Figure 2. Different sensitivities of existing and unknown polysaccharides to chondroitinase ACII.
(A) Chromatogram of unsaturated disaccharides of clam CS (Fr. 4) obtained by Chase ACII. Clam CS (2.5 μg) in reaction mixture (17.5 μl) was treated with Chase ACII at the specified concentrations, and resulting unsaturated disaccharides were analyzed by HPLC. (B) Clam CS has consecutive repeating unknown structures. After incubation of RT mix (17.5 μl) containing 2.5 μg of clam CS and 1.6 mU of Chase ACII, remaining polysaccharides and unsaturated disaccharides were separated using HiTrap™ Desalting column. The isocratic elution condition was as follows: eluent, 10 mM ammonium bicarbonate; flow rate, 1.0 ml/min. To obtain the remaining polysaccharide, 3 mg of CS (Fr. 4) was treated with 3 units of Chase ACII. (C) Chromatogram of unknown structure treated with Chase ACII. Remaining polysaccharides (2.5 μg) were treated with 12.5 mU of Chase ACII in RT mix (17.5 μl). To collect the unknown peaks (c) and (d), 200 μg of remaining polysaccharides was degraded and fractionated (see Supplementary Figure S2). Peaks: 1, ΔDi-0S; 2, ΔDi-4S; 3, ΔDi-6S; 4, ΔDi-diSE; a–d, unknown peaks.