Role of a flavonoid in the peroxide-dependent oxidation of glutathione catalysed by pea extracts

Crude pea extracts catalysed H₂O₂-dependent oxidation of glutathione but gel filtration through Sephadex G-25 abolished activity. Activity was restored by recombining the protein with a flavonoid [tentatively identified as kaempferol-3-(p-coumaroyltriglucoside)], isolated from peas. Protein fractions which supported peroxidase activity with pyrogallol as electron donor also supported H₂O₂-dependent oxidation of glutathione in the presence (but not in the absence) of the flavonoid with the concomitant consumption of O₂. In the absence of glutathione, active protein fractions also supported H₂O₂-dependent alteration of the spectral characteristics of the flavonoid. Some properties of these reactions were examined. It was concluded that these activities cannot be attributed to glutathione peroxidase and that a peroxidase belonging to EC class 1.11.1.7 is involved.     

Antioxidant defense enzymes in cell vacuoles of red beet roots

The vacuolar fraction isolated from red beet (Beta vulgaris L.) taproots was shown to contain the cyanide-sensitive Cu,Zn-activated superoxide dismutase (SOD; EC 1.15.1.1). The enzyme was represented by three isoforms located in the aqueous phase (in the vacuolar sap) without association to the membrane. Effective operation of SOD in plant cells, especially of its H₂O₂-sensitive molecular forms, is known to depend on peroxide-utilizing enzymes; this study revealed the existence of phenol-dependent peroxidase (EC 1.11.1.7) in the plant vacuoles. It was shown that the vacuolar peroxidase of red beet roots has a high affinity to benzidines and exhibits optimal activity at low pH (pH range 4–6 depending on substrate species). This peroxidase was represented by numerous molecular forms of acidic and basic nature. The isoenzyme composition of peroxidase in storage roots was highly labile: it depended on the duration of dormant period and comprised from 10 to 17 isoforms. The peroxidase isoforms were located both in the aqueous phase (vacuolar sap) and in the membrane, being weakly associated with the tonoplast. The presence of SOD and peroxidase in the vacuolar sap indicates the existence in vacuoles of an antioxidant defense system that protects vacuolar molecular structures against the impact of superoxide radicals and excessive amounts of H₂O₂.     

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.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

CYTOCHEMICAL LOCALIZATION OF PEROXIDATIC ACTIVITY OF CATALASE IN RAT HEPATIC MICROBODIES (PEROXISOMES)

Prominent staining of rat hepatic microbodies was obtained by incubating sections of aldehyde-fixed rat liver in a modified Graham and Karnovsky's medium for ultrastructural demonstration of peroxidase activity. The electron-opaque reaction product was deposited uniformly over the matrix of the microbodies. The microbodies were identified by their size, shape, presence of tubular nucleoids, and other morphologic characteristics, and by their relative numerical counts. The staining reaction was inhibited by the catalase inhibitor, aminotriazole, and by KCN, azide, high concentrations of H₂O₂, and by boiling of sections. These inhibition studies suggest that the peroxidatic activity of microbody catalase is responsible for the staining reaction. In the absence of exogenous H₂O₂ appreciable staining of microbodies was noted only after prolonged incubation. Addition of sodium pyruvate, which inhibits endogenous generation of H₂O₂ by tissue oxidases, or of crystalline catalase, which decomposes such tissue-generated H₂O₂, completely abolished microbody staining in the absence of H₂O₂. Neither diaminobenzidine nor the product of its oxidation had any affinity to bind nonenzymatically to microbody catalase and thus stain these organelles. The staining of microbodies was optimal at alkaline pH of 8.5. The biological significance of this alkaline pH in relation to the similar pH optima of several microbody oxidases is discussed. In addition to staining of microbodies, a heat-resistant peroxidase activity is seen in some of the peribiliary dense bodies. The relation of this reaction to the peroxidase activity of lipofuscin pigment granules is discussed.     

Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.)

Hydrogen peroxide (H₂O₂) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 50°C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent K_m = 40 millimolar) and H₂O₂ (apparent K_m = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed.     

Electron acceptor function of O2 in radical N-demethylation reactions catalyzed by hemeproteins

In aerobic solutions, O₂ consumption correlated well with N-demethylation of N,N-dimethyl-$\text{p}$-toluidine catalyzed by horseradish peroxidase, in the presence or absence of H₂O₂. In the absence of added H₂O₂, superoxide dismutase stimulated, and catalase inhibited, both reactions; in the presence of H₂O₂, argon inhibition of formaldehyde production increased with increasing concentration of horseradish peroxidase. These results provide evidence for competing reactions of the enzymatically-generated substrate radical: oxidation by O₂ increases formaldehyde production, while radical dimerization decreases the yield of this product. Implications of these findings for similar reactions catalyzed by microsomal cytochrome P-450 are suggested.     

