HIGH-PERFORMANCE SIZE-EXCLUSION CHROMATOGRAPHY OF PROTEOGLYCANS EXTRACTED FROM BOVINE ARTICULAR-CARTILAGE

The applicability of high-performance size-exclusion chromatography fo r determining the relative molecular weight of proteoglycans was teste d with a new type of column. The method is extremely reproducible, pre cise and rapid and allows molecular weight determinations up to 3 mill ions, even done in the presence of considerable impurities of low mole cular weight. This technique offers important advantages over the trad itional techniques, such as light scattering, sedementation velocity a nd equilibrium ultracentrifugation and viscosity. The peak position me thod was used to calibrate our system using pullulan standards. For th e pullulan standards in the molecular weight range between 1.0 X 10(4) and 8.5 X 10(5), an almost linear relationship between log M(p) and t he ratio between the retention time of the standard and that of the ex cluded salt was found. For the lower molecular weight standards, even at the lowest measured concentration, the curve deviated sharply for r atios above 0.80. The influence of the flow-rate on this ratio was neg ligible. We were able to separate the proteoglycan aggregates from the proteoglycan monomers in the KCl extracts. In the CaCl2 extract howev er, we could not separate the monomers from the aggregates, since thes e were present in a much higher concentration than in the KCl extract. After the addition of hyaluronidase from bovine testes, the proteogly can aggregates were broken down by lysis of the hyaluronic acid molecu les, who are essential for the formation of these aggregates.