Isolation and complete amino acid sequence of osteocalcin from canine bone

J Bone Miner Res. 1993 Jun;8(6):733-43. doi: 10.1002/jbmr.5650080612.


Osteocalcin was purified in high yield and to homogeneity from the diaphysis of dog femora by the following steps: (1) acid demineralization of bone powder, (2) solid-phase extraction of acid-soluble proteins on Sep-Pak C18 cartridges, (3) gel filtration on Sephadex G-50, and (4) fast protein liquid chromatography on an Accell-QMA anion-exchange column. Starting from 30 g washed bone powder, approximately 7-10 mg pure protein was obtained in 2 days. The key step is the initial solid-phase extraction of osteocalcin from a large volume of a demineralized bone solution. The primary structure was established by automated sequence analyses of two tryptic peptides, of two endoproteinase Glu-C carboxy-terminal peptides, and of the first 30 amino acid residues of the intact protein. Dog osteocalcin contains 49 amino acids, has a molecular mass of 5654 daltons, contains no Thr, Met, Hyp, or Trp, has a disulfide bond between Cys 23 and 29, and is fully gamma-carboxylated at residues 17, 21, and 24. Dog osteocalcin does not contain a pair of basic amino acids found at positions 43-44 in most other osteocalcins from mammals and birds. A computer search for homology indicated 88, 90, 84, 88, 66, and 57% sequence identity of dog osteocalcin with human, bovine, cat, monkey, chicken, and swordfish osteocalcin, respectively, and weaker homologies with the gamma-carboxylated domains of blood-clotting proteins and the Pro-rich N-terminal extensions of myosin light-chain A1 and beta-crystalline B1. The possible relevance of these homologies to the structure and potential functions of osteocalcin is discussed.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acids / analysis
  • Animals
  • Bone and Bones / chemistry
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Dogs
  • Electrophoresis, Polyacrylamide Gel
  • Endopeptidases
  • Glutamates / isolation & purification
  • Glutamic Acid
  • Humans
  • Molecular Sequence Data
  • Osteocalcin / chemistry
  • Osteocalcin / isolation & purification*
  • Peptide Fragments / isolation & purification
  • Spectrum Analysis
  • Trypsin


  • Amino Acids
  • Glutamates
  • Peptide Fragments
  • Osteocalcin
  • Glutamic Acid
  • Endopeptidases
  • Trypsin