Background: A core sequence (the 9 C-terminal residues) of calcification-associated peptide (CAP-
1) isolated from the exoskeleton of the red swamp crayfish was previously shown to control calcium carbonate
precipitation with chitin. In addition, a modified core sequence in which the phosphorylated serine at the N
terminus is replaced with serine exhibits was also previously shown to alter precipitation characteristics with
Objectives: We focused on calcium carbonate precipitation and attempted to elucidate aspects of the mechanism
underlying mineralization. We attempted to evaluate in detail the effects of modifying the N-terminus in the
core sequence on calcium carbonate mineralization without chitin.
Methods: The peptide modifications included phosphorylation, dephosphorylation, and a free or acetylated Nterminus.
The peptides were synthesized manually on Wang resin using the DIPCI-DMAP method for the first
residue, and Fmoc solid phase peptide synthesis with HBTU-HOBt for the subsequent residues. Prior to calcium
carbonate precipitation, calcium carbonate was suspended in MilliQ water. Carbon dioxide gas was bubbled into
the stirred suspension, then the remaining solid CaCO3 was removed by filtration. The concentration of calcium
ions in the solution was determined by standard titration with ethylenediaminetetraacetate. Calcium carbonate
precipitation was conducted in a micro tube for 3 h at 37°C. We used the micro-scale techniques AFM (atomic
force microscopy) and TEM (transmission electron microscopy), and the macro-scale techniques chelate titration,
HPLC, gel filtration, CD (circular dichroism) and DLS (dynamic light scattering).
Results: We determined the morphologies of the calcium carbonate deposits using AFM and TEM. The pS
peptide provided the best control of the shape and size of the calcium carbonate round particles. The acetylated
peptides (Ac-S and Ac-pS) provided bigger particles with various shapes. S peptide provided a mixture of bigger
particles and amorphous particles. We verified these findings using DLS. All the peptide samples produced
nanostructures of the expected size in agreement with the AFM and TEM results. We estimated the abilities of
these peptides to precipitate calcium carbonate by determining the residual calcium hydrogen carbonate concentration
by standard titration with ethylenediaminetetraacetate after calcium carbonate precipitation. The Ac-pS
peptide showed the lowest residual calcium hydrogen carbonate concentration whereas the S peptide showed the
highest, suggesting that the precipitating activities of these peptides towards calcium carbonate correlated with
peptide net charge. Then the gel filtration results showed a large oligomer peak and a small oligomer/monomer
peak for all peptide samples in agreement with the AFM, TEM and DLS results. CD measurements showed that
all the peptides formed random-coil-like structures. Thus, we used both macro- and micro-observation techniques
such as chelate titration, DLS, AFM and TEM to show that the calcium carbonate precipitating activities
of four derivatives of the core sequence of CAP-1 may correlate with the peptide net charge.
Conclusion: These peptides mainly act as a catalyst rather than as a binder or component of the calcium carbonate
deposits (as a template). On the other hand, the morphologies of the calcium carbonate deposits appeared
to be dependent on the ability of the peptide to assemble and act as a template. Consequently, elucidating the
relationship between peptide sequence and the ability of the peptide to assemble would be indispensable for
controlling precipitate morphologies in the near future. This knowledge would provide important clues for elucidating
the relationship between peptide sequence and mineralization ability, including deposit morphology
and precipitating activity, for use in nanobiochemistry and materials chemistry research.