The application of the Milner-Avigad method for the quantitative determination of endouronidase activities

The Milner-Avigad method (Milner, Y. and Avigad, G. (1967) Carbohydr. Res. 4, 359-361) has been shown to have high specificity for measuring the reducing powers of free hexuronic acids; its applicability in the quantitative determination of endouronidase activity was the purpose of this investigation. Among the constituent monosaccharides of glycosaminoglycans, hexuronic acids showed high color yield by this method, while xylose, galactose and N-acetylhexosamine recorded negligible color yield. Among the monosaccharide residues at the reducing terminals of oligosaccharides, only hexuronic acids exhibited color yield. However, the color yield was less than that of free hexuronic acids. Gel filtration chromatography of reaction products revealed that the cleavage of the oligosaccharide chains and the resultant exposure of new reducing terminals were not caused by the reaction procedures involved in this method. These data indicate that the Milner-Avigad method is useful for determining the presence of hexuronic acid residues preferentially at reducing terminals of glycosaminoglycan moieties. Thus, it supported the conclusion that the Milner-Avigad method is applicable for the quantitative determination of endouronidase activity using glycosaminoglycan as a substrate.