Horseradish peroxidase luminol oxidation

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). 

Figure 5.11. Reactive oxidant production by human neutrophils. In (a), neutrophils (5 x lOVml) were suspended in buffer containing 10 pM luminol in the presence and absence of a mixture of the extracellular oxidants scavengers SOD (1 /ig/ml), catalase (2 pg/ml) and methionine (0.25 mg/ml, to scavenge HOC1). In (b), neutrophils (1 x 106/ml) were suspended in buffer containing 75 jUM cytochrome c (to measure Of production) or 4 jUM scopoletin plus 5 /ig/ml horseradish peroxidase (to measure H202 production). In (c), neutrophils (2 x lOfyml) were placed in the chamber of a Clark-type 02 electrode. All measurements were made at 37 °C cell suspensions were stimulated by the addition of 1 /

The oxidation reactions of luminol and lucigenin can be used to assay for H Oj. For example, analysis of glucose in biological systems can be achieved using a three-enzyme system of mutarotase, glucose oxidase and horseradish peroxidase by correlation with the amount of HjOj released. Similarly, cholesterol can be measured using cholesterol oxidase. The fact that the rate of luminol oxidation depends on the concentration of the catalyst can be used as a method for determination of Co +, Fe +, Cr + and Mn + and other catalysts.Some examples of the use of luminol, isolumi-nol and their derivatives in immunoassays are shown in Table 3.11. ... 

ECL detects horseradish peroxidase-conjugated antibodies through oxidation of luminol, in the presence of hydrogen peroxide and a phenolic enhancer under alkaline conditions. ECL reagents are... 

Chemiluminescence of oxidized luminol has been the basis of several lumino-metric methods of estimation of TAC (Table 1). The mostcommon is to measure the induction time of the reaction. Often the chemiluminescence is first induced by an oxidant and then attenuated by addition of a sample, and the time to recover the initial fluorescence is measured. The enhanced chemiluminescent assay introduced a decade ago is based on the oxidation of luminol by perborate or by hydrogen peroxide in a reaction catalyzed by horseradish peroxidase. Enhancement (and stabilization) of luminescence is achieved by addition of p-iodophenol. The original procedure used a commercial reagent kit (ECL Anti-oxidant Detection Pack... 

As in the case of horseradish peroxidase, several synthetic metalloporphyrins in the presence of H2O2 have been found to be potent catalysts for the chemiluminescent oxidation of luminol or isoluminol. The microperoxidases, mainly MP8 and MPll, have been shown to act as functional peroxidase enzyme models. " However, they are readily inactivated within one min of catalytic turnover, and incorporation into a molecular sieve... 

Although the precise mechanism for the enhancement remains unknown, Thorpe and Kricka (T8) have proposed that enhancers render the sequence of events for unenhanced luminol oxidation (see Section 3.1.3) more efficient. This would be consistent with kinetic studies on the reactivity of phenol enhancers with the horseradish peroxidase intermediates Compounds I and II (Hll, VIO). Specifically, the hypothesis is that (a) horseradish peroxidase Compounds I and II... 

Since the reports on enhanced chemiluminescence in the mid-1980s (e.g., TIO, T12-T14, T16, T17), numerous assays have been described in the literature. Table 5 represents a summary of the types of analyte that have more recently proved amenable to detection by enhanced luminol oxidation via horseradish peroxidase labels. This table is meant to be illustrative rather than comprehensive. 

Horseradish peroxidase (HRP) is also used for the detection of toxic compounds. A chemiluminescence test based on the reaction of luminol and an oxidant in the presence of the enzyme HRP has been developed to indicate the presence of toxins in a sample. The HRP-catalyzed reaction produces light that is measured by a lumi-nometer or a luminescence transducer. This enzyme has been used to detect a range of compounds such as phenols, amines, heavy metals, or compounds that interact with the enzyme, reduce light output, and indicate contamination. Test kits such as the Eclox Water Test Kit (Seven Trent Services, U.K.) is based on the use of HRP in the test format described earlier. This type of test is designed for the qualitative assessment of water samples for a range to compounds that inhibit the HRP activity. 

In several cases the chemiluminescence reaction is initiated by addition of an oxidant such as hydrogen peroxide. Certain stabilized dioxetanes can be chemically cleaved to produce chemiluminescence. Some dioxetane derivatives show thermoluminescence. When simply heated, they are transferred to an excited state and exhibit luminescence. The peroxyoxa-late chemiluminescence reaction consists of different steps. The oxalate ester forms upon treatment with hydrogen peroxide an energy-rich dioxetane intermediate, which can transfer its energy to excite an acceptor fluorophore, which emits light. An increase in chemiluminescence emission in the luminol system was achieved by using certain catalysts as enhancers . Such enhanced chemiluminescence systems use, for example, horseradish peroxidase or certain phenohc molecules as enhancers. 

The detection is usually carried out in a two-step process. In step one, the membrane is incubated (30 min-overnight) in a dilute solution of the primary antibody, which is spedfic for the target protein. The membrane is subsequently rinsed to remove unbound antibody, and another antibody is applied in step two. This secondary antibody is often linked to a reporter enzyme such as horseradish peroxidase (HRP). HRP catalyzes oxidation of luminol by peroxide to produce luminescence light that can be captured with a photographic film or a camera. 

The Emneus group in Denmark has reported the sensing of atrazine in surface water using microfluidic enzyme immunoassay and chemiluminescence as the transduction technique [19]. Silicon microchips were functionalized with proteins A and G via a hydrophilic polymer layer. An affinity capture competitive immunoassay was performed using a mixture of enzyme tracer [horseradish peroxidase (HRP)], atrazine sample, and antiatra-zine antibody which was subsequently injected on the microchannel, followed by addition of the substrate mixture [luminol/p-iodophenol (PIP)/H202]. Chemiluminescent oxidation of luminol/PIP/H202 in the presence of HRP enzyme was monitored via PMT. 

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