ANALYSES OF EGG WHITE PROTEINS FROM DIFFERENT SPECIES BY CHROMATOGRAPHIC AND ELECTROPHORETIC METHODS

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1 ANALYSES OF EGG WHITE PROTEINS FROM DIFFERENT SPECIES BY CHROMATOGRAPHIC AND ELECTROPHORETIC METHODS M. Miguel, M. A. Manso, R. López-Fandiño, M. Ramos Instituto de Fermentaciones Industriales (CSIC) Juan de la Cierva, 3, Madrid, Spain Keywords : egg white protein analysis, RP-HPLC, SDS-PAGE, capillary electrophoresis Abstract This work describes the use of different high-resolution techniques for the study of egg white proteins from hen, quail, duck, pheasant and ostrich. For this purpose, RP- HPLC, SDS-PAGE, SDS-capillary gel electrophoresis (SDS-CGE) and free zone capillary electrophoresis using an uncoated capillary at ph 10.0 (CZE) were compared for egg white protein analysis. The major proteins: lysozyme, ovalbumin, ovomucoid and ovotransferrin were separated and identified using the four techniques, although differences were noted regarding the resolution provided by the different systems. Qualitative and quantitative differences were observed among the individual proteins of the animal species investigated. Resumé Cet article présente différentes techniques d analyse hautement résolutives pour l étude des protéines du blanc d oeuf de poule, de caille, de canard, de faisan et d autruche. La chromatographie liquide en phase inverse (RP-HPLC), l électrophorèse en milieu SDS (SDS-PAGE), l électrophorèse capillaire sur gel de SDS (SDS-CGE) et l électrophorèse capillaire de zone (CZE) à ph 10 ont ainsi été comparées. Les protéines majeures : lysozyme, ovalbumine, ovomucoide et ovotransferrine ont été séparées et identifiées avec les quatre techniques, bien que des différences aient été observées entre les systèmes en terme de résolution.. Nous avons également observé des différences qualitatives et quantitatives entre les protéines individuelles des espèces étudiées. 819 CONTENTS 819

2 Introduction Chicken eggs constitute one of the major protein sources of the diet (Powrie and Nakai, 1986). Egg white (albumen) accounts for about 58% of the entire egg mass and contains 10-12% proteins, mainly ovalbumin, ovomucoid, globulins, conalbumin and lysozyme. In addition to their nutritional importance, egg white proteins possess multiple functional properties such as foaming, emulsification and heat setting. This makes egg white an essential ingredient for the food industry (Mine et al., 1995). Furthermore, new uses of egg-white proteins are increasingly being found in the production of health-promoting products, which contribute to enhance their traditional food value. Nevertheless, and despite its importance, hen egg, and particularly egg white, is surprisingly uncharacterized (Desert et al., 2001), and even less is known on egg proteins from other poultry species. Several chromatographic and electrophoretic techniques have been applied to the study of protein components of egg white (Takeuchi et al., 1992; Chen and Tusak, 1994; Desert et al., 2001; Recio et al., 2001). This work compares different highresolution techniques, such as RP-HPLC, SDS-PAGE, SDS-capillary gel electrophoresis (SDS-CGE) and capillary zone electrophoresis (CZE) for the analysis of egg white proteins from different poultry species. Materials and Methods Samples Egg white samples were obtained in the laboratory from fresh shell eggs of different poultry species (hen, pheasant, quail, duck and ostrich), lyophilized and stored frozen until used. Their nitrogen contents were determined by the Kjeldahl method and protein contents were calculated by using a conversion factor of Egg white protein standards were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Molecular weight markers were from Pharmacia (Uppsala, Sweden). RP-HPLC RP-HPLC separations were carried out on a Beckman System Gold HPLC equipment with a System Gold Software data acquisition program 7.11 (Beckman Instruments, Fullerton, CA, USA), with a Hi Pore C 18 column (250 x 4.6 mm, BioRad Laboratories, Hercules, CA, USA). Solvent A was 0.1% (v/v) TFA in HPLC grade water and solvent B was 0.1 % (v/v) TFA in HPLC grade acetonitrile. Elution was at flow rate of 0.8 ml min -1, at room temperature, with a linear gradient from 2% to 65% of solvent B in 60 min. Absorbance was monitored at 214 nm 820 CONTENTS 820

