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Eur Food Res Technol (2005) 221:542546 DOI 10.



Marta Miguel M. Asuncin Manso Rosina Lpez-Fandio Mercedes Ramos

Comparative study of egg white proteins from different species by chromatographic and electrophoretic methods
Received: 18 June 2004 / Revised: 22 February 2005 / Published online: 14 July 2005  Springer-Verlag 2005

Abstract This work describes the use of different highresolution techniques to study egg white proteins from hen, quail, duck, pheasant and ostrich. For this purpose, reversed-phase high-performance liquid chromatography, sodium dodecyl sulfatepolyacrylamide gel electrophoresis and isoelectric focusing (IEF) were used to compare egg white proteins from these poultry species. The major proteins, lysozyme, ovalbumin, ovomucoid and ovotransferrin, were separated and identified using the three techniques. Isoforms of ovotransferrin and ovalbumin in some species were observed when IEF with gels of pH 4 6 were used. Qualitative and quantitative differences were observed among the individual proteins of the different species investigated. Keywords Egg white protein analysis Reversed-phase high-performance liquid chromatography Sodium dodecyl sulfatepolyacrylamide gel electrophoresis Isoelectric focusing Poultry species

Chicken eggs constitute one of the major protein sources of our diet [1]. Egg white (albumen) accounts for about 58% of the entire egg mass and has a protein content of 1012%, comprising mainly ovalbumin, ovotransferrin, ovomucoid, globulins and lysozyme. In addition to their nutritional importance, egg white proteins present multiple functional properties, such as foaming, emulsification and heat-setting. This makes egg white an essential ingredient for the food industry [2]. Furthermore, new uses of egg white proteins are increasingly being found for the production of health-promoting products, which contribute to enhancing their traditional food value. NeverM. Miguel M. A. Manso R. Lpez-Fandio M. Ramos ()) Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva, 3, 28006, Madrid, Spain e-mail: mramos@ifi.csic.es Tel.: +34-91-5622900 Fax: +34-91-5644853

theless, despite their nutritional and technological importance, hen egg white proteins are surprisingly less studied than other protein sources such as milk proteins or soya proteins [3]. Moreover, egg white proteins from other less known poultry species, such as quail, duck, pheasant and ostrich, are proving to be very useful and could, in some cases, present certain advantages over hen egg white proteins [4, 5]. Interspecies variability may explain particular characteristics concerning technological, functional and biological performance of egg proteins from different avian origins. Comparative studies of egg proteins from these poultry species are, therefore, necessary, in order to define qualitative and quantitative differences between them. The aim of this work was to perform preliminary studies to analyse and compare egg white proteins from different avian species by chromatographic and electrophoretic techniques (reversed-phase high-performance liquid chromatography, RP-HPLC, sodium dodecyl sulfatepolyacrylamide gel electrophoresis, SDS-PAGE, and isoelectric focusing, IEF) in order to investigate potential new egg white properties.

Materials and methods

Samples Egg white samples were obtained in the laboratory from fresh eggs of different poultry species (hen, pheasant, quail, duck and ostrich), lyophilised and stored at 20 C until use. Their nitrogen contents were determined by the Kjeldahl method and protein contents were calculated by using a conversion factor of 6.0 [6]. Chicken egg white protein standards were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Molecular weight markers were from Amersham Biosciences (Uppsala, Sweden). Reversed-phase high-performance liquid chromatography RP-HPLC separations were performed according to the method of Itoh et al. [7], with some modifications. The proteins were separated on Beckman System Gold HPLC equipment with System

