New enhancers for the chemiluminescent peroxidase catalysed chemiluminescent oxidation of pyrogallol and purpurogallin

The effects of various boronate compounds, 4-biphenylboronic acid, 4-bromobenzeneboronic acid, trans-4-(3-propionic acid)phenylboronic acid and 4-iodophenylboronic acid, on the horseradish peroxidase (HRP) catalysed chemiluminescent oxidation of pyrogallol and purpurogallin by peroxide were investigated. trans-4-(3-Propionic acid)phenylboronic acid produced a 13.7-fold enhancement in the peak light emission from the chemiluminescent HRP catalysed pyrogallol reaction (detection limit for HRP < 1.25 fmol). At low enhancer concentration a single peak of light emission was observed and as the enhancer concentration increased the time to peak light emission became progressively longer. The chemiluminescence showed two peaks at higher concentrations (> 54.3 μmol/L) and the individual peak times depended upon the concentration of the enhancer. All of the boronates enhanced peak light emission in the chemiluminescent HRP catalysed purpurogallin reaction. 4-Biphenylboronic acid was the most effective and it enhanced peak light emission 314-fold. The practical detection limit for HRP (Type VIA) using this enhancer was 4.18 pmol (peak emission at 20 minutes). This compound also enhanced peak light emission 232-fold from a chemiluminescent HRP-purpurogallin reaction in which molecular oxygen replaced peroxide as the oxidant.     

Enhanced chemiluminescence in the peroxidase-luminol-H2O2 system: Anomalous reactivity of enhancer phenols with enzyme intermediates

Phenols which markedly enhance chemiluminescence in the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide show anomalously high reactivity (by factors of ～10² compared with published Hammett correlations) in the reduction of the enzyme intermediates, Compound I and Compound II. The results support the hypothesis that efficient production of phenoxy radicals from phenols is a necessary criterion for chemiluminescence enhancer action.     

Tropolone as a substrate for horseradish peroxidase

Tropolone (2,4,6-cycloheptatrien-1-one), in the presence of hydrogen peroxide but not in its absence, can serve as a donor for the horseradish peroxidase catalysed reaction. The product formed is yellow and is characterized by a new peak at 418 nm. The relationship between the rate of oxidation of tropolone (ΔA at 418 nm/min) and various concentrations of horseradish peroxidase, tropolone and hydrogen peroxide is described. The yellow product obtained by the oxidation of tropolone by horseradish peroxidase in the presence of hydrogen peroxide was purified by chromatography on Sephadex G-10 and its spectral properties at different pHs are presented. The M, of the yellow product was estimated to be ca 500, suggesting that tropolone, in the presence of horseradish peroxidase and hydrogen peroxide is converted to a tetratropolone.     

Enzymatic enhancement of the catalytic rate of sulfhydryl oxidase

The rate of oxidation of glutathione by solubilized sulfhydryl oxidase was significantly enhanced in the presence of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7). This enhancement was proportional to the amount of active peroxidase in the assay, but could not be attributed solely to the oxidation of glutathione catalyzed by the peroxidase. A change in the Soret region of the horseradish peroxidase spectrum was observed when both glutathione and peroxidase were present. Moreover, addition of glutathione to a sulfhydryl oxidase/horseradish peroxidase mixture resulted in a rapid shift of the absorbance maximum from 403 nm to 417 nm. This shift indicates the oxidation of horseradish peroxidase. Spectra for three isozyme preparations of horseradish peroxidase, two acidic and one basic, all underwent this red-shift in the presence of sulfhydryl oxidase and glutathione. Cysteine and N-acetylcysteine could replace glutathione. Addition of catalase had no effect on the oxidation of peroxidase, indicating that the peroxide involved in the reaction was not derived from that released into the bulk solution by sulfhydryl oxidase-catalyzed thiol oxidation. Further evidence for a direct transfer of the hydrogen peroxide moiety was obtained by addition of glutaraldehyde to a sulfhydryl oxidase/horseradish peroxidase/N-acetylcysteine mixture. Size exclusion chromatography revealed the formation of a high-molecular-weight species with peroxidase activity, which was completely resolved from native horseradish peroxidase. Formation of this species was absolutely dependent on the presence of both the cysteine-containing substrate and sulfhydryl oxidase. The observed enhancement of sulfhydryl oxidase catalytic activity by the addition of horseradish peroxidase supports a bi uni ping-pong mechanism proposed previously for sulfhydryl oxidase.     

