Analytical and Preparative Strong Anion Exchange HPLC of Heparan Sulfate and Heparin Saccharides

Jeremy E. Turnbull

1. Introduction

Studies on the structure-function relationships of the complex linear polysaccharides, known as glycosaminoglycans (GAGs), are becoming increasingly important as biological functions are established for them. However, structural analysis of GAGs presents a difficult technical problem, particularly in the case of the N-sulfated GAGs heparan sulfate (HS) and heparin, which display remarkable structural diversity (1). A widely used and effective approach is to degrade the chains into smaller saccharide units that can then be separated and analyzed. In this regard, strong anion-exchange (SAX) HPLC techniques have proved particularly useful for both the analysis of disac-charide composition (2,3) and the separation of complex mixtures of larger saccharides (3-6). However, in many methods the columns used have been silica-based and suffer from drawbacks related to poor stability of the support (e.g., inconsistency of run times, peak broadening, and short column life). There is clearly a need for improvements in column performance, especially for the purification of larger saccharides to homogeneity for sequencing and bioactivity testing. This chapter describes how a single type of polymer-based SAX column, ProPac PA1, can be used to provide high-resolution separations of both disaccharides and larger oligosaccharides derived from HS and heparin, with consistent elution times and excellent column performance characteristics (see Notes 1 and 2). Disaccharides from chondroitin sulfate and dermatan sulfate can also be separated (see Note 3). The improved resolution of saccharides compared to other SAX-HPLC methods, combined with the versatility and longevity of the columns, makes them a valuable tool for purification and structural analysis of HS/heparin and other GAG saccharides.

2. Materials

1. Gradient HPLC system (capable of mixing at least two solutions).

2. ProPac PA1 analytical columns (4 x 250 mm; Dionex).

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

3. Double-distilled water, pH 3.5: pH water to 3.5 using hydrochloric acid (high-purity HCl such as Aristar grade from BDH-Merck).

4. Sodium chloride solution (1 M): dissolve 58.4 g of HPLC-grade sodium chloride (e.g., HiPerSolv grade from BDH Merck) in 1L distilled water, and pH to 3.5 using HCl. Filter using a sintered glass filter or a 0.2 ^m bottle-top filter.

5. Potassium dihydrogen phosphate solution (1 M, pH 4.6): dissolve 136.1 g of HPLC-grade KH2PO4 (e.g., HiPerSolv grade from BDH Merck) in 1L distilled water and filter using a sintered glass filter or a 0.2-^m bottle-top filter.

6. Unsaturated disaccharide standards (for both HS/heparin and chondroitin/dermatan sulfate) and heparitinases/chondroitinases are available from Seikagaku Kogyo (Tokyo).

3. Methods

3.1. Separation of Unsaturated HS/Heparin Disaccharides

A simple and commonly used method to assess the structural composition of HS or heparin is to depolymerize the chains to disaccharides with a mixture of bacterial lyases. They can then be separated with reference to commercially available disaccha-ride standards of known structure (see Fig. 1). Note that the eliminative cleavage mechanism of the lyases results in unsaturated hexuronate residues in the resulting disaccharides (see Note 4).

1. Prepare unsaturated disaccharides from heparin/HS by bacterial lyase treatment with heparitinase I, heparitinase II, and heparinase; see ref. 7 for details.

2. After equilibration of the Pro-Pac PA1 column in mobile phase (double-distilled water adjusted to pH 3.5 with HCl) at 1 mL/min, inject the sample.

3. After 1 min injection time, elute the disaccharides with a linear gradient of sodium chloride (0-1 M over 45 min) in the same mobile phase.