H2O2 production and cytochrome c peroxidase activity in mitochondria isolated from the trypanosomatid hemoflagellate Crithidia fasciculata

H₂O₂ production by coupled mitochondrial fractions from the protozoan, Crithidia fasciculata, has been measured spectrophotometrically by the formation of the stable enzyme-substrate complex with yeast cytochrome c peroxidase. H₂O₂ formation was observed with succinate, l-α-glycerophosphate, l-proline, α-ketoglutarate, and with endogenous substrate. The maximum rate of H₂O₂ generation obtained with each substrate in the presence of antimycin A was about 10% of the state 4 rate of O₂ respiration, and only 1–2% of the carbonylcyanide m-fluorophenylhydrazone-uncoupled respiratory rate. Therefore, excess O₂ uptake due to the formation of H₂O₂ cannot satisfactorily account for the low ADP:O ratios previously reported.Cytochrome c peroxidase activity was measured in mitochondrial preparations by recording the decrease in absorbance at 550 nm during the oxidation of horse heart ferrocytochrome c which was observed after addition of H₂O₂. The distribution of activity after sonic disruption of mitochondrial preparations was that expected for a soluble enzyme. The activity was proportional to the amount of enzyme protein added, and was abolished by heating at 100 °C for 3 min. Total cytochrome c peroxidase activity in mitochondrial fractions isolated from C. fasciculata was calculated to be 0.3% that of isolated yeast mitochondria, but it is suggested that the in vivo activity may be considerably higher than this estimate.     

Light-dependent reduction of hydrogen peroxide by ruptured pea chloroplasts

Ruptured pea (Pisum sativum cv. Massey Gem) chloroplasts exhibited ascorbate peroxidase activity as determined by H₂O₂-dependent oxidation of ascorbate and ascorbate-dependent reduction of H₂O₂. The ratio of ascorbate peroxidase to NADP-glyceraldehyde 3-phosphate dehydrogenase activity was constant during repeated washing of isolated chloroplasts. This indicates that the ascorbate peroxidase is a chloroplast enzyme. The pH optimum of ascorbate peroxidase activity was 8.2 and the K_m value for ascorbate was 0.6 millimolar. Pyrogallol, glutathione, and NAD(P)H did not substitute for ascorbate in the enzyme catalyzed reaction. The enzyme was inhibited by NaN₃, KCN, and 8-hydroxyquinoline but not ZnCl₂ or iodoacetate. The ascorbate peroxidase activity of sonicated chloroplasts was inhibited by light but not in the presence of substrate concentrations of ascorbate.     

Analysis of peroxidase activity of rice (Oryza sativa) recombinant hemoglobin 1: Implications for in vivo function of hexacoordinate non-symbiotic hemoglobins in plants

In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H₂O₂ system at pH 6.0 and compared it to that from horseradish peroxidase (HRP). Results showed that the affinity of rice Hb1 for H₂O₂ was 86-times lower than that of HRP (K_m = 23.3 and 0.27 mM, respectively) and that the catalytic efficiency of rice Hb1 for the oxidation of guaiacol using H₂O₂ as electron donor was 2838-times lower than that of HRP (k_cat/K_m = 15.8 and 44 833 mM⁻¹ min⁻¹, respectively). Also, results from this work showed that rice Hb1 is not chemically modified and binds CO after incubation with high H₂O₂ concentration, and that it poorly protects recombinant Escherichia coli from H₂O₂ stress. These observations indicate that rice Hb1 inefficiently scavenges H₂O₂ as compared to a typical plant peroxidase, thus indicating that non-symbiotic Hbs are unlikely to function as peroxidases in planta.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Apolar distal pocket mutants of yeast cytochrome c peroxidase: Hydrogen peroxide reactivity and cyanide binding of the TriAla,TriVal, and TriLeu variants

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H₂O₂ compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H₂O₂ and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H₂O₂ and HCN while features that inhibit substrate ionization slow the reactions.     

