Edward Conrad

1. Introduction

All glycosaminoglycans contain an amino sugar in every second position in their linear chains. In most cases these amino sugars are N-acetylated, e.g., N-acetyl-d-galactosamine (GalNAc) in chondroitin sulfates and dermatan sulfates, or N-acetyl-d-glucosamine (GlcNAc) in heparins, heparan sulfates, and hyaluronic acids. Heparins and heparan sulfates contain only glucosamine residues, and, in a high percentage of these, the N-acetyl substituents are replaced by N-sulfate groups. The N-sulfated glucosamines (GlcNSO3), found only in heparin-like glycosaminoglycans, are sites that are unique in their susceptibility to facile cleavage by nitrous acid at room temperature and at low pH (~1.5). Thus, when heparins or heparan sulfates are treated with nitrous acid, they are specifically cleaved into fragments with ranges of molecular weights that depend on the distributions of the GlcNSO3 residues in the chain. Since N-acetylated amino sugars are not affected by the nitrous acid treatment, only the heparin-like structures are cleaved when they are present in mixtures containing other glycosaminoglycans.

Interestingly, glycosaminoglycans containing amino sugars that have unsubstituted amino groups can be cleaved specifically with nitrous acid at a higher pH (~4), again at room temperature. Under these conditions, the GlcNSO3 residues do not react. Since a small number of N-unsubstituted glucosamine residues (GlcN) occur in heparan sulfates (1), cleavage occurs at these positions.

N-acetylated amino sugars do not react with nitrous acid under any conditions. However, by hydrazinolysis, it is possible to remove N-acetyl groups from N-acety-lated amino sugars under conditions that do not remove N-sulfate groups or otherwise alter the glycosaminoglycan structures (2). Thus, following hydrazinolysis, nitrous acid (at pH 4) cleaves heparins and heparan sulfates specifically at the glucosamine residues that were originally N-acetylated, yielding fragments with ranges of molecular weights that depend on the distributions of the GlcNAc residues in the chains. In a mixture of glycosaminoglycans, the hydrazinolysis/nitrous acid procedure yields the variously sized fragments from the heparin-like glycosaminoglycans, but converts all other glycosaminoglycans completely to disaccharides (since all other glycosaminoglycans contain N-acetylated amino sugars at every second residue).

The cleavage of the glycosidic bonds of the amino sugars with nitrous acid is an elimination reaction not a hydrolysis reaction. In all cases, the reaction yields fragments in which the amino sugar at the site of cleavage is converted to a reducing terminal anhydrosugar—in the case of d-glucosamine, 2,5-anhydro-d-mannose is formed; in the case of d-galactosamine, 2,5-anhydro-D-talose is formed. In order to stabilize the products, it is helpful to reduce the aldehyde groups of these anhydrosugars to alditols using NaBH4. The nitrous acid, hydrazinolysis, and NaBH4 reactions have been discussed in detail elsewhere (3).

2. Materials

2.1. Chemicals

1. Heparin.

2. Heparan sulfate.

6. Reacti-Vials (Pierce Chemical Co., Rockford, IL).

10. Water bath.

11. Hydrazine, anhydrous.

12. Hydrazine SO4.

2.2. Reference Standards

Heparin and heparan sulfate can be obtained from Sigma Chemical Co. Stock solutions of these glycosaminoglycans contain 20 mg/mL water and are stored frozen.

2.3. pH 1.5 Nitrous Acid Reagent

Solutions of 0.5 M H2SO4 and 0.5 M Ba(NO2)2 (114 mg/mL) are prepared and cooled separately to 0°C in an ice bath. A mixture containing 1 mL of each solution (0.5 mmol of each reagent) is prepared at 0°C, and the mixture is centrifuged in a clinical centrifuge to pellet the BaSO4 precipitate. The supernatant is drawn off with a Pasteur pipet, and stored on ice. This reagent should be prepared when needed and used immediately (see Note 1).

2.4. pH 4.0 Nitrous Acid Reagent pH 4 Nitrous acid is generated by adding 5 mL of 5.5 M NaNO2 to 2 mL of 0.5 M H2SO4. This reagent should be prepared when needed and used immediately. When glycosaminoglycans are hydrolyzed in 0.5 M H2SO4, the pH 4 nitrous acid is generated in situ by adding 5 ^L of the 5.5 M NaNO2 solution to 2 ^L of the hydrolysate (see Notes 2 and 3).

