Biotin availability

One advantage of indirect avidin-biotin method is an amplification of the detection system. For example, after biotin conjugated 2° antibody, avidin is incubated, binding to the available biotins. After the remaining free avidin is rinsed off, a biotin conjugated to 488 fluorophore is added and it binds to the remaining sites on the avidin. With this method, amplification occurs because any avidin can be attached to multiple biotins conjugated with either fluorescent or enzyme. This method requires two additional incubation steps of the indirect immunocytochemistry with fluorescent-labeled 2°. 

We have also observed that using the streptavidin-biotin interaction as the basis for array fabrication confers an additional major advantage the very high affinity of the streptavidin-biotin interaction means that we quickly start to saturate the available biotin-binding sites on the surface this means that a crude normalisation of protein loading can be achieved without pre-adjusting the concentrations of the crude lysates to... 

This method (70) takes advantage of the formation of a complex between avidin and HABA (Fig. 10), for which a three-dimensional structure is now available. Biotin, which has a higher affinity than HABA for avidin, quantitatively displaces the dye from the complex. The titration is generally monitored spectrophotometri-cally at 500 nm. This method is easy to use, but its sensitivity is low (around 1 Bg)-... 

Extraction of total biotin is obtained by acidic hydrolysis (2—3 M HCl at 100°C or 1—3 M H2SO4 by autoclaving at 121°C) that breaks bonds with proteins and totally converts D-biocytin into D-biotin [41,42], Possible losses of vitamin depend on both the acid concentration and the duration of autoclaving. Enzymatic digestion with papain for 18 hr leaves biocytin intact and allows the determination of available biotin (biotin plus biocytin) takadiastase is added for starchy foods, such as cereals [42]. 

RECOMMENDED DAILY ALLOWANCE OF BIOTIN. It is difficult to obtain a quantitative requirement for biotin, for the reason that intestinal microflora make a significant contribution to the body pool of available biotin often humans excrete via the feces and urine considerably more biotin than they have ingested. However, the estimated safe and adequate intakes of biotin are given in the section on VITAMIN(S), Table V-5, Vitamin Table. 

The biotin market is divided between agricultural and human use, with —90% of biotin used in the animal health care market and —10% for the human nutritional market. The major producers of biotin are Hoffmann-La Roche, Lon2a, E. Merck-Darmstadt, Rhc ne-Poulenc, Sumitomo Pharmaceutical, E. Sung, and Tanabe Seiyaku (100). Worldwide production of biotin in 1994 was approximately 60 metric tons. The Hst price for pure biotin in 1995 was — 7.00/g whereas, the Hst price for technical feed-grade biotin was — 5.50/g. Biotin is used in various pharmaceutical, food, and special dietary products, including multivitamin preparations in Hquid, tablet, capsule, or powder forms. One of the commercially available products of i7-biotin is Britrit-1, which is a 1% biotin trituration used in food premixes. 

Several elegant synthetic strategies have been devised for biotin (1) this chapter describes one of the total syntheses developed at Hoffmann-La Roche. This insightful synthesis employs a derivative of L-cysteine, a readily available member of the chiral pool,2 as the starting material, and showcases the powerful intramolecular nitrone-olefin [3+2] cycloaddition reaction. 

Example Optically active aldehyde (10) was needed for a synthesis of biotin. The compound has a 1,1-dlX disconnection (10a) clearly available and a C-N (amide) disconnection leaving (11) which has the same skeleton as the amino acid cysteine (12). 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Recently, SETA BioMedicals has developed a new near-infrared squaraine-based label Seta-633, which can be used to study the interaction between low-molecular-weight analytes and proteins using fluorescence lifetime as the readout parameter [19]. This label exhibits lower quantum yields and shorter fluorescence lifetimes when free in solution, but these values substantially increase upon interaction with proteins, which is contrary to tracers like Cy5 or Alexa 647. It was demonstrated in a model assay that a biotinylated Seta-633 binds to anti-biotin with high specificity. Importantly, the lifetime of Seta-633-biotin increases about 2.76 fold upon binding to a specific antibody (anti-biotin, MW =160 kDa), while the titration with BSA or nonspecific antibody does not result in a noticeable change in lifetime (Fig. 13). The label is compatible with readily available light sources (635 nm or 640 nm lasers) and filter sets (as for Cy5 or Alexa 647) and its... 

Although MTS-ATF-biotin and MTS-ATF-LC-biotin are available commercially (Thermo Fisher and Toronto Research), they are relatively new and don t have the publications or applications backing up their use as sulfo-SBED. A protocol for the use of these compounds in the study of protein interactions can be found in Chapter 28, Section 3.2. 

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.

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