Hydroxamate-activated peroxidases in potato tuber callus. Interaction with the determination of the cytochrome and the alternative pathways

In potato (Solatium tuberosum L. cv. Bintje and Doré) callus a very active hydrox-amate-stimulated NADH-dependent O₂-uptake develops during the growth of the callus, which is caused by a peroxidase. More than 95% of the peroxidase activity is found in the 40000 g supernatant. The total activity may be as high as 1000 times the respiratory acitivity of the callus tissue. At least two fractions, obtained by Sephadex gel filtration, can be distinguished showing this peroxidase activity, one of about 15 kDa and one > 50 kDa. The main properties of both fractions are: a) Hydroxamate at 0.2–0.5 mM gives half-maximal stimulation. Maximal stimulation is observed with 1–3 mM benzhydroxamate (BHAM) and 1–15 mM salicylhydroxamate (SHAM). Higher concentrations, especially of BHAM, give less or no stimulation. b) Hydroxamates are not consumed during the reaction. c) Both NADH and NADPH can serve as the electron donor for the reaction. The affinity for NAD(P)H is very low (K_m near 10 mM). In the absence of hydroxamates NAD(P)H is only slowly oxidized, with an even lower affinity. d) The peroxidase can carry out two reactions: an O₂-consuming and a H₂O₂-consuming reaction. In both reactions one NAD(P)H is consumed. In the first reaction H₂O₂ is formed which can be consumed in the second reaction, resulting in an overall stoichiometry of 2 NADH consumed for each O₂ molecule and in the production of H₂O. e) The reaction is completely blocked by cyanide, superoxide dismutase (EC 1.15.1.1) and (excess) catalase (EC 1.11.1.6), but not by antimycin A or azide. This peroxidase-mediated O₂-uptake might interfere with respiratory measurements. In experiments with isolated mitochondria this interference can be prevented by the addition of catalase to the reaction mixture. The use of high concentrations of hydroxamate is not allowed because of inhibitory effects on the cytochrome pathway. In intact callus tissue hydroxamates only stimulate O₂-uptake in the presence of exogenous NADH. In vivo the peroxidase does not appear to function in O₂-uptake, probably because of its localization (at least partly in the cell wall) and/or its low affinity for NADH. The use of hydroxamates in the determination of cytochrome and alternative pathway activity is discussed.     

The free amino acid tyrosine enhances the chlorinating activity of human myeloperoxidase

A key function of neutrophil myeloperoxidase (MPO) is the synthesis of hypochlorous acid (HOCl), a potent oxidizing agent that plays a cytotoxic role against invading bacteria and viruses at inflammatory sites and in phagosomes. MPO displayed a chlorinating activity preferably at acidic pH but at neutral pH MPO catalyzes mainly reactions of the peroxidase cycle. In the present work effects of tyrosine on the chlorinating activity of MPO were studied. At pH 7.4 we detected an increased HOCl production in the presence of tyrosine not only by the MPO-H₂O₂-Cl^- system but also in suspensions of zymosan-activated neutrophils. An excess of H₂O₂ is known to cause an accumulation of compound II of MPO blocking the generation of HOCl at neutral pH. As evidenced by spectral changes, tyrosine-induced activation of MPO to synthesize HOCl was due to the ability of tyrosine to reduce compound II back to the native state, thus accelerating the enzyme turnover. MPO-induced oxidation of tyrosine is relevant to what can be in vivo; we detected MPO-catalyzed formation of dityrosine in the presence of plasma under experimental conditions when tyrosine concentration was about three magnitudes of order less than the Cl⁻ concentration. At acidic pH formation of compound II was impaired in the presence of chloride and dityrosine couldn't be detected in plasma. In conclusion, the ability of tyrosine to increase the chlorinating activity of MPO at neutral pH and enhanced values of H₂O₂ may be very effective for the specific enhancement of HOCl production under acute inflammation.     

Intracellular catalase function: analysis of the catalatic activity by product formation in isolated liver cells

Intracellular catalase activity was measured in isolated rat hepatocytes by adding H₂O₂ under anaerobic conditions and measuring O₂ evolution. Hydrogen peroxide was introduced either by continuous infusion or by pulse injection. Continuous infusion at a rate similar to the endogenous H₂O₂ production rate provided results that 60–70% of the H₂O₂ was metabolized by the catalatic reaction. Comparison of rates of O₂ evolution to estimated rates of H₂O₂ metabolism obtained by the methanol-titration method (H. Sies and B. Chance, 1970, FEBS Lett.11, 172–176) indicated that the contribution of the peroxidatic reaction of catalase was small. The intracellular activity of glutathione peroxidase was estimated as the catalase-independent metabolism and used to determine the rate of intracellular H₂O₂ metabolism by the peroxidase. The results provide a quantitative basis for analysis of the physiological and toxicological aspects of H₂O₂ metabolism by liver.     

