Biotin measurement

Dose-response curve for biotin measured by FILIA-bioluminescence (BL) system. (Each point represents the mean of three measurements error bars represent 1 standard deviation.) The inset shows a linear fit to the central data. 

In an extensive SFA study of protein receptor-ligand interactions, Leckband and co-workers [114] showed the importance of electrostatic, dispersion, steric, and hydrophobic forces in mediating the strong streptavidin-biotin interaction. Israelachvili and co-workers [66, 115] have measured the Hamaker constant for the dispersion interaction between phospholipid bilayers and find A = 7.5 1.5 X 10 erg in water. 

Direct measurement of the interaction potential between tethered ligand (biotin) and receptor (streptavidin) have been reported by Wong et al [16] and demonstrate the possibility of controlling range and dynamics of specific biologic interactions via a flexible PEG-tether. 

The avidin-biotin complex, known for its extremely high affinity (Green, 1975), has been studied experimentally more extensively than most other protein-ligand systems. The adhesion forces between avidin and biotin have been measured directly by AFM experiments (Florin et al., 1994 Moy et al., 1994b Moy et al., 1994a). SMD simulations were performed on the entire tetramer of avidin with four biotins bound to investigate the microscopic detail of nnbinding of biotin from avidin (Izrailev et al., 1997). 

Add 0.3 mg of sulfo-NHS-SS-biotin (Thermo Fisher) to each ml of the antibody solution. To measure out small amounts of the biotinylation reagent, it may be first dissolved in water at a concentration of at least 1 mg/ml. Immediately transfer the appropriate amount to the antibody solution. This level of sulfo-NHS-SS-biotin addition represents about an 8-fold molar excess over the amount of antibody present. This should result in a molar incorporation of approximately 2-4 biotins per immunoglobulin molecule. 

Figure 18.15 NHS-chromogenic-PEG3-biotin contains an amine-reactive NHS ester that can be used to label biomolecules through an amide linkage. The chromogenic bis-aryl hydrazone group within the spacer arm of the reagent allows the degree of biotinylation to be quantified by measuring its absorbance at 354 nm. The compound also contains a hydrophilic PEG spacer, which provides greater water solubility.

Purify the modified protein from unreacted biotinylation reagent and reaction by-products using dialysis or gel filtration. Complete removal of the excess reagent is necessary to provide accurate measurement of the biotin incorporation level by absorptivity. 

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). 

It is often important to determine the extent of biotin modification after a biotinylation reaction is complete. Measuring biotin incorporation into macromolecules can aid in optimizing a particular (strept)avidin-biotin assay system. It also can be used to assure reproducibility in... 

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. 

Figure 28.11 Sulfo-SBED is a label transfer agent that contains a water-soluble sulfo-NHS ester to label bait proteins and a phenyl azide group for photoreactive capture of a prey protein. The biotin label can be used for detection or isolation of protein-protein conjugates using (strept)avidin reagents. The stars indicate the atoms that were used to measure the indicated molecular dimensions.

Thiamine has also been measured by bioassay, a marine yeast being used as the assay organism [474,475]. Marine bacteria [476,477], marine yeasts [475], and dinoflagellates [478] have been used for the assay of biotin. 

Several qualitative and quantitative immunochemical methods for CAP analysis in biological matrices of animal origin have been described [101,102, 104,105] (see Table 3). Van de Water et al. [ 102] described an ELISA that detected CAP in swine muscle tissue with an IC50 value of 3 ng mL1. This immunoassay was improved and subsequently optimized incorporating the streptavidin-biotin amplification system. There are also several commercially available test kits (see Table 4). RIDASCREEN is a competitive enzyme immunoassay for the quantitative analysis of CAP residues in milk, eggs, and meat in a microtiter plate. The measurement is made photometrically, obtaining a LOD of 100 ng L 1 in meat and eggs and 150 ng L 1 in milk. The test has been also applied to the analysis of tetracyclines. 

Immunosensors have been developed commercially mostly for medical purposes but would appear to have considerable potential for food analysis. The Pharmacia company has developed an optical biosensor, which is a fully automated continuous-flow system which exploits the phenomenon of surface plasmon resonance (SPR) to detect and measure biomolecular interactions. The technique has been validated for determination of folic acid and biotin in fortified foods (Indyk, 2000 Bostrom and Lindeberg, 2000), and more recently for vitamin Bi2. This type of technique has great potential for application to a wide range of food additives but its advance will be linked to the availability of specific antibodies or other receptors for the various additives. It should be possible to analyse a whole range of additives by multi-channel continuous flow systems with further miniaturisation. 

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. 

Figure 2.9. Differential pharmacological effect elicited by vector-mediated delivery of a VIP analogue. The organ blood flow in brain and salivary gland was measured in conscious rats after i.v. administration of vehicle (saline), the brain delivery vector OX26-SA, the VIP peptide alone, or the chimeric peptide. While cerebral blood flow increased in the chimeric peptide group by 60% compared to the saline control, the increase in salivary gland blood flow seen with the peptide alone was abolished by coupling to the vector. The VIP analogue was biotinylated with a non-cleavable 14-atom spacer (biotin-XX) for coupling to the vector. Data from reference [95].

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