Comparative study of substrates of fungal laccase

Coriolus versicolor, Pycnoporus cinnabarinus and Pycnoporus coccineus were grown under conditions to produce extracellular laccase. Prior to estimating enzyme activity, culture fluids were pretreated with catalase to destroy hydrogen peroxide and hence minimize peroxidase activity which might interfere with laccase determinations. Similar trends in enzyme assay were shown when colour reagents contained either syringaldazine or 3-dimethylaminobenzoic acid plus 3-methyl-2-benzothiazolone hydrasone as laccase substrates. Use of 2,2'-azino-bis-(3-ethylbenzothiazoline)-6-sulphonate (ABTS) as laccase substrate showed a different trend which was attributed to peroxidatic activity of the catalase using hydrogen peroxide generated by fungal oxidases. Peroxidatic activity was not observed with the other substrates.     

Kinetics and mechanisms of catalase in peroxisomes of the mitochondrial fraction

1. The primary intermediate of catalase and hydrogen peroxide was identified and investigated in peroxisome-rich mitochondrial fractions of rat liver. On the basis of kinetic constants determined in vitro, it is possible to calculate with reasonable precision the molecular statistics of catalase action in the peroxisomes. 2. The endogenous hydrogen peroxide generation is adequate to sustain a concentration of the catalase intermediate (p(m)/e) of 60-70% of the hydrogen peroxide saturation value. Total amount of catalase corresponds to 0.12-0.15nmol of haem iron/mg of protein. In State 1 the rate of hydrogen peroxide generation corresponds to 0.9nmol/min per mg of protein or 5% of the mitochondrial respiratory rate in State 4. 3. Partial saturation of the catalase intermediate with hydrogen peroxide (p(m)/e) in the mitochondrial fraction suggests its significant peroxidatic activity towards its endogenous hydrogen donor. A variation of this value (p(m)/e) from 0.3 in State 4 to 0 under anaerobic conditions is observed. 4. For a particular preparation the hydrogen peroxide generation rate in the substrate-supplemented State 4 corresponds to 0.17s(-1) (eqn. 6), the hydrogen peroxide concentration to 2.5nm and the hydrogen-donor concentration (in terms of ethanol) to 0.12mm. The reaction is 70% peroxidatic and 30% catalatic. 5. A co-ordinated production of both oxidizing and reducing substrates for catalase in the mitochondrial fraction is suggested by a 2.2-fold increase of hydrogen peroxide generation and a threefold increase in hydrogen-donor generation in the State 1 to State 4 transition. 6. Additional hydrogen peroxide generation provided by the urate oxidase system of peroxisomes (8-12nmol of uric acid oxidized/min per mg of protein) permits saturation of the catalase with hydrogen peroxide to haem occupancy of 40% compared with values of 36% for a purified rat liver catalase ofk(1)=1.7x10(7)m(-1).s(-1) and k'(4)=2.6x10(7)m(-1). s(-1)(Chance, Greenstein & Roughton, 1952). 7. The turnover of the catalase ethyl hydrogen peroxide intermediate (k'(3)) in the peroxisomes is initially very rapid since endogenous hydrogen peroxide acts as a hydrogen donor. k'(3) decreases fivefold in the uncoupled state of the mitochondria.     

Purification,characterization, and identification of a novel bifunctional catalase-phenol oxidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.     

Horseradish peroxidase-catalyzed aerobic oxidation and peroxidation of indole-3-acetic acid. I. Optical spectra.

A study of the indole-3-acetate reaction with horse-radish peroxidase, in the absence or presence of hydrogen peroxide, has been performed, employing rapid scan and conventional spectrophotometry. We present here the first clear spectral evidence, obtained on the millisecond time scale, indicating that at pH 5.0 and for high [enzyme/substrate] ratios peroxidase compound III is formed. Most, if not all, of the compound III is formed by oxygenation of the ferrous peroxidase. There is an inhibitory effect of superoxide dismutase and histidine on compound III formation which indicates the involvement of the active oxygen species superoxide and singlet oxygen. It is concluded that the oxidation of indole-3-acetate by horseradish peroxidase at pH 5.0 proceeds through compound III formation to the catalytically inactive forms P-670 and P-630. A reaction path in which the enzyme is directly reduced by indole-3-acetate might be involved as an initiation step. Rapid scan spectral data, which indicate differences in the formation and decay of enzyme intermediate compounds at pH 7.0, in comparison with those observed at pH 5.0, are also presented. At pH 7.0 compound II is a key intermediate in oxidation--peroxidation of substrate. Mechanisms of reactions consistent with the experimental data are proposed and discussed.     

