CRYSTAL GROWTH & DESIGN
Eggshell Membrane Removal for Subsequent Extraction of Intermineral and Intramineral Proteins
2002 VOL. 2, NO. 6 529-532
Maggie Cusack*,† and Alex C. Fraser†,‡ Division of Earth Sciences, and Veterinary School, University of Glasgow, Glasgow, U.K. Received July 31, 2002;
Revised Manuscript Received August 19, 2002
ABSTRACT: Biominerals are composite materials with organic and inorganic components. The organic components influence nucleation, growth, and the physical and material properties of the biomineral. Calcium minerals account for about 50% of all biominerals, and polymorphic calcium carbonate is dominant with calcite and aragonite being the most common natural forms. The eggshell of the domestic fowl Gallus gallus comprises calcite, in association with organic components, precipitated on membranes. Environmental scanning electron microscopy (E-SEM) analysis displays this intimate mineral-organic intergrowth between the inner membrane and the calcite mammillary caps. Before analysis of organic components within the eggshell, the inner membrane must be removed. Membrane removal is achieved by either plasma ashing (etching), sodium hypochlorite, or acid (HCl) treatment. These three methods are compared with the aim of subsequent extraction of intra- and intermineral proteins. Plasma ashing is suitable for subsequent SEM of the inner surface of the eggshell but entirely unsuitable for subsequent protein extraction. Removal of the inner membrane by sodium hypochlorite or acid (HCl) treatment are both suitable methods. Sodium hypochlorite treatment is the preferred method since the protein yield is highest here, and, unlike acid treatment, it does not remove part of the calcite fraction. Introduction The minerals of living systems all have organic material intimately associated with the inorganic phase. The organic components decrease the nucleation energy required for mineral precipitation and control the size and shape of the biomineral.1-3 By close association with the mineral, the organic material also influences the properties of the biomineral producing stronger materials with, for example, different fracture properties from the wholly inorganic counterpart.4-6 In a wide range of biomineralized systems such as eggshells, bones, teeth, and invertebrate exoskeletons, proteins are influential in the formation of biominerals.7-11 The organicinorganic composite structures produced by living organisms have very different properties from their wholly inorganic equivalents. The microlaminate composite of the molluscan shell is about 3000 times stronger than the crystals themselves.12 The occlusion of proteins within mineral structures results in conchoidal rather than brittle fracture.6 Calcium minerals account for about 50% of all biominerals,13 and polymorphic calcium carbonate is dominant with calcite and aragonite being the most common natural forms. In consequence, calcium carbonate biogenesis is employed by a wide range of phyla to produce biominerals that fulfill specific functions. Table 1 provides some examples. Avian eggshells are a readily available source of calcium carbonate for study of calcium carbonate biomineralization. The eggshell of the domestic fowl Gallus gallus comprises calcite in association with several soluble proteins of 10-116 kDa.14 Many of these egg* To whom correspondence should be addressed. Division of Earth Sciences, Gregory Building, University of Glasgow, Lilybank Gardens, Glasgow G12 8QQ U.K. Tel: +44 (0)141 330 5461. FAX: +44 (0)141 330 4817. http://www.earthsci.gla.ac.uk. † Division of Earth Sciences. ‡ Veterinary School.
Table 1. Examples of Occurrence and Functions of Calcite and Aragonite Biominerals function
examples of occurrence
embryonic chambers protective exoskeleton
avian eggshell shell of molluscs, brachiopods, and bryozoans otoconia in reptiles, amphibians, birds, and mammals otolith and otoconia, respectively, in teleost fish
gravity perception sound and gravity reception
shell proteins have been identified, e.g., ovalbumin,15 ovocleidin-17,16 osteopontin,17 and ovotransferrin.18 Nucleation of the eggshell occurs on membranes (Figure 1). These membranes must be removed before the intra- and intermineral proteins can be extracted and characterized. The outer membrane is easily removed manually. The inner membrane has a tighter association with the mammillary caps (Figure 2). Removal of the inner membrane is achieved using a variety of methods. In studies requiring membrane removal prior to assessment of the mammillary caps by scanning electron microscopy (SEM), plasma etching is employed.19,20 Before extraction of intra- and intermineral proteins, membranes are removed by scraping of the calcite of the mammillary caps,18,21 acid etching of the calcite of the mammillary caps, or oxidation of the membrane by sodium hypochlorite.22,15 No comprehensive comparison of each method has been made. In this study, the outer membrane was removed manually in all cases. The inner membrane was removed by plasma etching, sodium hypochlorite, or acid (HCl) treatment. The result of each treatment was assessed by SEM. Guanidine-HCl (GnHCl) extractable material (intermineral) and ethanediaminetetraacetic acid (EDTA)-soluble (intramineral) components from each treatment were quantified by amino acid analysis to identify which method of membrane removal is most suitable for subsequent extraction of inter- and intramineral proteins.
