Peroxidase-catalyzed Reactions Research Articles

Controlled Delivery of H2O2: A Three-Enzyme Cascade Flow Reactor for Peroxidase-Catalyzed Reactions.

Peroxidases are promising catalysts for oxidation reactions, yet their practical utility has been hindered by the fact that they require hydrogen peroxide (H2O2), which at high concentrations can cause deactivation of enzymes. Practical processes involving the use of peroxidases require the frequent addition of low concentrations of H2O2. In situ generation of H2O2 can be achieved using oxidase-type enzymes. In this study, a three-enzyme cascade system comprised of a H2O2 generator (glucose oxidase (GOx)), H2O2-dependent enzymes (chloroperoxidase (CPO) or horseradish peroxidase (HRP)), and a H2O2 scavenger (catalase (CAT)) was deployed in a flow reactor. Immobilization of the enzymes on a graphite rod was achieved through electrochemically driven physical adsorption, followed by cross-linking with glutaraldehyde. Modeling studies indicated that the flow in the reactor was laminar (Reynolds number, R e < 2000) and was nearly fully developed at the midplane of the annular reactor. Immobilized CAT and GOx displayed good stability, retaining 79% and 84% of their initial activity, respectively, after three cycles of operation. Conversely, immobilized CPO exhibited a considerable reduction in activity after one use, retaining only 30% of its initial activity. The GOx-CAT-GRE system enabled controlled delivery of H2O2 in a more stable manner with a 4-fold enhancement in the oxidation of indole compared to the direct addition of H2O2. Using CPO in solution coupled with GOx-CAT-GRE yields of 90% for the oxidation of indole to 2-oxyindole and of 93% and 91% for the chlorination of thymol and carvacrol, respectively.

Green production of low-molecular-weight xylooligosaccharides from oil palm empty fruit bunch via integrated enzymatic polymerization and membrane separation for purification

For xylooligosaccharides (XOS) production from agricultural residues, highly concentrated xylan liquor (116 g/L) was produced by crushing an oil palm empty fruit bunch (EFB) via continuous hydrothermal treatment and subsequent membrane-based concentration on a pilot scale. The yield of xylan extracted from the raw EFB was 53.7%. To produce XOS with a low degree of polymerization, xylanase-catalyzed hydrolysis was performed and the sum of xylobiose and xylotriose to extracted xylan was 81.6% under lab-scale optimal conditions and 69.3% under pilot-scale conditions. In addition, the crude XOS could be purified through a novel approach involving enzyme-mediated radical polymerization and membrane-based separation, while minimizing XOS loss and waste generation. As a result, 50.2% of the total phenolic compounds in the crude XOS material could be polymerized and precipitated through peroxidase-catalyzed reactions, and an additional 22.6% were removed using the membrane. Furthermore, the amount of activated carbon consumed and the XOS recovery rate after phenolic compound removal via the adsorption method using activated carbon were evaluated and compared with the integrated enzyme-membrane purification results. Consequently, this study provides an ecofriendly biomass refinery process for low-molecular-weight XOS production and purification that produces few toxic substances and little waste.

Exploration and quantification of ascorbate affecting peroxidase-catalyzed chromogenic reactions with a recirculating-flow catalysis detection system.

The use of a recirculating-flow catalysis detection system (RFCD) explored competition and the influence of ascorbic acid (AsA) in peroxidase (POD)-catalyzed reactions. The study identified that AsA is neither the inhibitor of POD nor could directly deplete H2O2; it directly reacts with chromogenic products to form colorless intermediates, which can react with H2O2 to again rapidly re-generate the chromogenic products. If using the reactions (trinder reactions or enzyme-linked reactions) to determine POD activity (EPOD), substrates or analytes, the interference of concomitant AsA should be removed and the conclusions have significance for oxidase/POD catalyzed reactions. In addition, the RFCD system was also used to simultaneously determine EPOD and AsA.

Flavonoid composition, cellular antioxidant activity and (myelo)peroxidase inhibition of a Bryonia alba L. (Cucurbitaceae) leaves extract.

