Analysis of Disaccharides and Tetrasaccharides Released from Glycosaminoglycans

H. Edward Conrad1

1 University 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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Table of Contents

  • 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
  • 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 17.13; also see protocol 3) or nitrous acid–degraded glycosaminoglycan (unit 17.22)
  • recipePaper chromatography or paper electrophoresis system appropriate to saccharide mixture to be analyzed (see Table 17.22.1 and Table 17.22.2; also see recipe in Reagents and Solutions)
  • Apparatus for paper electrophoresis
NOTE: [14C]glucose can be added as an internal standard. See unit 17.22 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 17.22) or lyase cleavage (unit 17.13)
  • 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)
  • recipeHPLC solvents (see recipe and Tables 17.22.1 and 17.22.5)
  • Gradient solutions for HPLC (Tables 17.22.1 17.22.5)
  • 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 7.2.3   Materials   Separation by Strong Ion‐Exchange HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment with or Without Prior Hydrazinolysis   Separation by Strong Ion‐Exchange HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment with or Without Prior Hydrazinolysis   Separation by Isocratic Ion‐Pairing HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment,With or Without Prior Hydrazinolysis m   Separation by Isocratic Ion‐Pairing HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment,With or Without Prior Hydrazinolysis   Gradient Conditions for Oligosaccharide Separation by Reversed‐Phase Ion‐Pairing HPLC   Gradient Conditions for Oligosaccharide Separation by Reversed‐Phase Ion‐Pairing HPLC

    Oligosaccharide Eluant (mM KH 2PO 4) Retention time (min)
    Disaccharides
    GlcA‐AMan R and IdoA AMan R 40 4.5
    AMan R 40 4.5
    AMan R(SO 4) 40 7.0
    GlcA(SO 4)‐AMan R 40 19.5
    GlcA‐AMan R(SO 4) 40 23.0
    IdoA‐AMan R(SO 4) 40 26.5
    IdoA(SO 4)‐AMan R 40 30.0
    GlcA(SO 4)‐AMan R(SO 4) 185 14.0
    IdoA(SO 4)‐AMan R(SO 4) 185 21.5
    GlcA‐AMan R(3,6diSO 4) 185 25.5
    Tetrasaccharides l
    t1  GlcA‐GlcNAc‐GlcA‐AMan R 20 28
    t2  IdoA‐GlcNAc‐GlcA‐AMan R 20 33.5
    t3  GlcA‐GlcNAc(SO 4)‐GlcA‐AMan R 100 31.5
    t4  IdoA(SO 4)‐GlcNAc‐GlcA‐AMan R 100 37.0
    t5  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R 100 42.5
    t6 200 25.0
    t7  IdoA(SO 4)‐GlcNAc‐GlcA‐AMan R(SO 4) 200 30.0
    t8  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) 200 32.5
    t9  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3‐SO 4) 185 53.0
    t10 320 23.0
    t11 320 30.0
    t12 320 32.0
    t13 350 23.5
    t14  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3,6‐diSO 4) 350 38.5
    t15  IdoA(SO 4)‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) 350 42.0
    t16 400 30.0
    Oligosaccharide Solvent n  Retention time (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(SO 4) A:B = 94:6 12.0
    IdoA(SO 4)‐AMan R A:B = 94:6 17.0
    GlcA(SO 4)‐AMan R A:B = 86:14 22.0
    GlcA‐AMan R(SO 4) A:B = 86:14 22.0
    IdoA‐AMan R(SO 4) A:B = 86:14 31.0
    GlcA‐AMan R(3,6diSO 4) A:B = 86:14 23.5
    IdoA(SO 4)‐AMan R(SO 4) A:B = 61:39 26.0
    GlcA(SO 4)‐AMan R(SO 4) A:B = 61:39 31.5
    Tetrasaccharides
    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(SO 4)‐GlcNAc‐GlcA‐AMan R A:C = 84:15 29.0
    t5  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R A:C = 84:15 33.0
    t3  GlcA‐GlcNAc(SO 4)‐GlcA‐AMan R A:C = 84:15 38.0
    t9  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3‐SO 4) A:C = 74:26 29.0
    t8  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) A:C = 74:26 33.0
    t13  IdoA(SO 4)‐RC(SO 4)IdoA(SO 4)‐AMan R A:C = 60:40 12.0
    t14  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3,6‐diSO 4) A:C = 60:40 15.0
    t16  IdoA‐RC(SO 4)IdoA(SO 4)‐AMan R(SO 4) 16.0
    Disaccharides Tetrasaccharides
    Time interval for solvent change (min) % Solvent B in time interval Time interval for solvent change (min) % Solvent C
    0→5 6 0→13 6
    5→6 6→14 13→14 6→11
    6→20 14 14→64 11
    20→40 14→20 64→65 11→25
    40→41 20→39 65→110 25
    41→81 39 110→111 25→31
    81→83 39→60 111→156 31 83→100
    157→192 39
    192→193 39→70
    193→220 70