A kinetic analysis of the catalase activity of myeloperoxidase

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.     

A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 x 10(6) M(-1) s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH(2) reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.     

Prostaglandin H synthase and hydroperoxides: peroxidase reaction and inactivation kinetics

The reaction kinetics of the peroxidase activity of prostaglandin H synthase have been examined with 15-hydroperoxyeicosatetraenoic acid and hydrogen peroxide as substrates and tetramethylphenylenediamine as cosubstrate. The apparent Km and Vmax values for both hydroperoxides were found to increase linearly with the cosubstrate concentration. The overall reaction kinetics could be interpreted in terms of an initial reaction of the synthase with hydroperoxide to form an intermediate equivalent to horseradish peroxidase Compound I, followed by reduction of this intermediate by cosubstrate to regenerate the resting enzyme. The rate constants estimated for the generation of synthase Compound I were 7.1 X 10(7) M-1 s-1 with the lipid hydroperoxide and 9.1 X 10(4) M-1 s-1 with hydrogen peroxide. The rate constants estimated for the rate-determining step in the regeneration of resting enzyme by cosubstrate were 9.2 X 10(6) M-1 s-1 in the case of the reaction with lipid hydroperoxide and 3.5 X 10(6) M-1 s-1 in the case of reaction with hydrogen peroxide. The intrinsic affinities of the synthase peroxidase for substrate (Ks) were estimated to be on the order of 10(-8) M for lipid hydroperoxide and 10(-5) M for hydrogen peroxide. These affinities are quite similar to the reported affinities of the synthase for these hydroperoxides as activators of the cyclooxygenase. The peroxidase activity was found to be progressively inactivated during the peroxidase reaction. The rate of inactivation of the peroxidase was increased by increases in hydroperoxide level, and decreased by increases in peroxidase cosubstrate. The inactivation of the peroxidase appeared to occur by a hydroperoxide-dependent process, originating from synthase Compound I or Compound II.     

Identification of uronic acid oxidase in plant peroxidase preparations

Commercial plant peroxidase preparations contained a uronic acid oxidase, separable from the peroxidase activity by ion exchange chromatography. The partially purified enzyme, devoid of peroxidase, oxidized hexuronic acids, with the greatest activity for D-glucuronic acid, whereas other aldoses were not substrates. The immediate products of reaction of D-glucuronic acid with oxygen were hydrogen peroxide and a D-glucarolactone, which was a very strong inhibitor of β-glucuronidase and believed to be the 1,5-lactone. The sensitivity to sulphite inhibition suggests that the enzyme is a flavoprotein.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Decrease in heart mitochondrial creatine kinase activity due to oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.     

Glutathione peroxidase in lens and a source of hydrogen peroxide in aqueous humour

1. Glutathione peroxidase has been demonstrated in cattle, rabbit and guineapig lenses. 2. The enzyme will oxidize GSH either with hydrogen peroxide added at the start of the reaction or with hydrogen peroxide generated enzymically with glucose oxidase. 3. No product other than GSSG was detected. 4. Oxidation of GSH can be coupled with oxidation of malate through the intermediate reaction of glutathione reductase and NADPH₂. 5. Traces of hydrogen peroxide are present in aqueous humour: it is formed when the ascorbic acid of aqueous humour is oxidized. 6. Hydrogen peroxide will diffuse into the explanted intact lens and oxidize the contained GSH. The addition of glucose to the medium together with hydrogen peroxide maintains the concentration of lens GSH. 7. Glutathione peroxidase in lens extracts will couple with the oxidation of ascorbic acid. 8. It is suggested that, as there is only weak catalase activity in lens, glutathione peroxidase may act as one link between the oxygen of the aqueous humour and NADPH₂.     

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase‐like reactivity of Bacillus pumilus catalase monitored by X‐ray crystallography and EPR spectroscopy

Heme‐containing catalases and catalase‐peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase‐peroxidase led us to investigate the enzyme for comparison with other catalase‐peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 20 s^?1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2‐chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low‐spin conversion of the Fe^III high‐spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.     

