N-[3H]Acetyl-labeling, a convenient method for radiolabeling of glycosaminoglycans.
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abstract
A method for the introduction of N-[3H]acetyl groups into glycosaminoglycans is described. The procedure is based on [3H]acetylation of N-unsubstituted hexosamine residues by treating the polysaccharides with [3H]acetic anhydride. Preparations of heparin and heparin sulfate were found to contain significant numbers of N-unsubstituted hexosamine residues, as isolated. In contrast, such units could not be detected in chondroitin sulfate, dermatan sulfate, or hyaluronic acid. These polysaccharides were therefore subjected to partial N-deacetylation by reaction with hydrazine in the presence of hydrazine sulfate. After treatment with [3H]acetic anhydride, the specific activities of the resulting labeled polysaccharide preparations ranged between 0.1 106 and 0.6 106 cpm 3H/g of uronic acid. The 3H-labeled polysaccharide preparations did not differ significantly from the corresponding unlabeled starting materials with regard to polyanion properties (chromatography on DEAE-cellulose) or polymer chain size (gel chromatography). Further, the radiolabeled polysaccharide derivatives were susceptible to specific enzymatic degradation (chondroitinase ABC and mammalian heparitinase) and retained their ability to interact specifically with certain proteins-for example, [3H]heparin with antithrombin [3H]hyaluronic acid oligosaccharides with chondroitin sulfate proteoglycan (Cleland, R. L. Biochem. Biophys. Res. Commun. 87, 1140-1145 (1979)). These findings indicate that the labeling procedures did not induce any major structural derangement of the polysaccharide molecules. The method developed should be useful in providing labeled glycosaminoglycans for metabolic and enzymatic experiments as well as for studies on the interaction between glycosaminoglycans and other biological macromolecules. 1982.
