Proteoglycans of Adult Bovine Compact Bone

Abstract

Proteoglycans of bovine compact bone were purified by chromatography of the formic acid precipitate of an EDTA extract. The sequential chromatographic steps consisted of gel filtration on Sepharose CL-6B in 4-M guanidine HC1, ion-exchange chromatography on DEAE-Sephacel in 4-M urea and rechromatography on Sepharose CL-6B in 4-M guanidine HC1. The preparation consisted of a relatively small proteoglycan (Kav = 0.4 on Sepharose CL-6B) containing about 40% protein, 21% hexuronic acid, 23% galactosamine and lesser amounts of other monosaccharides. The core protein was shown by gradient NaDodS0₄ gel electrophoresis, electrotransfer and immunodetection to be monodispersed with an Mr = 45,000. Analysis of glyco-peptides obtained after papain digestion of the proteoglycan and separation from glycosaminoglycan chains by gel chromatography, indicated that both AMinked and 0-linked oligosaccharides were present. The glycosaminoglycan chains liberated by papain digestion eluted from Sepharose CL-6B as a broad peak with Kav = 0.50, slightly ahead of the position of elution of bovine nasal cartilage glycosaminoglycans (Kav = 0.52); the bone glycosaminoglycans are thus slightly larger than those from cartilage and smaller than the ones attached to fetal bone proteoglycans. These chains were totally susceptible to chondroitinase AC II, a procedure that yielded unsaturated disaccharides corresponding predominantly to chondroitin-4-sulfate, and to a lesser extent chondroitin-6-sulfate.