| Pacific abalone, Haliotis discus hannai Ino(HD), is a highly valued marine shellfish. An uronic acid-containing polysaccharide(UACP) from abalone gonad, named as AGSP, was elucidated by graded acid hydrolysis followed by PMP(1-phenyl-3-methyl-5-pyrazolone)-HPLC-MSn and NMR analysis. In addition, UACPs in HD and its cheaper replacer, Volutharpa ampullacea perryi(VA), were characterized and compared.AGSP was hydrolyzed by 0.05 M, 0.2 M, 0.5 M, and 2.0 M TFA at 100 ℃ for 1 h, sequentially. Every hydrolysate was ultrafiltrated to collect oligo- and monosaccharides, and the final retentate was further hydrolyzed with 2.0 M TFA at 110 ℃ and 121℃ for 2 h, respectively. PMP derivatization followed by HPLC-PAD-MSn analysis was applied to detect the sugar residues in these hydrolysates and proved the distribution of these residues in the AGSP structure. The retentate after 0.5 M TFA hydrolysis was confirmed as the polysaccharide backbone, and it was further analyzed by 1D and 2D NMR spectroscopy. Thus, the structural elucidation of AGSP was accomplished, and its backbone consisted of →4)-β-GlcA(1→2)-α-Man(1→ repeating unit.Acid disaccharides produced by acid hydrolysis were analyzed by PMP-HPLC-MS to propose original UACPs in tissues of HD and VA. GlcA-(1→2)-Man, GlcA-(1→3)-GalN, and another disaccharide consisting of a hexuronic acid linked to a hexose were found in the hydrolysates, which indicated the existence of AGSP, AAP with the backbone composed of →3)-GalNAc-(1→2)-GlcA-(1→3)-GalNAc-(1→4)-GlcA-(1→ unit, and an unidentified DS1 P containing a hexuronic acid linked to a hexose unit. Taking together with the monosaccharide analysis results, it could be concluded that polysaccharides in pleopods of HD and VA are similar, while those in their gonads and viscera differ greatly. The method demonstrated could characterize UACPs in biological samples quickly. In addition, chemical colour reaction revealed neutral polysaccharides and sulfated polysaccharides in the two gastropods. |
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