Abstract
1. Two proteodermatan sulphate species from bovine sclera (fractions PG-I and PG-II) separable by gel chromatography were studied by isopycnic centrifugations in CsCl and Cs2SO4 both in an analytical and a preparative mode. 2. In CsCl, fraction PG-I formed a broad band at a density ρ=1.75g/ml whereas fraction PG-II banded sharply at ρ=1.64g/ml. However, in Cs2SO4, fraction PG-II banded at ρ=1.51g/ml and fraction PG-I at ρ=1.40g/ml, a reversal of the relative banding positions of the two species in CsCl. 3. Preparative isopycnic centrifugations in the two caesium salts permitted further subfractionation of fractions PG-I and PG-II. In both CsCl and Cs2SO4 fraction PG-I was split into subfractions that varied greatly in uronate/protein ratios but had very similar uronate composition. In contrast, isopycnic centrifugation of fraction PG-II in Cs2SO4 gave rise to subfractions with similar uronate/protein ratios but markedly different uronate composition (iduronate content, 88–42%). 4. Subfractions of fractions PG-I and PG-II obtained in preparative centrifugations in CsCl or Cs2SO4 were examined in the analytical ultracentrifuge. These subfractions banded at discrete positions in the gradient. Estimations of apparent molecular weight for these subfractions from data from the analytical isopycnic centrifugations gave values that were much higher (around 1×106) than were those obtained previously for their `monomeric' states (fraction PG-I 160000–410000, and fraction PG-II 70000–130000). 5. In CsCl, fraction PG-I may be subfractionated according to the number of side chains in the molecule. Fraction PG-I increased its net solvation (i.e. lowered its buoyant density) to a larger extent than did fraction PG-II in going from CsCl to Cs2SO4 (i.e. from lower to higher water activity). It is proposed that the presence of large amounts of iduronate in fraction PG-II makes these molecules relatively less solvated in Cs2SO4. Thus the uronate composition may be an important factor in determining the banding position of proteodermatan sulphates in density-gradient centrifugations.
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Selected References
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