543 Gold Software data acquisition program 7.11 (Beckman Instruments, Fullerton, CA, USA), with a Hi Pore C18 column (2504.6 mm, BioRad Laboratories, Hercules, CA, USA). Solvent A was 0.1% (v/v) trifluoroacetic acid (TFA) in HPLC-grade water and solvent B was 0.1% (v/v) TFA in HPLC-grade acetonitrile. Elution was performed at room temperature, at a flow rate of 0.8 ml min1 and with a linear gradient from 2 to 65% of solvent B in 60 min. Absorbance was monitored at 214 nm. Before injection, the samples were filtered through 0.45-mm filters (Millipore Corporation, Bedford, MA, USA). SDS-PAGE and IEF SDS-PAGE and IEF analyses were performed using a PhastSystem electrophoresis apparatus (Amersham Biosciences). SDS-PAGE separations were run on commercial 20% polyacrylamide precast gels, Homogeneous 20, using PhastGel SDS buffer strips (Amersham Biosciences). Electrophoretic and staining conditions with PhastGel Blue R and Silver stain followed the procedures recommended by the manufacturer. Approximately 3 mg of sample was dissolved in 450 ml of 10 mM tris(hydroxymethyl)aminomethaneHCl (Sigma Chemicals, St Louis, MO, USA) sample buffer (pH 8.0), containing 2.5% SDS (Merck, Darmstadt, Germany), 10 mM EDTA (Merck) and 5% 2-mercaptoethanol (Merck), and was heated at 95 C for 10 min. A 1-ml aliquot of each sample was applied at the cathode of the apparatus. A mixture of low molecular weight marker proteins ranging from 14 to 94 kDa was included in each run. IEF was performed using commercial precast IEF gels 39 and IEF gels 46 (Amersham Biosciences). Each sample was previously desalted with a hydrophilic 10,000-Da cut-off membrane (Centriprep, Amicon, Beverly, MA, USA) by centrifugation at 1,900g for 40 min. Retentates from ultrafiltration were lyophilised and stored at 20 C until use. One microlitre of sample (approximately 1 mg ml1) was directly placed on each well of the sample applicator 6/4 (Amersham Biosciences-LKB) with a micropipette. Electric conditions and staining followed the procedures recommended by the manufacturer.

Results and discussion

Determination of nitrogen concentration (Table 1) showed that all species present a similar protein content, ranging from 71 to 82%. Ostrich albumen showed the lowest protein concentration (61%). The highest protein concentration was observed in quail and duck albumen, which contained more than 80% protein. From a quantitative point of view, eggs from the latter species could be interesting sources of protein for different uses. Different chromatographic and electrophoretic methods, SDS-PAGE (Fig. 1), IEF (Figs. 2, 3) and RP-HPLC (Fig. 4), were used to analyse egg white proteins of various avian species. When analysed by SDS-PAGE (Fig. 1), egg white proteins presented a wide range of relative molecular masses, present in very different concentrations. The main hen egg white proteins, ovoalbumin, ovotransferrin, ovomucoid and lysozyme, constitute, respectively, 54, 12, 11 and 3.4% of total hen egg white proteins [2]. Figure 1A shows the standards of several hen egg white proteins. These standards allowed similar proteins to be identified in the egg white of other species. The majority of egg white proteins are glycoproteins, which should be taken into account when they are analysed by electrophoretic methods in the presence of SDS, as the carbohydrate moieties lead to an overestimation of molecular weight. As observed in Fig. 1B, SDS-PAGE revealed that the electrophoretic profile is qualitatively and quantitatively very similar in hen and pheasant egg white, which is in accordance with their phylogenetic proximity (order Galliforms). Ovalbumin was the major protein and was observed in all species, with some differences. Hen, pheasant, quail

Table 1 Nitrogen and protein contents of lyophilised egg white samples from different poultry species. The results are means of two determinations the standard deviation. Species Hen Pheasant Quail Duck Ostrich Nitrogen (%p/p) 11.970.06 12.600.11 13.360.38 13.600.03 10.240.00 Protein (%p/p) 71.82 75.60 80.16 81.60 61.44

Fig. 1 Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of egg white proteins. a 1 Pooled standard albumen proteins from Sigma Chemicals, 2 ovalbumin, 3 ovotransferrin, 4 ovomucoid , 5 avidin, 6 ovoflavoprotein, 7 lysozyme, 8 low molecular weight calibration kit (LMWK) (with Mr): phosphorylase b (94,000), bo-

vine serum albumin (76,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100), a-lactalbumin (14,400). b 1 Pooled standard proteins from Sigma Chemicals, 2 hen, 3 pheasant, 4 quail, 5 duck and 6 ostrich albumen, 7 LMWK


