Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition

Abstract

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemes, we isolated 14 apparently homogeneous components varying in molecular weight from .apprx. 150K to .apprx. 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from .apprx. 4% to .apprx. 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of .apprx. 150K, .apprx. 90K, and .apprx. 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the .apprx. 90- and .apprx. 60-kilodalton (kDa) and of the .apprx. 150- and .apprx. 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphorprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.