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Analysis of Disaccharides and Tetrasaccharides Released from Glycosaminoglycans

H. Edward Conrad¹

¹University of Illinois, Urbana, Illinois

Publication Name:

Current Protocols in Molecular Biology

Unit Number:

Unit 17.22B

DOI:

10.1002/0471142727.mb1722bs32

Online Posting Date:

May, 2001

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Abstract

Glycosaminoglycans (GAGs) are converted to disaccharides by various methods. This unit describes separation of individual disaccharides by paper chromatography or paper electrophoresis or high-performance liquid chromatography (HPLC). Lyase-released disaccharides can also be monitored by UV absorbance. Support protocols describe mild conditions for reduction of alkali-labile disaccharides obtained by cleavage of GAGs with lyases, scintillation counting of samples obtained from HPLC separation of radiolabeled saccharides, and calculations for the quantitation of disaccharides.

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Unit Introduction
Basic Protocol 1: Analysis of Disaccharides and Oligosaccharides from Glycosaminoglycans by Paper Chromatography and Paper Electrophoresis
Basic Protocol 2: Analysis of Disaccharides and Oligosaccharides from Glycosaminoglycans by HPLC
Support Protocol 1: Borohydride Reduction of Alkali-Labile Disaccharides Obtained by Cleavage with Lyases
Support Protocol 2: Scintillation Counting of Fractions from HPLC Analysis of Saccharides Released from Glycosaminoglycans
Support Protocol 3: Calculations for Quantitation of Disaccharides
Reagents and Solutions
Commentary
Bibliography
Tables

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Materials

Basic Protocol 1: Analysis of Disaccharides and Oligosaccharides from Glycosaminoglycans by Paper Chromatography and Paper Electrophoresis

Materials

Sample of lyase-degraded (unit 17.13B; also see Support Protocol 1) or nitrous acid–degraded glycosaminoglycan (unit 17.22A)
Paper chromatography or paper electrophoresis system appropriate to saccharide mixture to be analyzed (see Table 17.22B.1 and Table 17.22B.2; also see recipe in Reagents and Solutions)
Apparatus for paper electrophoresis

NOTE: [¹⁴C]glucose can be added as an internal standard. See unit 17.22A for details.

Basic Protocol 2: Analysis of Disaccharides and Oligosaccharides from Glycosaminoglycans by HPLC

Materials

Sample: mixture of saccharides obtained from glycosaminoglycan by nitrous acid cleavage (unit 17.22A) or lyase cleavage (unit 17.13B)
4.5-mm × 25-cm Partisil SAX strong anion-exchange column (Whatman) or 4-mm × 30-cm Varian MicroPak AX-5 weak anion-exchange column (Varian Analytical)
HPLC solvents (see recipe and Tables 17.22B.1-17.22B.4 and 17.22B.5)
Gradient solutions for HPLC (Tables 17.22B.1 - 17.22B.5)
Bio-Gel P-10 gel filtration column (Bio-Rad)
4.6 × 250–mm Hi-Chrom S-5 ODS C-18 column (Regis Technology)
Fraction collector or in-line radioactivity flow detector (e.g., Packard Instrument)

Additional reagents and equipment for gel-filtration chromatography (unit 10.9), ion-exchange HPLC (unit 10.13), and reversed-phase HPLC (unit 10.12)

Table 17.22B.3 Separation by Strong Ion-Exchange HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment with or Without Prior Hydrazinolysis^a^,^b

Oligosaccharide	Eluant (mM KH₂PO₄)	Retention time (min)

Disaccharides
GlcA-AMan_R and IdoA AMan_R	40	4.5
AMan_R	40	4.5
AMan_R(SO₄)	40	7.0
GlcA(SO₄)-AMan_R	40	19.5
GlcA-AMan_R(SO₄)	40	23.0
IdoA-AMan_R(SO₄)	40	26.5
IdoA(SO₄)-AMan_R	40	30.0
GlcA(SO₄)-AMan_R(SO₄)	185	14.0
IdoA(SO₄)-AMan_R(SO₄)	185	21.5
GlcA-AMan_R(3,6diSO₄)	185	25.5
Tetrasaccharides^c
t1 GlcA-GlcNAc-GlcA-AMan_R	20	28
t2 IdoA-GlcNAc-GlcA-AMan_R	20	33.5
t3 GlcA-GlcNAc(SO₄)-GlcA-AMan_R	100	31.5
t4 IdoA(SO₄)-GlcNAc-GlcA-AMan_R	100	37.0
t5 IdoA-GlcNAc(SO₄)-GlcA-AMan_R	100	42.5
t6	200	25.0
t7 IdoA(SO₄)-GlcNAc-GlcA-AMan_R(SO₄)	200	30.0
t8 IdoA-GlcNAc(SO₄)-GlcA-AMan_R(SO₄)	200	32.5
t9 IdoA-GlcNAc(SO₄)-GlcA-AMan_R(3-SO₄)	185	53.0
t10	320	23.0
t11	320	30.0
t12	320	32.0
t13	350	23.5
t14 IdoA-GlcNAc(SO₄)-GlcA-AMan_R(3,6-diSO₄)	350	38.5
t15 IdoA(SO₄)-GlcNAc(SO₄)-GlcA-AMan_R(SO₄)	350	42.0
t16	400	30.0

