FIGURE 3.

Identification of novel sulfotransferase reaction products. Enzymatic reactions were carried out using Chn polymer as an acceptor substrate under the incubation conditions described under “Experimental Procedures.” Reaction products were isolated by gel filtration using a syringe column packed with Sephadex G-25 and then digested with chondroitinase ABC (a), chondroitinase ABC and chondro-4-O-sulfatase (b), or chondroitinase ABC and chondro-6-O-sulfatase (c). The digests were analyzed by anion exchange HPLC using an amine-bound silica PA03 column as described under “Experimental Procedures.” The eluate was collected after 14 min of injection at 30-s intervals for radioactivity measurement by liquid scintillation counting. The numbered arrows indicate the elution positions of the authentic unsaturated disaccharides or inorganic sulfate: 1, ΔHexUAα1–3GalNAc(6-O-sulfate); 2, ΔHexUAα1–3GalNAc(4-O-sulfate); 3, inorganic [³⁵S]sulfate.