Long-Term Chemiluminescent Signal Is Produced in the Course of Luminol Peroxidation Catalyzed by Peroxidase Isolated from Leaves of African Oil Palm Tree

Optimal conditions were found for the oxidation of luminol by hydrogen peroxide in the presence of peroxidase isolated from leaves of the African oil palm tree Elaeis guineensis (AOPTP). The pH range for maximal chemiluminescence intensity (8.3-8.6) is similar for AOPTP, horseradish, and Arthromyces ramosus peroxidases and slightly different from that for tobacco peroxidase (9.3). Increasing the buffer concentration decreases the chemiluminescence intensity. As in the case of other anionic peroxidases, the catalytic efficiency of AOPTP does not depend on the presence of enhancers (4-iodophenol and 4-hydroxycinnamic acid) in the reaction medium. The detectable limit of AOPTP assayed by luminol peroxidation is 2·10^–12 M. The long-term chemiluminescence signal produced during AOPTP-dependent luminol peroxidation is a characteristic feature of the African oil palm enzyme. This feature in combination with its very high stability suggests that AOPTP will be a promising tool in analytical practice.     

Luminol activity of horseradish peroxidase mutants mimicking a proposed binding site for luminol in Arthromyces ramosus peroxidase.

To enhance the oxidation activity for luminol in horseradish peroxidase (HRP), we have prepared three HRP mutants by mimicking a possible binding site for luminol in Arthromyces ramosus peroxidase (ARP) which shows 500-fold higher oxidation activity for luminol than native HRP. Spectroscopic studies by (1)H NMR revealed that the chemical shifts of 7-propionate and 8-methyl protons of the heme in cyanide-ligated ARP were deviated upon addition of luminol (4 mM), suggesting that the charged residues, Lys49 and Glu190, which are located near the 7-propionate and 8-methyl groups of the heme, are involved in the specific binding to luminol. The positively charged Lys and negatively charged Glu were introduced into the corresponding positions of Ser35 (S35K) and Gln176 (Q176E) in HRP, respectively, to build the putative binding site for luminol. A double mutant, S35K/Q176E, in which both Ser35 and Gln176 were replaced, was also prepared. Addition of luminol to the HRP mutants induced more pronounced effects on the resonances from the heme substituents and heme environmental residues in the (1)H NMR spectra than that to the wild-type enzyme, indicating that the mutations in this study induced interactions with luminol in the vicinity of the heme. The catalytic efficiencies (V(max)/K(m)) for luminol oxidation of the S35K and S35K/Q176E mutants were 1.5- and 2-fold improved, whereas that of the Q176E mutant was slightly depressed. The increase in luminol activity of the S35K and S35K/Q176E mutants was rather small but significant, suggesting that the electrostatic interactions between the positive charge of Lys35 and the negative charge of luminol can contribute to the effective binding for the luminol oxidation. On the other hand, the negatively charged residue would not be so crucial for the luminol oxidation. The absence of drastic improvement in the luminol activity suggests that introduction of the charged residues into the heme vicinity is not enough to enhance the oxidation activity for luminol as observed for ARP.     

Study and immunoanalytical applications for peroxidase from Arthromyces ramosus

Some biochemical and catalytic properties of peroxidase from Arthromyces ramosus (EC 1.11.7.1) in chemiluminescent reaction of luminol oxidation by hydrogen peroxide were investigated. The second order rate constants were determined by the stopped-flow technique. Optimal conditions to quantity the enzyme were found, the detection limit being 5.10(-13) M. The peroxidase was used as a marker in the human IgG immunoassay.     

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.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Enhancer effect of fluorescein on the luminol–H2O2–horseradish peroxidase chemiluminescence: energy transfer process

The chemiluminescence of the luminol–H₂O₂–horseradish peroxidase system is increased by fluorescein. Fluorescein produces an enhancement of the luminol chemiluminescence similar to that of phenolphthalein, by an energy transfer process from luminol to fluorescein. The maximum intesity and the total chemiluminescence emission (between 380 and 580 nm) of luminol with fluorescein was more than three times greater than without fluorescein; however, the emission duration was shorter. The emission spectra in the presence of fluorescein had two maxima (425 and 535 nm) and the enhancement was dependent on pH and fluorescein concentration. A mechanism is proposed to explain these effects. © 1997 John Wiley & Sons, Ltd.     

