Singlet oxygen production by soybean lipoxygenase isozymes

The oxidation of linoleic acid catalyzed by soybean lipoxygenase isozymes was accompanied by 1268 nm chemiluminescence characteristic of singlet oxygen. The recombination of peroxy radicals as first proposed by Russell (Russell, G.A. (1957) J. Am. Chem. Soc. 79, 3871-3877) is a plausible mechanism for the observed singlet oxygen production. Lipoxygenase-3 was the most active isozyme. Under the optimal aerobic conditions of p2H 7, 100 micrograms/ml lipoxygenase-3, 100 microM linoleic acid, 100 microM 13-hydroperoxylinoleic acid, and air-saturated buffer, the yield of singlet oxygen was 12 +/- 0.4 microM or 12% of the amount predicted by the Russell mechanism. High yields of singlet oxygen required the presence of 13-hydroperoxylinoleic acid. Systems containing lipoxygenase-2 and lipoxygenase-3 produced comparable yields of singlet oxygen without added 13-hydroperoxylinoleic acid, since the lipoxygenase-2 served as an in situ source of hydroperoxide. Lipoxygenase-1 was active only at low oxygen concentrations. Its singlet oxygen-producing capacity was greatly increased by the addition of acetone to the system. Lipoxygenase-2 did not produce detectable quantities of singlet oxygen.     

Indole-3-methanol as an intermediate in the oxidation of indole-3-acetic acid by peroxidase

During oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase, indole-3-aldehyde and 3-hydroxymethayloxindole cease to be produced a few minutes after initiation of the reaction even though IAA is still being consumed. At the same time an increased accumulation of indole-3-methanol is observed and the ratio of oxygen to indole-3-acetic acid consumed becomes less than unity. Indole-3-niethanol can be a substrate for horseradish peroxidase provided that H₂O₂ is present. In this reaction, indole-3-aldehyde but not 3-hydroxymethyloxindole is formed. H₂O₂ is not merely an activating agent for the enzyme but also a true oxidant because it is consumed stoichiometrically (1 mol of H₂O₂ per mol of indole-3-methanol) and the reaction is independent of the presence of oxygen. Indole-3-methanol is proposed as an intermediate in the process of oxidation of indole-3-acetic acid into indole-3-al-denyde, the second step of which requires peroxide as an oxidant.     

The Mechanism of the Scopoletin-induced Inhibition of the Peroxidase Catalyzed Degradation of Indole-3-acetate

The naturally occurring coumarin, scopoletin, has been found to modify horseradish peroxidase rapidly to give a stable, spectroscopically distinguishable form of the enzyme. Peroxidase treated with scopoletin is less active in reactions with molecular oxygen and indole-3-acetic acid. Kinetic data for the degradation of this growth regulator were obtained with a continuously monitored fluorometric procedure. Lineweaver-Burk plots of the reciprocal rate of degradation against the reciprocal substrate concentration were markedly curved in the presence of the inhibitor, scopoletin. Excess indole-3-acetate restored the scopoletin-treated enzyme to a reactive state. In the presence of molecular oxygen, concentrations of indole-3-acetic acid which were at least 10-fold greater than the inhibitor concentration led to the rapid oxidation of the coumarin and converted peroxidase to compound III as expected from previous studies. This form of the enzyme is the catalytically active species in the oxidative degradation of the growth regulator. The kinetically preferential reaction of scopoletin or related coumarins with peroxidase and the suppression of indole-3-acetic acid degradation may provide a possible control mechanism over the oxidative degradation of indole-3-acetate by this plant enzyme.     

Near infrared emission of singlet oxygen generated in the dark

Singlet oxygen generation is reported from (1) enzymatic reaction and (2) electron transfer reactions of the superoxide anion measured directly with an ultrasensitive near-IR emission spectrophotometer by monitoring the O₂(¹Δ_g) → O₂ (³Σ_g^?) transition at 1268 nm. Near-IR emission spectra from the myeloperoxidase and lactoperoxidase enzymatic systems show only emission of singlet oxygen at 1268nm. The lipoxygenase/Na–linoleate enzymatic reaction exhibits two emissions, 1268 nm and 1288 nm. The latter emission is identified as originating from a peroxy radical. Spectral and kinetic data giving evidence of singlet oxygen generation is obtained from the reaction of potassium superoxide solubilized by 18-crown-6-ether in acetonitrile with a series of organometallic coordination compounds.     