     JAbbreviations: AMan, anhydro‐D‐mannose; GlcA, D‐glucuronic acid; GlcN, D‐glucosamine; GlcNAc, N‐acetyl‐D‐glucosamine; IdoA, L‐iduronic acid.
     kHPLC separations are obtained using step gradients. Details of gradients used for disaccharides and oligosaccharides are given in protocol 2; the KH 2PO 4 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 ( ) for details.
     lTetrasaccharide designations (t1‐t16) are described in Bienkowski and Conrad, . 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 ).
    Table 7.2.4   Materials   Separation by Strong Ion‐Exchange HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment with or Without Prior Hydrazinolysis   Separation by Strong Ion‐Exchange HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment with or Without Prior Hydrazinolysis   Separation by Isocratic Ion‐Pairing HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment,With or Without Prior Hydrazinolysis m   Separation by Isocratic Ion‐Pairing HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment,With or Without Prior Hydrazinolysis   Gradient Conditions for Oligosaccharide Separation by Reversed‐Phase Ion‐Pairing HPLC   Gradient Conditions for Oligosaccharide Separation by Reversed‐Phase Ion‐Pairing HPLC

    Oligosaccharide Eluant (mM KH 2PO 4) Retention time (min)
    Disaccharides
    GlcA‐AMan R and IdoA AMan R 40 4.5
    AMan R 40 4.5
    AMan R(SO 4) 40 7.0
    GlcA(SO 4)‐AMan R 40 19.5
    GlcA‐AMan R(SO 4) 40 23.0
    IdoA‐AMan R(SO 4) 40 26.5
    IdoA(SO 4)‐AMan R 40 30.0
    GlcA(SO 4)‐AMan R(SO 4) 185 14.0
    IdoA(SO 4)‐AMan R(SO 4) 185 21.5
    GlcA‐AMan R(3,6diSO 4) 185 25.5
    Tetrasaccharides l
    t1  GlcA‐GlcNAc‐GlcA‐AMan R 20 28
    t2  IdoA‐GlcNAc‐GlcA‐AMan R 20 33.5
    t3  GlcA‐GlcNAc(SO 4)‐GlcA‐AMan R 100 31.5
    t4  IdoA(SO 4)‐GlcNAc‐GlcA‐AMan R 100 37.0
    t5  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R 100 42.5
    t6 200 25.0
    t7  IdoA(SO 4)‐GlcNAc‐GlcA‐AMan R(SO 4) 200 30.0
    t8  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) 200 32.5
    t9  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3‐SO 4) 185 53.0
    t10 320 23.0
    t11 320 30.0
    t12 320 32.0
    t13 350 23.5
    t14  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3,6‐diSO 4) 350 38.5
    t15  IdoA(SO 4)‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) 350 42.0
    t16 400 30.0
    Oligosaccharide Solvent n  Retention time (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(SO 4) A:B = 94:6 12.0
    IdoA(SO 4)‐AMan R A:B = 94:6 17.0
    GlcA(SO 4)‐AMan R A:B = 86:14 22.0
    GlcA‐AMan R(SO 4) A:B = 86:14 22.0
    IdoA‐AMan R(SO 4) A:B = 86:14 31.0
    GlcA‐AMan R(3,6diSO 4) A:B = 86:14 23.5
    IdoA(SO 4)‐AMan R(SO 4) A:B = 61:39 26.0
    GlcA(SO 4)‐AMan R(SO 4) A:B = 61:39 31.5
    Tetrasaccharides
    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(SO 4)‐GlcNAc‐GlcA‐AMan R A:C = 84:15 29.0
    t5  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R A:C = 84:15 33.0
    t3  GlcA‐GlcNAc(SO 4)‐GlcA‐AMan R A:C = 84:15 38.0
    t9  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3‐SO 4) A:C = 74:26 29.0
    t8  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) A:C = 74:26 33.0
    t13  IdoA(SO 4)‐RC(SO 4)IdoA(SO 4)‐AMan R A:C = 60:40 12.0
    t14  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3,6‐diSO 4) A:C = 60:40 15.0
    t16  IdoA‐RC(SO 4)IdoA(SO 4)‐AMan R(SO 4) 16.0
    Disaccharides Tetrasaccharides
    Time interval for solvent change (min) % Solvent B in time interval Time interval for solvent change (min) % Solvent C
    0→5 6 0→13 6
    5→6 6→14 13→14 6→11
    6→20 14 14→64 11
    20→40 14→20 64→65 11→25
    40→41 20→39 65→110 25
    41→81 39 110→111 25→31
    81→83 39→60 111→156 31 83→100
    157→192 39
    192→193 39→70
    193→220 70