Fig. 2 Isoelectric focusing (IEF) with gels of pH 39 of egg white proteins of various poultry species: (a) Coomassie blue staining; (b) Silver staining. 1 Pooled standard proteins of hen albumen, 2 hen, Fig. 3 IEF with gels of pH 46 of egg white proteins of various poultry species: 1 pooled standard proteins of hen albumen, 2 hen, 3 pheasant, 4 quail, 5 duck and 6 ostrich albumen, 7 isoelectric point calibration kit (Amersham Biosciences)

3 pheasant, 4 quail, 5 duck and 6 ostrich albumen, 7 isoelectric point calibration kit (Amersham Biosciences)

and duck ovalbumin had similar electrophoretic profiles, with an estimated molecular mass of approximately 45 kDa. However, duck ovalbumin was less concentrated. Ostrich ovalbumin presented a distinct pattern, with a very slow migration compared with that of the other species, and with a higher molecular mass of approximately 58 kDa, which might indicate a higher degree of glycosylation. Therefore, there are differences between the ovalbumin of avian species. Eggs are among the foods that most frequently cause allergies, principally hen eggs and particularly the ovalbumin. Anibarro et al. [8] demonstrated that the ovalbumin was responsible for a case of food allergy after consumption of egg from duck and goose in an adult without hen egg allergy, and suggested that the antigenic determinant of this protein was specific to the Anseriformes order and it was not present in the ovalbumin of the Galliformes order. Ovotransferrin was present in all species with a similar electrophoretic profile and had a molecular mass of approximately 70 kDa. Moreover, hen, pheasant and quail ovotransferrin had similar concentrations, and were higher than those of duck and ostrich ovotransferrin. Ostrich ovotransferrin also presented a different molecu-

lar mass (approximately 75 kDa). The diffuse band, of approximately 3539 kDa, corresponded to ovomucoid and flavoprotein. This profile was similar in hen, pheasant and ostrich. However, the electrophoretic profiles of quail and duck were different, showing a lower molecular mass (3033 kDa), and especially the duck albumen presented a very high relative concentration of ovomucoid. This protein has a molecular mass of 28 kDa [9]. These results could be explained by the high glycosylation of the ovomucoid (approximately 25%) and also indicate that hen, pheasant and ostrich ovomucoid could present a greater degree of glycosylation than quail and duck ovomucoid. The high concentration of ovomucoid in duck egg white has been used in biospecific chromatography, to protect insulin against the action of proteolytic enzymes. The biospecific interaction of the polysaccharide component of ovomucoid with lectins leads to the targeting transport of hydrogel particles onto the small intestine wall [4]. Besides, the employment of avian ovomucoid was proposed to purify toxins by affinity chromatography, because the ovomucoid fraction from certain avian species contains antigenic activity against some types of toxins [10]. The protective effect of ovomucoid


from several species (turkey, duck and chicken) against salmon calcitonin metabolism by serine proteases has been compared. Turkey and duck ovomucoid were shown to be the most effective [11]. Lysozyme (approximately 14 kDa) showed the highest concentration in hen egg white, and was observed in all species, but was not found in ostrich albumen. It is known that two, radically different, types of lysozyme occur in bird egg white. The C-type has long been known to be present at a high concentration in egg white of the domestic chicken and many other species in the Galliformes order as well as in egg white of some species of the waterfowl order Anseriformes [12, 13, 14]. The existence of a different type of lysozyme, called G-type, in egg white from domestic geese has also been described [15, 16]. C-type and G-type lysozymes differ radically in amino acid sequence, molecular weight, and enzymatic properties, and are also antigenically distinct. Prager and Wilson [17] confirmed the presence of G-type lysozyme in several avian egg whites, including ostrich. The presence of G-type lysozyme in duck and ostrich albumen could explain the differences with the lysozyme of other species. Duck lysozyme showed a molecular mass of approximately 15 kDa, and the band corresponding to lysozyme in ostrich albumen was not identified. This protein could migrate in a different way in SDS-PAGE. The study of different types of lysozyme could permit to discover the sequences responsible for the enzymatic activity and to construct proteins with more potent activities [18]. Avidin was observed (Fig. 1, lane 1) with an estimated molecular mass of 18 kDa by SDS-PAGE, although its theoretical molecular mass is 68.3 kDa [5]. According to Korpela [19], avidin is fragmented into four monomers of 16 kDa under reducing conditions. Proteins such as ovomucin and ovostatin, with a molecular mass higher than 100 kDa [20], cannot be observed using this type of polyacrylamide gel because it only permits resolutions between 14 and 97 kDa. IEF has been widely used to separate proteins on the basis of differences in their isoelectric points. The major protein bands were identified by comparison with standard individual hen egg white proteins. (Fig. 2) Moreover, in some cases this method allowed the separation of G2 and G3 globulins (pI 5.5 and 5.8, respectively). Similar results has been obtained by Li-Chan and Nakai [21], using conventional IEF. In Fig. 2A, lanes 2, 3, 4 and 5, it should be noted that lysozyme and avidin, with a high pI, both over 10, did not appear in the commercial gels with a pH ranging between 3 and 9. Differences were noted among the ovalbumins from the different poultry species, as ostrich ovalbumin exhibited the most acidic pI (approximately 3.98), while quail and pheasant ovalbumin showed the most basic pI (approximately 4.54.6). Hen and pheasant ovotransferrins were very similar (lanes 2 and 3, pI 6.8). The other species showed small differences in their pI. The lowest concentration was observed in duck and ostrich albumen. With IEF other bands can be observed belonging to proteins with pI between 4.5 and

