Biotin hydrazide

Biotin-hydrazide also may be used to couple with carboxylate-containing molecules. Hydrazidcs can be coupled with carboxylic acid groups by using the carbodiimide reaction (Chapter 3, Section 1.1). The carbodiimide activates a carboxylate to an o-acylisourea intermediate. Biotin-hydrazide can react with this intermediate via nucleophilic addition to form a stable covalent bond. 

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). 

Biotin-hydrazide can be used to label aldehyde-containing molecules, creating hydrazone bonds. 

The increased length of this spacer (24.7 A) provides more efficient interaction potential with avidin or streptavidin probes, possibly increasing the sensitivity of assay systems. The reactions of biotin-LC-hydrazide are identical to those of biotin-hydrazide. 

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). 

Add biotin-hydrazide or biotin-LC-hydrazide to a final concentration of 5 mM. 

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). 

Figure 20.9 Polysaccharide groups on antibody molecules may be oxidized with periodate to create aldehydes. Modification with biotin-hydrazide results in hydrazone linkages. The sites of modification using this technique often are away from the antibody-antigen binding regions, thus preserving antibody activity.

Prepare bisulfite modification solution consisting of 3 M concentration of a diamine (i.e., ethylenediamine), 1M sodium bisulfite, pH 6. The use of the dihydrochloride form of the diamine avoids having to adjust the pH down from the severe alkaline pH of the free-base form. Note The optimum pH for transaminating biotin-hydrazide to cytosine residues using bisulfite is 4.5 (see Section 2.3, this chapter). 

Biotin-Hydrazide Modification of Bisulfite-Activated Cytosine Groups... 

Dissolve biotin-hydrazide in water at a concentration of lOmg/ml. 

Figure 27.13 Biotin-hydrazide may be incorporated into cytosine bases using a bisulfite-catalyzed transamination reaction.

Reisfeld, A., Rothenberg, J.M., Bayer, E.A., and Wilchek, M. (1987) Nonradioactive hybridization probes prepared by the reaction of biotin hydrazide with DNA. Biochem. Biophys. Res. Comm. 142, 519-526. 

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). 

The biochemical MS assay performance was studied for various biotin derivatives, such as biotin [m/z 245), N-biotinyl-6-aminocaproic acid hydrazide (m/z 372), biotin-hydrazide (m/z 259), N-biotinyl-L-lysine (m/z 373) and biotin-N-succinimi-dylester m/z 342). These five different bioactive compounds were consecutively injected into the biochemical MS assay. Figure 5.12 shows triplicate injections in the biochemical MS-based system of the different active compounds. Each compound binds to streptavidin, hence the MS responses of peaks of the reporter ligand (fluorescein-biotin, m/z 390) are similar. The use of SIM allows specific components to be selected and monitored, e.g. protonated molecule of the biotin derivatives. In this case, no peaks were observed for biotin-N-succinimidylester (m/z 342), because under the applied conditions fragmentation occurred to m/z 245. In combination with full-scan MS measurements, the molecular mass of active compounds can be determined simultaneously to the biochemical measurement. 

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