Potassium Dihydrogen Phosphate Gradient

Improved separation of monosulfated disaccharides can be obtained using a gradient of phosphate (see Figure 2c and Note 8).

1. Using two ProPac PA1 columns in series, equilibrate in H2O, pH 6.0.

2. Inject samples and elute with a linear gradient of 0-1 M KH2PO4 buffer, pH 4.6 over 90 min.

3. Monitor 3H radioactivity as under Subheading 3.2.1.

3.3. Separation of HS/Heparin Oligosaccharides

In addition to separation of disaccharides, much larger HS and heparin oligosac-charides can also be resolved on ProPac PA1 using a linear NaCl gradient (see Note 9).

Fig. 2. Separation of HNO2-derived HS/heparin disaccharides on a ProPac PA1 column. These profiles show the separation of the major disaccharides released from HS/heparin by low-pH nitrous acid treatment followed by 3H-borohydride end labeling (3), or from 3H-biosyn-thetically labeled samples (8). The structures of the 3H-labeled standards were: 1, IM/GM; 2, M6S (monosaccharide); 3, G2SM; 4, I2SM; 5, GM3S; 6, GM6S; 7, IM6S; 8, I2SM6S; 9, G2SM6S; and 10, GM3,6S (where G and I are glucuronic and iduronic acids, respectively; M is 2,5-anhydromannitol; and 2S, 3S, and 6S are 2-O-, 3-O-, and 6-O-sulfate groups, respectively). (A, B) show the first and last 60 min of the run time using a NaCl gradient as described under Subheading 3.2.1. (C) shows the separation of non- and monosulfated disaccharides using an alternative KH2PO4 gradient as described under Subheading 3.2.2.

Fig. 2. Separation of HNO2-derived HS/heparin disaccharides on a ProPac PA1 column. These profiles show the separation of the major disaccharides released from HS/heparin by low-pH nitrous acid treatment followed by 3H-borohydride end labeling (3), or from 3H-biosyn-thetically labeled samples (8). The structures of the 3H-labeled standards were: 1, IM/GM; 2, M6S (monosaccharide); 3, G2SM; 4, I2SM; 5, GM3S; 6, GM6S; 7, IM6S; 8, I2SM6S; 9, G2SM6S; and 10, GM3,6S (where G and I are glucuronic and iduronic acids, respectively; M is 2,5-anhydromannitol; and 2S, 3S, and 6S are 2-O-, 3-O-, and 6-O-sulfate groups, respectively). (A, B) show the first and last 60 min of the run time using a NaCl gradient as described under Subheading 3.2.1. (C) shows the separation of non- and monosulfated disaccharides using an alternative KH2PO4 gradient as described under Subheading 3.2.2.

Figure 3 shows examples of separations performed on complex natural mixtures of hexa-, octa-, and decasaccharides derived from porcine mucosal HS by treatment with heparitinase I. It is noteworthy that the saccharides eluted in sets of resolved or partially resolved peaks, which presumably have similar overall levels of anionic charge but variant patterns of sulfation and disaccharide sequence. This method is particularly useful for purification of saccharides to the degree necessary for sequencing (12) or testing in biological assays (13). Preparative scale separations are also possible (see Note 10).

0 20 40 60 80 100

Minutes

Fig. 3. Separation of HS oligosaccharides on a ProPac PA1 column. Porcine mucosal HS was depolymerised with heparitinase I and hexa-, octa-, and deca-saccharide pools prepared by gel filtration on Bio-Gel P6 (9) and resolved on two ProPac PA1 columns connected in series as described under Methods. (A), hexasaccharides; (B), octasaccharides; (C), decasaccharides. In each case, 100 nmol of each saccharide mixture was loaded (approximately 150, 200, and 250 ^g each, respectively).

1. Prepare HS and heparin oligosaccharides (tetrasaccharides and larger) as described previously—for example, by partial depolymerization with heparitinase I and gel filtration on Bio-Gel P6 (7-9).

2. Inject samples onto two ProPac PA1 columns in series, using the same mobile phase described under Subheading 3.1.

3. Elute with a modified salt elution gradient (e.g., 0-1 M NaCl over 100 min).

4. Monitor the eluant in-line for UV absorbance at 232 nm.

4. Notes

1. During development of improved HPLC methods for separation of GAG saccharides we found that a polymer-based column, ProPac PA1, originally designed primarily for

SAX of proteins, provided excellent separations of disaccharides derived from the glucosaminoglycans HS and heparin. Figure 1 shows the separation of the major unsaturated disaccharides (detected by absorbance at 232 nm) derived by digestion of these polysac-charides with a mixture of bacterial heparitinases. They elute as very sharp, symmetrical peaks at highly reproducible elution times, and good baselining between all the peaks was evident. These features of the separation provide more accurate identification of peaks and sensitive quantitation than possible with previous methods. Note that 3H or 35S radiolabeled disaccharides (derived from biosynthetically labeled polysaccharide) can also be separated using this method, using in-line monitoring of radioactivity (see Fig. 2) or counting of collected fractions.

2. Overall column performance is excellent. Elution times are highly consistent, and the columns exhibit very good stability and longevity, which outweighs their initial high cost compared to traditional silica columns. In one case a single column was used for more than 250 runs over a 2-yr period without significant loss of resolution. These properties are presumably dependent on the column resin, which is formed by 0.2m anion exchange microbeads being bonded to a 10-^m nonporous polystyrene/divinylbenzene polymeric resin.

