Glycoconjugates labeling with

Figure 9.11 This carbohydrazide-containing fluorescein derivative can be used to modify aldehyde-containing molecules. Glycoconjugates may be labeled with this reagent after treatment with sodium periodate to produce aldehydes.

Lectins, or proteins with specific binding sites for carbohydrates, can be used as targeting molecules to localize particular glycoconjugates such as glycoproteins or glycolipids on cell surfaces (Fig. 373). Labeled with gold particles, lectins are important probes for detection of cell surface components and intracellular receptors and in immunological or biochemical assay procedures (Bog-Hansen et al., 1978 Kimura et al., 1979 Nicolson, 1978 Roth, 1983 Benhamou et al., 1988 Nakajima et al., 1988). 

The rapid expansion of lectin-based applications for the detection and quantification of glycoconjugates has been led by the development of commercially available, purified and chemically derivatized lectins, and in some cases, anti-lectin antibodies. Over 50 purified plant lectins are sold commercially by a number of producers and vendors, with this number growing annually. Equally important is the ease by which investigators can obtain lectins labeled with various fluorescent dyes, haptenic moieties, biotin, and radioactive atoms, as well as conjugated to enzymes and solid-phase supports. These derivatized lectins are useful for either direct or indirect detection and quantification techniques, or for the physical separation of particulate-bound or soluble glycoconjugates. Table 4 lists many of the commercially available lectin reagents and sources. 

Polysaccharides, glycoproteins, and other glycoconjugates therefore may be specifically labeled on their carbohydrate portions by creating aldehyde functionalities and subsequently derivatizing them with another molecule containing an amine or a hydrazide group. This route of derivatization is probably the most common way of modifying carbohydrates. 

Figure 5.35 ABH reacts with aldehyde-containing compounds through its hydrazide end to form hydrazone linkages. Glycoconjugates may be labeled by this reaction after oxidation with sodium periodate to form aldehyde groups. Subsequent photoactivation with UV light causes transformation of the phenyl azide to a nitrene. The nitrene undergoes rapid ring expansion to a dehydroazepine that can couple to nucleophiles, such as amines.

The hydrazide derivative of AMCA can be used to modify aldehyde- or ketone-containing molecules, including cytosine residues using the bisulfite activation procedure described in Chapter 27, Section 2.1. AMCA-hydrazide reacts with these target groups to form hydrazone bonds (Figure 9.26). Carbohydrates and glycoconjugates can be labeled specifically at their polysaccharide portion if the required aldehydes are first formed by periodate oxidation or another such method (Chapter 1, Section 4.4). 

Spiegel, S., Wilchek, M., and Fishman, P.H. (1983) Fluorescent labeling of cell surface glycoconjugates with Lucifer Yellow CH. Biochem. Biophys. Res. Comm. 112, 872-877. 

Wilchek, M., and Bayer, E.A. (1987) Labeling glycoconjugates with hydrazide reagents. In Methods in Enzymology (V. Ginsburg, ed.), Vol. 138, pp. 429-442. Academic Press, Orlando Florida. 

A method for selective, radioactive labelling of sialic acids, especially in cell membranes, by mild oxidation with periodate followed by reduction with borotritide, has heen described by Gahmberg and Andersson.200 This procedure can be used either for isolation and characterization of the labelled, cell-surface glycoconjugates (see, for example, Ref. 201) or for autoradiography of tissues and cells (for example, erythrocytes).142... 

The pathway of the biosynthesis of Neu5Ac demonstrates the origin of sialic acids from the cellular hexose and hexosamine pools. These sugars are, therefore, suitable components for the study of the biosynthesis of sialic acid. However, only ManNAc has been shown to be a relatively specific precursor of sialic acids, as may be seen from the distribution of radioactivity between the individual monosaccharides of glycoconjugates after incubation. Injections of radioactive ManNAc into animals, or incubation of surviving tissue slices or individual cells with this compound, give incorporation of label mainly into the sialic acids.226 227... 

Schmidt, J. Peters, W. (1987). Localization of glycoconjugates at the tegument of the tapeworms Hymenolepis nana and H. microstoma with gold labelled lectins. Parasitology Research, 73 80-6. 

---