10.1021/cg0255624 CCC: $22.00 © 2002 American Chemical Society Published on Web 10/08/2002
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Cusack and Fraser
Figure 1. Digital SEM image of a transverse section of an eggshell of the domestic fowl Gallus gallus. Image depicts the inner membrane, mammillary caps (MA), palisade layer (PA), vertical crystal layer (VCL), cuticle (C), and pore (PO).
Figure 2. Digital E-SEM image of eggshell mammillary surface at low vacuum (5.6 Torr) and 85% relative humidity depicting the intimate interaction between calcite nucleation sites (mammillary caps) and the inner membrane. Scale bar represents 20 µm.
Experimental Section Eggs were purchased commercially from one source. For examination of eggshell and membrane by environmental scanning electron microscopy (E-SEM), eggs were opened, their contents discarded, and the outer membrane removed before 2 cm2 pieces were examined in an XL30 Philips E-SEM, initially at 90% relative humidity. Humidity was then decreased to monitor the result of membrane desiccation in real time. For membrane removal, 10 eggs were used for each treatment, and each treatment was carried out in duplicate. For acid and hypochlorite treatment, empty eggshells were cut longitudinally and each cleaned half placed in a bed of sand for support. The eggshell halves were then filled with either sodium hypochlorite (37% active chlorine) for 20 min or HCl (1 N) for 5 min. The eggshells were then emptied and rinsed in 18 MΩ water. For sodium hypochlorite treatment, several half shells were partially filled to allow SEM analysis of treated and untreated inner surface. For amino acid analysis, only completely filled shells were used. For plasma etching, cleaned shells were broken into 2 cm2 pieces and placed membrane side uppermost in the chamber, and organic membrane was removed by volatalization for 4 h in a Nanotech plasma etching unit in an oxygen atmosphere of 133.3 Pa made reactive by applying a radio frequency of 100 Ω. Intermineral proteins were extracted using guanidine hydrochloride (GnHCl) + protease inhibitors (PI).23 GnHCl was removed by ultrafiltration on a Minitan system, and the extract was further concentrated using a Centriprep 10 and then a Microcon 10 system. Subsequently, intramineral proteins were extracted by EDTA dissolution of the mineral.
Figure 3. Digital SEM images of gold coated mammillary surface of eggshells. (A) mammillary surface after plasma etching at 100 Ω for 4 h. (B) Mammillary surface with (bottom left) and without (top right) incubation with sodium hypochlorite (37% active chlorine) for 20 min. (C) Eroded mammillary surface following incubation with HCl (1 N) for 5 min. Scale bars represent 200 µm. EDTA was removed, and the extracts were concentrated as for the intermineral proteins. For amino acid analysis, aliquots of each EDTA and GnHCl extract were hydrolyzed by manual hydrolysis. Lyophilized samples in hydrolysis tubes were placed in hydrolysis vials containing 500 µL of HCl (6 N). Vials were purged with argon at 2-3 psi for 30 s, and vials were closed and heated at 165 °C for 1 h for vapor-phase hydrolysis. Amino acid concentrations were determined on a 420 amino acid analyzer from Perkin-Elmer-Applied Biosystems.
Eggshell Membrane Removal for Proteins
Figure 4. Concentration of eggshell amino acids. Amino acid concentration of intramineral and intermineral proteins from avian eggshells with outer membrane removed manually and inner membrane removed by plasma etching, sodium hypochlorite, or acid (HCl) treatment. Amino acids expressed as pmol/mg of shell. Triplicate analyses from duplicate preparations.
