Biotin derivative

Isotope Dilution Assay. An isotope dilution assay for biotin, based on the high affinity of avidin for the ureido group of biotin, compares the binding of radioactive biotin and nonradio active biotin with avidin. This method is sensitive to a level of 1—10 ng biotin (82—84), and the radiotracers typically used are p C]biotin (83), [3H]biotin (84,85) or an I-labeled biotin derivative (86). A variation of this approach uses I-labeled avidin (87) for the assay. 

The DMTr group was selectively introduced into a biotin derivative 0 0... 

Patterning of enzyme monolayers on a solid surface was carried out by photoactivation of immobilized monolayer of caged -biotin derivatives in selected areas. Specific oriented binding of enzyme-avidin conjugates could be readily made to the photoactivated zones [42]. Oriented immobilization of G-protein-coupled receptors on a solid surface was also made possible on a biotinylated surface by first immobilizing streptavidin, followed by the immobilization of biotinylated G-protein-coupled receptor [43]. 

The following sections discuss some of the more common biotinylation reagents available for modification of proteins and other biomolecules. Each biotin derivative contains a reactive portion (or can be made to contain a reactive group) that is specific for coupling to a particular functional group on another molecule. Careful choice of the correct biotinylation reagent can result in directed modification away from active centers or binding sites, and thus preserve the activity of the modified molecule. 

The protocol for modifying DNA probes with photobiotin can be found in Chapter 27, Section 2.3. It is based on the method of Forster et al. (1985). The following method is a suggested protocol for the modification of proteins using a photoreactive biotin derivative. Some optimization may be necessary to obtain the best incorporation levels. 

Figure 11.16 The aminophenyl group of this biotin derivative can be transformed into a diazonium reactive group by treatment with sodium nitrite in dilute HC1.

Incubate the membrane with the biotin derivative at a ratio of 1 ml/cc3 of membrane. 

Biotinylated liposomes usually are created by modification of PE components with an amine-reactive biotin derivative, for example NHS-LC-Biotin (Chapter 11, Section 1). The NHS ester reacts with the primary amine of PE residues, forming an amide bond linkage (Figure 22.19). A better choice of biotinylation agent may be to use the NHS-PEG -biotin compounds (Chapter 18), because the hydrophilic PEG spacer provides better accessibility in the aqueous environment than a hydrophobic biotin spacer. Since the modification occurs at the hydrophilic end of the phospholipid molecule, after vesicle formation the biotin component protrudes out from the liposomal surface. In this configuration, the surface-immobilized biotins are able to bind (strept)avidin molecules present in the outer aqueous medium. 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

Figure 1. Chemical structures of representative ligands investigated A) biotin, B) 2-(4 -hydroxyazobenzene) benzoic acid (HABA), C) charged (X=CH2) and neutral (X=NH2+) carboxylate MMP inhibitors, D) TIBO scaffold, E) sustiva, and F) hydroxyethylamine scaffold. The biotin derivatives," MMP inhibitors,19 TIBO analogs,21 and cathepsin D inhibitors22 derived from structures A), C), D), and F), respectively, have been published elsewhere.

Roffman, E., Meromsky, L., Ben-Hur, H., Bayer, E. A., and Wilchek, M. (1986) Selective labeling of functional groups on membrane proteins or glycoproteins using reactive biotin derivatives and 1-streptavidin. Biochem. Biophys. Res. Comm. 136, 80-85. 

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. 

Not only biotinylated proteins, but also nucleic acids or peptides synthesized in the presence of biotin derivatives or carbohydrate-biotin conjugates, are separated by immobilized avidin and streptavidin, respectively, or are identified by avidin-enzyme or avidin-gold conjugates. 

Mosdy the accessibility of an immobilized ligand to its Hgate is increased if the ligand is bound to the matrix via a spacer. If steric hindrance is suspected, use a lengthened biotin derivative as N-6-biotinylamidohexanoyl hydroxysuccinimide (Biotin-LC-NHS). 

Avidin technology can also be applied to the isolation of proteins and other materials from cells. Because the irreversibility of the binding of biotin may be a problem, photocleavable biotin derivatives have been developed. In the following structure, the biotin derivative has been joined to a protein (as in the first equation in this box) and is ready for separation, perhaps on a column containing immobilized avidin or streptavidin. After separation the biotin together with the linker and photocleavable group are cut off by a short irradiation with ultra-... 

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