An improved assay for the N-acetyl-D-glucosamine reducing ends of polysaccharides in the presence of proteins

Hyaluronan concentration and hyaluronidase activity can be assayed by using different techniques including turbidimetry, viscosimetry, ELISA, chromatography, and colorimetry, The most popular colorimetric method is that of J. Reissig et al. (1955, J. Biol. Chem. 217, 959-966), in which the color results from a reaction between the Ehrlich's reagent (DMAB) and the N-acetyl-D-glucosamine reducing end of each hyaluronan chain. Nevertheless, there are problems with this method when proteins are present in the medium. Here we propose a new interpretation of the Reissig signal for estimating such reducing ends in media containing enzymes or other proteins. This interpretation is based on the fact that the absorbance obtained by using the Reissig method results from two factors: a turbidity due to the formation of polysaccharide-protein complexes and a color resulting fi-om the action of DMAB on the reducing end of the polysaccharide chains. The turbidity at 585 nm, the wavelength at which the color intensity is maximal, may be estimated by curve fitting the spectrum between 450 and 650 nm. Subtracting the turbidity from the absorbance gives the colorimetric intensity which represents the concentration of polysaccharide chains. Moreover, the turbidity may give additional information about the existence of polysaccharide-protein complexes and their nature. (C) 2001 Academic Press.