Peroxidase bromo

Recent work in our laboratory (Kompella, Mathias, and Lee, unpublished observation) has revealed that activation of the cAMP-regulated Cl channels in the conjunctiva also enhances the transcytosis of horseradish peroxidase. 8-Bromo-cAMP (a membrane-permeable analog of cAMP) and terbutaline (a p2-adrenergic agonist known to increase intracellular levels of cAMP in other epithelial tissues [238]), at 0.5 mM, were found to enhance the transport of 100 pg/ mL HRP from the mucosal side to the serosal side of the pigmented rabbit conjunctiva by a factor of 4 (Fig. 11). 

Aminophthalate anion Atmospheric pressure active nitrogen Analyte pulse perturbation-chemiluminescence spectroscopy Arthromyces rasomus peroxidase Ascorbic acid Adenosine triphosphate Avalanche photodiode 5-Bromo-4-chloro-3-indolyl 2,6-Di-t< r/-bu(yl-4-mclhyl phenol Bioluminescence Polyoxyethylene (23) dodecanol Bovine serum albumin Critical micelle concentration Calf alkaline phosphatase Continuous-addition-of-reagent Continuous-addition-of-reagent chemiluminescence spectroscopy Catecholamines Catechol... 

Enzymes. Enzymes too are useful labels and several have been employed, with peroxidase and alkaline phosphatase being the most popular to date. One important feature of this technique is for the enzyme to be able to convert a soluble substrate into an insoluble product in order to localize the antigen properly. Several suitable substrates are available with 3 3 -diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) being used with peroxidase and 5-bromo-4-chloro-3-indoylphosphate/nitroblue tetrazolium (BCIP/NBT) with alkaline phosphatase. 

Enzymes requiring vanadium for catalytic activity. Perhaps the best studied of these are the vanadium-dependent nitrogenases [EC 1.18.6.1]. Other vanadium-dependent enzymes include vanadium haloperoxidase, vanadium chloroperoxidase, and vanadium bromoper-oxidase. In the vanadium chloroperoxidase and bromo-peroxidase reactions, the vanadium(V) is coordinated in a trigonal bipyramidal site to a histidyl residue, three nonprotein oxygens, and, presumably, to a hydroxide. 

Substrate products can be classified as either soluble or precipitating. Soluble peroxidase substrates include o-phenylenediamine, which is converted into a yellow product 2,2 -azino-(3-ethyl)-benzothiazoline-sulfonic acid, which is converted into a green product and tetramethylbenzidine, which is converted into a blue product. Precipitating substrates for peroxidase include 4-chloronaphthol, which yields a blue precipitate and aminoethylcarbizole, which forms a red precipitate. Alkaline phosphatase is most frequently used with p-nitrophenyl phosphate to give a yellow-orange soluble product, or with 5-bromo-4-chloro-3-indo-lyl-phosphate p-toluidine salt to yield an insoluble blue product. 

Common chromogen systems currently in use include diaminobenzidine (DAB), 3-amino-9-ethyl-carbazole (AEC), Hanker-Yates reagent, alpha-naphthol pyronin used with peroxidase as substrate fast blue, fast red, BCIP- (5-bromo-4-chloro-3-indolyl phosphate) NBT (nitroblue tetrazolium) used with alkaline phosphatase as substrate tetrazolium, tetranitroblue tetrazolium used with glucose oxidase as substrate, and immunogold with silver enhancement (Leong, 1993 Leong et al, 1997a). 

By far, the most studied chlorinating peroxidase is CPO (in particular that from Caldariomyces fumago, which is involved in the biosynthesis of the chlorometa-bolite caldariomycin). CPO is able to halogenate (Cl, Br) activated C-H bonds, such as those of 1,3-dicarbonyl compounds. The reaction can afford both the monohalogenated compound and the dihalogenated product (and also mixed chloro/bromo compounds) [89-92] (Fig. 6.8a). 

We have considered several other possibilities for vanadium bromo-peroxidase. A cis-(dioxo) arrangement, even with only two ligands at 1.72 A, gives an oxidation state by BVS of 6.30, which is also inconsistent with the known valence (5+) of the metal. Other six-coordinate arrangements with the sixth ligand as H20 at a long distance (2.2-2.3 A) produce similar results. However, a five-coordinate geometry, with the 2.11 A bonds as either V-O or V-N, has a calculated valence of 4.83 and 5.08, respectively, consistent with the known oxidation state of 5+. 

Schiff base complexes have been extensively used to mimic the molecular reaction of bromo-peroxidases by treating the pre-formed complex with hydrogen peroxide. Decreasing the... 

The chlorination, bromination, and iodination of various nitrogen-containing heterocycles catalyzed by chloroperoxidase from Caldariomyces jumago and bromo-peroxidase from Corallina pilulifera were compared by Itoh et al.[14. ... 

Albert, C. J., A. K. Thukkani, R. M. Heuertz, A. Slungaard, S. L. Hazen, and D. A. Ford. 2003. Eosinophil peroxidase-derived reactive brominating species target the vinyl ether bond of plasmalogens generating a novel chemoattractant, alpha-bromo fatty aldehyde. J Biol Cfem 278(1 n 8942-.50. 

There are many commercially available labeled secondary antibody products. The most common conjugated enzymes are alkaline phosphatase and horseradish peroxidase. Alkaline phosphatase, discussed earlier in the section on histochemistry, dephosphorylates the substrate 5-bromo-4-chloro-3-indolyl phosphate which can be oxidized by nitroblue tetrazolium. The resulting signal would appear as a blue/ purple dye. Horseradish peroxidase can catalyze the oxidation of 4-(chloro-l-naphthol), in the presence of hydrogen peroxide, into an insoluble blue dye. Apart from using antibody-enzyme conjugates, secondary antibodies can be labeled with to permit detection by autoradiography. 

Winter, G,E,M, and Butler, A. (1996) Inactivation of vanadium bromo-peroxidase formation of 2-oxohistidine. Biochemistry, 35, 11805-11811. 

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