Phenol derivatives as enhancers and inhibitors of luminol–H2O2–horseradish peroxidase chemiluminescence

Systematic studies on phenol derivatives facilitates an explanation of the enhancement or inhibition of the luminol–H₂O₂–horseradish peroxidase system chemiluminescence. Factors that govern the enhancement are the one-electron reduction potentials of the phenoxy radicals (PhO^•/PhOH) vs. luminol radicals (L^•/LH⁻) and the reaction rates of the phenol derivatives with the compounds of horseradish peroxidase (HRP-I and HRP-II). Only compounds with radicals with a similar or greater reduction potential than luminol at pH 8.5 (0.8 V) can act as enhancers. Radicals with reduction potentials lower than luminol behave in a different way, because they destroy luminol radicals and inhibit chemiluminescence. The relations between the reduction potential, reaction rates and the Hammett constant of the substituent in a phenol suggest that 4-substituted phenols with Hammett constants (σ) for their substituents similar or greater than 0.20 are enhancers of the luminol–H₂O₂–horseradish peroxidase chemiluminescence. In contrast, those phenols substituted in position 4 for substituents with Hammett constants (σ) lower than 0.20 are inhibitors of chemiluminescence. On the basis of these studies, the structure of possible new enhancers was predicted. © 1998 John Wiley & Sons, Ltd.     

A comparative study of peroxidases from horse radish and Arthromyces ramosus as labels in luminol-mediated chemiluminescent assays.

The properties of a peroxidase from Arthromyces ramosus (ARP) in the chemiluminescent reaction of luminol oxidation have been studied. These were compared with the properties of horse radish peroxidase (HRP) in the cooxidation of luminol and p-iodophenol, the enhanced chemiluminescence (ECL) reaction. By means of the stop-flow technique, ARP was shown to have an enzymatic activity toward luminol higher than that toward HRP. ARP can efficiently catalyze luminol oxidation in the absence of substrate enhancer. pH and substrate concentrations were optimized to determine ARP with the highest sensitivity. The detection limit of ARP was 5 x 10(-13) M, the same as that for HRP in the ECL reaction. The data on the use of ARP as a label in enzyme immunoassay of human IgG are presented. ARP was shown to have all the advantages of HRP as a label in chemiluminescent enzyme immunoassays: (i) high signal intensity, (ii) slow decay of luminescence, (iii) high signal/noise ratio, and (iv) as a consequence of (i)-(iii), high detection sensitivity. However, the low thermostability of ARP can limit the potential fields of its application.     

Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts

The light output of the peroxidase-catalyzed luminol chemiluminescent oxidation reaction can be greatly increased by incorporating different enhancers. Such an increase is attributed to the preferential oxidation of the enhancer by peroxidase intermediates and the rapid formation of enhancer radicals that, in turn, quickly oxidize luminol to its radical anion. These enhancers, which include substituted phenols, substituted boronic acids, indophenols, and N-alkyl phenothiazines, behave as electron transfer mediators. A further, very significant increase in light output was also observed by the addition of nucleophilic acylation catalyst to the enhancer/luminol/oxidant substrate. The effect of the new component is general and applicable to many of the known enhancers but is much more remarkable in association with phenothiazine enhancers (up to 10-fold light output). The addition of a nucleophilic acylation catalyst to these substrates lowered the limit of detection for horseradish peroxidase from 50 to 8 amol. Similar improvements were observed in “sandwich” enzyme-linked immunosorbent assays and Western blot assays.     

4-phenylylboronic acid: A new type of enhancer for the horseradish peroxidase catalysed chemiluminescent oxidation of luminol

4-Phenylylboronic acid enhances the light emission from the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. Optimization studies showed that the greatest enhancement was obtained using micromolar concentrations of the new enhancer. The largest degree of enhancement was found with the basic isoenzyme of horseradish peroxidase (Type VIA), and lesser degrees of enhancement were obtained with Type VII and Type IX horseradish peroxidase. The enhancer was also effective in the peroxidase catalysed oxidation of isoluminol by peroxide.     

