-X2 resin to the same volume of solution. Therefore, the most appropriate amount of AG1-X2 resin for releasing iron from 100 mL of holo-ovotransferrin was around 0.6 g. Because the iron separated from or bound to holo-ovotransferrin could not be removed completely by the first addition of 0.6 g of AG1-X2 resin/100 mL of ovotransferrin solution (6 mg/ mL), a second treatment with 0.6 g of AG1-X2 resin/100 mL of ovotransferrin solution was needed.
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). As in previous reports, citrate played an important role in iron release from holo-ovotransferrin. In this study, citric acid was added to adjust the pH of the holo-ovotransferrin solution to pH 4.7. The added citrate formed ferric citrate with iron released from ovotransferrin. The ferric citrate could be easily removed by AG1-X2 resin. Although some residual citrate can remain in the final apo-ovotransferrin solution, the amount of residual citrate did not affect iron-binding capacity of ovotransferrin. Therefore, it can be concluded that the dialysis step to remove residual citrate was not necessary.
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Iron-bound ovotransferrin (holo-form) in supernatant was stable at alkaline pH (Figure 7). When the pH of the solution was changed to 4.7 by citric acid for iron removal, however, most of the holo-form of ovotransferrin was changed to apo-form. The apo-ovotransferrin formed in acidic conditions was denatured easily by ethanol in the solution. Therefore, it was very difficult to measure the concentration of ovotransferrin in the supernatant containing ethanol, and the yield was measured only at the final stage in which iron-bound ovotransferrin was precipitated with ethanol. The ethanol precipitate was dissolved with distilled water and used to determine the yield of ovotransferrin by measuring iron-binding capacity.
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). Also, the total yield of ovotransferrin using the protocol in Figure 7
was around 99% (Table 4), indicating that almost all ovotransferrin in egg white was recovered through the ethanol extraction method. Also, the purity of the separated ovotransferrin was >80% (Figure 7). Ovalbumin comprising about 50% of total egg white proteins displayed the broadest band on SDS-PAGE (lane 3). Almost all ovotransferrin was precipitated by 59% of ethanol (lane 6), but some proteins still remained in the supernatant (lane 5). The purity of apo-ovotransferrin solution could be increased by reextracting the final holo-ovotransferrin preparation using ethanol before iron removal.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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