Proteoglycans: Biological Roles and Strategies
361
5.3.4
Disaccharide Composition and Identification of HS and Heparin
Specific degradation of heparin and HS to ~-di- and ~-oligosaccharide can be
achieved by using the various heparin lyases. Heparin lyase I, commonly referred to
as heparinase (EC 4.2.2.7), heparin lyase II (heparinase II, no EC number) and heparin lyase III, commonly referred to as heparitinase (EC 4.2.2.8), are all commercially available and each one of them has different specificity in respect to the uronic
acid moiety and sulfation (Fig. 24.5). When these lyases are used in combination,
the polymeric chain is cleaved to a large extent (> 90 %) to ~-disaccharides and the
disaccharide composition can therefore be obtained. This approach has recently
been used to separate and determine all commercially available heparin- and HSderived ~-disaccharides by a sensitive HPLC method (Karamanos et al. 1997).
Using CE in a reversed polarity mode and phosphate buffer (pH 3.5) all twelve
known isomeric ~-disaccharides are separated within 15 min (Fig. 24.1GB)
(Karamanos et al. 1996). This method is useful for determining structural differences between heparin and HS as well as for distinguishing them from each other
in biologic samples by analyzing the ~ -disaccharides obtained by their separate
depolymerization with heparin lyases.
References
Arvaniti A, Karamanos NK, Dimitracopoulos G, Anastassiou ED (1994) Isolation and characterization of a novel 20-kDa sulfated polysaccharide from the extracellular slime layer of Staphylococcus
epidermidis. Arch Biochem Biophys 308: 432-438
Bourdon MA (1990) Extracellular matrix genes. In: Sandell L, Boyd C (eds) Academic press, San
Diego, USA
Carlsson M (1968) Structure and immunological properties of oligosaccharides isolated from pig submaxillary mucins. J Bioi Chern 243: 616-626
Cheng F, Heinegard D, Malmstrom A, Schmidtchen A, Yoshida K, Fransson LA (1994) Patterns of uronosyl epimerization and 4-/6-0-sulphation in chondroitin/dermatan sulphate from decorin and
biglycan of various tissues. Glycobiology 4: 685-696
Cooper EH, Rathobone BJ (1990) Clinical significance of immunometric measurement of hyaluronic
acid. Ann Clin Biochem 27: 444-451
David G (1993) Integral membrane heparan sulfate proteoglycans. FASEB J 7: 1023-1030
EI Rassi, Z (1996) High-performance capillary electrophoresis of carbohydrates. Beckman Instruments, Fullerton, CA, USA
EI Rassi Z, Mechref Y (1996) Recent advances in capillary electrophoresis of carbohydrates. Electrophoresis 17: 275-301
Gioldassi X, Karamanos NK (1999) Determination of phosphorylated and sulfated linkage-region oligosaccharides in chondroitin/dermatan and heparan sulfate proteoglycans by high-performance
liquid chromatography. J Liq Chrom & Rei Technol 23 "in press"
Grimshaw J (1997) Analysis of glycosaminoglycans and their oligosaccharide fragments by capillary
electrophoresis. Electrophoresis 18: 2408-2414
Hascall VC, Kimura JH (1982) Proteoglycans: isolation and characterization. Methods Enzym 82:
769-800
Hascall VC, Calabro A, Midura RJ, Yanagishita M (1994) Isolation and characterization of proteoglycans. Methods Enzym 230: 390-417
Hardingham T, Fosang AJ (1992) Proteoglycans: many forms and many functions. FASEB J 6: 861-870
Hjerpe A, Karamanos NK (1998) Capillary electrophoresis in structural analysis of proteoglycans and
disease diagnosis. In: Karamanos NK (ed) European training program in microseparation techniques (ECOSEP1). Typorama, Patras, pp 136-154
Hjerpe A, Antonopoulos CA, Classon B, Engfeldt B (1980) Separation and quantitative determination
of galactosamine and glucosamine at the nanogram level by sulphonyl chloride reaction and highperformance liquid chromatography. J Chromatogr 202: 453-459
361
5.3.4
Disaccharide Composition and Identification of HS and Heparin
Specific degradation of heparin and HS to ~-di- and ~-oligosaccharide can be
achieved by using the various heparin lyases. Heparin lyase I, commonly referred to
as heparinase (EC 4.2.2.7), heparin lyase II (heparinase II, no EC number) and heparin lyase III, commonly referred to as heparitinase (EC 4.2.2.8), are all commercially available and each one of them has different specificity in respect to the uronic
acid moiety and sulfation (Fig. 24.5). When these lyases are used in combination,
the polymeric chain is cleaved to a large extent (> 90 %) to ~-disaccharides and the
disaccharide composition can therefore be obtained. This approach has recently
been used to separate and determine all commercially available heparin- and HSderived ~-disaccharides by a sensitive HPLC method (Karamanos et al. 1997).
