Enzyme tags

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

With enzyme-multiplied immunoassay technique (EMIT) assays, enzyme tags are used instead of radiolabels. The antibody binding alters the enzyme characteristics,... 

It may be possible to build up a two- or three-dimensional architecture composed of proteins using avidin and biotin-labeled enzymes as building blocks. If we use enzymes tagged with more than two biotin residues, an enzyme multilayer illustrated in Figure 10 would be constructed because avidin contains four biotin-binding sites per molecule. Fortunately, the biotin-binding sites are located in two pairs on the opposed faces of the avidin molecule. Thus, the use of biotin-labeled enzyme... 

And cytosolic proteins that are damaged or otherwise destined for degradation are enzymically tagged with the activated ubiquitin. 

The method is based on the covalent immobilization of enzyme onto the immunosensor surface, circumventing the use of enzyme-tagged antibody and alleviating the signal instability from low-affinity binding. The electrochemical signal can be maintained even if... 

Antibody-masking enzyme tag immunoassay Adenosine 5 -monophosphate S-Acetylmercaptosuccinic anhydride Alkaline phosphatase anti-alkaline phosphatase (enzyme-antibody) complex Alkaline phosphatase 5-Aminosalicylic acid Adenosine 5 -triphosphate Aa-Benzoyl-L-arginine ethyl ester (-f-)-Biotin bromoacetyl hydrazide (-b)-Biotin Y-aminocaproic acid A-hydroxy-succinimide ester Bis-diazotized benzidine -Galactosidase (-I- )-Biotin hydrazide (-I- )-Biotin-A-hydroxysuccinimide ester (-I-)-Biotin p-nitrophenyl ester Bridged avidin-biotin (method)... 

Recently, evidence for large-scale associations between enzymes involved in the de novo pathway for purine synthesis has emerged. This pathway involves 10 steps that convert PRPP to inosine monophosphate. In higher eukaryotes, these steps are catalyzed by six enzymes one is trifiinctional and two are bifunctional. Versions of these enzymes tagged with green fluorescent protein (GFP) or orange fluorescent protein (OFP)... 

Tag polymerase (Stratagene 1-800-424-5444, TaqPlus DNA Polymerase cat. no. 600203). As with any enzyme. Tag should be stored at -20°C in a nonfrost-free freezer. 

Labeling with enzymes has been the most widely employed approach to increase sensitivity in chromatographic immunodetection, probably because of broad experience with the very well known enzyme-linked immunoassays. Enzyme tags have been used to form electroactive, fluorescent, and colored products. Assays developed by de Alwis and Wilson are landmarks in the field of immunodetection. Electrochemical detection of hydrogen peroxide formed by the reaction of a glucose oxidase tag in both sandwich [58] and competitive [59] formats allowed detection of subpicomoles of bovine and human IgG in serum. 

Genomic and transcriptomic technologies have been used to rapidly identify biosynthetic steps. There are currently over 40,000 expressed enzyme tags (ESTs) generated fi om alkaloid-producing plants that have been used to isolate genes involved in the alkaloid pathway [7]. Some alkaloid biosynthetic steps occur as spontaneous chemical reactions without the use of enzymes, for example, conversion of the intermediate neopine into codeinone in the morphine biosynthetic pathway. Also, some enzymes may catalyze two or more separate reactions in the pathway, for example, hyoscyamine 6-hydroxylase, which carries out two consecutive steps in the scopolamine biosynthetic pathway. Alkaloid biosynthesis also involves compartmentalization. Tissue-specific localization studies have shown that sequential biosynthetic enzymes can occur in distinct cell types [8, 9]. During the biosynthesis of the indole alkaloids vinblastine and vincristine in Catharanthus roseus, different enzymatic steps are carried out in different cellular compartments (Fig. 8.5) [10]. Various steps in the pathway are carried out in different types of cell. This requires the intercellular transport of metabolic intermediates. Similarly, scopolamine biosynthesis also involves two different cell types. 

Indeed, this idea has been shown to be powerful for the immobilization of enzymes using nickel NTA-linkers on sepharose for coordinatively trapping enzymes tagged with a His-tag. 

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