Biotinylation enzymes

FIG. 6 Successive coupling of two different biotinylated compounds with the DNA-STV conjugates 2 [33]. In a first step, a macromolecular functional component (FC, represented by the shaded ellipse), such as a biotinylated enzyme or oligonucleotide, is coupled. In a second step, a biotinylated low-molecular-weight modulator M, represented by the shaded sphere) is coupled to the remaining free biotin-binding sites. The modulator is used to modify the conjugate s hybridization properties or to supplement its functionality. 

Figure 23.10 Glycoproteins may be oxidized with sodium periodate to generate aldehyde residues. These may be specifically labeled using a hydrazide-streptavidin derivative through hydrazone bond formation. Subsequent detection may be done using biotinylated enzymes.

This biotinylated enzyme [EC 4.1.1.3] catalyzes the decarboxylation of oxaloacetate to pyruvate and carbon dioxide. 

MECHANISM FIGURE 16-16 The role of biotin in the reaction catalyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the e-amino group of a Lys residue, forming biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed by separate active sites on the enzyme as exemplified by the pyruvate carboxylase reaction. In the first phase (steps to ), bicarbonate is converted to the more activated C02, and then used to carboxylate biotin. The bicarbonate is first activated by reaction with ATP to form carboxyphosphate (step ), which breaks down to carbon dioxide (step ). In effect, the... 

Structures of biotinyl enzyme and N -carboxybiotin. The reactive portions of the coenzyme and the active intermediate are shown in red. In carboxylase enzymes, biotin is covalently bonded to the proteins by an amide linkage between its carboxyl group and a lysyl- -NH2 group in the polypeptide chain. 

Biotinylated enzymes can be used as detection reagents in avidin—biotin assay procedures. Particularly, in the bridged avidin—biotin (BRAB) approach or the ABC technique (Chapter 13, Section 2), a biotin-labeled enzyme is used as the signaling agent... 

The low-molecular-weight vitamin biotin is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) antibody can be used in the avidin-biotin methods. In the labeled avidin method the tracer is attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-labeled antibody. 

Deposition of biotin (30 pi, 10 mM) dissolved in 0.1M phosphate buffer pH — 7 onto electrodes and incubation for 20 min.This saturation of the binding sites of immobilized avidin is carried out before the immunoreaction in order to eliminate any possible immobilization of biotinylated enzymes on the first avidin layer. 

Enzyme kinetics were evaluated in a PDMS-glass chip using a continuous-flow system. A biotinylated enzyme (HRP or (5-galactosidase) was coupled to streptavidin-coated beads via the amide coupling of an aminocaproyl spacer. These beads (15.5 pm) were retained by a weir in the chip. The channel wall was passivated by 1 mg/mL BSA. The apparent enzyme kinetic parameters were evaluated using the Lilly-Homby model, as developed for the packed-bed enzymatic reactor systems. It was found that the apparent Michaelis constant (Km) approached the tme Km value of the free enzyme at zero-flow rate of a homogeneous reaction [845]. 

Avidin Biotin-containing enzymes and biotinylated enzymes... 

However, the same basic principles can be applied (with appropriate modifications) to the labeling of antigens with other enzymes (peroxidase, alkaline phosphatase, -galactosidase). This is particularly true for those labeling methods involving the use of biotinylated antigens, since biotinylated enzymes as well as avidin-enzyme conjugates are available from numerous commercial sources. 

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