A convenient, high-throughput method for enzyme-luminescence detection of dopamine released from PC12 cells

This protocol represents a novel enzyme-luminescence method to detect dopamine sensitively and rapidly with high temporal resolution. In principle, dopamine is first oxidized with tyramine oxidase to produce H(2)O(2), and then the produced H(2)O(2) reacts with luminol to generate chemiluminescence in the presence of horseradish peroxidase (POD). We applied this method successfully to perform real-time monitoring of dopamine release from PC12 cells using a luminescence plate reader upon stimulation with several drugs (e.g., acetylcholine, bradykinin). The results indicated that the dopamine release from PC12 cells was modulated by these drugs in a way similar to that found by using several conventional analytical techniques, such as HPLC-electrochemical detector (ECD). Unlike other assays, this assay technique is simple, rapid, highly sensitive and thus useful for assessment of effects of drugs on the nervous system. The dopamine release assay takes only < or =1 h once reagent setup and culture plates' preparation are finished.     

Improvement of mimetic peroxidase activity of gold nanoclusters on the luminol chemiluminescence reaction by surface modification with ethanediamine

Peroxidase is a commonly used catalyst in luminol–H₂O₂ chemiluminescence (CL) reactions. Natural peroxidase has a sophisticated separation process, short shelf life and unstable activity, therefore it is important to develop peroxidases that have both high catalytic activity and good stability as alternatives to the natural enzyme. Gold nanoclusters (Au NCs) are an alternative peroxidase with catalytic activity in the luminol–H₂O₂ CL reaction. In the present study, ethanediamine was modified on the surface of Au NCs forming cationic Au NCs. The zeta potential of the cationic Au NCs maintained its positive charge when the pH of the solution was between 4 and 9. The cationic Au NCs showed higher catalytic activity in the luminol–H₂O₂ CL reaction than did unmodified Au NCs. A mechanism study showed that the better performance of cationic Au NCs may be attributed to the generation of ¹O₂ on the surface of cationic Au NCs and a positive surface charge, for better affinity to luminol. Cationic Au NC, acting as a peroxidase mimic, has much better stability than horseradish peroxidase over a wide range of temperatures. We believe that cationic Au NCs may be useful as an artificial peroxidase for a wide range of potential applications in CL and bioanalysis.     

Luminometric determination of oxidase activity in peroxisomal fractions of rat liver: glycolate oxidase

The feasibility of using the H2O2-mediated chemiluminescence for determination of the activity of oxidases in peroxisomes of rat liver has been investigated. In an assay medium containing luminol, horseradish peroxidase, and azide with glycolate as substrate, a linear relationship is obtained between the amount of peroxisomal protein used and the luminescence signal. In comparison with other techniques available for measuring the activities of peroxisomal oxidases the luminometric approach described here is 5-10 times more sensitive than the spectrophotometric methods and 100 times more efficient than the polarographic determination of O2. Under the optimal assay conditions the glycolate oxidase activity can be determined in amounts as low as 0.5 micrograms peroxisomal protein.     

Analysis of horseradish peroxidase-amplified chemiluminescence produced by human neutrophils reveals a role for the superoxide anion in the light emitting reaction

When polymorphonuclear leukocytes and soluble or particulate matter interact, the cells produce chemiluminescence, which is linked to activation of the oxidative metabolism of the cells. A luminol chemiluminescence assay in which the reaction mixture contains a relatively large amount of horseradish peroxidase combined with sodium azide has been proposed to quantitate H2O2 produced by human neutrophils during the respiratory burst (M.P. Wymann, V. von Tscharner, D. A. Deranleau, and M. Baggiolini (1987) Anal. Biochem. 165, 371-378). We found, when comparing the response to concanavalin A and a formylated peptide (formylmethionyl-leucyl-phenylalanine), that neutrophils produce H2O2 that is not detected as chemiluminescence by the horseradish peroxidase-azide-luminol system. Furthermore, the horseradish peroxidase-amplified chemiluminescence response obtained from granule-depleted neutrophil cytoplasts is inhibited by superoxide dismutase, an O2- scavanger. Based on these results, we question the specificity of the described technique for H2O2. The usefulness of the technique in the determining the extracellular and intracellular production of oxidative metabolites is discussed.     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

Improvements to enhanced horseradish peroxidase detection sensitivity

At very low horseradish peroxidase (HRP) concentrations, the enhanced chemiluminescence reaction is often characterized by a lag time between initiation of the reaction and beginning of light output. In this study, four treatments of luminol solution were examined in an effort to remove the lag time and to improve chemiluminescence light output. Addition of ammonium persulphate stimulated light output more than tenfold. Ultraviolet irradiation and photoactive dye pretreatment of luminol solution both increased light output fourfold. Luminol purity was the most important factor affecting detection sensitivity. Recrystallization of luminol from base improved the detection limit 13-fold although there was an improvement in the detection limit from 13 attomoles per millilitre to 5 attomoles per millilitre with highly purified luminol when photoactive dye pretreatment was utilized. The results are consistent with a simple interference mechanism whereby enhancer radicals produced by the enzyme are preferentially quenched by contaminants present in the luminol, in the enhancer and in the solvent used to dissolve the enhancer. Consumption of these interferences prior to light emission results in a lag time and a less favourable HRP detection limit.     