     mAbbreviations: AMan, anhydro‐D‐mannose; GlcA, D‐glucuronic acid; GlcN, D‐glucosamine; IdoA, L‐iduronic acid; RC, ring‐contraction product.
     nHPLC separations are obtained using a C‐18 reversed‐phase column using isocratic elution conditions obtained by mixing solvents A, B, and C (see reciperecipes in ) in the ratios shown.
     All 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 17.22.5.
     Tetrasaccharide designations are described in Bienkowski and Conrad, .
    Table 7.2.5   Materials   Separation by Strong Ion‐Exchange HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment with or Without Prior Hydrazinolysis   Separation by Strong Ion‐Exchange HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment with or Without Prior Hydrazinolysis   Separation by Isocratic Ion‐Pairing HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment,With or Without Prior Hydrazinolysis m   Separation by Isocratic Ion‐Pairing HPLC of Oligosaccharides Released from Heparin by Nitrous Acid Treatment,With or Without Prior Hydrazinolysis   Gradient Conditions for Oligosaccharide Separation by Reversed‐Phase Ion‐Pairing HPLC   Gradient Conditions for Oligosaccharide Separation by Reversed‐Phase Ion‐Pairing HPLC

    Oligosaccharide Eluant (mM KH 2PO 4) Retention time (min)
    Disaccharides
    GlcA‐AMan R and IdoA AMan R 40 4.5
    AMan R 40 4.5
    AMan R(SO 4) 40 7.0
    GlcA(SO 4)‐AMan R 40 19.5
    GlcA‐AMan R(SO 4) 40 23.0
    IdoA‐AMan R(SO 4) 40 26.5
    IdoA(SO 4)‐AMan R 40 30.0
    GlcA(SO 4)‐AMan R(SO 4) 185 14.0
    IdoA(SO 4)‐AMan R(SO 4) 185 21.5
    GlcA‐AMan R(3,6diSO 4) 185 25.5
    Tetrasaccharides l
    t1  GlcA‐GlcNAc‐GlcA‐AMan R 20 28
    t2  IdoA‐GlcNAc‐GlcA‐AMan R 20 33.5
    t3  GlcA‐GlcNAc(SO 4)‐GlcA‐AMan R 100 31.5
    t4  IdoA(SO 4)‐GlcNAc‐GlcA‐AMan R 100 37.0
    t5  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R 100 42.5
    t6 200 25.0
    t7  IdoA(SO 4)‐GlcNAc‐GlcA‐AMan R(SO 4) 200 30.0
    t8  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) 200 32.5
    t9  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3‐SO 4) 185 53.0
    t10 320 23.0
    t11 320 30.0
    t12 320 32.0
    t13 350 23.5
    t14  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3,6‐diSO 4) 350 38.5
    t15  IdoA(SO 4)‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) 350 42.0
    t16 400 30.0
    Oligosaccharide Solvent n  Retention time (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(SO 4) A:B = 94:6 12.0
    IdoA(SO 4)‐AMan R A:B = 94:6 17.0
    GlcA(SO 4)‐AMan R A:B = 86:14 22.0
    GlcA‐AMan R(SO 4) A:B = 86:14 22.0
    IdoA‐AMan R(SO 4) A:B = 86:14 31.0
    GlcA‐AMan R(3,6diSO 4) A:B = 86:14 23.5
    IdoA(SO 4)‐AMan R(SO 4) A:B = 61:39 26.0
    GlcA(SO 4)‐AMan R(SO 4) A:B = 61:39 31.5
    Tetrasaccharides
    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(SO 4)‐GlcNAc‐GlcA‐AMan R A:C = 84:15 29.0
    t5  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R A:C = 84:15 33.0
    t3  GlcA‐GlcNAc(SO 4)‐GlcA‐AMan R A:C = 84:15 38.0
    t9  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3‐SO 4) A:C = 74:26 29.0
    t8  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(SO 4) A:C = 74:26 33.0
    t13  IdoA(SO 4)‐RC(SO 4)IdoA(SO 4)‐AMan R A:C = 60:40 12.0
    t14  IdoA‐GlcNAc(SO 4)‐GlcA‐AMan R(3,6‐diSO 4) A:C = 60:40 15.0
    t16  IdoA‐RC(SO 4)IdoA(SO 4)‐AMan R(SO 4) 16.0
    Disaccharides Tetrasaccharides
    Time interval for solvent change (min) % Solvent B in time interval Time interval for solvent change (min) % Solvent C
    0→5 6 0→13 6
    5→6 6→14 13→14 6→11
    6→20 14 14→64 11
    20→40 14→20 64→65 11→25
    40→41 20→39 65→110 25
    41→81 39 110→111 25→31
    81→83 39→60 111→156 31 83→100
    157→192 39
    192→193 39→70
    193→220 70

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

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

  Materials
  • Sample of lyase‐degraded glycosaminoglycan
  • 1 M Na 2CO 3, pH 9.0 (ice‐cold)
  • recipeSodium borohydride reagent (see recipe)
  • 3 M H 2SO 4
  • 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 β‐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 α‐L‐iduronidase, sulfoiduronate sulfatase, and β‐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.
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