Calcified Tissue International

, Volume 36, Issue 1, pp 259–265 | Cite as

Isolation of phosphophoryn from human dentin organic matrix

  • Yuzo Takagi
  • Arthur Veis
Clinical Investigations


Normal human dentin was demineralized in 0.6 N HCl and then extracted in 1.0 M NaCl, 0.5 M Tris/HCl, pH 7.6 in the presence of neutral protease inhibitors. All of the soluble phosphorus-containing proteins were extracted directly in the 0.6 N HCl demineralizing solution; none were collected in the 1.0 M NaCl neutral pH extraction. The principal phosphoprotein was precipitated from solution by 1.0 M CaCl2 and subjected to further chromatographic purification. This fraction proved to be a typical phosphophoryn with Asp and Ser+PSer, in near equimolar amounts, accounting for ∼75 residue percent of the protein. The second major organic phosphate-containing component was a peptide, M4 ∼2,000. It was calcium precipitable and its amino acid composition showed a relationship to phosphophoryn. The residual collagenous matrix, which also contained organic phosphate, was digested with CNBr and the phosphate-containing moiety isolated. This had a composition indicative of a complex of collagen and phosphophoryn. Thus, in spite of the reports by Leaver and colleagues that human dentin contains neither soluble nor matrixbound phosphophoryns, these data show that human dentin, like rat, hamster, rabbit, porcine, and bovine dentins, does contain a phosphophoryn as a major noncollagenous protein.