Results and Discussion E-SEM analysis makes possible observations of materials in their near native state since low vacuum and high humidity conditions can be maintained, and thus, the inner membrane is observed without desiccation. E-SEM analysis of the mammillary surface of untreated eggshells at 85% relative humidity (RH) depicts the inter-relationship between the calcite nucleation sites (mammillary caps) and the inner membrane (Figure 2). The mammillary surface of eggshells treated by plasma etching, sodium hypochlorite, and HCl, as viewed by SEM, are presented in Figure 3. Acid etching is an efficient means of removing the inner membrane by dissolution of a portion of the mammillary caps. This removal of a portion of the shell from subsequent analyses is not suitable for assessment of mammillary features or extraction of inter- or intramineral protein extraction since proteins included with that fraction would be lost. Plasma etching and sodium hypochlorite treatment are both very efficient methods for membrane removal as determined by SEM imaging of the mammillary surface (Figure 3). For assessment of mammillary features, plasma etching and sodium hypochlorite incubation are both suitable methods for membrane removal. Amino acid analysis of the inter- and intramineral extract quantifies these fractions. The concentrations of amino acids in the inter- and intramineral extract obtained from the three methods are very different (Figure 4). In all cases, there is a higher concentration of EDTA-soluble (intramineral) amino acids than there is GnHCl-extractable (intermineral) amino acids. The highest concentration of EDTA-soluble amino acids is obtained following sodium hypochlorite treatment (4051 ( 138 pmol of amino acid/ mg of eggshell). Acid etching reduces the concentration
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of amino acids (1986 ( 252 pmol of amino acid/mg of eggshell) relative to the sodium hypochlorite treatment since the organic-rich mammillary caps have been removed. The lowest concentration of EDTA-soluble amino acids was extracted from eggshells where the membrane had been removed by plasma etching (420 ( 32 pmol of amino acid/mg of eggshell). The heating incurred during plasma etching is likely to denature the proteins of the EDTA- soluble fraction such that a chaotropic agent like GnHCl is required to solubilize some of these proteins. However, even with GnHCl, only a tiny fraction of the proteinaceous material is extracted. In conclusion, oxidation of the inner membrane by sodium hypochlorite treatment removes the inner membrane in a simple and efficient manner. This approach is ideal for further assessment of the mammillary surface (Figure 3). Since it does not alter or remove any calcite, the entire calcite fraction is available for subsequent analysis, and the intermineral and intramineral fraction can be subsequently released by treatment with GnHCl and EDTA, respectively. Higher concentrations of intra- and intermineral amino acids were extracted following membrane removal by sodium hypochlorite than by either HCl treatment or plasma etching. Acknowledgment. Cusack and Fraser thank BBSRC Grant 17/D09549. Both authors are most grateful to Dr. Jim Buckman of the Department of Petroleum Engineering, Heriot-Watt University, Edinburgh, for Environmental Scanning Electron Microscopy Analysis. References (1) Mann, S. Struct. Bonding 1983, 54, 125-174. (2) Mann, S. New Sci. 1990, 125, 42-47. (3) Weiner, S.; Addadi, L. Trends Biochem. Sci. 1991, 16, 252256. (4) Weiner, S.; Traub, W. Philosophical Transactions of the Royal Society of London, Series B 1984, 304, 425-434. (5) Berman, A.; Hanson, J.; Leiserowitz, L.; Koetzle, T. F.; Weiner, S.; Addadi, L. Science 1993, 259, 776-779. (6) Addadi, L.; Aizenberg, J.; Albeck, S.; Berman, A.; Leiserowitz, L.; Weiner, S. Mol. Cryst. Liq. Cryst. 1994, 248, 185198. (7) Addadi, L.; Berman, A.; Moradian-Oldak, J.; Weiner, S. Croat. Chem. Acta 1990, 63, 539-544. (8) Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Nature 1996, 381, 56-58. (9) Falini, G.; Albeck, S.; Weiner, S.; Addadi, L. Science 1996, 271, 67-69. (10) Cusack, M.; Walton, D.; Curry, G. B. In Treatise on Invertebrate Paleontology: Part H, Brachiopoda; Kaesler, R., Ed.; Geol. Soc. Am. and University Kansas Press: New York & Lawrence, 1997; Vol. 1, Chapter 4, pp 243-266. (11) Cusack, M.; Laing, J. H.; Brown, K.; Walton, D. Trends Comp. Biochem. Physiol. 2000, 6, 47-56. (12) Currey, J. D. Proc. R. Soc. London, B. 1977, 196, 443-463. (13) Lowenstam, H. A.; Weiner, S. On Biomineralization; Oxford University Press: New York, 1989. (14) Nys, Y.; Hincke, M. T.; Arias, J. L.; Garcia-Ruiz, J. M.; Solomon, S. E. Poult. Avian Biol. Rev. 1999, 10, 143-166. (15) Hincke, M. T. Connect. Tissue Res. 1995, 31, 227-233. (16) Hincke, M. T.; Tsang, C. P. W.; Courtney, M.; Hill, V.; Narbaitz, R. Calcif. Tissue Res. 1995, 56, 578-583. (17) Pines, M.; Knopov, V.; Bar, A. Matrix Biol. 1995, 14, 765771. (18) Gautron, J.; Hincke, M. T.; Dominguez-Vera, J. M.; GarciaRuiz, J. M.; Nys, Y. In Proceedings of the European Poultry Meat and Egg Quality Symposium; Ed. Kijoski, J. Poznan, 1997; pp 172-181. (19) Reid, J. Br. Poult. Sci. 1983, 24, 233-235.
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(20) Solomon, S. E.; Hughes, B. O.; Gilbert, A. B. Br. Poult. Sci. 1987, 28, 585-588. (21) Panheleux, M.; Bain, M.; Fernandez, M. S.; Morales, M. S.; Gautron, J.; Arias, J.; Solomon, S.; Hincke, M. T.; Nys, Y. Br. Poult. Sci. 1999, 40, 240-252. (22) Hincke, M. T.; Bernard, A. M.; Lee, E. R.; Tsang, C. P. W.; Narbaitz, R. Br. Poult. Sci. 1992, 33, 505-516.
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