Biotinyl hydrazides

Protein modifications by HNE, ONE, and MDA have been characterized on specific proteins, but there are few global characterizations, which could potentially provide the big picture necessary to determine the role oxidative stress plays in disease etiology. Most recently, Condreanu et al. (2009) conducted a study on protein damage in RKO, human colorectal carcinoma, cells treated with either 50 or 100 iM HNE. Michael adducts of HNE were biotinylated by reaction with biotinamidohexanoic acid hydrazide and captured with streptavidin. The biotinylated hydrazide reacts with the residual... 

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). 

An analog of this biotinylation reagent with a longer spacer arm also exists. Biotin-LC-hydrazide contains a 6-aminocaproic acid extension off its valeric acid group (Thermo Fisher). 

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. 

Hydrazide-containing PEG-biotinylation reagents provide reactivity with carbonyl groups (e.g., aldehydes) to label carbohydrates or glycoproteins via hydrazone bond formation (Figures 18.19 and 18.20). The hydrazide group also may be coupled with carboxylate-containing... 

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.

The following protocol describes a method for the periodate oxidation of a glycoprotein followed by biotinylation of the resultant aldehydes using hydrazide-PEG4-biotin. Chapter 1, Section 4.6 describes an alternative protocol for the modification of glycans at their reducing ends with hydrazide compounds. 

Figure 23.10 Glycoproteins may be oxidized with sodium periodate to generate aldehyde residues. These may be specifically labeled using a hydrazide-streptavidin derivative through hydrazone bond formation. Subsequent detection may be done using biotinylated enzymes.

Leteux, C., Childs, R.A., Chai, W., Stoll, M.S., Kogelberg, H., and Feizi, T. (1998) Biotinyl-L-3-(2-naph-thyl)-alanine hydrazide derivatives of N-glycans Versatile solid-phase probes for carbohydrate-recognition studies. Glycobiology 8, 227-236. 

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. 

V-(+)-Biotinyl-6-aminocaproyl hydrazide (biotin-6-aminohexanoic hydrazide) [1109276-34-8] M 371.5, m 189-191°, 210 , [a]E) +23 (c 1, Me2NCHO). Suspend in ice-water (lOOmg/ml), allow to stand overnight at 4°, filter and dry the solid in a vacuum. Recrystd from isoPrOH. Rp 0.26 on SiO2 plate using CHCl3-MeOH (7 3) as eluent. [O Shannessy et al. AB 163 204 7987]. 

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