Phosphoprotein analysis of sequential extracts of human dentin and the determination of the subsequent remineralization potential of these dentin matrices.
Phosphoprotein appears to play an important role in the mineralization of dentin during tooth development and remineralization after demineralization by dental caries. To better understand this role, we describe the extraction and characterization of phosphoprotein from immature, human root apex dentin during and after EDTA demineralization. The extraction procedure included dissociation of the demineralized dentin matrix by guanidine hydrochloride (Gdn.HCl) followed by subsequent digestion with cyanogen bromide (CNBr) and collagenase. Characterization of these extracts included 'Stains-All' staining of SDS polyacrylamide gels (SDS-PAGE) and amino acid, protein and phosphorus analyses. The ability of these matrices to remineralize was determined by TEM and measuring calcium levels in the remineralized tissue by atomic absorption spectroscopy. The staining of SDS-PAGE gels and amino acid analysis showed that an intact phosphophoryn was extracted from the dentin of the immature apices during EDTA demineralization and that it had an apparent Mr approximately 140,000. In the subsequent extracts and digests, the phosphoprotein has a range of molecular weights, some of which may have been degraded products of the intact phosphoprotein. A greater quantity of phosphoprotein was found in the EDTA-demineralized dentin matrices than in dentin after Gdn.HCl, CNBr and collagenase digests. These EDTA-demineralized matrices also remineralized to a greater extent than those dissociated with Gdn.HCl. The differences in both the quantity and the quality, as defined by the amino acid residue profile, of the phosphoprotein in the sequential extracts of the root apex dentin may be important in affecting the ability of this tissue to remineralize.
Department of Cariology, Restorative Sciences and Endodontics, University of Michigan, Ann Arbor, Mich., USA. bricla@umich.edu
B H
BH
Clarkson
S R
SR
Chang
G R
GR
Holland
D000293:Adolescent; D000596:Amino Acids / Q000032:analysis; D002118:Calcium / Q000032:analysis; D002648:Child; D017364:Collagenases; D004396:Coloring Agents; D003488:Cyanogen Bromide; D003731:Dental Caries / Q000378:metabolism / Q000503:physiopathology; D003804:Dentin / Q000737:chemistry* / Q000378:metabolism; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006801:Humans; D008854:Microscopy, Electron; D008970:Molecular Weight; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010758:Phosphorus / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012967:Sodium Dodecyl Sulfate; D013054:Spectrophotometry, Atomic; D019227:Tooth Apex / Q000737:chemistry / Q000378:metabolism; D014074:Tooth Calcification / Q000502:physiology; D017001:Tooth Demineralization / Q000378:metabolism / Q000503:physiopathology; D014089:Tooth Remineralization
D000596:Amino Acids; D004396:Coloring Agents; D010750:Phosphoproteins; D011506:Proteins; C017630:phosphophoryn; D010758:Phosphorus; D012967:Sodium Dodecyl Sulfate; D004492:Edetic Acid; D017364:Collagenases; D019791:Guanidine; D003488:Cyanogen Bromide; D002118:Calcium