|M.Sc Student||Akiva Anat|
|Subject||Molecular Details of ACC Stabilization: NMR Insights into|
Cherax Quadricarinatus Gastroliths
|Department||Department of Chemistry||Supervisor||Professor Asher Schmidt|
|Full Thesis text|
Crystalline and amorphous calcium carbonates (ACC) are the most abundant building blocks of biomineralization from which biomaterials with a wide variety of functionalities are created. Highly bioavailable calcium reservoirs are maintained by some crustaceans, in particular freshwater crayfish, by stabilizing ACC within a pair of reservoir organs - gastroliths, serving to replenish its newly formed exoskeleton. Despite the key scientific and biomedical importance of the in situ molecular-level picture of biogenic ACC and its stabilization in a bioavailable form, such a description has eluded efforts to date.
Herein, using solid state and solution NMR, in situ molecular level characterization of biogenically stabilized ACC within intact gastroliths of the crayfish Cherax quadricarinatus is demonstrated. In addition to the known CaCO3, chitin scaffold and inorganic phosphate (Pi), we identify within the gastrolith two primary metabolites, citrate and phosphoenolpyruvate (PEP) and quantify their abundance by applying solution NMR techniques to the gastrolith "soluble matrix". The long-standing question on the physicochemical state of ACC stabilizing, P-bearing moieties within the gastrolith is answered directly by the application of solid state REDOR and TEDOR NMR to the intact gastroliths: Pi and PEP are found molecularly dispersed throughout the ACC as a solid solution. Citrate carboxylates are found <5Å from a phosphate (intermolecular CP distance) an interaction that must be mediated by Ca. The high abundance and extensive interactions of these molecules with the ACC matrix identify them as the central constituents stabilizing the bioavailable form of calcium. This study further emphasizes that it is imperative to characterize the intact biogenic CaCO3. Solid state NMR spectroscopy is shown to be a robust and accessible means of determining composition, internal structure and molecular functionality in situ.