Purification and Partial Amino Acid Sequence of Osteogenin, a Protein Initiating Bone Differentiation

TLDR

Osteogenin was purified from bovine bone matrix using 6 M urea extraction followed by heparin‑Sepharose, hydroxyapatite, and Sephacryl S‑200 chromatography, and further refined by preparative SDS‑PAGE without reduction. The purified protein exhibited bone‑inducing activity in a 30–40 kDa zone, its tryptic peptides were sequenced, but reduction and alkylation in 7 M guanidine hydrochloride abolished activity, and reducing SDS‑PAGE revealed a broad 22 kDa band.

Abstract

Osteogenin was purified from bovine bone matrix and its activity monitored by an in vivo bone induction assay. The purification method utilized extraction of the bone-inducing activity with 6 M urea, followed by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Active fractions were further purified by preparative sodium dodecyl sulfate gel electrophoresis without reduction. Osteogenin activity was localized in a zone between 30 and 40 kDa. The amino acid sequences of a number of tryptic peptides of the gel-eluted material were determined. Reduction and alkylation of purified osteogenin in 7 M guanidine hydrochloride resulted in the total loss of biological activity. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a broad band with an apparent molecular mass of 22 kDa.