Spectral similarities and kinetic differences of two tomato plant peroxidase isoenzymes

The kinetic and spectral properties of peroxidases A and B from the dwarf tomato plant were compared. The absolute absorption spectra were essentially the same for peroxidases A and B and their derivatives. Peroxidases A and B had different pH optima with guaiacol as the hydrogen donor but essentially the same optimum when pyrogallol was the substrate. The substrate concentrations required for optimum activity were different not only for the different substrates but also for each isoenzyme. When pyrogallol was used as the substrate, peroxidases A and B were 80% active when assayed under conditions optimal for the other isoenzyme. When guaiacol was used as the substrate, peroxidase A was completely inactive when assayed under conditions optimal for peroxidase B. In this case the pH was not optimum and the H₂O₂ concentration was inhibitory. Similarly, peroxidase B retained only 9% of its peroxidase activity toward guaiacol when assayed under conditions optimum for peroxidase A. In this case the pH was not optimum and the H₂O₂ was limiting. A possible role for peroxidase isoenzymes is discussed.     

Comparison of Indole-3-acetic Acid Oxidation in Peroxidases from Rust-Infected Resistant Wheat Leaves

Seven peroxidase isozyme fractions were isolated from rust-infectedresistant wheat leaves by means of ammonium sulfate fractionation,pH precipitation, ion exchange chromatography and gel filtration.Three isozymes showed a single peroxidative band in the electrophoreticgels. The catalytic activity of the enzymes on non-physiologicalsubstrates was comparable to that of commercial horseradishperoxidase. When compared to isozyme 9, isozyme 10 had twicethe activity on guaiacol and eugenol but only one-fifth theoxidative activity on p-phenylenediamine and o-dianisidine.The IAA oxidation activity was compared among purified enzymefractions. Isozyme 10 was the only enzyme which could destroythe auxin without phosphate and manganese cofactors. All theother enzymes, including isozyme 9, showed the activity onlywhen both cofactors were present. The possible involvement ofthese peroxidases in IAA destruction in the resistant tissueis discussed. (Received June 14, 1984; Accepted October 15, 1984)     

Root growth inhibition by aluminum is probably caused by cell death due to peroxidase-mediated hydrogen peroxide production

Summary. The effect of aluminum on hydrogen peroxide production and peroxidase-catalyzed NADH oxidation was studied in barley roots germinated and grown between two layers of moistened filter paper. Guaiacol peroxidase activity significantly increased after 48h and was approximately two times higher after 72h in Al-treated roots. The oxidation of NADH was also significantly increased and, like guaiacol peroxidase activity, it was two times higher in Al-treated roots than in controls. Elevated H₂O₂ production was observed both 48 and 72h after the onset of imbibition in the presence of Al. Separation on a cation exchange column allowed the detection of two peaks with NADH peroxidase and H₂O₂ production activity. However, a difference between control and Al-treated plants was found only in one fraction, in which four times higher guaiacol peroxidase activity and five times higher NADH peroxidase activity were expressed and about three times more H₂O₂ was produced. One anionic peroxidase and three cationic peroxidases were detected in this fraction by native polyacrylamide gel electrophoresis. The anionic peroxidase was activated in the Al-treated root tips and also oxidized NADH but was detectable only after a long incubation time. Two of the cationic peroxidases were capable of oxidizing NADH and producing a significant amount of H₂O₂, but only one of these was activated by Al stress. The role of these peroxidases during Al stress in barley root tips is discussed.Correspondence and reprints: Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, 845 23 Bratislava, Slovakia.     

Iaa oxidase and polyphenol oxidase activities of peanut peroxidase isozymes

Four anionic isozymes (A₁, A₂, A₄ and A₅) from peanut cells in suspension medium possessed IAA oxidase and polyphenol oxidase activities. The specific activities of each of the enzymes differed among the 4 isozymes. The pH optima established in these assays for peroxidase was acidic, for IAA oxidase neutral and for polyphenol oxidase alkaline. All 4 isozymes had different K_m and V_max for the enzyme activities of peroxidase and polyphenol oxidase. The sigmoid kinetics from the IAA oxidase assays for the isozymes probably indicates an allosteric nature.     