An electron spin resonance study of free radical intermediates in the oxidation of indole acetic acid by horseradish peroxidase

The oxidation of indole-3-acetic acid by horseradish peroxidase was studied using the spin traps t-nitrosobutane and 5,5-dimethyl-1-pyrroline N-oxide to trap free radical intermediates. The major free radical metabolite of indole acetic acid was unambiguously determined by the use of indole-3-[2,2-2H2]acetic acid to be the skatole carbon-centered free radical. In the presence of oxygen, superoxide was also trapped.     

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.     

Catalysis of singlet oxygen production in the reaction of hydrogen peroxide and hypochlorous acid by 1,4-diazabicyclo[2.2.2]octane (DABCO)

The kinetics of the singlet oxygen production in the hydrogen peroxide plus hypochlorous acid reaction were studied by measuring the time course of the singlet oxygen emission at 1268 nm. The addition of 1,4-diazabicyclo[2.2.2]octane (DABCO) increased the peak intensity of the chemiluminescence, but decreased its duration. The increased rate of singlet oxygen production likely accounts for the enhancement of singlet oxygen dimol emission reported in 1976 by Deneke and Krinsky (J. Am. Chem. Soc. 98, 3041-3042). This phenomenon was not seen when singlet oxygen was generated with the reaction of hypobromous acid and hydrogen peroxide. Thus, the enhancement of red chemiluminescence by DABCO should not be regarded as a general test for the production of singlet oxygen in complex biochemical systems.     

Proposed Model for the Peroxidase-Catalyzed Oxidation of Indole-3-acetic Acid in the Presence of the Inhibitor Ferulic Acid

Linear increments in ferulic acid concentration produce logarithmic increases in the ferulic acid-induced lag periods prior to the peroxidase-catalyzed oxidation of indole-3-acetic acid in a system containing 2,4-dichlorophenol and MnCl₂ in acetate buffer at pH 5.6. Maintaining the ratio of indole-3-acetic acid to ferulic acid constant at 100 while linearly raising the ferulic acid concentration results in linear increases in the lag period. Both indole-3-acetic acid and ferulic acid are substrates of horseradish peroxidase in the presence of H₂O₂, and indole-3-acetic acid competitively inhibits the oxidation of ferulic acid. A model for the enzymatic oxidation of indole-3-acetic acid catalyzed by peroxidase is proposed.     

The Role of Indole-3-Acetic Acid in Peroxidase Oxidation of Ascorbic Acid Catalyzed by Horseradish Peroxidase

We studied stationary kinetics of ascorbic acid oxidation in the presence of indole-3-acetic acid catalyzed by horseradish peroxidase. The catalytic (k_cat and K_m) and inhibition (K_i) constants were determined for pH from 4.5 to 7.0. The auxin proved to competitively inhibit the enzyme when a single ascorbic acid molecule is bound, while a non-competitive inhibition by IAA is observed for peroxidase oxidation of two or more substrate molecules. A mechanism of ascorbic acid oxidation in the presence of indole-3-acetic acid is proposed.     

Glucuronidation of oxygenated benzo(a)pyrene derivatives by UDP-glucuronyl transferase of nuclear envelope

The variation of the spectra and its reactivity towards 2-methylpropanal, indole-3-acetic acid and malonaldehyde of solutions of horseradish peroxidase in dimethyl sulfoxide-water mixtures has been studied. A broad pattern of changes was observed in the CD spectra of peroxidase, especially in the 400 nm region. These variations influenced strongly the excited triplet acetone emission from the 2-methylpropanal system which is generated in the active site of the enzyme protected from external quenching. This means that presumably the active site is more uncovered in the presence of dimethyl sulfoxide than the native form. Energy transfer parameters indicate that in fact there is a conformational effect produced by dimethyl sulfoxide in the horseradish peroxide active site. Dimethyl sulfoxide appears to be an important conformational probe in biochemistry.     