The aim of the present study consisted in the isolation of flavonoids from the leaves of Bryonia alba L. and evaluation of their antioxidant activity and inhibition on peroxidase-catalysed reactions. Flavonoids were isolated by preparative HPLC-DAD and their structures were elucidated by MS and NMR. Inhibitory effect was tested by the horseradish peroxidase and the myeloperoxidase assays. Cellular antioxidant assays consisted in testing the inhibitory activity on the reactive oxygen species released upon activation of neutrophils freshly isolated ex vivo from equine blood and of human monocytes-derived macrophages in vitro. Whole organism toxicity was assessed on zebrafish larvae. Four flavonoids (lutonarin, saponarin, isoorientin and isovitexin) were isolated. The performed assays showed significant antioxidant activity and inhibition for the peroxidase-catalysed reactions. Absence of cellular and zebrafish toxicity was confirmed. Bryonia alba L. leaves are particularly interesting for their flavonoids content and showed significant inhibitory effect on peroxidase-catalysed oxidation of substrates (Amplex Red and L012), as well as antioxidant/antiradical activity, proving that this species has a medicinal potential. Moreover, the present study highlights the absence of the toxicity of these leaves and offers though a novel perspective on the species, previously known as being toxic.

Influence of NOM and SS on the BPA removal via peroxidase catalyzed reactions: Kinetics and pathways

Abstract In the present study, horseradish peroxidase (HRP) was employed to removal bisphenol A (BPA) with the presence of natural organic matter (NOM) and suspended solids (SS). Humic acid (HA) and kaolin have been selected as the typical NOM and SS in the experiment. According to the results, HA has shown a very interesting performance, which has both inhibitory and facilitation effects on BPA removal. For the low enzyme concentration (20 u/L and 50 u/L), small amount of HA could facilitate the BPA removal, while large amount of HA could inhibit the BPA removal. For the higher enzyme concentration (100–300 u/L), the addition of HA reduced the removal transformation of BPA and the inhibition effect of HA raised with the increase of HA concentrations. The facilitation nature of HA on enzyme catalytic processes can be attributed to the mitigation of enzyme inactivation, which has been proven by HRP inactivation rate. The inhibitory effect could be caused by the inhibition effect of HA on the enzyme activity, which has been suggested by both kinetic study and enzyme inactivation analysis. Kaolin showed a negative effect on BPA removal by HRP catalyzed reactions due to the inhibition effect of kaolin on HRP activity. The results have important implications for the engineering design by employing HRP in water and waste water treatment.

Organoselenium Compounds as Potential Neuroprotective Therapeutic Agents

Selenium plays its physiological chemistry in mammalian living cells as the selenol group of a selenocysteinyl residue found in few numbers of selenoproteins. These proteins have antioxidant properties and catalyze redox reactions, for instance, the selenol-mediated decomposition of peroxides (glutathione peroxidase catalyzed reactions) or the selenol-thiol-mediated reduction of disulfide bonds (the thioredoxin reductase catalyzed reactions). Organochalcogens can mimic the glutathione peroxidase activity (GPx-like activity) by diverse mechanisms. Ebselen and diphenyl diselenide are two types of organoselenium compounds that have been extensively studied in the literature because they posses interesting biochemical and pharmacological properties. They can interact with reactive species (peroxynitrite, peroxides) and have anti-inflammatory properties. Ebselen was used with borderline efficacy in human trials associated with brain ischemia/reperfusion. Experimental models confirmed the neuroprotective effects of ebselen and diselenides in a variety of in vitro and in vivo models of neurotoxicity, including those associated with brain ischemia and acidosis. Ebselen is registered as a safe drug, but it has not yet been approved for the treatment of brain pathologies. In fact, the major problem with organoselenium compounds is that they have no specific target, but modulate general physiological/pathological processes (e.g., inflammatory response/oxidative stress). Thus, the future of organoselenium compounds as potential therapeutic agent will require the synthesis of target-directed molecules. Keywords: Organochalcogens, neuroprotection and lipid peroxidation inhibition.

Corking Nitrogen-Doped Carbon Nanotube Cups with Gold Nanoparticles for Biodegradable Drug Delivery Applications.

Carbon nanomaterials have been proposed as effective drug delivery devices; however their perceived biopersistence and toxicological profile may hinder their applications in medical therapeutics. Nitrogen doping of carbon nanotubes results in a unique "stacked-cup" structure, with cups held together through van der Waals forces. Disrupting these weak interactions yields individual and short-stacked nanocups that can subsequently be corked with gold nanoparticles, resulting in sealed containers for delivery of cargo. Peroxidase-catalyzed reactions can effectively uncork these containers, followed by complete degradation of the graphitic capsule, resulting in effective release of therapeutic cargo while minimizing harmful side effects. The protocols reported herein describe the synthesis of stacked nitrogen-doped carbon nanotube cups followed by effective separation into individual cups and gold nanoparticle cork formation resulting in loaded and sealed containers.

