Properties of dissociatively extracted fetal tooth matrix proteins. II. Separation and purification of fetal bovine dentin phosphoprotein.
After an initial 4 M guanidine HCl extraction to remove enamel contaminants, subsequent guanidine HCl/EDTA extraction of fetal bovine molar dentin removes almost all of the high molecular weight phosphoprotein from the tissue in a single step. The fetal bovine dentin phosphoprotein is then purified by a series of ion exchange (DEAE-cellulose or DEAE-Se-phacel) and gel filtration (Sepharose CL-6B) chromatographic steps, all under denaturing elution conditions in 7 M urea (ion exchange) or 4 M guanidine HCl (gel filtration). The final purified phosphoprotein product was chromatographically and electrophoretically homogenous. The apparent molecular weight for this fetal bovine dentin phosphoprotein was approximately 100,000 both by 4 M guanidine HCl gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This value is considerably higher than those previously reported for any other dentin phosphoprotein preparation. Roughly half of the total serine residues in the protein were phosphorylated and these residues together with aspartic acid comprised approximately 80% of the total polypeptide backbone. The purified fetal bovine dentin phosphoprotein was also glycosylated, containing approximately 6 galactosamine, 2 to 3 glucosamine, and 2 to 3 sialic acid residues/mol.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002850:Chromatography, Gel; D003746:Dental Enamel Proteins / Q000302:isolation & purification*; D003804:Dentin / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005333:Fetus; D006146:Guanidines; D008970:Molecular Weight; D009306:Natal Teeth / Q000032:analysis*; D010750:Phosphoproteins / Q000302:isolation & purification*; D011247:Pregnancy; D014094:Tooth, Deciduous / Q000032:analysis*
D000596:Amino Acids; D002241:Carbohydrates; D003746:Dental Enamel Proteins; D006146:Guanidines; D010750:Phosphoproteins