Enzymatic analysis with chondrosulfatases of constituent disaccharides of sulfated chondroitin sulfate and dermatan sulfate isomers by high-performance liquid chromatography.

Various under-sulfated, monosulfated, and over-sulfated chondroitin sulfate and dermatan sulfate isomers were analyzed in terms of disaccharide units before or after desulfation with chondrosulfatases in addition to digestion with chondroitinases. The unsaturated disaccharides were separable by a high-performance liquid chromatography (HPLC) method using a resin made from a sulfonized styrene-divinylbenzene copolymer. The retention times of the parent sulfated unsaturated disaccharides and newly generated unsaturated mono- or nonsulfated disaccharides were reproducible. On desulfation of the parent sulfated unsaturated disaccharides with chondrosulfatases, almost all delta Di-S showed the same retention times as those of standard delta Di-S from known components. Following digestion of delta Di-diSB with chondro-4-sulfatase as well as delta Di-diSD or delta Di-diSG with chondro-6-sulfatase, three delta Di-monoS with the same retention time were detected with the HPLC method. These newly generated delta Di-monoS2 showed that the structure is N-acetyl-D-galactosamine, uronic acid 2-sulfate.