Horseradish peroxidase: a modern view of a classic enzyme

Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism, cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed.     

The inhibition of peroxidase and indole-3-acetic acid oxidase activity by British Anti-Lewisite

British Anti-Lewisite (BAL) binds to horseradish peroxidase in a manner which results in inhibition of both peroxidatic and oxidative functions of the enzyme. BAL competes with hydrogen peroxide for binding on peroxidase, and the inhibition of peroxidatic activity is irreversible. Solutions of purified horseradish peroxidase and individually resolved peroxidase isozymes show a gradual loss of peroxidatic activity with time when incubated with BAL. In these same treatments, however, the inhibition of indole-3-acetic acid (IAA) oxidase activity is immediate. With increasing amounts of enzyme in the incubation mixture, IAA oxidase activity is not completely inhibited and is observed following a lag period in the assay which shortens with longer incubation times. Peroxidase activity during this same time interval shows a lag period which increases with longer incubation times. Lowering the pH removed the lag period for oxidase activity, but did not change the pattern of peroxidase activity. These results suggest that the sites for the oxidation of indole-3-acetic acid and for peroxidatic activity may not be identical in horseradish peroxidase isozymes.     

Singlet oxygen production by human eosinophils

Human eosinophils, stimulated with phorbol myristate acetate, were found to produce 1268 nm chemiluminescence characteristic of singlet oxygen. Singlet oxygen generation required the presence of bromide ion. A bromide ion concentration of 100 microM, comparable to the total bromine content of whole blood, was sufficient for the eosinophils to generate measurable amounts of singlet oxygen. For the conditions used (10(7) cells/ml and 10 micrograms/ml phorbol myristate acetate), the duration of the singlet oxygen generation was brief, about 5 min, and the total yield of singlet oxygen was modest, 1.0 +/- 0.1 microM. The cells remained viable after the singlet oxygen production ceased. This is the first demonstration of singlet oxygen production from living cells. The singlet oxygen generated by eosinophils likely results from a peroxidase-catalyzed mechanism, since a purified eosinophil peroxidase-hydrogen peroxide-bromide system was also shown to produce singlet oxygen. The unique properties of eosinophil peroxidase are illustrated by the fact that at p2H 7.0 and with 100 microM bromide, eosinophil peroxidase generated 20 +/- 2% of the theoretical yield of singlet oxygen, whereas under identical conditions, myeloperoxidase and lactoperoxidase produced only 1.0 +/- 0.1% and -0.1 +/- 0.1%, respectively.     

Singlet oxygen production from the peroxidase catalyzed formation of styrene glutathione adducts

Recently, Stock et al. (J. Biol. Chem. 261, 15915-15922 [1986]) described a model enzyme system composed of horseradish peroxidase, hydrogen peroxide, phenol, glutathione and styrene. This system forms glutathione-styrene conjugates. Glutathione radicals and carbon-centered radicals are intermediates in this process. In the present study, this model enzyme system was also shown to generate singlet oxygen, probably via a Russell mechanism. No singlet oxygen was generated in the absence of styrene. Thus, contrary to prior suggestions, the reaction of glutathione radical with oxygen to produce a thiyl peroxyl radical is not a significant source of singlet oxygen.     

Singlet oxygen production by biological systems

Singlet oxygen (1 delta g) is a highly reactive, short-lived intermediate which readily oxidizes a variety of biological molecules. The biochemical production of singlet oxygen has been proposed to contribute to the destructive effects seen in a number of biological processes. Several model biochemical systems have been shown to produce singlet oxygen. These systems include the peroxidase-catalyzed oxidations of halide ions, the peroxidase-catalyzed oxidations of indole-3-acetic acid, the lipoxygenase-catalyzed oxidation of unsaturated long chain fatty acids and the bleomycin-catalyzed decomposition of hydroperoxides. Results from these model systems should not be uncritically extrapolated to living systems. Recently, however, an intact cell, the human eosinophil, was shown to generate detectable amounts of singlet oxygen. This result suggests that singlet oxygen may be shown to be a significant biochemical intermediate in a few biological processes.     