Key Words

Human dentin Phosphophoryn Phosphopeptides Mineralization 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Veis A, Stetler-Stevenson W, Takagi Y, Sabsay B, Fullerton R (1981) The nature and localization of the phosphorylated proteins of mineralized dentin. In: Veis A (ed) The chemistry and biology of mineralized connective tissues. Elsevier/North-Holland, New York, p 377Google Scholar
  2. 2.
    Termine JD, Kleinman HK, Whitson WS, Conn KM, McGarvey ML, Martin GR (1981) Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26:99–105CrossRefPubMedGoogle Scholar
  3. 3.
    Price PA, Lothringer JW, Nishimoto SK (1980) Absence of the vitamin K-dependent bone protein in fetal rat mineral: evidence for another γ-carboxyglutamic acid-containing component in bone. J Biol Chem 255:2938–2942PubMedGoogle Scholar
  4. 4.
    Dimuzio MT, Veis A (1978) Phosphophoryns: major noncollagenous proteins of rat incisor dentin. Calcif Tissue Int 25:169–178CrossRefGoogle Scholar
  5. 5.
    Weinstock M, Leblond CP (1973) Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentin of the rat incisor. J Cell Biol 56:838–845CrossRefPubMedGoogle Scholar
  6. 6.
    Dimuzio MT, Veis A (1978) The biosynthesis of phosphophoryns and dentin collagen in the continuously erupting rat incisor. J Biol Chem 253:6845–6852PubMedGoogle Scholar
  7. 7.
    Lee SL, Veis A, Glonek T (1977) Dentin phosphoprotein: an extracellular calcium-binding protein. Biochemistry 16:2971–2979CrossRefPubMedGoogle Scholar
  8. 8.
    Zanetti M, Bernard B de, Jontell M, Linde A (1981) Ca2+-binding studies of the phosphoprotein from rat-incisor dentine. Eur J Biochem 113:541–545CrossRefPubMedGoogle Scholar
  9. 9.
    Nawrot CF, Campbell DJ, Shroeder JK, von Valkenburg M (1976) Dental phosphoprotein-induced formation of hydroxyapatite during in vitro synthesis of amorphous calcium phosphate. Biochemistry 15:3445–3449CrossRefPubMedGoogle Scholar
  10. 10.
    Banks E, Nakajima S, Shapiro LC, Tilevitz O, Alonzo JR, Chianelli RR, (1977) Fibrous apatite grown on modified collagen. Science 198:1164–1166PubMedGoogle Scholar
  11. 11.
    Linde A, Bhown M, Butler WT (1980) Noncollagenous proteins of dentin-A re-examination of proteins from rat incisor dentin utilizing techniques to avoid artifacts. J Biol Chem 255:5931–5942PubMedGoogle Scholar
  12. 12.
    Termine JD, Belcourt AB, Miyamoto MS, Conn KM (1980) Properties of dissociatively extracted fetal tooth matrix proteins. II. Separation and purification of fetal bovine dentin. J Biol Chem 255:9769–9772PubMedGoogle Scholar
  13. 13.
    Lyaruu DM, Belcourt A, Fincham AG, Termine JD (1982) Neonatal hamster molar tooth development: extraction and characterization of amelogenins, enamelins and soluble dentin proteins. Calcif Tissue Int 34:86–96PubMedGoogle Scholar
  14. 14.
    Butler WT (1972) Dentinal phosphoproteins. In: Slavkin HC (ed) The comparative molecular biology of extracellular matrices. Academic Press, New York, p 225Google Scholar
  15. 15.
    Leaver AG, Thomas M, Holbrook IB (1977) Glycoproteins of human dentine. Calcif Tissue Res 22:347–349CrossRefPubMedGoogle Scholar
  16. 16.
    Thomas M, Leaver AG (1977) The less-acidic glycoproteins of the organic matrix of human dentine. Arch Oral Biol 22:545–549CrossRefPubMedGoogle Scholar
  17. 17.
    Leaver AG, Price R, Smith AJ (1978) The insoluble fraction isolated after digestion of demineralized human dentine matrix with collagenase. Arch Oral Biol 23:511–513CrossRefPubMedGoogle Scholar
  18. 18.
    Smith AJ, Leaver AG (1978) The effects of periodate degradation and collagenase digestion on the organic matrix of human dentine. Arch Oral Biol 23:535–542CrossRefPubMedGoogle Scholar
  19. 19.
    Kuboki Y, Fujisawa R, Aoyama K, Sasaki S (1979) Calcium-specific precipitation of dentin phosphoprotein: a new method of purification and the significance for the mechanism of calcification. J Dent Res 58:1926–1932PubMedGoogle Scholar
  20. 20.
    Scott PG, Veis A (1976) The cyanogen bromide peptides of bovine-soluble and insoluble collagens. II. Tissue specific cross-linked peptides of insoluble skin and dentin collagen. Connect Tissue Res 4:117–129PubMedCrossRefGoogle Scholar
  21. 21.
    Butler WT, Bhown M, Dimuzio MT, Linde A (1981) Noncollagenous proteins of dentin. Isolation and partial characterization of rat dentin proteins and proteoglycans using a three-step preparative method. Coll Res 1:187–199Google Scholar
  22. 22.
    Green MR, Pastewka JV, Peacock AC (1973) Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Anal Biochem 56:43–51CrossRefPubMedGoogle Scholar
  23. 23.
    Kirkpatrick DS, Bishop SH (1971) Simplified wet ash procedure for total phosphorus analysis of organophosphonates in biological samples. Anal Chem 43:1707–1709CrossRefPubMedGoogle Scholar
  24. 24.
    Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  25. 25.
    Curley-Joseph J, Veis A (1979) The nature of covalent complexes of phosphoproteins with collagen in bovine dentin matrix. J Dent Res 58:1625–1633PubMedGoogle Scholar
  26. 26.
    Stetler-Stevenson WG, Veis A (1983) Bovine dentin phosphophoryn: composition and molecular weight. Biochemistry 22:4326–4335CrossRefPubMedGoogle Scholar
  27. 27.
    Dickson IR, Dimuzio MT, Volpin D, Ananthanarayanan S, Veis A (1975) The extraction of phosphoproteins from bovine dentin. Calcif Tissue Res 19:51–56PubMedGoogle Scholar
  28. 28.
    Partridge SM, Davis HF (1950) Preferential release of aspartic acid during the hydrolysis of proteins. Nature 165:62–63PubMedGoogle Scholar
  29. 29.
    Schultz J, Allison H, Grice M (1961) Specificity of the cleavage of proteins by dilute acid. I. Release of aspartic acid from insulin, ribonuclease and glucagon. Biochemistry 1:694–698CrossRefGoogle Scholar
  30. 30.
    Weiner S, Hood L (1975) Soluble protein of the organic matrix of mollusk shells. A potential template for shell formation. Science 190:987–989PubMedGoogle Scholar
  31. 31.
    Krippner RD, Nawrot CF (1977) The distribution of aspartic acid residues in bovine dentine phosphoprotein. J Dent Res 56:873PubMedGoogle Scholar
  32. 32.
    Lechner JH, Veis A, Sabsay B (1981) Domain sequences in dentin phosphophoryn. In: Veis A (ed) The chemistry and biology of mineralized connective tissue. Elsevier/North Holland, New York, p 395Google Scholar

Copyright information

© Springer-Verlag 1984

Authors and Affiliations

  • Yuzo Takagi
    • 1
  • Arthur Veis
    • 1
  1. 1.Department of Oral BiologyNorthwestern University Dental SchoolChicagoUSA

Personalised recommendations