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Procedure for oxidizing the ligand

Ligands containing cis-diols are oxidized with sodium metaperiodate to introduce aldehyde groups before immobilization.

Oxidation of cis-diols to aldehydes with periodate is a simple and well characterized method of introducing aldehyde groups into monosaccharide residues ¹. This method is suitable for the majority of polysaccharides and glycoconjugates.

Materials

100 mM sodium acetate buffer pH 5.5 (buffer A)
10 mM sodium acetate buffer pH 4.0 (buffer B)
50 mM freshly prepared sodium metaperiodate (NaIO₄) in buffer A
Desalting column (e.g. NAP-5)

Procedure

1. Prepare a cold solution of the ligand to be oxidized in buffer A at 1 mg/ml.
2. For oxidation of sialic acid, add 1/50 volume of sodium metaperiodate solution (final periodate concentration 1 mM). Use higher concentrations (10 mM) for oxidation of other sugars with adjacent hydroxyl groups.
3. Incubate for 20 minutes on ice.
4. Stop the reaction by desalting the mixture on a desalting column eluted with buffer B. Store the oxidized ligand in the refrigerator.

The degree of oxidation is most easily estimated by testing immobilization on a hydrazide-activated sensor chip surface. Unoxidized ligand is preferably used as negative control. If the results indicate insufficient oxidation, increase the time of oxidation, the periodate concentration or the oxidation temperature.

Enzymatic oxidation of terminal galactose and N-acetyl-D-galactose residues with galactose oxidase is an alternative method applicable to many glycoproteins ². In many cases, penultimate galactose residues can be exposed by treatment with neuraminidase before being oxidized with galactose oxidase.

¹ O’Shannessy, D. J. and Wilchek, M. (1990) Analyt. Biochem. 191, 1-8.
² Avigad, E., Amaral, D., Asensio, C. and Horecker, B.L. (1962) J. Biol. Chem. 237, 2736.

See also

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