2.5. Hydrazine Reagent

Hydrazine sulfate (100 mg) is dissolved in 3 mL of distilled water. Anhydrous hydrazine (7 mL) is added to this solution to give a solution containing 1% hydrazine sulfate in 30% aqueous hydrazine. Hydrazine is a toxic and corrosive reagent and should be handled accordingly.

2.6. Sodium Borohydride Reagent

3. Methods

3.1. Cleavage of N-Sulfated Glycosaminoglycans with Nitrous Acid at pH 1.5

1. Cool a 5-mL aliquot of a solution of heparin or heparan sulfate (20 mg/mL of water) to 0°C and add 20 mL of the cold pH 1.5 nitrous acid reagent.

2. Let the mixture warm to room temperature; deamination is complete within 10 min following nitrous acid addition.

3. Adjust the pH of the deaminated product to 8.5 with 1 M Na2CO3.

4. Reduce the sample with NaBH4 as described under Subheading 3.3.

5. The reduced sample may be separated into its individual components by gel filtration to separate di-, tetra-, hexasaccharides, etc., and then by ion-exchange chromatography, high-pressure liquid chromatography, or capillary electrophoresis to separate the oligosaccharides according to charge (2,4-14). Since the nitrous acid cleavage products are complex mixtures of oligosaccharides, multiple separation steps are required to obtain individual components in a pure form.

3.2. Hydrazinolysis of N-Acetylated Glycosaminoglycans and Cleavage with pH 4 Nitrous Acid

1. Place 15 ^L of a solution of heparin or heparan sulfate (20 mg/mL) in a 100-^L Reacti-Vial and dry the sample in a stream of air.

2. Redissolve the dried sample in 20 ^L of hydrazine reagent.

3. Cap the vial and place it in a 100°C water bath for 4 h.

4. Cool the sample, dry it in a stream of air; and lyophilize the partially dried sample to remove as much of the hydrazine as possible.

5. Due to residual hydrazine SO4 and hydrazine, the pH of this solution is actually alkaline (pH 8-10). Add 5-10 ^L of 3 M H2SO4 to the sample to bring the pH to 4.0, as measured with pH paper.

6. Dry the pH-adjusted sample in a stream of air.

7. Add 20 ^L of the pH 4 nitrous acid reagent to the dried sample.

8. After 15 min, the cleavage reaction is complete.

9. Adjust the pH of the reaction mixture to 8.5

10. Reduce the cleavage products with NaBH4 as described below and separate the individual components by gel filtration and ion-exchange chromatography (above).

3.3. Reduction of Cleavage Products with NaBH4

1. Treat the pH 8.5 solutions of the nitrous acid-cleaved products with 10 mL of the sodium borohydride reagent and incubate at room temperature for 15 min. Since H2 gas is evolved in this reaction, all NaBH4 reductions should be carried out in a fume hood.

2. Destroy excess NaBH4 by addition of ~5 mL of 3 M H2SO4 to give a slightly acidic solution. Evaporate the sample to dryness. Redissolve the sample in water and again evaporate the solution to dryness to remove as much H2 as possible.

3. Redissolve the sample in water for separation of the individual components.

4. Notes

1. Once the nitrous acid is prepared, there is a series of complex reactions of the resulting oxides of nitrogen. Within a short time, the active species of "nitrous acid" undergoes changes that result in the loss of the capacity of the reagent to cleave the glycosidic bonds of the amino sugars [for a discussion, see ref. 15)]. Consequently, the nitrous acid reagents must be used within a few minutes after their preparation. This is of less concern for the pH 4 reagent than for the pH 1.5 reagent, because the pH 4 reagent is more highly concentrated in nitrite.

2. The pH of these reactions is important for maintaining the selectivity of the cleavage. Although there is good selectivity for N-unsubstituted GlcN's and N-sulfated GlcN's at pH 4 and 1.5, respectively, the glycosidic bonds of both types of GlcN residues are cleaved at a pH between these values. Thus, it is desirable to let the reactions at the respective pH proceed only for 10-15 min. Also, when samples are derived from buffered solutions, it is necessary to check the pH with pH paper before addition of the nitrous acid reagent. In fact, it is desirable to dialyze the glycosaminoglycan solution to remove all salts before beginning the cleavage step. An important reason for the predialysis is that NaBH4 is catalytically destroyed by oxyanions, such as PO43 (16).

