Isolation of dermatan sulfate proteoglycans from mature bovine articular cartilages

Two species of dermatan sulfate proteoglycans, called DS-PGI and DS-PGII, have been isolated from mature bovine articular cartilages. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis at low ionic strength in 0.01 M phosphate the dermatan sulfate proteoglycans appeared as a single polydisperse species whose molecular weight ranged from 80,000 to 140,000. The dermatan sulfate proteoglycans eluted as a single peak on Sepharose CL-4B chromatography in 4 M guanidine hydrochloride and showed no tendency to separate into two components. Following chondroitinase AC and ABC digestion, a core protein was obtained whose molecular weight was 45,000. However, what appeared to be a single dermatan sulfate proteoglycan was consistently separated into two species of distinctly different mobilities by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at high ionic strength in 0.375 M Tris. The molecular weight of the smaller species (DS-PGII) ranged from 87,000 to 120,000. The molecular weight of the larger species (DS-PGI) ranged from 165,000 to 285,000. DS-PGI self-associates in 0.375 M Tris, while DS-PGII does not. This phenomenon was exploited to separate DS-PGI and DS-PGII by preparative electrophoresis on 5 to 20% gradient slab gels. The immunological identities of the individual species, DS-PGI and DS-PGII, were examined by enzyme-linked immunosorbent assay using polyclonal antiserum to cartilage-specific proteoglycan monomer from bovine articular cartilage and polyclonal and monoclonal antibodies to DS-PGII. The polyclonal antiserum to cartilage-specific proteoglycan monomer did not react with DS-PGI or DS-PGII, indicating that DS-PGI and DS-PGII possess different core proteins from cartilage-specific proteoglycan monomer. Polyclonal and monoclonal antibodies raised against the mixture of DS-PGI and DS-PGII reacted strongly with DS-PGII, but weakly or not at all with DS-PGI. These results suggest that DS-PGI and DS-PGII possess different core proteins and may represent two different species of dermatan sulfate proteoglycans.