4. Monitor the eluant in-line for UV absorbance at 232 nm (see Note 5).

5. Identify peaks by reference to standards separated under the same run conditions.

3.2. Separation of HS/heparin Disaccharides Derived by Nitrous Acid Degradation

A further class of disaccharides, those derived by treatment of HS or heparin with nitrous acid, are more difficult to resolve by SAX-HPLC. However, in contrast to the lyase-derived saccharides, they have the advantage of containing intact hexuronate residues (see Note 6). The most widely reported method for their separation uses a silica-based SAX Partisil column with a KH2PO4 gradient separation system (3). In our hands this method gives very broad and inadequately resolved peaks, which limits the sensitivity of detection and the accuracy of peak identification and quantitation. In marked contrast, these disaccharides can be resolved well on a ProPac PA1 column using appropriate shallow NaCl gradients. Resolution is improved slightly by use of two columns connected in series (see Figs. 2A,B), but adequate results can also be obtained using a single column. An alternative separation using a phosphate gradient is described under Subheading 3.2.2. 3.2.1. Sodium Chloride Gradient

1. Prepare reduced 3H-labeled disaccharides from heparin/HS by low-pH nitrous acid treatment, either from unlabeled samples using 3H-borohydride end-labeling (3) or from samples radiolabeled biosynthetically using 3H-glucosamine (8).

Auto Correlation

Fig. 1. Separation of lyase-derived HS/heparin disaccharides on a ProPac PA1 column. The profile shows the separation of the eight major unsaturated disaccharides released from these polysaccharides by treatment with a combination of the bacterial lyases heparitinase I, heparitinase II, and heparinase. The NaCl gradient is indicated by the dashed line. The structures of the standards and the amounts loaded were: 1, A-HexA-GlcNAc, 1 nmol; 2, A-HexA-GlcNSO3 , 2 nmol; 3, A-HexA-GlcNAc(6S), 0.5 nmol; 4, A-HexA(2S)-GlcNAc, 1.5 nmol; 5, A-HexA-GlcNSO3(6S), 1 nmol; 6, A-HexA(2S)-GlcNSO3, 1.5 nmol; 7, A-HexA(2S)-GlcNAc(6S), 2 nmol; 8, A-HexA(2S)-GlcNSO3(6S), 1.5 nmol. Abbreviations: GlcNAc, N-acetyl glucosamine; GlcNSO3, N-sulfated glucosamine; 2S, 3S, and 6S are 2-O-, 3-O-, and 6-O-sulfate groups, respectively; A-HexA, unsaturated hexuronate residue formed at non-reducing end of disaccharides and oligosaccharides by eliminative lyase scission.

Fig. 1. Separation of lyase-derived HS/heparin disaccharides on a ProPac PA1 column. The profile shows the separation of the eight major unsaturated disaccharides released from these polysaccharides by treatment with a combination of the bacterial lyases heparitinase I, heparitinase II, and heparinase. The NaCl gradient is indicated by the dashed line. The structures of the standards and the amounts loaded were: 1, A-HexA-GlcNAc, 1 nmol; 2, A-HexA-GlcNSO3 , 2 nmol; 3, A-HexA-GlcNAc(6S), 0.5 nmol; 4, A-HexA(2S)-GlcNAc, 1.5 nmol; 5, A-HexA-GlcNSO3(6S), 1 nmol; 6, A-HexA(2S)-GlcNSO3, 1.5 nmol; 7, A-HexA(2S)-GlcNAc(6S), 2 nmol; 8, A-HexA(2S)-GlcNSO3(6S), 1.5 nmol. Abbreviations: GlcNAc, N-acetyl glucosamine; GlcNSO3, N-sulfated glucosamine; 2S, 3S, and 6S are 2-O-, 3-O-, and 6-O-sulfate groups, respectively; A-HexA, unsaturated hexuronate residue formed at non-reducing end of disaccharides and oligosaccharides by eliminative lyase scission.

2. Separate samples on two ProPac PA1 columns in series, using the same mobile phase described under Subheading 3.1., but with a two-step linear NaCl gradient (0-150 mM over 50 min followed by 150-500 mM over 70 min).

3. Monitor 3H radioactivity either with an in-line radioactivity detector or by collecting fractions for scintillation counting (e.g., in Optiphase HiSafe III scintillant, Wallac). See Note 7.

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