Study on the factors yielding high color in the carbazole reaction with hexuronic acid-containing substances

The color yields with hexuronic acids and hexuronic acid-containing substances were studied by means of the carbazole method of Bitter and Muir with or without 0.025 M borate. The carbazole-borate to carbazole (CB/C) ratios thus obtained indicated not only the degree of the borate effects on the color yields with these materials but also the anomalous nature of some of these substances in the carbozole reaction. The present data indicate that the high color yield with heparin in the carbozole reaction may be due to the production of free amino groups from acid-labile sulfamino groups in the early stage of the reaction, resulting in suppression of protonation on the alpha-D-glucosaminidic oxygen atoms attached to the hexuronic acid residues in the molecule. Moreover, it is suggested that the degree of the unusual color yields in the carbazole reaction with glycosaminoglycans is greatly influenced by the anomeric configuration of the hexosaminyl linkages attached to the hexuronic acid residues in these polymers.