Analysis of the peroxidatic mode of action of catalase

Catalase is an enzyme which can function either in the catabolism of hydrogen peroxide or in the peroxidatic oxidation of small substrates such as ethanol, methanol, or elemental mercury (Hg0). It has been reported that native catalase can peroxidatically oxidize larger organic molecules (e.g. L-dopa) and that catalase maintained at alkaline pH for various lengths of time demonstrates an increase in peroxidase activity using guaiacol as substrate. We have shown, by using two distinct methods of H2O2 introduction for measuring peroxidase activity, that native catalase shows no peroxidatic activity toward these larger organic molecules. We have also shown, through the use of these peroxidase assays and by enzyme absorption spectra, that the peroxidase activity attributed to catalase maintained at alkaline pH is a catalytic but not enzymatic activity associated with a hematin group attached to a denatured catalase monomer. Possible mechanisms for the catalytic and peroxidatic modes of action of catalase involving hydride-ion transfer are discussed.     

A Fluorometric Assay for Detection of Lysyl Oxidase Enzyme Activity in Biological Samples

Lysyl oxidase catalyzes the final known enzymatic step required for collagen and elastin cross-linking in the biosynthesis of normal mature functional insoluble extracellular matrices. In addition, lysyl oxidase has been identified as a possible tumor suppressor. Lysyl oxidase activity in biological samples is traditionally and most reliably assessed by tritium release end-point assays using radiolabeled collagen or elastin substrates involving laborious vacuum distillation of the released tritiated water. In addition, a less sensitive fluorometric method exists that employs nonpeptidyl amine lysyl oxidase substrates and measures hydrogen peroxide production with horseradish peroxidase coupled to homovanillate oxidation. The present study describes a more sensitive fluorescent assay for lysyl oxidase activity that utilizes 1,5-diaminopentane as substrate, and released hydrogen peroxide is detected using Amplex red in horseradish peroxidase-coupled reactions. This method allows the detection of 40 ng of enzyme per 2 ml assay at 37 degrees C and is 7.5 times more sensitive than the currently available fluorometric assay for enzyme activity. This method eliminates the interference that occurs in some biological samples and can be successfully used to detect lysyl oxidase activity in cell culture experiments.     

ON THE INACTIVATION OF ASCORBIC ACID OXIDASE

1. In the absence of protective agents, highly purified ascorbic acid oxidase is rapidly inactivated during the enzymatic oxidation of ascorbic acid under optimum experimental conditions. This inactivation, called reaction inactivation to distinguish it from the loss in enzyme activity that frequently occurs in diluted solutions of the oxidase prior to the reaction, is indicated by incomplete oxidation of the ascorbic acid as measured by oxygen uptake; i.e., "inactivation totals." 2. A minor portion of the reaction inactivation appears to be due to environmental factors such as rate of shaking of the manometers, pH of the system, substrate concentration, and oxidase concentration. The presence of inert protein (gelatin) in the system ameliorates the environmental inactivation to a considerable extent, and variation of the above factors in the presence of gelatin has much less effect on the inactivation totals than in the absence of gelatin. 3. A major portion of the reaction inactivation of the oxidase appears to be due to some factor inherent in the ascorbic acid-ascorbic acid oxidase-oxygen system, possibly a highly reactive "redox" form of oxygen other than H₂O₂ or H₂O. The inactivation cannot be attributed to dehydroascorbic acid, the oxidation product of ascorbic acid. 4. Small amounts of native catalase, native peroxidase, native or denatured methemoglobin, and hemin when added to the system, markedly protect the oxidase against inactivation. Cytochrome c has no such protective action. Likewise proteins such as egg albumin, gelatin, denatured catalase, or denatured peroxidase show no such protective action. 5. None of the protective agents mentioned above affect the initial rate of oxygen uptake or change the total oxygen absorbed for complete oxidation of the ascorbic acid, and hence do not act by removal of hydrogen peroxide, per se. 6. Sodium azide and hydroxylamine hydrochloride which inhibit catalase and peroxidase activity also inhibit the protective action of these iron-porphyrin enzymes.     

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.     