Partial purification and kinetics of indoleacetic Acid oxidase from tobacco roots

Extracts from roots of Nicotiana tabacum L var. Bottom Special contain oxidative enzymes capable of rapid degradation of indoleacetic acid (IAA) in the presence of Mn²⁺ and 2, 4-dichlorophenol. Purification of IAA oxidase was attempted by means of ammonium sulfate fractionation and elution through a column of SE-Sephadex. Two distinct fractions, both causing rapid oxidation of IAA in the absence of H₂O₂, were obtained. One fraction exhibited high peroxidase activity when guaiacol was used as the electron donor; the other did not oxidase guaiacol. Both enzyme fractions caused similar changes in the UV spectrum of IAA; absorption at 280 mμ was reduced, while major absorption peaks appeared at 254 and 247 mμ. The kinetics of IAA oxidation by both fractions were followed by measuring the increase in absorption at 247 mμ. The peroxidase-containing fraction showed no lag or a slight lag which could be eliminated by addition of H₂O₂ (3 μmoles/ml). The peroxidase-free fraction showed a longer lag, but addition of similar amounts of H₂O₂ inhibited the rate of IAA oxidation and did not remove the lag. With purified preparations, IAA oxidation was stimulated only at low concentrations of H₂O₂ (0.03 μmole/ml). A comparison of K_m values for IAA oxidation by the peroxidase-containing and peroxidase-free fractions suggests that tobacco roots contain an IAA oxidase which may have higher affinity for IAA and may be more specific than the general peroxidase system previously described from other plant sources. A similar oxidase is present in commercial preparations of horseradish peroxidase. It is suggested that oxidation of IAA by horseradish peroxidase may be due to a more specific component.     

Partial purification and characterization of a copper-induced anionic peroxidase of sunflower roots

Treatment of 14-day-old sunflower seedlings with a toxic amount of copper (50 μM of CuSO₄) during 5 days caused significant increase in peroxidase activity in roots. Qualitative analysis of soluble proteins using native anionic PAGE followed by detection of peroxidase activity with guaïacol as electron donor in the presence of H₂O₂ revealed five stimulated peroxidases, named A1, A2, A3, A4, and A5. These peroxidases had differential behavior during the period of treatment. A1, A2, A3 and A4 were stimulated in the first period of stress, but rapidly suppressed at 72 h. A5 showed a progressive stimulation which was even increased at 120 h. A1 was partially purified, identified using liquid chromatography coupled to mass spectrometry (LC-MS/MS), and characterized. Effects of pH and temperature on its activity were determined with guaïacol as electron donor. Optima were obtained at pH 8 and at 40 °C. Analysis of substrate specificity showed that A1 was active on coniferyl alcohol but not on IAA. Enzymatic activity was inhibited by a high concentration of H₂O₂.     

Cadmium-induced microsomal membrane-bound peroxidases mediated hydrogen peroxide production in barley roots

The effect of cadmium on microsomal membrane-bound peroxidases and their involvement in hydrogen peroxide production was studied in barley roots. One anionic and two cationic peroxidases were detected, which were strongly activated by Cd treatment. Positive correlation was found between root growth inhibition and increased peroxidase, NADH oxidase activity and H₂O₂ generation in root microsomal membrane fraction of Cd-treated barley roots.     

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.     

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.     

Peroxidases of Solanum melongena leaves

Three major peroxidases, designated as A, B₂ and B₂ from Solanum melongena leaves have been reported. Peroxidases-A, -B₂ and -B₂ were considered to be true peroxidases on the basis of k₁:k₄ ratio. The pH optima for the three enzymes were found to be 7·0, 6·0 and 6.0 respectively. These peroxidases differ in their k₁:k₄ ratio, in the effect of pH on this ratio and in the uric acid/guaiacol and o-dianisidine/guaiacol activity ratio.     

Inertness of horseradish peroxidase to 2,4-dichlorophenoxyacetic acid

The possibility of mutual effects of 2,4-D and horseradish (Armoracia lapathifolia L.) peroxidase on each other has been explored by four procedures. (i) Compounds I, II, and III of horseradish peroxidase (HRP) and H₂O₂ were exposed to 2,4-D. (ii) Extracts from batchwise operations of HRP + H₂O₂ and 2,4-D were analyzed for oxidation products by means of thin layer chromatography. (iii) The velocity of the IAA oxidase reaction with HRP as catalyst, and (iv) K_m and V_s of the overall peroxidation of guaiacol by HRP + H₂O₂, were determined in the absence and presence of 2,4-D. The results failed to show any effect of 2,4-D; only at very high concentrations did 2,4-D slightly inhibit the oxidation of IAA by one isoperoxidase. It is concluded that 2,4-D does not promote growth in plants by hampering a peroxidase-catalyzed IAA oxidation. It seems probable that 2,4-D perturbs the isoperoxidase pattern by acting at some step prior to the release of the enzyme from its site of synthesis.     

