Carbohydrates biotin-hydrazide

Biotin-hydrazide has been used to biotinylate antibodies at their oxidized carbohydrate residues (O Shanessy et al., 1984, 1987 O Shanessy and Quarles, 1985 Hoffman and O Shannessy, 1988), to modify the low-density lipoprotein (LDL) receptor (Wade et al., 1985), to biotinylate nerve growth factor (NGF) (Rosenberg et al., 1986), and to modify cytosine groups in oligonucleotides to produce probes suitable for hybridization assays (Reisfeld et al., 1987) (Chapter 27, Section 2.3). 

The following protocol describes the use of biotin-hydrazide to label glycosylated proteins at their carbohydrate residues. Control of the periodate oxidation level can result in specific labeling of sialic acid groups or general sugar residues (Chapter 1, Section 4.4). 

The reactivity and use of biocytin-hydrazide is similar to that described for biotin-hydrazide in Section 3, this chapter. The following protocol for labeling glycoproteins at oxidized carbohydrate (galactose) sites is from Bayer and Wilchek (1992). 

If a free amino group forms a portion of the protein that is essential for activity (e.g., the antigen-combining site for antibody), biotinylation with the succinimide ester will lower or destroy the activity of the protein, and other methods of labeling should be tried. Biotin hydrazide has been used to modify the carbohydrate moieties of antibodies (10,11). Other alternatives are the thiol-reactive biotin maleimide (12) or biotin iodoacetamide (13). 

Conjugation with Biotin Hydrazide at the Carbohydrate Site... 

In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden). 

Biotinylation of Antibody Carbohydrates With Biotin Hydrazide Derivatives... 

Biotin hydrazides will react with antibody carbohydrate moieties after oxidation with sodium metaperiodate. This step opens the sugar rings between vicinal hydroxides and converts them to aldehydes that will react with hydrazides. 

In some cases, the ability to modify glycans at the reducing end without reduction preserves the carbohydrate s native structure sufficiently to allow interactions with proteins that would otherwise not interact if the bond were reduced. Therefore, depending on the ultimate use of the biotinylated carbohydrate, using a hydrazide mediated conjugation process can have advantages over the use of amine-biotin compounds. 

Figure 18.19 Biotin-PEG4-hydrazide is a hydrophilic biotinylation reagent that can be used to modify glycans or carbohydrates at their reducing end or after periodate oxidation to create aldehydes.

Carbohydrates are easily oxidized by periodate to aldehydes which react with primary amines or hydrazines. If the formed hydrazide carries a specific ligand, e.g., biotin or digoxigenin, these ligands immobilized via the blotted macromolecule are very sensitively detected by the respective enzyme conjugates. 

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