Using CE in a reversed polarity mode and phosphate buffer (pH 3.5) all twelve
known isomeric ~-disaccharides are separated within 15 min (Fig. 24.1GB)
(Karamanos et al. 1996). This method is useful for determining structural differences between heparin and HS as well as for distinguishing them from each other
in biologic samples by analyzing the ~ -disaccharides obtained by their separate
depolymerization with heparin lyases.
References
Arvaniti A, Karamanos NK, Dimitracopoulos G, Anastassiou ED (1994) Isolation and characterization of a novel 20-kDa sulfated polysaccharide from the extracellular slime layer of Staphylococcus
epidermidis. Arch Biochem Biophys 308: 432-438
Bourdon MA (1990) Extracellular matrix genes. In: Sandell L, Boyd C (eds) Academic press, San
Diego, USA
Carlsson M (1968) Structure and immunological properties of oligosaccharides isolated from pig submaxillary mucins. J Bioi Chern 243: 616-626
Cheng F, Heinegard D, Malmstrom A, Schmidtchen A, Yoshida K, Fransson LA (1994) Patterns of uronosyl epimerization and 4-/6-0-sulphation in chondroitin/dermatan sulphate from decorin and
biglycan of various tissues. Glycobiology 4: 685-696
Cooper EH, Rathobone BJ (1990) Clinical significance of immunometric measurement of hyaluronic
acid. Ann Clin Biochem 27: 444-451
David G (1993) Integral membrane heparan sulfate proteoglycans. FASEB J 7: 1023-1030
EI Rassi, Z (1996) High-performance capillary electrophoresis of carbohydrates. Beckman Instruments, Fullerton, CA, USA
EI Rassi Z, Mechref Y (1996) Recent advances in capillary electrophoresis of carbohydrates. Electrophoresis 17: 275-301
Gioldassi X, Karamanos NK (1999) Determination of phosphorylated and sulfated linkage-region oligosaccharides in chondroitin/dermatan and heparan sulfate proteoglycans by high-performance
liquid chromatography. J Liq Chrom & Rei Technol 23 "in press"
Grimshaw J (1997) Analysis of glycosaminoglycans and their oligosaccharide fragments by capillary
electrophoresis. Electrophoresis 18: 2408-2414
Hascall VC, Kimura JH (1982) Proteoglycans: isolation and characterization. Methods Enzym 82:
769-800
Hascall VC, Calabro A, Midura RJ, Yanagishita M (1994) Isolation and characterization of proteoglycans. Methods Enzym 230: 390-417
Hardingham T, Fosang AJ (1992) Proteoglycans: many forms and many functions. FASEB J 6: 861-870
Hjerpe A, Karamanos NK (1998) Capillary electrophoresis in structural analysis of proteoglycans and
disease diagnosis. In: Karamanos NK (ed) European training program in microseparation techniques (ECOSEP1). Typorama, Patras, pp 136-154
Hjerpe A, Antonopoulos CA, Classon B, Engfeldt B (1980) Separation and quantitative determination
of galactosamine and glucosamine at the nanogram level by sulphonyl chloride reaction and highperformance liquid chromatography. J Chromatogr 202: 453-459