Collagenase-Released Non-Collagenous Proteins of Cortical Bone Matrix

Two distinct groups of non-collagenous components were isolated from rat cortical bone gelatin which had previously been digested with purified bacterial col-lagenase. One component was disulfide-bonded, strongly acidic, trypsin-labile glycoprotein aggregate with a molecular mass of more than 100, 000 daltons. When reduced with β-mercaptoethanol this protein disaggregated into subunits with a molecular mass of about 60, 000 daltons. The other components consisted of a group of polypeptides with a molecular mass of about 5, 000 daltons. The latter group was present in collagenase digests prepared from normal bone gelatin but was hardly detectable or absent in digests of gelatin prepared from either autolyzed, trypsinized or lathyritic bone, or from the residue of neutral salt extracted rat tail tendon.

A recently discovered group of non-collagenous proteins is tightly bound to the highly crosslinked insoluble structure of collagen fibrils of bone and dentin. Dische et al ¹ predicted the existence of these proteins in bone by analyzing the products of KOH hydrolysis of the EDTA-insoluble residues of cortical bone collagen. Comparable products were separated following digestion of bone matrix with collagenase by Herring², and oxidation of dentin matrix by alkaline sodium metaperiodate by Shuttleworth and Veis.³ Leaver et al ⁴ proposed the name collagenase-released proteins (CRP) for non-collagenous proteins obtained from the EDTA-insoluble residue of bone and dentin. There are at least two limitations in information about CRP in the above-cited reports. The first is that the collagenases were not certified to be protease-free. The second is that EDTA-insoluble residues of cortical bone were only partly digestable by bacterial collagenase. We report here isolation and partial characterization of CRP from a special preparation of bone matrix gelatin which is quantitatively digested by a protease-free bacterial collagenase-gelatinase purified by the method of Peterkofsky and Diegelmann.^{5, 6}