Physicochemical Changes of Few-Layer Graphene in Peroxidase-Catalyzed Reactions: Characterization and Potential Ecological Effects.

The environmental implications of graphene have received much attention, however, little is known about how graphene affects or may be affected by the enzymatic reactions that are critically involved in natural organic matter transformation processes. We conducted experiments to examine the role of few-layer graphene (FLG) in the reaction system of tetrabromobisphenol A (TBBPA) mediated by horseradish peroxidase (HRP). We found that TBBPA was transformed by HRP into two products that were likely formed from coupling of two TBBPA radicals via interaction of an oxygen atom on one radical and a propyl-substituted aromatic carbon atom on the other. Presence of FLG greatly increased the reaction rate by protecting HRP from inactivation. Direct reactions between TBBPA radicals and FLG were unequivocally evidenced using (14)C labeling and the characteristic photoelectron response of bromine contained in TBBPA. The thickness, size, and aggregation profile of FLG was modified by the reaction as shown by multiple characterization tools. Assessment using Daphnia magna revealed a substantial decrease in the bioaccumulation and toxicity of the FLG after being modified. The data provides the first evidence that FLG can be modified in HRP-mediated reactions and indicates that such modifications may have strong implications in its ecological effects.

Scavenging of Free-Radical Metabolites of Aniline Xenobiotics and Drugs by Amino Acid Derivatives: Toxicological Implications of Radical-Transfer Reactions

We investigated a novel scavenging mechanism of arylamine free radicals by poly- and monoaminocarboxylates. Free radicals of arylamine xenobiotics and drugs did not react with oxygen in peroxidase-catalyzed reactions; however, they showed marked oxygen uptake in the presence of an aminocarboxylate. These free-radical intermediates were identified using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and electron paramagnetic resonance (EPR) spectrometry. Diethylenetriaminepentaacetic acid (DTPA), a polyaminocarboxylate, caused a concentration-dependent attenuation of N-centered radicals produced by the peroxidative metabolism of arylamines with the subsequent formation of secondary aliphatic carbon-centered radicals stemming from the cosubstrate molecule. Analogously, N,N-dimethylglycine (DMG) and N-methyliminodiacetate (MIDA), but not iminodiacetic acid (IDA), demonstrated a similar scavenging effect of arylamine-derived free radicals in a horseradish peroxidase/H2O2 system. Using human promyelocytic leukemia (HL-60) cell lysate as a model of human neutrophils, DTPA, MIDA, and DMG readily reduced anilinium cation radicals derived from the arylamines and gave rise to the corresponding carbon radicals. The rate of peroxidase-triggered polymerization of aniline was studied as a measure of nitrogen-radical scavenging. Although, IDA had no effect on the rate of aniline polymerization, this was almost nullified in the presence of DTPA and MIDA at half of the molar concentration of the aniline substrate, whereas a 20 molar excess of DMPO caused only a partial inhibition. Furthermore, the yield of formaldehyde, a specific reaction endproduct of the oxidation of aminocarboxylates by aniline free-radical metabolites, was quantitatively determined. Azobenzene, a specific reaction product of peroxidase-catalyzed free-radical dimerization of aniline, was fully abrogated in the presence of DTPA, as confirmed by GC/MS. Under aerobic conditions, a radical-transfer reaction is proposed between aminocarboxylates and arylamine free radicals via the carboxylic group-linked tertiary nitrogen of the deprotonated amino acid derivatives. These findings may have significant implications for the biological fate of arylamine xenobiotic and drug free-radical metabolites.

Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine

Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5 × 10(-7) M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory.

Photo-oxidation-induced inactivation of the selenium-containing protective enzymes thioredoxin reductase and glutathione peroxidase