Role of peroxidase in lignification of tobacco cells : I. Oxidation of nicotinamide adenine dinucleotide and formation of hydrogen peroxide by cell wall peroxidases

The two peroxidase isoenzyme groups (G_I and G_III) localized in the cell walls of tobacco (Nicotiana tabacum L.) tissues were compared with respect to their capacity for NADH-dependent H₂O₂ formation. Peroxidases of the G_III group are slightly more active than those of the G_I group when both are assayed under optimal conditions. This difference is probably not of major regulatory importance. NADH-dependent formation of H₂O₂ required the presence of Mn²⁺ and a phenol as cofactors. The addition of H₂O₂ to the reaction mixture accelerated subsequent NADH-dependent H₂O₂ formation. In the presence of both cofactors or Mn²⁺ alone, catalase oxidized NADH. However, if the cofactors were absent or if only dichlorophenol was present, catalase inhibited NADH oxidation. No H₂O₂ accumulation occurred in the presence of catalase. Superoxide dismutase inhibited NADH oxidation quite significantly indicating the involvement of the superoxide radical in the peroxidase reaction. These results are interpreted to mean that the reactions whereby tobacco cell wall peroxidases catalyze NADH-dependent H₂O₂ formation are similar to those proposed for horseradish peroxidase (Halliwell 1978 Planta 140: 81-88).     

Comparison of some kinetic parameters of peroxidase and IAA oxidase in the course of growth and differentiation of plant cells

An attempt is made to characterize the functional activity of the protein moleculo possessing both peroxidase and IAA oxidase activity by comparing the kinetic parameters for the two types of enzyme activity with regard to the following substrates: H₂O₂, benzidine, guaiacol and IAA. The curves expressing the dependence of the enzyme reaction velocity on the concentration of the enzyme or the substrate are different depending on the enzyme extract origin and the type of the substrate. It is established that the K_m of peroxidase for IAA decreases while its K_m for H₂O₂ increases during cell development. Both types of enzyme activity show similar pH and temperature dependence. The presented data show that IAA oxidase activity of the peroxidase develops as extension and differentiation of the root cells proceed. This is one of the possible mechanisms through which peroxidase may participate in the regulation of growth and differentiation of the primary root cells of maize (Zea mays L.)     

Isolation and characterization of wheat peroxidase isoenzyme B1

Two pure peroxidase isoenzymes B1 and D4 were isolated from the upper parts of 10-day-old wheat seedlings by means of gel and ion-exchange chromatography. Their MWs were 85000 and 24000 respectively. B1 was unstable and under various conditions it was converted to another isoenzyme, electrophoretically identical with D4. B1 contains about 40% of neutral sugars: 17.2% arabinose, 15.3% galactose, 5% glucose and traces of mannose. D4 is free of neutral sugars. None of the isoenzymes contained amino sugars. B1 oxidizes ferulic and p-coumaric acids. This oxidation has two pH optima of 4.4 and 5.4–5.6 and is inhibited by high concentrations of substrates, cyanide and azide. B1 oxidizes IAA in the presence of phenolic cofactor and Mn²⁺ ions. IAA oxidation has two pH optima of 4.5 and 5.6 and is inhibited by high substrate concentration, cyanide and azide, and by a number of indole derivatives. The main products of IAA oxidation are 3-methyleneoxindole and indole-3-methanol. o- and p- diphenols induce a lag period prior to IAA oxidation. Ferulic acid is oxidized during this lag period, probably to a dimer. B1 is able to produce H₂O₂ from oxygen. Mn²⁺ ions, a phenolic cofactor and an electron donor (IAA or NADH) are needed. B1 oxidizes α-keto-γ- methylmercaptobutyric acid to ethylene. D4 has a low peroxidatic activity and is inactive as an IAA oxidase. Thus B1 is probably an active cell wall-bound peroxidase isoenzyme, whereas D4 is its decomposition product.     

Gallic acid oxidation by turnip peroxidase

Both ellagic and gallic acids non competitively inhibited guaiacol oxidation by turnip peroxidase. The K_i values were 3 and 26 μm for ellagic and gallic acid respectively. Enzymatic oxidation of gallic acid by the isolated major turnip peroxidase was characterized with respect to spectral behaviour, affinity constant and pH effect. The K_m for H₂O₂ and gallic acid are 2.5 and 8.0 mM for turnip peroxidase. The pH optimum for gallic acid oxidation is about 6.5 and the rate constant k₄ decreased with the increase of pH in presence of both guaiacol and Gallic acid. When the gallic acid oxidation products were subjected to chromatographic analysis, it was found to be converted mainly to ellagic and an unknown quinone.     

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.     