DNA strand scission in E.coli by electronically excited state molecules generated by enzymatic systems

$\text{E.}\text{coli}$ containing pAT 153 plasmid undergoes strand scission when exposed to the indole-3-acetic acid/peroxidase/D₂ system. Neither the initial components of this reaction nor the final stable products are responsible for this effect. Indole-3-aldehyde in its triplet state and singlet oxygen have been recently identified in this system. That singlet oxygen is one of the species acting on the plasmid in $\text{E.}\text{coli}$ cells was suggested by protective effect of histidine and guanosine which are singlet oxygen quenchers. Similar effect on plasmid with malonaldehyde/peroxidase/O₂ system was observed, which is an excellent singlet oxygen generator. This is the first report of a biological system where it is possible to detect a DNA scission in the intact cell by a bioenergized process. This presumably is related to spontaneous mutagenesis.     

Chemiluminescent oxidation of ribose catalyzed by horseradish peroxidase in presence of hydrogen peroxide

The reaction of ribose with horseradish peroxidase in the presence of H₂O₂ is accompanied by light emission. The detection of horseradish peroxidase Compound II (FeO²⁺) indicates that the enzyme participates in a normal peroxidatic cycle. Hydrogen peroxide converts horseradish peroxidase into Compound I (FeO³⁺) which in turn is converted into Compound II by abstracting a hydrogen atom from ribose forming a ribosyl radical. In aerated solutions oxygen rapidly adds to the ribosyl radical. Based on the spectral characteristics and the enhancement of the chemiluminescence by chlorophyll-a, xanthene dyes, D₂O and DABCO, it is suggested that the excited species, apparently triplet carbonyls and ¹O₂, are formed from the bimolecular decay of the peroxyl radicals via the Russell mechanism.     

Halogenated indole-3-acetic acids as oxidatively activated prodrugs with potential for targeted cancer therapy

Substituted indole-3-acetic acid (IAA) derivatives, plant auxins with potential for use as prodrugs in enzyme-prodrug directed cancer therapies, were oxidised with horseradish peroxidase (HRP) and toxicity against V79 Chinese hamster lung fibroblasts was determined. Rate constants for oxidation by HRP compound I were also measured. Halogenated IAAs were found to be the most cytotoxic, with typical surviving fractions of <10(-3) after incubation for 2h with 100 microM prodrug and HRP.     

Promotion of peroxidase activity in the cell wall of Nicotiana

Peroxidase catalyzes the oxidation of indole-3-acetic acid. The primary products of this reaction stimulate growth in plants. Therefore, our concept is that an increase in peroxidase activity will increase the effect of indole-3-acetic acid as a growth hormone. Our objective was to study the effect of 2,3,5-triiodobenzoic acid, a growth regulator, on isoperoxidases in the cell wall and cytoplasm of Nicotiana. Isoperoxidases from the cell wall and cytoplasmic fractions were separated by acrylamide gel electrophoresis. We found that 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase peroxidase activity in the cell wall. Since both 2,3,5-triiodobenzoic acid and indole-3-acetic acid increase the activity of the same isoperoxidase, we conclude that 2,3,5-triiodobenzoic acid synergizes rather than antagonizes auxin action, and we suggest that this increase in indole-3-acetic acid oxidase activity sensitizes plant tissues to auxin.     

Singlet oxygen production by chloroperoxidase-hydrogen peroxide-halide systems

Singlet oxygen production in the chloroperoxidase-hydrogen peroxide-halide system was studied using 1268 nm chemiluminescence. With chloride or bromide ions, singlet oxygen is produced by the mechanism (formula; see text) (formula; see text) where X- is chloride or bromide ion. Under conditions where there is high enzyme activity and when Reaction B is fast relative to Reaction A, singlet oxygen is produced in near stoichiometric amounts. In contrast, when Reaction A is fast relative to Reaction B, oxidized halogen species (chlorine and hypochlorous acid for chloride ion; bromide, tribromide ion, and hypobromous acid for bromide ion) are the principle reaction products. With iodide ion, no 1268 nm chemiluminescence was detected. Past studies have shown that iodine and iodate ion are the major end products of this system.