Oxidation of 4-tert-butylcatechol and dopamine by hydrogen peroxide catalysed by horseradish peroxidase.

The catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-tert-butylcatechol and dopamine catalysed by this enzyme. The different polarity of the side chains of both o-diphenol substrates could help in the understanding of the nature of the rate-limiting step in the oxidation of these substrates by the enzyme. The procedure used is based on the experimental data to the corresponding steady-state equations and permitted evaluation of the more significant individual rate constants involved in the corresponding reaction mechanism. The values obtained for the rate constants for each of the two substrates allow us to conclude that the reaction of horseradish peroxidase compound II with o-diphenols can be visualised as a two-step mechanism in which the first step corresponds to the formation of an enzyme-substrate complex, and the second to the electron transfer from the substrate to the iron atom. The size and hydrophobicity of the substrates control their access to the hydrophobic binding site of horseradish peroxidase, but electron density in the hydroxyl group of C-4 is the most important feature for the electron transfer step.     

Sensitivity limitations encountered in enhanced horseradish peroxidase catalysed chemiluminescence

Conditions for the enhanced horseradish peroxidase (HRP) catalysed reaction between luminol and hydrogen peroxide were optimized to determine detection limits for HRP conjugated to antibody fragment (HRP-Fab) in solution phase. Light output was linear with respect to HRP-Fab concentration but became nonlinear at low HRP-Fab concentrations when an accelerator (enhancer) of the reaction was used. para-Phenylphenol was a more effective enhancer than p-iodophenol at HRP-Fab concentrations below 20 pmol/l. The detection limit for HRP-Fab was 1.2 femtomoles in the absence of p-phenylphenol and 0.08 femtomole in the presence of p-phenylphenol. The acceleration of peroxidase activity at the lowest HRP-Fab concentrations occurred after an incubation time period of up to five minutes. This lag time limited the sensitivity and the mechanism for it was sought. Preincubation experiment results indicated that the lag time phenomenon may involve a reversible alteration in HRP catalytic activity and that enhancer, peroxide, luminol and HRP-Fab had to be incubated together some time before maximum activation could occur.     

Enhancement of the horseradish peroxidase-catalyzed chemiluminescent oxidation of cyclic diacyl hydrazides by 6-hydroxybenzothiazoles

6-Hydroxybenzothiazole, 2-cyano-6-hydroxybenzothiazole, and 2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid (dehydroluciferin) dramatically enhance light emission from the horseradish peroxidase conjugate catalyzed oxidation of luminol, isoluminol, N-(6-aminobutyl)-N-ethyl isoluminol, and 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide by either peroxide or perborate. Light emission is enhanced by up to 1000-fold, which is an improvement over the enhancement previously observed using firefly luciferin (4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)thiazole-4-carboxylic acid). Enhancement is influenced by enhancer concentration and pH. Spectral scans of light emitted in enhanced and unenhanced reactions are similar, suggesting that aminophthalate products, and not the enhancers, are the emitters.     

