To understand the role of eggshell organic matrix on the biomineralization process, we have tested the influence of different purified fractions of the eggshell organic matrix on calcium carbonate (CaCO3) precipitation. Purification was carried out after successive anion-exchange chromatography, hydrophobic interaction chromatography and gel filtration chromatography of two different prepurified eggshell extracts (A) and (B); the purified fractions (named g, h, n and r) and (c′, g′, i′, k′) respectively were diluted to 50 μg/ml before being tested in vitro and analysed by the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) procedure and mass spectrometry. The precipitation experiments were carried out by the method of vapour diffusion on crystallization mushrooms. Each purified fraction showed a different effect on CaCO3 precipitation. Some of them exhibited a strong inhibitory effect on nucleation, thus suppressing the precipitation of CaCO3 almost totally while the others did not produce any notable effect. However, all fractions favoured the precipitation of calcite over the other CaCO3 polymorphs. Additionally, all fractions modified in a different manner the size and morphology of the precipitated calcite crystals.

This work has been partially financed by the Projects ‘‘EggDefence’’ of the CE VFP, PIE 200630l133 of Spanish CSIC and MAT2006/11701 of the Spanish Ministry of Education and Science. The Spanish team acknowledge also to the Factoría de Cristalización (Consolider-Ingenio 2010) for logistic support. AHH acknowledges a fellowship from CONACYT (México). ARN acknowledges financial support through the Program Ramo´ n y Cajal (Spain). We thank Dr. J. Cartwright for English corrections.

6 pages, 3 figures, 1 table.

Peer reviewed