Schulich Faculty of Chemistry and Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Haifa 32000 Israel.
Proc Natl Acad Sci U S A. 2011 Sep 6;108(36):14763-8. doi: 10.1073/pnas.1102608108. Epub 2011 Aug 22.
Bioavailable calcium is maintained by some crustaceans, in particular freshwater crayfish, by stabilizing amorphous calcium carbonate (ACC) within reservoir organs--gastroliths, readily providing the Ca(2+) needed to build a new 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, its description has eluded efforts to date. Herein, using multinuclear NMR, we accomplish in situ molecular-level characterization of ACC within intact gastroliths of the crayfish Cherax quadricarinatus. In addition to the known CaCO(3), 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 physico-chemical state of ACC stabilizing, P-bearing moieties within the gastrolith is answered directly by the application of solid state rotational-echo double-resonance (REDOR) and transferred-echo double-resonance (TEDOR) NMR to the intact gastroliths: Pi and PEP are found molecularly dispersed throughout the ACC as a solid solution. Citrate carboxylates are found < 5 Å from a phosphate (intermolecular CP distance), an interaction that must be mediated by Ca(2+). 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 CaCO(3). Solid state NMR spectroscopy is shown to be a robust and accessible means of determining composition, internal structure, and molecular functionality in situ.
一些甲壳动物，特别是淡水小龙虾，通过在储器官——胃石中稳定无定形碳酸钙（ACC）来维持生物可利用的钙，这为构建新的外骨骼提供了所需的 Ca(2+)。尽管生物成因 ACC 的原位分子水平图像及其以生物可利用形式稳定的描述具有重要的科学和生物医学意义，但迄今为止，其描述一直难以实现。在此，我们使用多核 NMR 技术对小龙虾 Cherax quadricarinatus 的完整胃石中的 ACC 进行了原位分子水平的表征。除了已知的 CaCO(3)、几丁质支架和无机磷酸盐（Pi）外，我们还在胃石中鉴定出两种主要代谢物，柠檬酸和磷酸烯醇丙酮酸（PEP），并通过应用溶液 NMR 技术对胃石“可溶性基质”来定量它们的丰度。应用固态旋转回波双共振（REDOR）和转移回波双共振（TEDOR）NMR 直接回答了长期以来关于胃石中稳定 ACC 的物理化学状态的问题：Pi 和 PEP 被发现以固态溶液的形式在整个 ACC 中分子分散。柠檬酸羧酸盐被发现与磷酸盐（分子间 CP 距离）<5 Å，这种相互作用必须由 Ca(2+)介导。这些分子与 ACC 基质的高丰度和广泛相互作用表明它们是稳定生物可利用钙形式的主要成分。这项研究进一步强调，必须对完整的生物成因 CaCO(3)进行特征描述。固态 NMR 光谱被证明是一种强大且易于获取的原位确定组成、内部结构和分子功能的方法。
