Biotin allowances

Certain enzymes catalyze their reactions by way of a multisite mechanism in which the covalently linked intermediate is attached to a long arm that swings from one subsite to another subsite within the enzyme. In some cases, the covalently tethered intermediate can actually be transferred between subunits that form the active site. An example is Propionibacterium shermanii transcarboxylase an enzyme that catalyzes the biotin-dependent conversion of methylmalonyl-CoA and pyruvate to propionyl-CoA and oxaloacetate. Carboxylated biotin allows the two catalytic subsites to operate on the same reaction intermediate. 

Fig. 8.6. By utilizing a streptavidin-coupled immobilized matrix or gel-shift PAGE, the affinity tag biotin allows the RNA catalysts to be captured and separated from the noncatalytic RNA.

An application of biomolecule-nanoparticle hybrid supramolecular assemblies is the sensitive detection of p53, a tumor suppressor protein, by AuNP streptavidin conjugates capped with multiple ferrocene molecules.26 As shown in Figure 8.6, a mixed monolayer of double-stranded DNA and 1-hexanethiol on an Au electrode is used to capture p53, which binds to DNA containing the consensus site sequence. Subsequent conjugation of the captured p53 with biotin allowed for attachment of... 

Immunohistochemical staining can be direct or indirect. Direct immunohis-tochemical staining methods utilize only a primary antibody, which may be conjugated to horseradish peroxidase, biotin, alkaline phosphatase, or other chromogens. In the case of biotin-labeled primary antibodies, avidin or strep-tavidin linked to peroxidase binds to the biotin allowing detection of reactivity of the test antibody with the tissue. Indirect immunohistochemical staining methods utilize secondary, tertiary, or even quaternary antibodies, any of which may be linked either to biotin or enzyme (e.g., peroxidase). 

Applications. The most common application of electrofunctional polymers is in the development of new materials in the (bio)sensors field [78]. For instance, a pyrrole-modified biotin allows successive attachment of avidin and biotin-labeled glucose oxidase, resulting in an efficient glucose biosensor [77,79]. Oureghi et al. [80] developed an impedimetric immunosensor for... 

A method is described that allows the attachment of COPI vesicles and Golgi membranes to glass slides that can then be analyzed using electron microscopy (EM) and immuno-EM methods. Subpopulations of COPI vesicles can be bound selectively using recombinant golgins. Alternatively, COPI vesicles can be attached to prebound Golgi membranes. Marking these vesicles selectively with biotin allows their site of attachment to be identified. 

Vitamins are a well-known group of compounds that are essential for human health. Water-soluble vitamins include folate (vitamin B9) to create DNA. Folate also plays an important role in preventing birth defects during early pregnancy. Thiamine is the first vitamin of the B-complex (vitamin Bl) that researchers discovered. It allows the body to break down alcohol and metabolize carbohydrates and amino acids. Like many other B vitamins, riboflavin (vitamin B2) helps the body to metabolize carbohydrates, proteins, and fat. Niacin (vitamin B3) protects the health of skin cells and keeps the digestive system functioning properly. Pantothenic acid (vitamin B5) and biotin allow the body to obtain energy from macronutrients such as carbohydrates, proteins, and fats. Vitamin B6 (pyridoxine) acts as a coenzyme, which means it helps chemical reactions to take place. It also plays a vital role in the creation of nonessential amino acids. 

Because there is less information upon which to base dietary allowances for biotin and pantothenic acid, ranges of intake are provided, as in Table 8. 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

A variation on the theme of conventional assay uses both lanthanide-labeled and biotin-labeled single strands to form split probes for sequence of target strands (Figure 12).120 When both of these bind to DNA, the complex binds (via the biotin residue) to a surface functionalized with streptavidin, immobilizing the europium and allowing assay to be carried out. This approach is already very sensitive to DNA sequence, since both sequences must match to permit immobilization of the lanthanide, but can be made even more sensitive by using PCR (the polymerase chain reaction) to enhance the concentration of DNA strands. In this way, initial concentrations corresponding to as few as four million molecules can be detected. This compares very favorably with radioimmunoassay detection limits. 

On the other hand, the volume effects provided by e.g. hydrogels such as dextran layers14, 15 will allow an increased number of binding sites. In addition, Figure 9 shows that biotin streptavidin binding and the use of HIS-... 

Nitration of the tyrosine rings in the four binding pockets of avidin or streptavidin can be done to increase the steric hinderance within the biotin binding sites (Morag et al., 1996). This process yields chromogenic proteins that have reduced binding affinity for biotin, thus allowing elution of biotinylated molecules under mild conditions. 

Figure 6.3 Mts-Atf-Biotin can be used to label bait proteins at available thiol groups using the MTS group, which forms a disulfide linkage after reaction. The modified protein then is allowed to interact with a protein sample and photoactivated with UV light to cause a covalent crosslink with any interacting proteins. Cleavage of the disulfide bond effectively transfers the biotin label to the unknown interacting protein.

Figure 11.4 NHS-LC-biotin provides an extended spacer arm to allow greater distance between the biotin rings and a modified molecule. Reaction with amines forms amide linkages.

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