Ascidian (Chordata-Tunicata) Glycosaminoglycans: Extraction, Purification, Biochemical, and Spectroscopic Analysis

  • Mauro S. G. PavãoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1229)


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 words

Glycosaminoglycans 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.


  1. 1.
    Pavao MS, Mourao PA, Mulloy B, Tollefsen DM (1995) A unique dermatan sulfate-like glycosaminoglycan from ascidian. Its structure and the effect of its unusual sulfation pattern on anticoagulant activity. J Biol Chem 270:31027–31036PubMedCrossRefGoogle Scholar
  2. 2.
    Mourao PA, Pavao MS, Mulloy B, Wait R (1997) Chondroitin ABC lyase digestion of an ascidian dermatan sulfate. Occurrence of unusual 6-O-sulfo-2-acetamido-2-deoxy-3-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-beta-D-galactose units. Carbohydr Res 300:315–321PubMedCrossRefGoogle Scholar
  3. 3.
    Pavao MS, Aiello KR, Werneck CC, Silva LC, Valente AP, Mulloy B, Colwell NS, Tollefsen DM, Mourao PA (1998) Highly sulfated dermatan sulfates from Ascidians. Structure versus anticoagulant activity of these glycosaminoglycans. J Biol Chem 273:27848–27857PubMedCrossRefGoogle Scholar
  4. 4.
    Gandra M, Kozlowski EO, Andrade LR, de Barros CM, Pascarelli BM, Takiya CM, Pavao MS (2006) Collagen colocalizes with a protein containing a decorin-specific peptide in the tissues of the ascidian Styela plicata. Comp Biochem Physiol B Biochem Mol Biol 144:215–222PubMedCrossRefGoogle Scholar
  5. 5.
    Vicente CP, He L, Pavao MS, Tollefsen DM (2004) Antithrombotic activity of dermatan sulfate in heparin cofactor II-deficient mice. Blood 104:3965–3970PubMedCrossRefGoogle Scholar
  6. 6.
    Cavalcante MC, Allodi S, Valente AP, Straus AH, Takahashi HK, Mourao PA, Pavao MS (2000) Occurrence of heparin in the invertebrate styela plicata (Tunicata) is restricted to cell layers facing the outside environment. An ancient role in defense? J Biol Chem 275:36189-6PubMedCrossRefGoogle Scholar
  7. 7.
    Cavalcante MC, de Andrade LR, Du Bocage Santos-Pinto C, Straus AH, Takahashi HK, Allodi S, Pavao MS (2002) Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata). J Struct Biol 137:313–321PubMedCrossRefGoogle Scholar
  8. 8.
    de Barros CM, Andrade LR, Allodi S, Viskov C, Mourier PA, Cavalcante MC, Straus AH, Takahashi HK, Pomin VH, Carvalho VF, Martins MA, Pavao MS (2007) The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma. J Biol Chem 282:1615–1626PubMedCrossRefGoogle Scholar
  9. 9.
    Jaques LB, Ballieux RE, Dietrich CP, Kavanagh LW (1968) A microelectrophoresis method for heparin. Can J Physiol Pharmacol 46:351–360PubMedCrossRefGoogle Scholar
  10. 10.
    Dietrich CP, McDuffie NM, Sampaio LO (1977) Identification of acidic mucopolysaccharides by agarose gel electrophoresis. J Chromatogr 130:299–304PubMedCrossRefGoogle Scholar
  11. 11.
    Conrad HE (1993) Dissection of heparin – past and future. Pure Appl Chem 65:787–791CrossRefGoogle Scholar
  12. 12.
    Farndale RW, Buttle DJ, Barrett AJ (1986) Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 883:173–177PubMedCrossRefGoogle Scholar
  13. 13.
    Bitter T, Muir HM (1962) A modified uronic acid carbazole reaction. Anal Biochem 4:330–334PubMedCrossRefGoogle Scholar
  14. 14.
    Pavao MS, Albano RM, Lawson AM, Mourao PA (1989) Structural heterogeneity among unique sulfated L-galactans from different species of ascidians (tunicates). J Biol Chem 264:9972–9979PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Laboratório de Bioquímica e Biologia Celular de Glicoconjugados, Programa de Glicobiologia, Instituto de Bioquímica Médica Leopoldo de Meis, Hospital Universitário Clementino Fraga FilhoUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil

Personalised recommendations