Protein Protocols Protein Protocols

816 Hounsell, Davies, and Smith
10. Quantitate the sialic acids by comparison with known standards run on the same day.
11. When the baseline has stabilized, the system is ready for the next injection.
12. When the analyses have been completed, regenerate the column in eluant B, and flush
pumps with HPLC-grade H 2O (see Note 5).
4. Notes
1. If problems with contaminants are encountered, it may be necessary to wash the vials with
chromic acid overnight, wash them thoroughly with distilled water, and then treat with a
hydrophobic coating, such as repelcoat (see Chapter 109).
2. 0.01 M HCl will release sialic acids with intact N- or O-acyl groups, but without quantitative
release of the sialic acids. These can also be detected by HPAEC-PAD (3). At 0.1 M
HCl, quantitive release is achieved, but with some loss of O- and N-acylation. At this
concentration, some fucose residues may also be labile. Alternatively, the sialic acids can
be released by neuraminidase treatment, which can be specific for α2-6 or α2-3 linkage,
e.g., with α-sialidase of Arthrobacter ureafacians for α2-6 and α-sialidase of Newcastle
disease virus for α2-3 using the manufacturer’s instructions.
3. Use polypropylene reagent vessels as far as possible for HPAEC-PAD because of the corrosive
nature of the NaOH, and to minimize leaching of contaminants from the reservoirs.
4. For maximum efficiency of detection, always ensure that the PAD reference electrode is accurately
calibrated, the working electrode is clean, and the solvents are thoroughly degassed.
5. Failure to wash out the eluants from the pumps at the end of an analysis may result in
crystallization and serious damage to the pump heads.
References
1. Townsend, R. R. (1995) Analysis of glycoconjugates using high-pH anion-exchange chromatography.
J. Chromatog. Library 58, 181–209.
2. Manzi, A. E., Diaz, S., and Varki, A. (1990) HPLC of sialic acids on a pellicular resin
anion exchange column with pulsed amperometry. Anal. Biochem. 188, 20–32.