Protein Protocols Protein Protocols

Chemical Methods of Analysis 803
108
Chemical Methods of Analysis of Glycoproteins
Elizabeth F. Hounsell, Michael J. Davies, and Kevin D. Smith
1. Introduction
The first analysis of glycoconjugates that often needs to be carried out is to see if
they indeed contain sugar. For glycoproteins in gels or oligosaccharides in solution,
this can be readily achieved by periodate oxidation at two concentrations, the first to
detect sialic acids, and the second, any monosaccharide that has two free vicinal hydroxyl
groups (1). Periodate cleaves between the hydroxyl groups to yield reactive aldehydes,
which can be detected by reduction with NaB 3 H 4 or coupled to high sensitivity probes
available in commercial kits, e.g., from Boeringher Mannheim (Mannheim, Germany)
or Oxford GlycoSciences (Abingdon, UK). In solution, a quick spot assay can be carried
out for the presence of any monosaccharide or oligosaccharide having a C-2
hydroxyl group by visualization with charring by phenol/sulfuric acid reagent (2).
These methods are relatively specific for mono/oligosaccharides (3).
2. Materials
2.1. Periodate Oxidation
1. 0.1 M Acetate buffer, pH 5.5, containing 8 or 15 mM sodium periodate.
2. Ethylene glycol.
3. 0.1 M Sodium hydroxide.
4. Reducing agent: sodium borohydride, tritiated sodium borohydride, or sodium
borodeuteride.
5. Glacial acetic acid.
6. Methanol.
2.2. Phenol Sulfuric Acid Assay
1. H2O (HPLC-grade).
2. 4% Aqueous phenol.
3. Concentrated H2SO4. 4. 1 mg/mL galactose.
5. 1 mg/mL mannose.
3. Methods
3.1. Periodate Oxidation
1. Dissolve 0.1–1.0 mg glycoprotein in 2 mL of acetate buffer containing sodium periodate
(15 mM for all monosaccharides, 8 mM for alditols, and 1 mM specifically for oxidation
of sialic acids).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
803