Bovine dentin phosphophoryn: composition and molecular weight

Abstract

The molecular weight of phosphophoryn, an acidic phosphoprotein unique to dentin matrix, has been difficult to determine because of a combination of neutral protease activities in this tissue and the intrinsic high charge density of the molecule. In this study, bovine dentin phosphophoryn (BDPP) was isolated by a procedure designed to prevent proteolysis. Bovine unerupted third molar powder was demineralized by ethylenediaminetetraacetic acid (EDTA). The EDTA-soluble phosphophoryn fraction was isolated and purified by sequential calcium chloride precipitation, gel filtration in sodium dodecyl sulfate (NaDodSO4) containing buffer, anion-exchange chromatography, and finally gel filtration in 4 M guanidine hydrochloride (4 M Gdn.HCl) buffer. Sedimentation equilibrium, sedimentation velocity, and diffusion coefficient data, viscosity studies in a high ionic strength buffer, and NaDodSO4 gradient gel electrophoresis data gave consistent results for the molecular weight of BDPP, all being in the range of 151 000-167 000. This range is much higher than any previously reported value. An anomalous behavior was observed in nongradient NaDodSO4 gel electrophoresis. Dissociative analytical gel filtration chromatography in 4 M Gdn.HCl gave a molecular weight value of 100 000. This discrepancy was resolved by studying the viscosity of BDPP in 4 M Gdn.HCl which showed BDPP does not assume a true random-chain conformation in this solvent.