Bovine and rat dentin contain aspartylphosphoseryl-enriched collagen-associated phosphoproteins which represent 1-2% of the mineralfree dry weight. These phosphophoryn moieties are not extracted by saturated neutral EDTA, pH 7.4, nor by guanidine hydrochloride-EDTA, pH 7.4. Cyanogen bromide degradation of the dentin matrix does release a high molecular weight fragment containing hydroxyprolyl, hydroxylysyl, prolyl, and glycyl residues as well as high concentrations of aspartyl and phosphoseryl residues, the amounts of which indicate a 50% collagen-50% phosphophoryn nature. Gel filtration and ion exchange chromatography under dissociative, denaturing conditions, as well as in the presence of disulfide bond reducing reagents failed to separate the collagen and phosphophoryn moieties. Hydroxyapatite, which selectively absorbs phosphophoryn, also failed to separate the collagenous component, leading to the conclusion that the moieties represented a covalent conjugate. 3~p NMR spectroscopy showed the bovine collagen-phosphophoryn complex to contain only phosphomonoesters similar to soluble phosphophoryn. Reduction with [3H]NaBH4, followed by cross-link analysis, did not reveal any reduced aldimine cross-link amino acids. Of the 4 hydroxylysyl residues/1000 in the intact bovine collagenphosphophoryn complex, one-fourth are periodate resistant, indicating either O-or N-substitution. The periodate-resistant hydroxylysyl residues are located in bacterial collagenase-sensitive regions, and it is likely that these represent hydroxylysine Oglycosides. These data suggest that: (a) the collagenous component of the conjugate derives from a glycosylated peptide, probably c~2CB4, and (b) the Present address: Send offprint request~s to Dr. Arthur Veis at above address. association is covalent, but does not involve disulfides, phosphate-, hydroxylysine-, or reducible aldehyde-mediated covalent bonds.

Analogs of phosphophoryn, a calcium‐binding phosphorylated protein found in mineralized dentin, were synthesized by solid phase peptide synthesis. The dentin phosphophoryn appears to contain some sequence blocks of (Asp‐PhosphoSer)n. As this sequence is difficult to synthesize, polymers of (α‐L‐Glu‐L‐Ser) were prepared. The 30‐peptide, (α‐L‐Glu‐L‐Ser)15, was phosphorylated by reaction with orthophosphoric acid in the presence of trichloroacetonitrile in anhydrous dimethylsulfoxide. Calcium ion binding studies were carried out with both the 30‐peptide and phosphorylated 30‐peptide. The conformation of the original 30‐peptide, (Glu‐Ser)15, was examined, in the presence and absence of calcium ion, by circular dichroism measurements. Purified bovine phosphophoryn, previously studied by the same techniques, was partially dephosphorylated by alkaline phosphatase, and its calcium ion binding properties were determined. Dephosphorylation to 31% of the initial phosphorus content reduced the number of high affinity sites to ˜ 30% of the initial value. However, the stoichiometry of binding indicated that both phosphate and carboxylate groups participate in the high affinity binding and that the binding constant was decreased only slightly. Partial phosphorylation of the 30‐mer raised the calcium binding constant, Ka, from 2.1 times 102 to 3.3 × 103 M‐1 and increased the amount of binding from an electrostatic equivalent number of sites to a stoichiometric equivalent number. Concomitant with binding, there was a transition from random coil to ß‐like structure. These data suggest that the repetitive (Asp‐PhosphoSer)n regions in phosphophoryn and the (Glu‐PhosphoSer)n sequence of the synthetic polymer have special conformations which favor the unidentate binding of calcium to the carboxyl groups and phosphate groups, and which enhance the binding affinities of the carboxyl groups in such sequences in a cooperative fashion.