Formation of Transient Amorphous Calcium Carbonate Precursor in Quail Eggshell Mineralization:  An In Vitro Study

Abstract

To understand the mechanism of quail eggshell biomineralization, we have performed two CaCO(3) precipitation experiments. In the reprecipitation experiments, supersaturated Ca(HCO(3))(2) was prepared by bubbling CO(2) through a slurry of biogenic CaCO(3) obtained from bleach-treated eggshell followed by filtration to obtain a clear solution for crystallization experiments. The nucleated crystals were collected at various time intervals and analyzed. In the second experiment, the extracted SOM from the bleach-treated eggshell was added to the supersaturated clear solution of Ca(HCO(3))(2) solution obtained by bubbling CO(2) gas through a slurry of synthetic CaCO(3) followed by filtration. The crystals/precipitates collected at various time intervals were analyzed. Both experiments showed that amorphous CaCO(3) (ACC) was precipitated in the early stages, which then transformed to the most stable crystalline calcite phase. Amino acid analysis of the soluble organic matrixes (SOM) indicated the presence of high amounts of Glx and Asx amino acids. Ovomucoid--an acidic glycoprotein, and lysozyme--a basic protein, are the two major components along with a few low molecular weight peptides present in the SOM of quail eggshell matrix. Both ovomucoid and lysozyme did not induce precipitation of the ACC phase in in vitro conditions, while the fraction containing low molecular weight peptides induced the precipitation of ACC, suggesting that the latter play an important role in the eggshell biomineralization. Thus, organisms can produce inorganic minerals which assume nonequilibrium morphologies and intricate architecture by precipitating transient ACC, which then transformed into the crystalline phase. Altogether, these observations further demonstrate that this strategy may be common in both vertebrate and invertebrate mineralized structures.