Fig. 4 Reversed-phase high-performance liquid chromatography of egg white proteins of various poultry species: a hen, b pheasant, c quail, d duck and e ostrich albumen

6.5, but these proteins could not be identified. The Silver staining method (Fig. 2B) makes it possible to visualize better the bands than Coomassie brilliant blue staining, and it also allows to distinguish the globulins in hen, pheasant, quail and duck albumen. Besides, we observed other bands with pI between 6.8 and 7.4; these bands were probably from different forms of ovotransferrin. Desert et al. [3] identified seven proteins, together with an important number of other well-separated bands that remain to be characterised. This emphasizes the complexity of the minor protein components of egg white. Several minor egg white proteins have been characterized by Western blotting with monoclonal antibodies [3]. Similarly, differences have been reported in minor egg white proteins of different poultry species. Chicken avidin is a protein used in a wide variety of applications in life sciences owing to its strong affinity for biotin, and has a new and promising use in pretargeting cancer treatments. Other avian species offer pharmacokinetic and immunological advantages over chicken avidin in pretargeting applications [5]. In order to clarify the differences between the isoelectric points in these species, we repeated the isoelectric focusing in the commercial gels with a pH ranging from 4 to 6, because the majority of egg white proteins have their pI in this range (Fig. 3). This method permitted different isoforms of ovalbumin and ovotransferrin to be identified. The three ovalbumin isoforms with different degrees of phosphorylation may be observed in the case of hen, pheasant, quail and duck. Ovotransferrin was also ob-


served and three isoforms were identified. According to Desert et al. [3], this polymorphism reflects the existence of isoforms with no, one or two iron atoms per molecule. Using IEF in the commercial gels with a pH ranging from 4 to 6, we could use gradient to differentiate the proteins from eggs of different origins. RP-HPLC has also been used to separate proteins from different avian species. Figure 4, spectrum a shows a typical elution profile at 214 nm of hen egg white proteins. The method allowed to separate with different retention times ovomucoid, lysozyme, ovotransferrin and ovalbumin. The results obtained were similar to those obtained with SDS-PAGE and we can see that the profile of duck presents a very high relative concentration of ovomucoid and a lower content of ovotransferrin. The chromatogram profile that belongs to ostrich albumen (Fig. 4, spectrum e) showed the most important differences. RP-HPLC and SDS-PAGE using Phast System methods have been proved reliable methods to separate the principal proteins from different avian species. Both can be used as routine methods. In general terms the drawback which has often been associated with gel electrophoretic methods is the quantification of the bands. The use of IEF using different gradients permitted to differentiate isoforms of ovalbumin and ovotransferrin in the different avian species . In conclusion, all the techniques managed to separate the main egg white proteins, and qualitative and quantitative differences were observed between species. This variability between species suggests advantages in the use of proteins from different egg sources, which not only have a similar protein richness to that of hen egg but may also present other beneficial health properties. This work constitutes a preliminary study on the different characteristics of these proteins present in the albumen of different species. Nevertheless, more studies are required using proteomic techniques to characterize novel proteins and their properties.

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