Biotin biotinylated protein

A few particularly novel approaches have been developed to increase the sensitivity (and specificity) involved in the detection of bacteria by MALDI. The use of avadin-biotin technology to recover biotinylated proteins from... 

Biocytin is e-N-biotinyl-L-lysine, a derivative of D-biotin containing a lysine group coupled at its e-amino side chain to the valeric acid carboxylate. It is a naturally occurring complex of biotin that is typically found in serum and urine, and probably represents breakdown products of recycling biotinylated proteins. The enzyme biotinidase specifically cleaves the lysine residue and releases the biotin component from biocytin (Ebrahim and Dakshinamurti, 1986, 1987). 

Measure the absorbance of the biotinylated protein solution at 354 nm. Use the molar extinction coefficient for the chromogenic group (e = 29,000 M-1cm-1) to determine the concentration of biotin present. To determine the molar ratio of biotin-to-protein, divide the molar concentration of biotin by the molar concentration of protein present (which may be determined by using the Coomassie assay or the BCA assay methods). 

The presence of biotin labels on an antibody molecule provides multiple sites for the binding of avidin or streptavidin. If the biotin binding protein is in turn labeled with an enzyme, fluoro-phore, etc., then a very sensitive detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through multiple biotinylation sites provides an increase in detectability over antibodies directly labeled with a detectable tag. 

Since a biotinylated molecule potentially is able to interact with (strept)avidin at its biotin binding sites just as strongly as biotin in solution, the degree of biotinylation may be determined using the HABA method as well. Comparison of the response of a biotinylated protein, for example, with a standard curve of various biotin concentrations allows calculation of the molar ratio of biotin incorporation. 

To measure the response of the biotinylated protein sample, add 3 ml of the (strept)avidin solution plus 75 pi of the HABA dye to a cuvette. Mix well and measure the absorbance of the solution at 500 nm. Next, add a small amount of sample to this solution and mix. Record the absorbance at 500 nm. If the change in absorbance due to sample addition was not sufficient to obtain a significant difference from the initial (strept)avidin-HABA solution, add another portion of sample and measure again. Determine the amount of biotin present in the protein sample by using the standard curve. The number of moles of biotin divided by the moles of protein present gives the number of biotin modifications on each protein molecule. 

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. 

Mixture of D-penicillamine (pen, Fig. 22) and copper(II) chloride forms a complex of the formula [Cu(I)8Cu(II)6pen12]5-. The Ri value of this complex at 300 MHz and 23°C is 0.586 mM 1 s-1 [178]. The carboxylate groups on the cluster have been shown to react with aziridines such as XAMA-7 (Fig. 23) which in turn can be reacted with biotin [179]. The copper clusters can then be attached to antibodies or other biotinylated proteins via an avidin linker molecule. 

The avidin-biotin interaction has also been used to immobilize antibodies and proteins, especially in commercial systems based on surface plasmon resonance (SPR) measurements (e.g., the BIAcore). The extraordinary affinity (Kl 10-15 M) of avidin (or its bacterial relative, streptavidin) for the vitamin biotin is the basis of this immobilization procedure. A solid support (e.g., glass beads, sensor chip, optical fiber) covered with avidin can be used as an activated carrier for a very sturdy immobilization of previously biotinylated antibodies. In spite of the many methods for biotinylating proteins described in the literature, the use of biotinyl N-hydroxysuccinimide ester (BNHS) and similar derivatives, remains the most useful [65]. 

Fig. 4. Schematic of a single-step array fabrication process for in vivo biotinylated proteins. Step a A cmde lysate containing the desired biotinylated recombinant protein is printed onto a streptavidin-coated surface coderivatized with a polymer that resists nonspecific protein absorption. Step b Unbound proteins are washed away to leave the purified recombinant protein, specifically immobilized and oriented on the array surface via the biotin moiety on the BCCP tag.

The blocking and wash steps should remove all nonbiotinylated proteins from the array surface, while the biotin in the milk powder blocks any remaining biotinbinding sites on the streptavidin surface, such that any biotinylated proteins that do dissociate cannot rebind to the array. Arrays stored in p53 freeze mix at -20°C have been shown to be stable for >1 y. 

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