Analysis of Glycosaminoglycans with Polysaccharide Lyases

Robert J. Linhardt1

1 University of Iowa, Iowa City, Iowa
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.13B
DOI:  10.1002/0471142727.mb1713bs48
Online Posting Date:  May, 2001
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Abstract

Polysaccharide lyases are a class of enzymes useful for analysis of glycosaminoglycans (GAGs) and the glycosaminoglycan component of proteoglycans (PGs). These enzymes cleave specific glycosidic linkages present in acidic polysaccharides and result in depolymerization. The lyases are derived from a wide variety of pathogenic and nonpathogenic bacteria and fungi. This class of enzymes includes heparin lyases (heparinases), heparan sulfate lyases (heparanases or heparitinases), chondroitin lyases (chondroitinases), and hyaluronate lyases (hyaluronidases), all of which are described in this unit. Two protocols describe depolymerization of GAGs, and two support protocols describe assays to confirm and quantitate the activity of heparin and chondroitin ABC lyases.

     
 
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Table of Contents

  • Overview of Heparin Lyases
  • Basic Protocol 1: Complete Heparin Lyase–Catalyzed Depolymerization of an Unlabeled Sample
  • Alternate Protocol 1: Complete Heparin Lyase–Catalyzed Depolymerization of Very Small Amounts of Radiolabeled Glycosaminoglycans
  • Support Protocol 1: Assay of Heparin Lyase Activity
  • Support Protocol 2: Assay of Chondroitin ABC Lyase Activity
  • Reagents and Solution
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Complete Heparin Lyase–Catalyzed Depolymerization of an Unlabeled Sample

  Materials
  • Heparin‐ or heparan sulfate–containing sample
  • recipeSodium phosphate/NaCl buffer (see recipe)
  • recipeHeparin lyase I solution (see recipe)
  • Spectropor dialysis membrane, MWCO 1000 (Spectrum)
  • 500‐µl polypropylene microcentrifuge tubes
  • 30° and 100°C water baths
  • Additional reagents and equipment for polysaccharide dialysis ( appendix 3D), HPLC (unit 17.18), and gel‐filtration chromatography (units 10.9 & 17.1717.17)

Alternate Protocol 1: Complete Heparin Lyase–Catalyzed Depolymerization of Very Small Amounts of Radiolabeled Glycosaminoglycans

  • Radiolabeled heparin‐containing sample (unit 17.4)
  • recipe20 mg/ml chondroitin sulfate A solution (see recipe)
  • recipe20 mg/ml chondroitin sulfate C solution (see recipe)
  • recipe20 mg/ml dermatan sulfate solution (see recipe)

Support Protocol 1: Assay of Heparin Lyase Activity

  Materials
  • recipeSodium phosphate/NaCl buffer (see recipe)
  • recipeHeparin lyase I solution (see recipe)
  • recipe20 mg/ml heparin solution (see recipe)
  • UV spectrophotometer, temperature controlled
  • 1‐ml quartz cuvette with 1‐cm pathlength

Support Protocol 2: Assay of Chondroitin ABC Lyase Activity

  Materials
  • recipeTris⋅Cl/sodium acetate buffer, pH 8.0 (see recipe)
  • recipeChondroitin ABC lyase solution (see recipe)
  • recipe20 mg/ml chondroitin sulfate A (see recipe), recipechondroitin sulfate C solution (see recipe), or recipedermatan sulfate solution (see recipe)
  • UV spectrophotometer, temperature controlled
  • 1‐ml quartz cuvette with 1‐cm path length
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Figures

Videos

Literature Cited

Literature Cited
   Desai, U.R., Wang, H.H., and Linhardt, R.J. 1993a. Substrate specificity of the heparin lyase from Flavobacterium heparinum Arch. Biochem. Biophys. 306:461‐468.
   Desai, U.R., Wang, H.M., and Linhardt, R.J. 1993b. Specificity studies on the heparin lyases from Flavobacterium heparinum. Biochemistry 32:8140‐8145.
   Grant, A.C., Linhardt, R.J., Fitzgerald, G., Park, J.J., and Langer, R. 1984. Metachromatic activity of heparin and heparin fragments. Anal. Biochem. 137:25‐32.
   Gu, K., Liu, J., Pervin, A., and Linhardt, R.J. 1993. Comparison of the activity of two chondroitin AC lyases on dermatan sulfate. Carbohydr. Res. 244:369‐377.
   Hiyama, K. and Okada, S. 1975. Crystallization and some properties of chondroitinase from Arthrobacter aurescens. J. Biol. Chem. 250:1824‐1828.
   Jandik, K.A., Gu, K., and Linhardt, R.J. 1994. Action pattern of polysaccharide lyases on glycosaminoglycans. Glycobiology 4:000‐000.
   Linhardt, R.J., Cooney, C.L., and Galliher, P.M. 1986. Polysaccharide lyases. Appl. Biochem. Biotechnol. 12:135‐177.
   Linhardt, R.J., Al‐Hakim, A., Liu, J., Hoppensteadt, D., Mascellani, G., Bianchini, P., and Fareed, J. 1991. Structural features of dermatan sulfates and their relationship to anticoagulant and antithrombotic activities. Biochem. Pharmacol. 42:1609‐1619.
   Lohse, D.L. and Linhardt, R.J. 1992. Purification and characterization of heparin lyases from Flavobacterium heparinum. J. Biol. Chem. 267:24347‐24355.
   Michelacci, Y.M. and Dietrich, C.P. 1975. A comparative study between a chondroitinase B and a chondroitinase AC from Flavobacterium heparinum. Biochem. J. 151:121‐129.
   Nakada, H.I. and Wolfe, J.B. 1961. Studies on the enzyme chondroitinase: Product structure and ion effects. Arch. Biochem. Biophys. 94:244‐251.
   Rautela, G.S. and Abramson, C. 1973. Crystallization and partial characterization of Staphylococcus aureus hyaluronate lyase. Arch. Biochem. Biophys. 158:687‐693.
   Rice, K.G. and Linhardt, R.J. 1989. Study of defined oligosaccharide substrates of heparin and heparan monosulfate lyases. Carbohydr. Res. 190:219‐233.
   Saito, H., Yamagata, T., Suzuki, S. 1968. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243:1536‐1542.
   Sasaki, I., Gotoh, H., Yamamoto, R., Tanaka, H., Takami, K., Yamashita, K., Yamashita, J., and Horio, T. 1982. Hydrophobic‐ionic chromatography: Its application to microbial glucose oxidase, hyaluronidase, cholesterol oxidase, and cholesterol esterase. J. Biol. Chem. 91:1555‐1561.
   Vesterberg, O. 1968. Studies on extracellular proteins from Staphylococcus aureus III. Investigations on the heterogeneity of hyaluronate lyase using the method of isoelectric focusing. Biochim. Biophys. Acta 168:218‐226.
   Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. 1968. Purification and properties of bacterial chondroitinases and chondrosulfatases. J. Biol. Chem. 243:1523‐1535.
Key References
   Desai et al., 1993a, b. See above.
  Describes the specificity of the heparin lyases in detail.
   Lohse and Linhardt, 1992. See above.
  Describes physical and catalytic properties of the heparin lyases.
   Michelacci and Dietrich, 1975. See above.
  Describes kinetic properties of chondroitin B lyase and chondroitin AC lyase.
   Seikagaku Company Product Literature, 1991.
  Describes general properties and assay conditions for all of the polysaccharide lyases.
   Saito et al., 1968. See above.
  Good example of how different chondroitin sulfates can be distinguished using chondroitin ABC lyase and chondroitin AC lyase.
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