3. Although the nitrous acid reactions cleave the bonds of p-linked amino sugars virtually stoichiometrically, the reaction of nitrous acid with a-linked amino sugars takes two pathways. In both cases treatment with nitrous acid leads to the loss of the amino group and the formation of a carbonium ion at carbon 2. In the most prominent further conversion, the a-glycosidic bond is cleaved with the formation of the reducing terminal anhydrosugar as described above. However, a significant proportion (~10%) of the carbonium ion undergoes a reaction in which the ring is contracted to a furanose ring without glycosidic bond cleavage. This "ring contraction reaction" is described elsewhere (3).

References

1. van den Born, J., Gunnarsson, K., Bakker, M. A. H., Kjellen, L., Kusche-Gullberg, M., Maccarana, M., Berden, J. H. M., and Lindahl, U. (1995) Presence of N-unsubstituted glucosamine units in native heparan sulfate revealed by a monoclonal antibody. J. Biol. Chem. 270, 31,303-31,309.

2. Guo, Y. and Conrad, H. E. (1989) The disaccharide composition of heparins and heparan sulfates. Anal. Biochem. 176, 96-104.

3. Conrad, H. E. (1998) Heparin-Binding Proteins, Academic Press, San Diego, CA.

4. Ampofo, S. A., Wang, H. M., and Linhardt, R. J. (1991) Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis. Anal. Biochem. 199, 249-255.

5. Bienkowski, M. J. (1984) Structure and metabolism of heparin and heparan sulfate, Ph.D. dissertation, University of Illinois, Urbana, IL.

6. Delaney, S. R., Leger, M., and Conrad, H. E. (1980) Quantitation of the sulfated dis-accharides of heparin by high performance liquid chromatography. Anal. Biochem. 106, 253-261.

7. Desai, U. R., Wang, H.-M., Ampofo, S. A., and Linhardt, L. J. (1993) Oligosaccharide composition of heparin and low-molecular-weight heparins by capillary electrophoresis. Anal. Biochem. 213, 120-127.

8. Desai, U. R., Wang, H.-M., Kelly, T. R., and Linhardt, R. J. (1993) Structure elucidation of a novel acidic tetrasaccharide and hexasaccharide derived from a chemically modified heparin. Carbohydr. Res. 241, 249-259.

9. Guo, Y. and Conrad, H. E. (1988) Analysis of oligosaccharides from heparin by reversed phase ion-pairing high pressure liquid chromatography. Anal. Biochem. 168, 54-62.

10. Kitagawa, H., Kinoshita, A., and Sugahara, K. (1995) Microanalysis of glycosami-noglycan-derived disaccharides labeled with the fluorophore 2-aminoacridone by capillary electrophoresis and high-performance liquid chromatography. Anal. Biochem. 232, 114-121.

11. Liu, J., Shworak, N. W., Fritze, L. M., Edelberg, J. M., and Rosenberg, R. D. (1996) Purification of heparan sulfate D-glucosaminyl 3-O-sulfotransferase. J. Biol. Chem. 271, 27,072-27,082.

12. Merchant, Z. M., Kim, Y. S., Rice, K. G., and Linhardt, R. J. (1985) Structure of heparin-derived tetrasaccharides. Biochem. J. 229, 369-377.

13. Murata, K., Murata, A., and Yosida, K. (1995) High-performance liquid chromatographic identification of eight constitutional disaccharides from heparan sulfate isomers digested with heparitinases. J. Chromatogr. B 670, 3-10.

14. Pervin, A., Al-Hakim, A., and Linhardt, R. J. (1994) Separation of glycosaminoglycan-derived oligosaccharides by capillary electrophoresis using reverse polarity. Anal. Biochem. 221, 182-188.

15. Shively, J. E., and Conrad, H. E. (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15, 3932-3942.

16. Conrad, H. E., James, M. E., and Varboncoeur, E. (1973) Qualitative and quantitative analysis of reducing carbohydrates by radiochromatography on ion exchange papers. Anal. Biochem. 51, 486-500.

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