Previous 31P cross-polarization and differential cross-polarization magic angle spinning (CP/MAS and DCP/MAS) solid-state NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix synthesized by osteoblasts in cell culture identified and characterized the major PO(-3)(4) phosphate components of the mineral phase. The isotropic and anisotropic chemical shift parameters of the minor HPO(-2)(4) component in bone mineral and in mineral deposited in osteoblast cell cultures were found to differ significantly from those of brushite, octacalcium phosphate, and other synthetic calcium phosphates. However, because of in vivo and in vitro evidence that phosphoproteins may play a significant role in the nucleation of the solid mineral phase of calcium phosphate in bone and other vertebrate calcified tissues, the focus of the current solid-state 31P NMR experiments was to detect the possible presence of and characterize the phosphoryl groups of phosphoproteins in bone at the very earliest stages of bone mineralization, as well as the possible presence of calcium-phosphoprotein complexes. The present study demonstrates that by far the major phosphate components identified by solid-state 31P NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with calcium. However, very small amounts of calcium-complexed protein phosphoryl groups as well as even smaller, trace amounts of apatite crystals were also present at the earliest phases of mineralization. These data support the hypothesis that phosphoproteins complexed with calcium play a significant role in the initiation of bone calcification.

Incubation of bovine articular cartilage with papain at 4°followed by several washes with 0.15 m NaCl removes most of the hexosamines, uronic acid, sialic acid, and noncollagen-bound hexoses. Subsequent extraction with 0.45 m NaCl (pH 7.0) causes 18-20% of the collagen to appear in solution. This collagen is still associated with significant amounts of glycosaminoglycans and can be considerably

Circular dichroism and Fourier transform infrared spectroscopic studies of the major amelogenin protein of developing bovine tooth enamel in solution and in the solid state suggest a unique secondary structure containing beta-sheet and repetitive beta-turn structures. The repetitive beta-turn structure at the C-terminal end results from the unique primary structure of amelogenin.

Amelogenins, a class of sparsely phosphorylated hydrophobic proteins characterized by a high concentration of Pro, Gln, Leu, and His have been implicated in the mineralization of tooth Recently a unique primary structure of bovine tooth enamel amelogenin was reported by Takagi et al.,3 characterized by repeating tripeptides Gln-Pro-X near the C-terminal region. Recent CD, Fourier transform infrared, and Chou-Fasman predictions of the secondary structure4 of the lower molecu- Lagant et al.7.' and Krimm and Seaton (see Refs. 9 and 10, and other references cited therein). In addition, Raman spectra provide information on the side-chain conformations of certain aromatic amino acid residues. EXPERIMENTALPreparation of the major highest molecular weight bovine amelogenin from developing premolar teeth has been previously de~cribed.~ Raman spectra were obtained with a Spex Industries Ramalog 5 Raman spectrometer equipped with the computer Spex SCAMP. A sample of amelogenin in aqueous solution was placed in a capillary tube and was illuminated using the 514.5-nm excitation line of an argon-ion laser (Spectraphysics model SP-164). A green interference filter was used to eliminate plasma lines of the laser. The signal was averaged from 10 scans by SCAMP data acquisition processor. Deuterated amelogenin was made by dissolving amelogenin in D,O and then by lyophilization to recover the solid. The sample was again dissolved in D,O for