Several mechanisms have been proposed to explain the interactions between proteins and mineral surfaces formed during biomineralization. To investigate the effect of the surface charge of proteins on calcium carbonate precipitation, a group of globular proteins with similar sizes and molecular weights but with different isoelectric points (iep) has been selected to be added to a CaCl2 solution in free-drift calcium carbonate precipitation experiments. These proteins are lysozyme (Lyz; theoretical iep 9.32), ribonuclease-A (Rib-A; theoretical iep 8.64), myoglobin (Myo; theoretical iep 7.36), and α-lactalbumin (α-La; theoretical iep 4.83). Depending on their isoelectric point and their concentration in the solution, these proteins affected the nucleation, growth, polymorphism and growth morphology of calcium carbonate in different manners, evidencing different types of protein−surface interactions. For the protein with an acidic isoelectric point (α-La), electrostatic interactions were predominant. For proteins with isoelectric points around neutrality or slightly higher (Myo and Rib-A), the foremost interactions were hydrophobic with a certain electrostatic contribution. For the protein with a basic isoelectric point (Lyz), there was not any notable effect on most of the analyzed precipitation properties, evidencing a weaker protein−surface interaction.

No abstract

The hen eggshell is formed in a three-stage sequential process, namely, the initial stage (nucleation of calcite crystals), the active growth phase (linear deposition), and the terminal stage (inhibition of crystal formation). During these phases, different proteins are sequentially expressed in the uterine fluid. Some of them are thought to regulate the shell mineralization and particularly crystal growth. To identify proteins that are actively involved in this process, we have analyzed and compared the effects on CaCO3 precipitation in vitro of some commercial egg white proteins, some noncommercial purified fractions of the eggshell organic matrix and, also of uterine fluids extracted from the hen uterus during each one of the stages of calcification. Uterine fluids strongly increased the nucleation density with respect to commercial proteins and purified fractions of the shell. A few identified acidic proteins (ovocleidin-17, ovocalyxin-32, osteopontin and ovocleidin-116) showed a strong affinity for the CaCO3 surfaces and were selectively removed from the solution during its precipitation. These proteins may have an active role on CaCO3 growth, aggregation, and inhibition. Other proteins, with very different isoelectric points, seem to regulate the chemical environment in which the precipitation takes place, that is, by buffering the pH favoring crystal growth as the couple ovocleidin-17 and ovocalyxin-21.

The nucleation and polymorphism of calcium carbonate have been studied using a microdevice named the crystallization mushroom. This setup allows carrying out precipitation experiments reproducibly by the vapor diffusion sitting drop technique. Within the range of concentrations investigated (from 10 to 500 mmol/L CaCl2 and from 1 to 25 mmol/L NH4HCO3), the dominant polymorph to appear first in the drops was calcite, or mixtures of calcite and vaterite followed by aragonite. Additionally, amorphous calcium carbonate (ACC) was not observed. The order of appearance of the polymorphs in the droplets is explained by intrinsic features of the crystallization mushroom, that is, the slow increase in the ionic activity product caused by slow diffusion of NH3 and CO2 gases, which favors the least soluble phase calcite to crystallize before other more soluble polymorphs. The appearance of calcite as the first nucleating dominant polymorph in the drops allowed us to calculate its surface free energy from induction time measurements assuming the mononuclear nucleation model. The experimentally calculated result of 35 mJ/m2 is lower than the value predicted for homogeneous nucleation. The cause is the existence of heterogeneous nucleation taking place at the air−solution and solution−support interfaces.

Calcium phosphate precipitation was carried out in the presence of L-aspartic acid (L-asp, iep = 2.77), L-alanine (L-ala, iep = 6.00), and L-arginine (L-arg, iep = 10.76) at different concentrations by using a vapor diffusion sitting drop method (VDSD) in microdroplets. Irrespective of the nature and the concentration of the amino acid used, the early stage in the precipitation consisted in the formation of a white viscous suspension composed of amorphous calcium phosphate (ACP) spherulites. After 1 week, different calcium phosphate phases were found depending on the amino acid nature and concentration. At higher concentrations of L-aspartic acid, brushite (dicalcium phosphate dihydrate, DCDP) platelets and a few needle-like carbonate-hydroxyapatite (HA) crystals were found. In the presence of higher concentrations of L-alanine, the precipitate was composed of both needle-like HA and octacalcium phosphate (OCP) platelets. Finally, at higher concentrations of L-arginine, we obtained carbonate-HA nanocrystals with length of 20À40 nm and a few OCP crystals, as in the blank experiment (without amino acids). The results are explained on the basis of the influence of these amino acids on the pH evolution of the solution and on the nature and strength of the interactions of the major charged species of the amino acids with the surface lattice ions of the apatite precursor phases (DCPD or OCP). A thermodynamic model, based on the temporal existence of OCP in the solution, is proposed to explain the formation of the HA nanocrystals.