Horseradish peroxidase also

It should be noted that a number of different enzyme preparations can now be purchased directly from manufacturing chemists. It must be emphasised that the activity of an enzyme, whether purchased or prepared in the laboratory, may vary between rather wide limits. The activity is dependent on the source of the enzyme, the presence of poisons and also on the temperature. It appears, for example, that the quality of horseradish peroxidase depends upon the season of the year at which the root is obtained from the ground. It cannot be expected therefore that all the experiments described below will work always with the precision characteristic of an organic reaction proceeding under accurately known conditions. 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

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). 

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

HTAC cationic micelles also markedly enhance the CL intensity of fluorescein (FL) in the oxidation of hydrogen peroxide catalyzed by horseradish peroxidase (HRP) [39], However, no CL enhancement was observed when anionic micelles of sodium dodecyl sulphate (SDS) or nonionic micelles of polyoxyethylene (23) dodecanol (Brij-35) were used (Fig. 9). CL enhancement is attributed to the electrostatic interaction of the anionic fluorescein with the HTAC micelles. The local concentration of fluorescein on the surface of the micelle increases the efficiency of the energy transferred from the singlet oxygen (which is produced in the peroxidation catalyzed by the HRP) to fluorescein. This chemiluminescent enhancement was applied to the determination of traces of hydrogen peroxide. The detection limit was three times smaller than that obtained in aqueous solution. 

Acyl nitroso compounds (3, Scheme 7.2) contain a nitroso group (-N=0) directly attached to a carbonyl carbon. Oxidation of an N-acyl hydroxylamine derivative provides the most direct method for the preparation of acyl C-nitroso compounds [10]. Treatment of hydroxamic acids, N-hydroxy carbamates or N-hydroxyureas with sodium periodate or tetra-alkyl ammonium periodate salts results in the formation of the corresponding acyl nitroso species (Scheme 7.2) [11-14]. Other oxidants including the Dess-Martin periodinane and both ruthenium (II) and iridium (I) based species efficiently convert N-acyl hydroxylamines to the corresponding acyl nitroso compounds [15-18]. The Swern oxidation also provides a useful alternative procedure for the oxidative preparation of acyl nitroso species [19]. Horseradish peroxidase (HRP) catalyzed oxidation of N-hydroxyurea with hydrogen peroxide forms an acyl nitroso species, which can be trapped with 1, 3-cyclohexanone, giving evidence of the formation of these species with enzymatic oxidants [20]. 

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