^aAbbreviations: AMan, anhydro-d-mannose; GlcA, d-glucuronic acid; GlcN, d-glucosamine; GlcNAc, N-acetyl-d-glucosamine; IdoA, l-iduronic acid.

^bHPLC separations are obtained using step gradients. Details of gradients used for disaccharides and oligosaccharides are given in Basic Protocol 2; the KH₂PO₄ concentrations are those at which each oligosaccharide emerges during the gradient. Columns are run at a flow rate of 1 ml/min; see Guo and Conrad (1988) for details.

^cTetrasaccharide designations (t1-t16) are described in Bienkowski and Conrad, 1985. Those tetrasaccharides for which no monosaccharide sequences are given (t6, t10-t13, and t16) are “ring-contraction tetrasaccharides,” which are formed in relatively low yields (see Commentary).

Table 17.22B.4 Separation by Isocratic Ion-Pairing HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment,With or Without Prior Hydrazinolysis^a

Oligosaccharide	Solvent^b	Retention time^c (min)

Mono- and Disaccharides
AMan_R	A:B = 94:6	5.0
GlcA-AMan_R	A:B = 94:6	6.5
IdoA-AMan_R	A:B = 94:6	9.0
AMan_R(SO₄)	A:B = 94:6	12.0
IdoA(SO₄)-AMan_R	A:B = 94:6	17.0
GlcA(SO₄)-AMan_R	A:B = 86:14	22.0
GlcA-AMan_R(SO₄)	A:B = 86:14	22.0
IdoA-AMan_R(SO₄)	A:B = 86:14	31.0
GlcA-AMan_R(3,6diSO₄)	A:B = 86:14	23.5
IdoA(SO₄)-AMan_R(SO₄)	A:B = 61:39	26.0
GlcA(SO₄)-AMan_R(SO₄)	A:B = 61:39	31.5
Tetrasaccharides^d
t1 GlcA-GlcNAc-GlcA-AMan_R	A:C = 94:6	9.0
t2 IdoA-GlcNAc-GlcA-AMan_R	A:C = 94:6	9.5
t4 IdoA(SO₄)-GlcNAc-GlcA-AMan_R	A:C = 84:15	29.0
t5 IdoA-GlcNAc(SO₄)-GlcA-AMan_R	A:C = 84:15	33.0
t3 GlcA-GlcNAc(SO₄)-GlcA-AMan_R	A:C = 84:15	38.0
t9 IdoA-GlcNAc(SO₄)-GlcA-AMan_R(3-SO₄)	A:C = 74:26	29.0
t8 IdoA-GlcNAc(SO₄)-GlcA-AMan_R(SO₄)	A:C = 74:26	33.0
t13 IdoA(SO₄)-RC(SO₄)IdoA(SO₄)-AMan_R	A:C = 60:40	12.0
t14 IdoA-GlcNAc(SO₄)-GlcA-AMan_R(3,6-diSO₄)	A:C = 60:40	15.0
t16 IdoA-RC(SO₄)IdoA(SO₄)-AMan_R(SO₄)	—	16.0

^aAbbreviations: AMan, anhydro-d-mannose; GlcA, d-glucuronic acid; GlcN, d-glucosamine; IdoA, l-iduronic acid; RC, ring-contraction product.

^bHPLC separations are obtained using a C-18 reversed-phase column using isocratic elution conditions obtained by mixing solvents A, B, and C (see recipes in Reagents and Solutions) in the ratios shown.

^cAll elution are performed at a flow rate of 1 ml/min. For separations of di- or tetrasaccharide mixtures containing the total mixtures of these oligosaccharides, see gradient conditions in Table 17.22B.5.

^dTetrasaccharide designations are described in Bienkowski and Conrad, 1985.