3 SDS-PAGE SDS-PAGE was performed using the PhastSystem Electrophoresis apparatus, Precast Phastgels Homogeneus 20%, and Phastgel SDS buffer strips (Pharmacia, Uppsala, Sweden). Electrophoretic and staining conditions with Phastgel Blue R followed the procedures of the manufacturer. Samples were dissolved in 10 mm Tris-HCl buffer, ph 8.0, containing 25 g L -1 SDS, 10mM EDTA, and 50 ml L -1 β-mercaptoethanol, and heated at 95 ºC for 10 min. Capillary Zone Electrophoresis Capillary zone electrophoresis (CZE) was performed with a Beckman P/ACE System MDQ, following the method described by Chen and Tusak (1994), with some modifications. Separations were carried out at ph 10, using an uncoated fused-silica capillary (NFL-06A, Composite Metal Services, LTD) of 0.40 m x 50 µm i.d. (0.30 m to detection point), with a slit opening of 100 x 800 μm. Migrations were run at 25ºC, with a linear voltage gradient of 0-10 kv in 3 min, followed by a constant voltage of 10 kv for 40 min. Absorbance was monitored at 214 nm. SDS-Capillary Gel Electrophoresis SDS-Capillary Gel Electrophoresis (SDS-CGE) followed Manso et al. (2002), with some modifications. Separations were carried out with a hydrophilic-coated fusedsilica capillary CElect 175 (Supelco, Bellefonte, PA, USA) of 0.27 m x 75 μm i.d. (0.20 m to detection point) and ecap TM SDS gel buffer (Beckman Instruments), as electrophoresis buffer. Migrations were run at 25ºC, with a linear voltage gradient of 2-8 kv in 1.7 min, followed at a constant voltage of 8 kv for 15 min. Absorbance was monitored at 214 nm. Results and discussion Table 1 shows the nitrogen and protein contents of egg white samples of different poultry species. Protein concentrations ranged for 75 to 85%, except for ostrich, which showed the lowest protein level (64%.) Table 1. Nitrogen and protein contents of egg white samples from different species. Results are means of two determinations ± SD. SPECIES NITROGEN (%) PROTEIN (%) HEN 11.97± PHEASANT 12.60± QUAIL 13.36± DUCK 13.60± OSTRICH 10.24± CONTENTS 821

4 Figures 1, 2, 3 and 4 show, respectively, RP-HPLC, SDS-PAGE, SDS-CGE and CZE separations of hen egg albumen standards and egg white proteins from different species. The four techniques allowed the separation of the main proteins: ovoalbumin, ovotransferrin, ovomucoid and lysozyme (which constitute 54%, 12%, 11% and 3.4% of hen egg white proteins, respectively, Mine, 1995). (a) (b) A214 (ua) (c) (d) A214 (ua) (e) (f) Time (minutes) Time (minutes) Figure 1. RP-HPLC of standard proteins of chicken egg white (A): a) pooled standard proteins, b) ovalbumin, c) ovotransferrin, d) ovomucoid, e) avidin, f) lysozyme, and egg white proteins of various poultry species (B): a) hen, b) pheasant c) quail, d) duck and e) ostrich. A B Lysozyme Avidin Ovomucoid, ovoflavoprotein Ovalbumin Ovotransferrin Figure 2. SDS-PAGE of chicken egg white protein standards (A): 1) pooled standard proteins, 2) ovalbumin, 3) ovotransferrin, 4) ovomucoid, 5) avidin, 6) ovoflavoprotein, 7) lysozyme, 8) molecular weight calibration kit (LMWK) and egg white proteins of various poultry species (B): 1) pooled standard proteins, 2) hen, b) pheasant, c) quail, d) duck, e) ostrich. LMWK: (with Mr) phosphorilase b (94,000), bovine serum 822 CONTENTS 822

