The Isolation and Characterization of a Family of Water-Soluble Proteins from Haliotis rufescens Raymond J. Clifford 1, Robert Hickey 1, Dante’ Tolbert 2, Tyson Wickboldt 2 Loyd D. Bastin 1 1 Department of Chemistry, Widener University, Chester, PA Joint Science Department, Claremont McKenna College, Claremont, CA Abstract Organisms have been producing mineralized parts for the last 550 million years. Hard parts have evolved to serve a variety of functions, and paramount to this evolution are the associated macromolecules that control nucleation, growth, shape, and physical strength of biominerals. The growth of biominerals play an important role in the growth and shape of endo- and exoskeletons in many organisms. Previous studies of a family of water-soluble proteins isolated from the outer calcite layer of Haliotis rufescens (Red Abalone) indicated that the proteins bind non- uniformly to growing calcite crystal surfaces. However, little is understood about the mechanism by which this binding occurs. To this end, we are studying the effect of these proteins on the growth of calcite and aragonite crystals. In order to elucidate the mechanism of habit modification within the red abalone, we are systematically studying the effect of protein fragments on crystal growth in order to determine the structurally important domain of the protein(s) involved in the mineralization process. Here we report the isolation of a two families of water-soluble proteins from the outer calcite layer and inner aragonite layer of the red abalone. Introduction The vast majority of life utilizes biomineralization, a process which converts ions into solid crystals, spanning across at least 55 phyla, and is widespread in both land and marine life. Many organisms have adapted biomineralization in parallel with one another, however it is possible to track the roots of a species by comparing their biomineralization processes. The main function of biologically controlled mineralization is to provide framework and support, mainly in the form of endo- and exoskeletons. There are a few different polymorphs of calcium carbonate. Calcite and aragonite are the two most common forms. Calcite is more thermodynamically stable, however aragonite does often form. It has been demonstrated that certain conditions can favor aragonite growth over calcite. Non-biologically, aragonite growth is seen in caves. The Red Abalone has specific proteins that induce the growth of aragonite. There are two proposed mechanisms for how this happens: the protein has an active site which substitutes on the crystal surface (Figure 8), or the protein influences the growth through coulombic and intermolecular binding (Figure 9). Morse et al. has shown the presence of 13 proteins in the shells of the Red Abalone. Six proteins (110, 48, 44, 35, 28, and 24 kDA) in the calcite layer while seven proteins (7,8,18,24,41,56, and 116 kDa) are present in the aragonite layer. However, the importance of each protein or the role they play in crystal formation was not investigated. Methods and Materials Aragonite growth was induced by allowing calcium carbonate crystals to grow on a thin layer of silk generated by a colony of silk worms. Calcite was removed from the abalone shells by sand blasting (Figure 5). The half-shell was ground up and dissolved in EDTA. Infrared spectroscopy was used to ensure purity of aragonite. The resulting solution was dialyzed extensively against deionized water Finally the water was removed by lyopholization and the protein solid was dissolved in minimal water to have a saturated protein solution. The Gel Electrophoresis showed five bands, one not previously reported: 82 kDa. UV Spectroscopy found significant absorbance at 280cm -1 for the protein solution Conclusions and Future Work The gel electrophoresis and the UV spectroscopy proves successful isolation of the proteins from the aragonite layer. Gel electrophoresis determined the approximate molecular weights to be 110, 82, and 58 kDa. These proteins will then be separated by gel filtration chromatography and inserted into a solution of growing calcium carbonate crystals with non specific dyes to observe the effects that the individual protein will have on aragonite growth. Also the proteins will be sequenced in order to determine the primary and secondary structure of each protein. Acknowledgements I would like to thank Dr. Loyd Bastin for incorporating me into his lab. Thanks to Dante’ Tolbert and Tyson Wickbodt for paving the way. Thanks to US Abalone for supplying the shells Also I would like to thank the Department of Chemistry and the Department of Biology of Widener University for funding and allowing us the use of equipment. This project was supported by funds from The Joint Science Department of The Claremont Colleges, Pitzer College, and Widener University. References Addad, L; Beikovitch-Yellin, Z.; Weissbuch, I; Van mil, J.; Shiman, L.J.W.; Lahav, M.; Leiserowitz, L. Angew. Chemie Int. Eng. Ed. 1985, 24, Belcher, A. M., Wu, X. H., Christensen, R. J., Hansma, P.K., Stucky, G. D., Morse, D. E. (1996). Control of crystal phase switching and orientation by soluble mollusk-shell proteins. Nature 381, Tolbert, Dante. Interactions of Proteins and Crystal Surfaces: The Mechanism of Biomineralization Formation in Haliotis rufescens. (2003). Senior Thesis in Biology, Claremont McKenna College. Zaremba, C.; Morse, D.; Mann, S.; Hansma, P.; Stucky, G.; Critical transitions in the biofabrication of abalone shells and flat pearls Chemistry of Materials (1998), 10(12), http ://belcher10.mit.edu/research/research.html Fig. 1 Aragonite side of Red Abalone shell. Fig. 3: Infrared Spectrum of Aragonite isolated from Red Abalone Shell. Fig. 7: Nanoscale structure of the aragonite layer in red abalone shell Figure 2 Calcite layer of Red Abalone Shell Figure 4: Infrared Spectrum of calcite isolated from abalone shell Figure 5: The original abalone shell’s calcite layer (left) is removed with a sandblaster (center) to reveal the aragonite layer underneath (right). Figure 6: Habit Modification significantly changes the crystal structure by altering the rates of growth for a particular side Figure 8, 9: The two theories of how proteins influence crystal growth: substitution mechanism (left) and intermolecular forces/coulombic binding (right) Figure 10,11: Unmodified calcium carbonate crystal (left) is a simple cube compared to calcium carbonate crystal modified by a biological organism (right) Figure 12: Gel electrophoresis of the aragonite layer (left) and the calcite layer (right).