Singlet oxygen (1O2) is a reactive oxygen species generated during photo-oxidation, inflammation, and via peroxidase-catalyzed reactions (e.g., myeloperoxidase and eosinophil peroxidase). 1O2 oxidizes the free amino acids Trp, Tyr, His, Cys, and Met, and those species present on peptides/proteins, with this resulting in modulation of protein structure and function. Impairment of the activity of antioxidant enzymes may be of relevance to the oxidative stress observed in a number of pathologies involving either light exposure or inflammation. In this study, the effects of 1O2 on glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) activity, including the mechanisms of their inactivation, were investigated. Exposure of GPx or TrxR, either as purified proteins or in cell lysates, to Rose Bengal and visible light (an established source of 1O2) resulted in significant, photolysis time-dependent reductions in enzyme activity (10–40%, P<0.05). More extensive inhibition (ca. 2-fold) was detected when the reactions were carried out in D2O, consistent with the intermediacy of 1O2. No additional inhibition was detected after the cessation of photolysis, eliminating a role for photo-products. Methionine, which reacts rapidly with 1O2 (k∼107M−1 s−1), significantly reduced photo-inactivation at large molar excesses, presumably by acting as an alternative target. Reductants (NaBH4, DTT, GSH, or NADPH) added after the cessation of 1O2 formation were unable to reverse enzyme inactivation, consistent with irreversible enzyme oxidation. Formation of nonreducible protein aggregates and/or fragments was detected for both photo-oxidized GPx and TrxR by SDS-PAGE. An oxidant concentration-dependent increase in protein carbonyls was detected with TrxR but not GPx. These studies thus demonstrate that the antioxidant enzymes GPx and TrxR can be irreversibly inactivated by 1O2.

Oscillating chemiluminescence systems: state of the art

Oscillating chemiluminescence (CL) was reported for the first time about 30 years ago. Since then several systems based on addition of a chemiluminescent reagent to a known oscillator system or based on the light emitting features of one component of the oscillating system, have been described. This information, scattered in the scientific literature, is compiled in the present paper. Several oscillating CL systems, including those based on Belousov-Zhabotinskii and Orban oscillators, or horseradish peroxidase-catalyzed reactions, among others, are critically presented. The application of this type of oscillatory systems is also discussed, in analytical chemistry and for educational purposes.

The role of hypothiocyanous acid (HOSCN) in biological systems

Hypohalous acids (HOX), produced by peroxidase-catalysed reactions of halide and pseudohalide ions with H2O2, play an important role in the human immune system. However, there is compelling evidence that these oxidants also mediate host tissue damage and contribute to the progression of a number of inflammatory diseases. Although it is well established that significant amounts of hypothiocyanous acid (HOSCN) are formed under physiological conditions, the reactions of this oxidant with host biological systems are relatively poorly characterized. It is generally accepted that HOSCN is a mild oxidant that reacts selectively with thiols. However, it is becoming increasingly recognized that this selectivity can result in the induction of significant cellular damage, which may contribute to disease. This review will outline the formation and reactivity of HOSCN and the role of this oxidant in biological systems.

Catalytic oxidative degradation of s-triazine and phenoxyalkanoic acid based herbicides with metalloporphyrins and hydrogen peroxide: Identification of two distinct reaction schemes

Oxidative degradation of the herbicides atrazine ( 1), atraton ( 2), ametryn ( 3) and mecoprop ( 4), was carried out with hydrogen peroxide and metalloporphyrins as catalysts. Two different reaction conditions were studied, the first involving Mn(TDCPP)Cl in an aprotic solvent with buffer ( S-I), and the second using Fe(TPFPP)Cl in a protic solvent ( S-II). Reaction products were characterized, and based on these it is shown that there are two distinct reaction schemes. In the case of the S-I conditions, it is suggested that the s-triazines were oxidized through hydroxylation of the alkyl side chains followed by dealkylation, while S-II was ineffective for these reactions. In contrast, mecoprop, was oxidized with high efficiency by S-II, leading to decarboxylation and further oxidation, while in the presence of S-I, low substrate conversion was observed, and reaction resulted mainly from oxidation at the benzyl position. Sulfoxidation of ametryn was observed with both systems. The different reactivity shown by the two systems supports the involvement of different reactive species, which we assign to the oxo and hydroperoxy complexes. These routes show similarities with metabolic pathways, with the reactivity pattern of S-I analogous to the reported metabolism of these pollutants with cytochrome P450 enzymes, while S-II catalyses mecoprop decarboxylation via a similar pathway to that seen with peroxidase catalysed reactions.

Effect of thione—thiol tautomerism on the inhibition of lactoperoxidase by anti-thyroid drugs and their analogues

The keto-enol type tautomerism in anti-thyroid drugs and their selenium analogues are described. The commonly used anti-thyroid drug methimazole exists predominantly in its thione form, whereas its selenium analogue exists in a zwitterionic form. To understand the effect of thione/thiol and selone/selenol tautomerism on the inhibition of peroxidase-catalysed reactions, we have synthesized some thiones and selones in which the formation of thiol/selenol forms are blocked by different substituents. These compounds were synthesized by a carbene route utilizing an imidazolium salt. The crystal structures of these compounds reveal that the C=Se bonds in the selones are more polarized than the C=S bonds in the corresponding thiones. The structures of selones were studied in solution by NMR spectroscopy and the 77Se NMR chemical shifts for the selones show large upfield shifts in the signals, confirming their zwitterionic structures in solution. The inhibition of lactoperoxidase by the synthetic thiones indicates that the presence of a free N-H moiety is essential for an efficient inhibition. In contrast, such moiety is not required for an inhibition by the selenium compounds.