Activation of indole-3-acetic acid oxidase from horseradish and Prunus by phenols and H2O2

The enzyme-catalysed oxidation of indole-3-acetic acid (IAA) was sytematically investigated with respect to enzyme source and cofactor influence using differential spectrophotometry and oxygen uptake measurement. Commercially-available horseradish peroxidase (HRP) and a peroxidase preparation from Prunus phloem showed identical catalytic properties in degrading IAA. There was no lag phase of IAA oxidation with any of the reaction mixtures tested. Monophenols exhibited a much stronger stimulatory effect than inorganic cofactors, but during the incubation of IAA the phenols were also gradually oxidised. Hydrogen peroxide (H₂O₂) in combination with monophenols accelerated peroxidation of the monophenol and IAA oxidation simutaneously. Since photometric determination of IAA was affected by oxidation products of dichlorophenol or phenol contamination of the enzyme preparation used, the standard IAA absorption measurements appear to be susceptible to methodological errors. Under certain incubation conditions a catalase-like activity of HRP during the course of IAA oxidation was noted and substrate inhibition was observed above 1.5 × 10^\s-4 M IAA. Some concepts concerning the mode of activation of the enzyme-catalysed IAA oxidation are deduced from the experimental results.     

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.     

Activities of Hydrogen Peroxide-Scavenging Enzymes during Low-Temperature Hardening of Potato Plants Transformed by the <Emphasis Type="Italic">desA</Emphasis> Gene of Δ12-Acyl-Lipid Desaturase

Activities of enzymes decomposing hydrogen peroxide (H₂O₂) under long exposure to hardening low temperatures and the effect of Δ12-acyl-lipid desaturase on these processes were studied on potato (Solanum tuberosum L., cv. Desnitsa), which typically represents cold-tolerant plants. We compared nontransformed plants (control) and the line transformed with the construction carrying the target desA gene of the mentioned desaturase from cyanobacterium Synechocystis sp. PCC (desA-licBM3 plants). The plants were hardened at 5°C for six days under illumination of 50 μmol/(m² s). The hardening was found to favor plant tolerance to the subsequent frost, and the desA-licBM3 plants exceed the controls in this property. Of the studied H₂O₂-scavenging enzymes, soluble type III peroxidases (guaiacol peroxidases) displayed the most activity, and type I peroxidase (ascorbate peroxidase) was the least active in the two potato lines over the hardening period. The activity of catalase increased twofold in the control and fourfold in the transformed plants in the first day of the hardening. However, the doubled catalase activity did not appear to compensate the H₂O₂ accumulation over this period. The recorded rise in catalase activity in the desA-licBM3 plants, together with the high activity of guaiacol peroxidases, favored lowering the hydrogen peroxide level in comparison with the initial values. For the first time, electrophoresis revealed two catalase isoforms, CAT1 and CAT2, in leaves of both potato lines. The significance of CAT1 was greater than that of CAT2 in the total catalase activity during the hardening period. It is concluded that, under the long-term cold hardening of potato plants, the content of hydrogen peroxide is determined by highly active guaiacol peroxidases and Class I catalase exerting energy-independent H₂O₂ decomposing. In this case, in the transformants that are rich in membrane lipids, where polyunsaturated fatty acids predominate, the activity of H₂O₂-scavenging enzymes increased significantly more than in the control, which is why the hardening of the transformants is more effective.     

Indole-3-Acetic Acid Control on Acidic Oat Cell Wall Peroxidases

Incubation of oat coleoptile segments with 40 μm indoleacetic acid (IAA) induced a decrease of 35–60% in peroxidase activity at the cell wall compartment. Treatment with IAA also produced a similar decrease in the oxidation of NADH and IAA at the cell wall. Isoelectric focusing of ionic, covalent, and intercellular wall peroxidase fractions showed that acidic isoforms (pI 4.0–5.5) were reduced preferentially by IAA treatment. Marked differences were found between acidic and basic wall isoperoxidases in relation to their efficacy in the oxidation of IAA. A peroxidase fraction containing acidic isoforms oxidized IAA with a V _max/s_0.5 value of 2.4 × 10⁻² min⁻¹· g fw⁻¹, 4.0 times higher than that obtained for basic peroxidase isoforms (0.6 × 10⁻² min⁻¹· g fw⁻¹). In contrast, basic isoforms were more efficient than acidic isoperoxidases in the oxidation of coniferyl alcohol or ferulic acid with H₂O₂ (5.6 and 2.1 times, respectively). The levels of diferulate and lignin in the walls of oat coleoptile segments were not altered by treatment with IAA. The decrease in cell wall peroxidase activity by IAA was related more to reduced oxidative degradation of the hormone than to covalent cell wall cross-linking. Received November 1, 1998; accepted December 14, 1998