Table 17.22B.5 Gradient Conditions for Oligosaccharide Separation by Reversed-Phase Ion-Pairing HPLC^a

Disaccharides		Tetrasaccharides

Time interval for solvent change (min)	% Solvent B in time interval	Time interval for solvent change (min)	% Solvent C

05	6	013	6
56	614	1314	611
620	14	1464	11
2040	1420	6465	1125
4041	2039	65110	25
4181	39	110111	2531
8183	3960	111156	31 83100
		157192	39
		192193	3970
		193220	70

^aOligosaccharides are chromatographed on a C-18 reversed-phase column using the ion-pairing systems described in Basic Protocol 2. Mixtures of standards are chromatographed with increasing percentages of solvent B (disaccharides) or solvent C (tetrasaccharides) in solvent A (see recipes for solvents A, B, and C in Reagents and Solutions). Details of the gradients used are described in Basic Protocol 2.

Support Protocol 1: Borohydride Reduction of Alkali-Labile Disaccharides Obtained by Cleavage with Lyases

Materials

Sample of lyase-degraded glycosaminoglycan
1 M Na₂CO₃, pH 9.0 (ice-cold)
Sodium borohydride reagent (see recipe)
3 M H₂SO₄
6 × 150–mm test tubes

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Literature Cited

Literature Cited
	Al-Hakim, A. and Linhardt, R.J. 1991. Capillary Electrophoresis for the analysis of chondroitin sulfate and dermatan sulfate-derived disaccharides. Anal. Biochem. 195:68-73.
	Bienkowski, M.J. and Conrad, H.E. 1985. Structural characterization of the oligosaccharides formed by depolymerization of heparin with nitrous acid. J. Biol. Chem. 260:356-365.
	Delaney, S.R., Conrad, H.E., and Glaser, J.H. 1980. A new approach for isolation and sequencing of pure chondroitin SO₄ oligosaccharides. Anal. Biochem. 108:25-34.
	Desai, U.R., Wang, H.-M., Ampofo, S.A., Linhardt, R.J. 1993. Oligosaccharide composition of heparin and low-molecular-weight heparins by capillary electrophoresis. Anal. Biochem. 213:120-127.
	Edge, A.S.B. and Spiro, R.G. 1985. Structural Elucidation of glycosaminoglycans through characterization of disaccharides obtained after fragmentation by hydrazine-nitrous acid treatment. Arch. Biochem. Biophys. 240:560-572.
	Glaser, J.G. and Conrad, H.E. 1979. Chick embryo liver B-glucuronidase. Comparison of activity on natural and artificial substrates. J. Biol. Chem. 254:6588-6597.
	Guo, Y. and Conrad, H.E. 1988. Analysis of oligosaccharides from heparin by reversed phase ion-pairing high performance liquid chromatography. Anal. Biochem. 168:54-62.
	Guo, Y. and Conrad, H.E. 1989. The disaccharide composition of heparins and heparan sulfates. Anal. Biochem. 176:96-104.
	Hopwood, J.J. and Elliott, H. 1983. Selective depolymerisation of keratan sulfate: Production of radiolabeled substrates for 6- O-sulfogalactose sulfatase and -D-galactosidase. Carbohydr. Res. 117:263-274.
	Hopwood, J.J. and Muller, V.J. 1983. Selective depolymerisation of dermatan sulfate: Production of radiolabelled substrates for -l-iduronidase, sulfoiduronate sulfatase, and -d-glucuronidase. Carbohydr. Res. 122:227-239.
	Linhardt, R.J., Rice, K.G., Kim, Y.S., Lohse, D.L., Wang, H.M., and Loganathan, D. 1988. Mapping and quantification of the major oligosaccharide components of heparin. Biochem. J. 254:781-787.
	Rice, K.G., Kim, Y.S., Grant, A.C., Merchant, Z.M., and Linhardt, R.J. 1985. High-performance liquid chromatographic separation of heparin derived oligosaccharides. Anal. Biochem. 150:325-331.
	Shaklee, P.N. and Conrad, H.E. 1986. The disaccharides formed by deaminative cleavage of N-deacetylated glycosaminoglycans. Biochem. J. 235:225-236.
	Shively, J.E. and Conrad, H.E. 1970. Stoichiometry of the nitrous acid deaminative cleavage of model amino sugar glycosides and glycosaminoglycuronans. Biochemistry 9:33-41.
	Shively, J.E. and Conrad, H.E. 1976. Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15:3932-3942.
Key References
	Bienkowski and Conrad, 1985. See above.
Describes the separation of heparin di- and tetrasaccharides on SAX columns.
	Guo and Conrad, 1988. See above.
Describes the separation of heparin di- and tetrasaccharides by reversed-phase ion-pairing HPLC.
	Shaklee and Conrad, 1986. See above.
Describes the separation of disaccharides from chondroitin sulfate, dermatan sulfate, and keratan sulfate formed by both nitrous acid and lyase cleavage.