| Popis: |
The applicability of high-performance size-exclusion chromatography for determining the relative molecular weight of proteoglycans was tested with a new type of column. The method is extremely reproducible, precise and rapid and allows molecular weight determinations up to 3 millions, even done in the presence of considerable impurities of low molecular weight. This technique offers important advantages over the traditional techniques, such as light scattering, sedementation velocity and equilibrium ultracentrifugation and viscosity. The peak position method was used to calibrate our system using pullulan standards. For the pullulan standards in the molecular weight range between 1.0 × 10 4 and 8.5 × 10 5 , an almost linear relationship between log M p and the ratio between the retention time of the standard and that of the excluded salt was found. For the lower molecular weight standards, even at the lowest measured concentration, the curve deviated sharply for ratios above 0.80. The influence of the flow-rate on this ratio was negligible. We were able to separate the proteoglycan aggregates from the proteoglycan monomers in the KCl extracts. In the CaCl 2 extract however, we could not separate the monomers from the aggregates, since these were present in a much higher concentration than in the KCl extract. After the addition of hyaluronidase from bovine testes, the proteoglycan aggregates were broken down by lysis of the hyaluronic acid molecules, who are essential for the formation of these aggregates. |