Coupling of Dopamine Oxidation (Monoamine Oxidase Activity) to Glutathione Oxidation Via the Generation of Hydrogen Peroxide in Rat Brain Homogenates

Abstract: Homogenates of perfused rat brain generated oxidized glutathione from reduced glutathione during incubation with dopamine or serotonin. This activity was blocked by pargyline. a monoamine oxidase inhibitor, or by catalase, a scavenger of hydrogen peroxide. These results demonstrate formation of hydrogen peroxide by monoamine oxidase and the coupling of the peroxide to glutathione peroxidase activity. Oxidized glutathione was measured fluorometrically via the oxidation of NADPH by glutathione reductase. In the absence of added dopamine or serotonin, a much smaller amount of reduced glutathione was oxidized: this activity was blocked by catalase, but not by pargyline. Therefore, endogenous production of hydrogen peroxide, not linked to monoamine oxidase activity, was present. These results indicate that glutathione peroxidase (linked to hexose monophosphate shunt activity) can function to eliminate hydrogen peroxide generated by monoamine oxidase and other endogenous sources in aminergic neurons.     

On the stoichiometry of the oxidase and monooxygenase reactions catalyzed by liver microsomal cytochrome P-450. Products of oxygen reduction

This laboratory has recently reported that, in a reconstituted enzyme system containing alcohol-induced isozyme 3a of liver microsomal cytochrome P-450, the sum of acetaldehyde generated by the monooxygenation of ethanol and of hydrogen peroxide produced by the NADPH oxidase activity is inadequate to account for the O2 and NADPH consumed. Studies on the stoichiometry have revealed the occurrence of an additional reaction involving an overall 4-electron transfer to molecular oxygen which is presumed to yield water: O2 + 2 NADPH + 2H+----2 H2O + 2 NADP+. The occurrence of a peroxidase reaction in which free H2O2 is reduced to water by NADPH was ruled out. When the 4-electron oxidase activity is taken into account, measurements of NADPH oxidation and O2 consumption are in accord with the amounts of products formed in the presence of various P-450 isozymes, either in the absence or presence of typical substrates, including those which undergo hydroxylation, N- or O-demethylation, or oxidation of hydroxymethyl to aldehyde groups. Of the substrates examined, some had no effect on the oxidase reaction yielding hydrogen peroxide or the 4-electron oxidase reaction, some were inhibitory, and some were stimulatory, but the same substrate did not necessarily have the same effect on the two reactions.     

Singlet oxygen formation by a peroxidase, H2O2 and halide system.

Evidence for singlet oxygen formation has been obtained for the lactoperoxidase, H2O2 and bromide system by monitoring 2,3-diphenylfuran and diphenylisobenzofuran oxidation, O2 evolution, and chemiluminescence. This could provide an explanation for the cytotoxic and microbicidal activity of peroxidases and polymorphonuclear leukocytes. Evidence for singlet oxygen formation included the following. (a) Chemiluminescence accompanying the enzymic reaction was doubled in a deuterated buffer and inhibited by singlet oxygen traps. (b) The singlet oxygen traps, diphenylfuran and diphenylisobenzofuran, were oxidized to their known singlet oxygen oxidation products in the presence of lactoperoxidase, hydrogen peroxide and bromide. (c) The rate of oxidation of diphenylfuran and diphenylisobenzofuran was inhibited when monitored in the presence of known singlet oxygen traps or quenchers. (d) Oxygen evolution from the enzymic reaction was inhibited by singlet oxygen traps but not by singlet oxygen quenchers. (e) The traps or quenchers which were effective inhibitors in the experiments above did not inhibit peroxidase activity, were not competitive peroxidase substrates and did not react with the hypobromite intermediate since they did not inhibit hydrogen peroxide consumption by the enzyme. Using these criteria, various biological molecules were tested for their reactivity with singlet oxygen. Furthermore, by studying their effect on oxygen release by the enzymic reaction, it could be ascertained whether they were acting as singlet oxygen traps or quenchers.     

Peroxide metabolism in the cestodes Hymenolepis diminuta and Moniezia expansa

The enzymes of hydrogen peroxide metabolism have been investigated in the cestodes H. diminuta and M. expansa. Neither catalase, lipoxygenase, glutathione peroxidase, NADH peroxidase nor NADPH peroxidase could be detected in homogenates of either species. However, both H. diminuta and M. expansa possessed a peroxidase which had a high affinity for reduced cytochrome c. The peroxidase was characterized by substrate and inhibitor studies and cell fractionation showed the enzyme to be located in the mitochondrial membrane fraction. The peroxidase could act as a substitute for catalase, by destroying metabolic hydrogen peroxide. Appreciable superoxide dismutase activity was found in M. expansa and H. diminuta and it is possible that this enzyme is the source of helminth hydrogen peroxide.