5 albumin (76,000), ovalbumin (43,000), carbonic anhidrase (30,000), soybean trypsin inhibitor (20,100), α-lactalbumin (14,400). Ovalbumin Ovotransferrin Reference Lysozyme Ovomucoid Figure 3. SDS-Capillary Gel Electrophoresis (SDS-CGE) of hen egg white protein. Ovalbumin Ovomucoid Globulins Ovotransferrin Lysozyme Figure 4. Capillary zone electrophoresis (CZE) of hen egg white proteins. RP-HPLC proved to be useful for the separation and identification of proteins from different species. Schäfer et al. (1995) used a C4 column to quantify albumen proteins during storage of chicken eggs. As expected, SDS-PAGE showed proteins in a wide range of relative molecular masses, present in very different concentrations (Figure 2). Desert et al. (2001) unambiguously identified 6 major proteins in the SDS-PAGE pattern of albumen, however an important number of other well separated bands remained to be characterized. This underlines the complexity of the minor protein components of egg white. SDS-CGE (Figure 3) affords less resolution than SDS-PAGE (Figure 2) for the separation of egg white proteins, but presents the advantage of providing shorter 823 CONTENTS 823

6 analysis times and thus, it offers the possibility of analyzing more samples in a similar period of time. In fact, Hiidenhovi et al. ( 1995) achieved the separation of the main albumen proteins of chicken eggs in only 7 min by SDS-CGE. In agreement with Chen et al. (1994), CZE provides a well-defined separation pattern (Figure 4). Lysozyme, a very basic protein, shows a short migration time. The use of a high ionic strength buffer minimizes its adsorption to the capillary wall. The other proteins migrate according to their isoelectric points. In addition, as compared with the other separation techniques assayed, CZE affords the separation of the albumen globulins (approximately 8%; Mine, 1995). Qualitative and quantitative differences were observed among the individual proteins of the animal species investigated. Hen and pheasant, which are very close phylogenetic species (Phasianidae family), showed very similar chromatographic and electrophoretic protein profiles (Figures 1 and 2). The duck albumen pattern was characterized for a very high relative concentration of ovomucoid. The ostrich albumen presented the most dissimilar pattern: lysozyme was absent, while ovalbumin presented a very slow migration as compared with the other species, which might be indicative of a higher glycosylation degree. References Chen, F.T. and Tusak, A., 1994, Characterization of food proteins by capillary electrophoresis, J. Chromatogr. A, 685: Desert, C., Gúerin-Dubiard, C. Nau, F., Jan, G., Val,.F. and Mallard, J., 2001, Comparison of different electrophoretic separations of hen egg white proteins, J. Agric. Food. Chem., 49: Hiidenhovi, J., Aro, H.S., Huopalahti, R., Suominen, I. and Kankare, V., 1995, Studies of egg white proteins by capillary gel electrophoresis, Proceedings of VI European Symposium on the Quality of Egg and Egg Products, RC Briz (ed), Manso, M., Cattaneo, T.M., Barzaghi, S., Olieman, C. and López-Fandiño, R., 2002, Determination of vegetal proteins in milk powder by sodium dodecyl sulfate-capillary gel electrophoresis: interlaboratory study. J. AOAC Int., 85: Mine, Y., 1995, Recent advances in the understanding of egg white protein functionality, Trends Food Sci. Technol., 6: CONTENTS 824

7 Powrie, W-D. and Nakai, S., in: Stadelman, W.J. and Cotterill, O.J. (Eds), 1986, Egg Science and Technology, Macmillan, London, Recio, I., Ramos, M. and López-Fandiño, R., 2001, Capillary electrophoresis for the analysis of food proteins of animal origin. Electrophoresis, 22: Schäfer, A., Drewes, W., Honikel, K.O. and Schwägele, 1995, Separation and quantification of egg white proteins from fresh and stored eggs by high performance liquid chromatography, Proceedings of VI European Symposium on the Quality of Egg and Egg Products, RC Briz (ed), Takeuchi, S., Saito, T. and Itoh, T., 1992, Rapid analysis of chicken egg white proteins via high-performance liquid chromatography. Anim. Sci. Technol., 63: CONTENTS 825