Anaerobic stopped-flow studies of indole-3-acetic acid oxidation by dioxygen catalysed by horseradish C and anionic tobacco peroxidase at neutral pH: catalase effect

The effect of order of reagent mixing in the absence and in the presence of catalase on the transient kinetics of indole-3-acetic acid (IAA) oxidation by dioxygen catalysed by horseradish peroxidase C and anionic tobacco peroxidase at neutral pH has been studied. The data suggest that haem-containing plant peroxidases are able to catalyse the reaction in the absence of exogenous hydroperoxide. The initiation proceeds via the formation of the ternary complex enzyme-->IAA-->oxygen responsible for IAA primary radical generation. The horseradish peroxidase-catalysed reaction is independent of catalase indicating a significant contribution of free radical processes into the overall mechanism. This is in contrast to the tobacco peroxidase-catalysed reaction where the peroxidase cycle plays an important role. The transient kinetics of IAA oxidation catalysed by tobacco peroxidase exhibits a biphasic character with the first phase affected by catalase. The first phase is therefore associated with the common peroxidase cycle while the second is ascribed to native enzyme interaction with skatole peroxy radicals yielding directly Compound II.     

Direct electron spin resonance detection of free radical intermediates during the peroxidase catalyzed oxidation of phenacetin metabolites

The oxidation of the phenacetin metabolites p-phenetidine and acetaminophen by peroxidases was investigated. Free radical intermediates from both metabolites were detected using fast-flow ESR spectroscopy. Oxidation of acetaminophen with either lactoperoxidase and hydrogen peroxide or horseradish peroxidase and hydrogen peroxide resulted in the formation of the N-acetyl-4-aminophenoxyl free radical. Totally resolved spectra were obtained and completely analyzed. The radical concentration was dependent on the square root of the enzyme concentration, indicating second-order decay of the radical, as is consistent with its dimerization or disproportionation. The horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of p-phenetidine (4-ethoxyaniline) at pH 7.5-8.5 resulted in the one-electron oxidation products, the 4-ethoxyaniline cation free radical. The ESR spectra were well resolved and could be unambiguously assigned. Again, the enzyme dependence of the radical concentration indicated a second-order decay. The ESR spectrum of the conjugate base of the 4-ethoxyaniline cation radical, the neutral 4-ethoxyphenazyl free radical, was obtained at pH 11-12 by the oxidation of p-phenetidine with potassium permanganate.     

The oxidation and decarboxylation of retinoic acid by horseradish peroxidase.

The decarboxylation of retinoic acid by horseradish peroxidase was investigated. A marked increase in the yield of products was obtained. However, the data indicated the reaction was a nonenzymatic, heme catalyzed peroxidation. Previously reported requirements for phosphate, oxygen and ferrous ion were eliminated when hydrogen peroxide was provided. Peroxide also eliminated the EDTA and cyanide induced inhibition of the phosphate dependent system. In the presence of hydrogen peroxide, horseradish peroxidase was not essential to the reaction; heme equivalent amounts of hemoglobin decarboxylated retinoic acid with equal facility. However, hemoglobin was ineffective in the absence of hydrogen peroxide. Attainment of 50--60% decarboxylation represented complete utilization of the available retinoic acid. Thus the products of the reaction can be divided into two groups, products of retinoic acid oxidation and products of an oxidative decarboxylation of retinoic acid.     

Influence of the reaction medium on the product distribution of peroxidase-catalysed oxidation of p-cresol

Summary p-Cresol was oxidized by hydrogen peroxide in a reaction catalysed by horseradish peroxidase and the low molecular weight products were investigated. In aqueous media Pummerer's ketone (I) was the dominating product but in organic media the product distribution was quite different; 2,2'-dihydroxy-5,5'-dimethyldiphenyl (II) was the main low molecular weight product. Similar product distributions were obtained with peroxidase adsorbed on a solid support and suspended in toluene and with peroxidase solubilized in a microemulsion containing the same solvent. The best selectivity for the formation of (II) was obtained when the enzyme was adsorbed on Celite and suspended in water-saturated chloroform with 0.5% (v/v) extra water added. The yield of low molecular weight products in this case was 28%; of this fraction, 95% was (II). Offprint requests to: P. Adlercreutz     

Luminol oxidation by hydrogen peroxide with chemiluminescent signal formation catalyzed by peroxygenase from the fungus Agrocybe aegerita V.Brig.

Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig. have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris-buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Detection limit of the enzyme in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.     

Spin trapping of the azidyl radical in azide/catalase/H2O2 and various azide/peroxidase/H2O2 peroxidizing systems

The azidyl radical is formed during the oxidation of sodium azide by the catalase/hydrogen peroxide system, as detected by the ESR spin-trapping technique. The oxidation of azide by horseradish peroxidase, chloroperoxidase, lactoperoxidase, and myeloperoxidase also forms azidyl radical. It is suggested that the evolution of nitrogen gas and nitrogen oxides reported in the azide/catalase/hydrogen peroxide system results from reactions of the azidyl radical. The azide/horseradish peroxidase/hydrogen peroxide system consumes oxygen, and this oxygen uptake is inhibited by the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, presumably due to the competition with oxygen for the azidyl radical. Although azide is used routinely as an inhibitor of myeloperoxidase and catalase, some consideration should be given to the biochemical consequences of the formation of the highly reactive azidyl radical by the peroxidase activity of these enzymes.     

Oxidation of luminol with peroxidase from royal palm leaves

We optimized the conditions for luminol oxidation by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3-8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.     

Kinetic evidence of horseradish peroxidase oxidation by compound I.

The kinetics of compound II formation, obtained upon mixing a highly purified horseradish peroxidase and hydrogen peroxide, was spectrophotometrically studied at three wavelengths in the absence of an added reducing agent. Our experiments confirm George's finding that more than one mole of compound II is formed per mole of hydrogen peroxide added. The new mechanism that we propose, contrary to the mechanism of George, is only valid when compound II is obtained in the absence of an added donor. Moreover, it is not inconsistent with the classical Chance mechanism of oxidation of an added donor by the system peroxidase -- hydrogen peroxide. According to this new mechanism, in the absence of an added donor, compound II formation involved two pathways. The first pathway is the monomolecular reduction of compound I by the endogenous donor, and the second pathway is the formation of two moles of compound II through the oxidoreduction reaction between one mole of peroxidase and one mole of compound I.     

Studies on Auxin Protectors: XI. Inhibition of Peroxidase-Catalyzed Oxidation of Glutathione by Auxin Protectors and o-Dihydroxyphenols

Commercial horseradish peroxidase, when supplemented with dichlorophenol and either manganese or hydrogen peroxide, will rapidly oxidize glutathione. This peroxidase-catalyzed oxidation of glutathione is completely inhibited by the presence of auxin protectors. Three auxin protectors and three o-dihydroxyphenols were tested; all inhibited the oxidation. Glutathione oxidation by horseradish peroxidase in the presence of dichlorophenol and Mn is also completely inhibited by catalase, implying that the presence of Mn allows the horseradish peroxidase to reduce oxygen to H₂O₂, then to use the H₂O₂ as an electron acceptor in the oxidation of glutathione. Catalase, added 2 minutes after the glutathione oxidation had begun, completely inhibited further oxidation but did not restore any gluthathione oxidation intermediates. In contrast, the addition of auxin protectors, or o-dihydroxyphenols, not only inhibited further oxidation of gluthathione by horseradish peroxidase (+ dichlorophenol + Mn), but also caused a reappearance of glutathione as if these antioxidants reduced a glutathione oxidation intermediate. However, when gluthathione was oxidized by horseradish peroxidase in the presence of dichlorophenol and H₂O₂ (rather than Mn), then the inhibition of further oxidation by auxin protectors or o-dihydroxyphenols was preceded by a brief period of greatly accelerated oxidation. The data provide further evidence that auxin protectors are cellular redox regulators. It is proposed that the monophenol-diphenol-peroxidase system is intimately associated with the metabolic switches that determine whether a cell divides or differentiates.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers

Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proehancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picornole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D -glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.