HABA, avidin

Add 100-p.L aliquots of the antibody dilutions in duplicate to separate 0.9 mL aliquots of the HABA-avidin or streptavidin solution and stir at room temperature for 5-10 min. If the DOL of some of the samples is very high, a precipitate may form during the incubation that should be removed by centrifugation prior to spectrophotomet-ric analysis. The decrease in A500 for each sample should then be recorded. The average value from each pair of samples is used to determine the nmol of biotin in the various aliquots of antibody... 

Simple chemical laboratory procedures for biotin are restricted to pure materials as described in pharmacopeia monographs using alkaline titration. Similarly, spectral methods for determining native biotin are unknown in most samples except for highly purified materials as it has no useful absorption above 210 nm. To obtain suitable sensitivity and selectivity, biotin must first be derivatized. For example, the disruption of a / -hydroxy-azobenzene-2 -carboxylic acid (HABA) avidin complex by biotin, which binds to avidin with higher affinity, can be followed titrimetrically or spectroscopically to give useful determinations of biotin. 

This technique has been developed by Aizawa and coworkers. The goal is to build a convenient and specific detector using an enzymatic activity as signal. In the case of biotin determination, avidin coupled with catalase is bound to a membrane bearing covalently linked HABA residues. Addition of biotin destroys quantitatively the HABA-avidin-catalase complex. Washing the membrane and measurement of the remaining catalase activity afforded a sensitive (0.5 ng) and convenient titration of biotin (95). 

An electrode for the determination of vitamin H (biotin) is based on the competitive affinity between biotin and its homologue HABA for the same protein, avidin [84]. The catalase-labelled HABA-avidin complex is immobilized on a p02 electrode to produce a biotin-sensitive sensor [255]. Biotin dissociares some of the HABA-avidin complexes by its strong affinity for avidin (Figure 6.1), leading to a reduction in enzymatic activity at the pC>2 electrode which can be related to the concentration ctf biotin. The coupling between biotin and avidin occurs more from bioaffinity than from an irmnunological process. 

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. 

Dissolve the HABA dye (Sigma) in 10mM NaOH at a concentration of 2.42mg/ml (lOmM). Prepare about 100 pi of the HABA solution for each 3 ml portion of (strept)avidin solution required. 

To construct a standard curve of various biotin concentrations, first zero a spectrophotometer at an absorbance setting of 500 nm with sample and reference cuvettes filled with 0.05M sodium phosphate, 0.15M NaCl, pH 6.0. Remove the buffer solution from the sample cuvette and add 3 ml of the (strept)avidin solution plus 75 pi of the HABA-dye solution. Mix well and measure the absorbance of the solution at 500nm. Next add 2 pi aliquots of the biotin solution to this (strept)avidin-HABA solution, mix well after each addition, and measure and record the resultant absorbance change at 500 nm. With each addition of biotin, the absorbance of the (strept)avidin-HABA complex at 500 nm decreases. The absorbance readings are plotted against the amount of biotin added to construct the standard curve. 

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. 

To prepare a standard curve, add 0.25 mL of HABA reagent to 10 mL of avidin solution. Incubate 10 min at room temperature and record the absorbance at 500 nm of 1 mL avidin-HABA complex with 0.1 mL buffer, pH 6.0 Distribute 1 mL of the avidin-HABA complex into six test tubes. Add to each the biotin solution in a range of 0 005-0.10 mL. Bring the final volume to 1.10 mL with pH 6.0 buffer, and record the absorbance at 500 nm of each concentration point. Plot a standard curve with the nanomoles of biotin vs the decrease in absorbance at 500 nm. An example of a standard curve is illustrated in Fig 3... 

To measure the degree of biotinylation of the sample, add an aliquot of biotinylated antibody of known concentration to 1 mL of avidin-HABA complex For example, add 0.05-0.1 mL of biotinylated antibody at 1 mg/mL to 1 mL of avidin-HABA mixture Bring the volume to 1 10 mL, if necessary, incubate for 10 min, and measure the decrease in absorbance at 500 nm. 

Fig. 3. Examples of standard curve for biotin assay with avidin-HABA reagent.

New rhenium(I) biotin complexes containing the extended planar diimine ligands dppz and dppn (17) have been prepared to explore bifunctional biological probes [44], These complexes bind to double-stranded calf thymus DNA by intercalation, as revealed by absorption and emission titrations. HABA assays show that all the complexes bind to avidin with a stoichiometry of 4 1 ([Re] [avidin]). Similar to other rhenium(I) diimine biotin complexes described above, the emission intensities... 

Four luminescent cyclometallated iridium(III) diimine complexes [Ir(ppy-spacer-biotin)2(NAN)]+ (NAN = Me4-phen, Ph2-phen) (38), each containing two biotin units, have been synthesised and characterised by Lo and co-workers [79], Photoexcitation of these iridium(III) diimine bis(biotin) complexes in fluid solutions at 298 K and in alcohol glass at 77 K results in intense and long-lived 3MLCT (djr(Ir) — ti (NaN))/3IL (ir —> ji ) (Me4-phen) emission. HABA assays and emission titrations indicate that the two biotin moieties of each complex are functional. RET-based emission-quenching experiments, microscopy studies using avidin-conjugated microspheres, and HPLC analysis all reveal that the complexes with a... 

Biotin HABA competition of HABA O2 electrode with biotin for avidin-catalase Ikariyama et al. (1983)... 

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