Purification, properties, and distribution of ascorbate peroxidase in legume root nodules

All aerobic biological systems, including N₂-fixing root nodules, are subject to O₂ toxicity that results from the formation of reactive intermediates such as H₂O₂ and free radicals of O₂. H₂O₂ may be removed from root nodules in a series of enzymic reactions involving ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase. We confirm here the presence of these enzymes in root nodules from nine species of legumes and from Alnus rubra. Ascorbate peroxidase from soybean nodules was purified to near homogeneity. This enzyme was found to be a hemeprotein with a molecular weight of 30,000 as determined by sodium dodecyl sulfate gel electrophoresis. KCN, NaN₃, CO, and C₂H₂ were potent inhibitors of activity. Nonphysiological reductants such as guaiacol, o-dianisidine, and pyrogallol functioned as substrates for the enzyme. No activity was detected with NAD(P)H, reduced glutathione, or urate. Ascorbate peroxidation did not follow Michaelis-Menten kinetics. The substrate concentration which resulted in a reaction rate of ½ V_max was 70 micromolar for ascorbate and 3 micromolar for H₂O₂. The high affinity of ascorbate peroxidase for H₂O₂ indicates that this enzyme, rather than catalase, is responsible for most H₂O₂ removal outside of peroxisomes in root nodules.     

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H₂O₂, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H₂O₂-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H₂O₂-induced liposome leakage was abolished by cyanide presumably competing with H₂O₂ for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H₂O₂ permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H₂O₂, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.     

Involvement of Molecular Oxygen in the Enzyme-Catalyzed NADH Oxidation and Ferric Leghemoglobin Reduction

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb⁺³), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O₂ was removed from the reaction system, but all were restored upon readdition of O₂. In the absence of exogenous electron carriers and in the presence of O₂ and excess NADH, FLbR catalyzed NADH oxidation with the generation of H₂O₂ functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction. The formation of H₂O₂ during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H₂O₂. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction were not constant but changed with time and with concentrations of NADH and O₂ in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb⁺³ by reaction intermediates. A study of the affinity of FLbR for O₂ showed that the enzyme required at least micromolar levels of dissolved O₂ for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.     

Purification and properties of a highly active catalase from cabbage loopers,Trichoplusia ni

Using ethanol-chloroform fractionation in conjunction with standard column chromatography techniques catalase has been purified to electrophoretic homogeneity from mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni. The specific activity of purified catalase was 2.2 × 10⁵ units (IU = 1 μmol H₂O₂ decomposed mg protein⁻¹ min⁻¹). The purified enzyme's native molecular weight was in the 247,000–259,000 Da range and was tetrameric with an apparent molecular weight of 63,000 Da for each subunit. In addition, biochemical properties of the enzyme were studied with emphasis on substrate specificity, kinetics, and the mechanism of inactivation by the irreversible inhibitor 3-amino-1,2,4-triazole (AT). The apparent K_m of the purified catalase for H₂O₂ was 54.2 mM and 50% of the maximal rate occurred at 16 mM H₂O₂. Purified catalase was ineffective in metabolizing organic hydroperoxides and, unlike other catalases, lacked peroxidase activity. Lastly, AT in the presence and absence of H₂O₂ was an effective inhibitor of catalase activity (I₅₀ = 100 mM) suggesting that a portion of the purified catalase was complexed with hydrogen peroxide in a compound 1 configuration.     

Hydrogen peroxide production by roots and its stimulation by exogenous NADH

H₂O₂ production by roots of young seedlings was monitored using a non-destructive in vivo assay at pH 5.0. A particularly high rate of H₂O₂ production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H₂O₂ production. In the soybean root H₂O₂ production can be stimulated 10-fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase-catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O₂ to H₂O₂. Comparative measurements showed that the NADH-induced H₂O₂ production of the roots resembles the H₂O₂-forming activity of horseradish peroxidase with respect to NADH and O₂ concentration requirements and sensitivity to inhibition by KCN, NaN₃, superoxide dismutase and catalase. NADH-induced H₂O₂ production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H₂O₂ production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH-induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O₂. It is concluded that the basal H₂O₂ production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O₂^?-producing oxidase.