Light microscopy and scanning electron microscopy (SEM) images show that aragonite precipitated as needles up to 20 μm long, and calcite as rhombohedrons () with an edge length of about 20 μm. The purity of the microcrystal powder measured by X-ray diffraction () was 97 wt % for aragonite (the remaining 3 wt % are calcite impurities) and 100 wt % for calcite, with an uncertainty of 1 wt %. Thus, the protocol used for crystallization produced different polymorphs with reasonable purities. Determination of the specific surface area of aragonite and calcite by evaluation of light microscopy images and gas adsorption (BET method, see, e.g., [4
]) both showed a roughly ten times larger specific surface area for aragonite. The actual values were different though, the BET method giving 4.07 m2
/g for argonite and 0.40 m2
/g for calcite, while the geometric estimate from the light microscopy images was 0.78 m2
/g to 1.72 m2
/g for aragonite and 0.10 m2
/g to 0.19 m2
/g for calcite. The values from the light microscopy images were estimated from 20 aragonite microcrystals assumed to be cylinders, neglecting top and bottom area, and ten calcite microcrystals assumed to be rhombohedrons with all twelve edges of equal length. The density for aragonite is 2.95 g/cm3
and 2.71 g/cm3
for calcite [5
Precipitated aragonite needles (left) and calcite rhombohedrons (right). Upper: light microscopy images. Lower: scanning electron microscopy images.
Figure 2 X-ray diffraction patterns of precipitated aragonite (upper) and calcite rhombohedrons (lower). Purity is 96–98 wt % for aragonite and 99–100 wt % for calcite as evaluated with the BRASS software suite . Indexing according to data from (more ...)
shows the result of the protein–crystal binding experiment. The proteins in the shell from Haliotis laevigata that were insoluble in 6% acetic acid were removed from the chitin core with an SDS/DTT/Tris buffer as described in the experimental section. This protein solution contained several proteins or protein fragments visible in lane P on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in . The protein solution was gel-filtered to remove the SDS and DTT. The obtained gel-filtered protein solution (lane gfP) shows the major bands on SDS-PAGE. The gel-filtered protein solution was incubated with aragonite or calcite microcrystals. After incubation, the supernatant liquid from the aragonite or calcite microcrystals was removed and subjected to SDS-PAGE at two different concentrations (AS, AS* and CS, CS*). The crystals were washed 3 times with a NaCl/Tris solution. The washing solution was retained and combined for aragonite (AW) and calcite (CW), respectively, and also subjected to SDS-PAGE. The most dominant bands were still observable. The washed crystals were dissolved in 6% acetic acid. The proteins that were attached to the aragonite crystals (A) or to the calcite crystals (C) were obtained by removing the acetic acid by dialysis.
Figure 3 SDS-PAGE of proteins after different preparational steps as follows. M: marker proteins of known size. P, P*: protein solution of proteins that are insoluble in 6% acetic acid (chitin associated proteins), at two different concentrations. Orange marked (more ...)
Lanes A and C show the important difference between aragonite and calcite. In contrast to lane C, there are still proteins detectable by SDS-PAGE in lane A. This indicates that more protein was attached to the aragonite microcrystals. Generally, this could be expected due to the greater specific surface area of the aragonite microcrystals used compared to the calcite microcrystals. Nonetheless a lysozyme control experiment shows that unspecific binding cannot be the only reason for the larger amount of protein in lane A (here lane A from ). This control experiment shows the influence of the greater specific surface area of aragonite on the SDS-PAGE results, assuming unspecific binding of lysozyme to both, aragonite and calcite. A difference in intensities in lanes A and C from due only to unspecific binding seems unlikely, since lanes A and C () show no difference in intensity.
Figure 4 SDS-PAGE of control experiment with lysozyme. M: marker proteins of known size. L, L*: high concentrated lysozyme solution, at two different concentrations. AS, CS, AS*, CS*: supernatant of incubated aragonite and calcite crystals, at two different concentrations. (more ...)
Verification of a specific binding would be most interesting, because this would be evidence that protein–mineral interaction guide polymorph selection and morphology of the calcium carbonate crystals. This would require a competitive assay. However, there is a suggestion of specific binding of one protein to aragonite in our results. Looking at the two protein bands indicated by the orange and green arrows in and then comparing the relative intensity of these two protein bands in lane P (entity of chitin associated proteins) and their relative intensity in lane A (aragonite associated proteins) shows an increased relative concentration of the one indicated by the orange arrow in lane A.
To estimate the detection limit for proteins in this experiment, different quantities of lysozyme were subjected to SDS-PAGE (data not shown). While for 50 ng lysozyme the band is still visible, it is not for 5 ng. So for calcite there was less than 50 ng of each protein on 10 μg crystal powder. In the protein–crystal binding experiment 15 to 20 μL of protein solution was subjected to SDS-PAGE per lane. Assuming that the detection limit for different proteins is the same, we estimate the minimum protein concentration in the subjected solution for a visible band to be 2.5 to 3.3 μg/mL.