Proteoglycans from Bovine Proximal Humeral Articular Cartilage

Abstract

Abstract To provide methods applicable to the study of human articular cartilage, we have studied the extraction, fractionation, and physical characterization of proteoglycans from bovine articular cartilage. Two dissociative solvents, MgCl2 and GuHCl, differ sharply in the amount of the proteoglycan extracted from articular cartilage and the macromolecular species present in the initially extracted crude proteoglycan. PG(MgCl2), the crude proteoglycan extracted with 3 m MgCl2, is obtained in a yield of 0.16 g per g of dry cartilage, and accounts for 50% of the uronate and 23% of the hexose of the whole cartilage. PG(GuHCl), the crude proteoglycan extracted with 4 m GuHCl, is obtained in a yield of 0.26 g per g of dry cartilage, and accounts for 81% of the uronate and 61% of the hexose of the whole cartilage. On analytical ultracentrifugation in associative solvents, PG(MgCl2) shows three components: a 4 S protein, a 16 S proteoglycan species, and a 60 to 70 S proteoglycan aggregate. Equilibrium density gradient centrifugation of PG(MgCl2) under associative conditions in 4 m CsCl results in a sharp separation of the 4 S protein into the top sixth of the gradient, and of the 16 S species and 70 S aggregate into the bottom sixth of the gradient. On analytical ultracentrifugation in associative solvents, PG(GuHCl) shows a 4 S protein, a 16 S proteoglycan species, and a 56 S proteoglycan aggregate. The proteoglycan aggregate in either PG(MgCl2) or PG(GuHCl) is completely and reversibly dissociable in either 3 m MgCl2 or 3 m GuHCl. However, while dissociation in 3 m MgCl2 yields the 16 S species, dissociation in 3 m GuHCl yields a 10.5 S proteoglycan subunit. The isolated 16 S proteoglycan species has an intrinsic viscosity of 184 ml per g, a partial specific volume of 0.594 ml per g, and a molecular weight of 1.6 x 106. PG(GuHCl) contains a high molecular weight complex, not present in PG(MgCl2). The complex differs from the 70 S proteoglycan aggregate in size, as indicated by its sedimentation properties; in composition, as indicated by its lower proteoglycan and higher protein and collagen contents; and, in the noncovalent bonds involved in its formation, as indicated by its dissociation in 3 m GuHCl but not in 3 m MgCl2. Analytical ultracentrifugation of the dissociated complex in 3 m GuHCl shows that a 2.7 S protein is a major component of the complex. None of the 2.7 S protein is detectable in preparations of PG(MgCl2) or of the 70 S aggregate. Two states of macromolecular organization of proteoglycans appear to exist in articular cartilage. The first, and lower level of organization is that of the 70 S proteoglycan aggregate. The second and higher level of organization is that of the complex formed by the noncovalent association of proteoglycans and collagen in the presence of the 2.7 S protein.