3. Good separations of unsaturated disaccharides derived by chondroitinase ABC digestion of the galactosaminoglycans chondroitin sulfate and dermatan sulfate can also be obtained using the ProPac-PA1 column. Similar results have been reported for the separation of this class of disaccharides on another polymer-based column, the CarboPac PA1 (10).

4. The eliminative cleavage mechanism of the lyases results in unsaturated hexuronate residues (double bond between carbons 4 and 5 of the sugar ring) in the resulting disaccha-rides (14). It is thus not possible to distinguish directly whether they resulted originally from glucuronate or iduronate residues. In contrast, cleavage with nitrous acid leaves these residues intact (see Note 6).

5. The limit of detection of unsaturated disaccharides by UV absorbance is approximately 20-50 pmol.

6. Nitrous acid cleaves between N-sulfated glucosamine residues and hexuronate residues. The resulting saccharides have intact hexuronate residues but the reducing-end glucosamines are converted to an anhydromannose residues (which are reduced to anhydromannitol by reduction during preparation of the disaccharides).

7. When detecting radiolabeled samples, if accurate quantitation is required, be aware of the potential for salt-dependent variability in counting efficiencies, depending on the scintillant being used.

8. With in-line radioactivity monitoring, baselining between peaks is sufficient in most cases to allow accurate quantitation of the individual major species. The exception is the partial separation of I2SM from the rare disaccharide G2SM (see Fig. 2a). Note that slight changes in run conditions (e.g., pH) can reverse their order of elution. However, conditions using an alternative KH2PO4 buffer separation system give the best resolution of the monosulfated disaccharide standards, including excellent baseline separations of I2SM and G2SM when required (see Fig. 2c).

9. This approach is effective for separating saccharides up to approximately dp14-16 in size, although the patterns become inherently more complex and resolution less efficient with increasing size. Improved resolution can be obtained using shallower gradients than described under Subheading 3.3. Additional examples of applications can be found in refs. 11-13.

10. Preparative separations are possible with loadings up to approximately 0.5-1.0 mg per run on the 4 x 250 mm analytical columns, and a fivefold scale-up should be achievable on 9 x 250 mm preparative columns that are available from Dionex.

References

1. Gallagher, J. T., Turnbull, J. E., and Lyon, M. (1992) Patterns of sulphation in heparan sulphate: polymorphism based on a common structural theme. Int. J. Biochem. 24, 553-556.

2. Yoshida, K., Miyauchi, S., Kikuchi, H., Tawada, A., and Tokuyasu, K. (1989) Analysis of unsaturated disaccharides from glycosaminoglycuronan by HPLC. Anal. Biochem. 177, 327-332.

3. Bienkowski, M. J. and Conrad, H. E. (1984) Structural characterisation of the oligosaccharides formed by depolymerisation of heparin with nitrous acid. J. Biol. Chem. 260, 356-365.

4. Guo, Y. and Conrad, H. E. (1988) Analysis of oligosaccharides from heparin by reversed-phase ion pairing HPLC. Anal. Biochem. 168, 54-62.

5. Linhardt, R. J., Gu, K., Loganathan, D., and Carter, S. R. (1989). Analysis of glycosami-noglycan-derived oligosaccharides using reverse-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection. Anal. Biochem. 181, 288-296.

6. Rice, K. G., Kim, Y., Grant, A., Merchant, Z., and Linhardt, R. J. (1985) HPLC separation of heparin-derived oligosaccharides. Anal. Biochem. 150, 325-331.

7. Turnbull, J. E. and Gallagher, J. T. (1991) Distribution of iduronate-2-sulphate residues in heparan sulphate: evidence for an ordered polymeric structure. Biochem. J . 273, 553-559.

8. Turnbull, J. E., Fernig, D., Ke, Y., Wilkinson, M. C., and Gallagher, J. T. (1992) Identification of the basic FGF binding sequence in fibroblast HS. J. Biol. Chem. 267, 10,337-10,341.

9. Walker, A., Turnbull, J. E., and Gallagher, J. T. (1994) Specific HS saccharides mediate the activity of basic FGF. J. Biol. Chem. 269, 931-935.

10. Midura, R. J., Salustri, A., Calabro, A., Yanagashita, M., and Hascall, V. C. (1994) Highresolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography. Glycobiology 4, 333-342.

11. Sanderson, R. D., Turnbull, J. E., Gallagher, J. T., and Lander, A.D. (1994) Fine structure of HS regulates cell syndecan-1 function and cell behaviour. J. Biol. Chem. 269, 13,100-13,106.

12. Turnbull, J. E., Hopwood, J., and Gallagher, J. T. (1999) A strategy for rapid sequencing of heparan sulphate/heparin saccharides. Proc. Nat. Acad. Sci. USA 96, 2698-2703

13. Guimond, S. E. and Turnbull, J. E. (1999) Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides. Current Biology 9, 1343-1346.

14. Linhardt R. J., Turnbull J. E., Wang H., Loganathan D., and Gallagher J. T. (1990) Examination of the substrate specificity of heparin and heparan sulphate lyases. Biochemistry 29, 2611-2617.

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