Ascidian (Chordata-Tunicata) Glycosaminoglycans: Extraction, Purification, Biochemical, and Spectroscopic Analysis
Sulfated polysaccharides with unique structures of the chondroitin/dermatan and heparin/heparan families of sulfated glycosaminoglycans have been described in several species of ascidians (Chordata-Tunicata). These unique sulfated glycans have been isolated from–ascidians and characterized by biochemical and spectroscopic methods. The ascidian glycans can be extracted by different tissues or cells by proteolytic digestion followed by cetylpyridinium chloride/ethanol precipitation. The total glycans are then fractionated by ion-exchange chromatography on DEAE-cellulose and/or Mono Q (HR 5/5) columns. Alternatively, precipitation with different ethanol concentrations can be employed. An initial analysis of the purified ascidian glycans is carried out by agarose gel electrophoresis on diaminopropane/acetate buffer, before or after digestion with specific glycosaminoglycan lyases or deaminative cleavage with nitrous acid. The disaccharides formed by exhaustive degradation of the glycans is purified by gel-filtration chromatography on a Superdex-peptide column and analyzed by HPLC on a strong ion exchange Sax-Spherisorb column. 1H or 13C nuclear magnetic resonance spectroscopy in one or two dimensions is used to confirm the structure of the intact glycans.
Key wordsGlycosaminoglycans Dermatan sulfate Heparin Agarose gel electrophoresis Polyacrylamide gel electrophoresis Ion-exchange chromatography Gel filtration chromatography 1H-NMR 13C-NMR
This work was supported by grants from CNPq, FAPERJ, Fundação do Câncer. M.S.G.P. is a research fellow from CNPq, FAPERJ.
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