Influence of different luminols on the characteristics of the chemiluminescence reaction in human neutrophils

In search for a luminol with very high output of light, 20 different luminol samples were tested for their ability to enhance the chemiluminescence reaction in phorbol myristate acetate activated human neutrophils. We found that the majority of luminols tested (17 samples) gave almost the same light output from neutrophils, and that the major part of the activity was from an intracellular origin. Owing to the fact that three isoluminol samples were unable to monitor respiratory burst activity taking place intracellularly, a very low level of chemiluminescence was obtained with these samples. Their light output was, however, greatly increased when horseradish peroxidase or myeloperoxidase was added, showing that the light-generating reaction with isoluminol as well as with luminol is peroxidase-dependent. The fact that isoluminol could also use myeloperoxidase as amplifying peroxidase, suggests that the lack of measurable intracellular activity in the presence of isoluminol is somehow related to a limited or restricted diffusion of the molecule to intracellular sites. The isoluminol system constitutes a sensitive system for measuring release of oxygen metabolites from phagocytic cells.     

Microwave effects on immobilized peroxidase chemiluminescence

Protein gels formed by crosslinking bovine serum albumin and horseradish peroxidase with glutaraldehyde were used to measure effects on peroxidase activity of 400-MHz (CW) radiofrequency radiation (RFR) at an average specific absorption rate (SAR) of 1.45 W/kg. The enzyme activity was measured by luminol chemiluminescence recorded on photographic film after hydrogen peroxide activation. Activity was measured during RFR exposure of gels or after exposure of gels polymerized in the RFR field. During exposure, a significant (P less than .05) reversible increase occurred in overall mean peroxidase activity of gels activated with 0.88 M H2O2 but not in those activated with 8.8 M H2O2. Gels containing solubilized luminol and formed in the field showed no overall mean increase in peroxidase activity, but did display a highly significant (P less than .001) alteration in the distribution of local activities when compared to unexposed gels. These results are apparently due to changes in the rate of diffusion (concentration equilibration) of hydrogen peroxide in the gel.     

Effect of reaction conditions on phenol removal by polymerization and precipitation using Coprinus cinereus peroxidase

The quantitative relationships between removal efficiency of phenol and reaction conditions were investigated using Coprinus cinereus peroxidase. The most effective ratio of hydrogen peroxide to phenol was nearly 1/1 (mol/mol) at an adequate enzyme dose. 12.2 U of the enzyme was needed to remove 1 mg of phenol when our peroxidase preparation was used. At an insufficient peroxidase dose, the optimum pH value was 9.0, and lowering the reaction temperature led to the improvement of removal efficiency. At an excess peroxidase dose, almost 100% removal of phenol was obtained over a wide range of pH (5-9) and temperature (0-60 degrees C). Despite the presence of culture medium components, it was shown that Coprinus cinereus peroxidase had the same phenol polymerization performance as horseradish peroxidase or Arthromyces ramosus peroxidase.     

Effect of Enhancers on the Pyridopyridazine–Peroxide–HRP Reaction

4-Substituted phenyl boronic acids (e.g., 4-iodo, 4-bromo, 4-phenyl) are effective enhancers of the horseradish peroxidase (Type VIA) catalysed chemiluminescent oxidation of various pyrido[3,4-d]pyridazine-1,4(2H,3H)dione derivatives. The most effective combination was 4-biphenylboronic acid and 8-amino-5-chloro-7- phenylpyrido[3,4-d]- pyridazine-1,4(2H,3H)dione. Generally, the intensity of light emission in the presence of peroxidase was higher with the pyridopyridazines than with sodium luminol. However, the blank light emission was much lower with sodium luminol than with the pyridopyridazines. A synergistic enhancement phenomenon was demonstrated for the combination of a 4-iodophenol and a 4-biphenylboronic acid enhancer with 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione. The combination of these two enhancers produced a light emission intensity in an assay for 5 fmol of peroxidase that was 25% higher than expected from the sum of the individual light intensities.