A dot-blot assay of metabolically radiolabeled hyaluronan

Glycosaminoglycans labeled in tissue culture in the presence of [3H]glucosamine were proteolytically solubilized and then precipitated with cetylpyridinium chloride onto a sheet of nitrocellulose using a dot-blot apparatus. The proportion of hyaluronan (HA) was calculated from parallel aliquots digested with Streptomyces hyaluronidase, an enzyme specifically degrading HA. A linear response of radioactivity was obtained for samples in the range of 50-10,000 cpm (1-1000 ng total HA) when the dots cut from the membrane were counted with liquid scintillation. Negligible interference from an excess of unincorporated precursor, chondroitin sulfate, and proteolytic tissue digest was observed. The assay was particularly convenient in measuring large numbers of samples with relatively low activity, such as testing the effects of different drug concentrations and analyzing chromatographic fractions.