Initiation and inhibition of free-radical processes in biochemical peroxide systems: A review

The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase- and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Coupled peroxidase-catalyzed oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (Ki, micromol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (alpha, M(-1)), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).

The prospects for peroxidase-based biorefining of petroleum fuels

Peroxidases have many potential uses for biotechnological processes. In this review, peroxidase-catalyzed reactions potentially applicable to the petroleum industry are described. Although peroxidases are attractive catalysts for desulfurization, aromatic oxidation and asphaltene transformation, there are important issues that must be overcome before any industrial application can be considered. The opportunities and challenges of enzymatic petroleum biorefining are documented and discussed, with emphasis on the available tools to design a biocatalyst with appropriate performance for the oil and other industries.

Major chorion proteins and their crosslinking during chorion hardening in Aedes aegypti mosquitoes

The chorion of Aedes aegypti eggs undergoes a hardening process following oviposition and individual chorion proteins become insoluble thereafter. Our previous studies determined that peroxidase-catalyzed chorion protein crosslinking and phenoloxidase-mediated chorion melanization are primarily responsible for the formation of a hardened, desiccation resistant chorion in A. aegypti eggs. To gain further understanding of peroxidase- and phenoloxidase-catalyzed biochemical processes during chorion hardening, we analyzed chorion proteins, identified three low molecular weight major endochorion proteins that together constituted more than 70% of the total amount of endochorion proteins, and assessed their insolubilization in relation to phenoloxidase- and peroxidase-catalyzed reactions under different conditions. Our data suggest that the three low molecular weight endochorion proteins undergo disulfide bond crosslinking prior to oviposition in A. aegypti eggs, and that they undergo further crosslinking through dityrosine or trityrosine formation by peroxidase-catalyzed reactions. Our data suggest that chorion peroxidase is primarily responsible for the irreversible insolubilization of the three major endochorion proteins after oviposition. The molecular mechanisms of chorion hardening are also discussed.

Anti-Thyroid Drugs and Thyroid Hormone Synthesis: Effect of Methimazole Derivatives on Peroxidase-Catalyzed Reactions

Syntheses and characterization of the selenium analogue (MSeI) of anti-thyroid drug methimazole and a series of organoselenium compounds bearing N-methylimidazole pharmacophore are described. In contrast to the sulfur compound that exists predominantly in its thione form, the selenium analogue exists in a selenol form, which spontaneously oxidizes in air to produce the corresponding diselenide. The reduction of the diselenide by GSH or NaBH(4) affords the biologically active selenol, which effectively inhibits the lactoperoxidase (LPO) activity in vitro. The monoselenides having N-methylimidazole moiety are found to be much less active than the selenol, suggesting that the presence of a selenol moiety is important for the LPO inhibition. The kinetic and mechanistic studies reveal that MSeI inhibits the LPO activity by reducing the H(2)O(2), providing a novel method to reversibly inhibit the enzyme. Although MSeI strongly inhibits LPO, the enzyme's activity can be completely recovered by increasing the H(2)O(2) concentration. On the other hand, the inhibition by methimazole (MMI), the sulfur analogue, cannot be reversed by increasing the H(2)O(2) concentration, leading to a complete inactivation of the enzyme. The reversible inhibition of LPO by some of the selenium derivatives is correlated with their glutathione peroxidase (GPx) activity, and the high GPx activity of the selenium compounds as compared with their sulfur analogues suggests that the selenium derivatives may protect the thyroid gland from oxidative damage.

Diamine oxidase is involved in H(2)O(2) production in the chalazal cells during barley grain filling.

The localization and activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) together with polyamine levels have been investigated in developing grains of barley (Hordeum vulgare L.). DAO (pH 7.5) is present mainly in vascular tissue and its neighbouring cells, namely chalazal cells and nucellar projection, while PAO (pH 6.0) is mainly localized in the chlorenchymatous cells of the crease and at the base of the vascular tissue. Activities of both these enzymes appear to be independently-regulated, as DAO activity increased steadily throughout grain development while PAO activity was higher during the early stages of grain filling, declined thereafter and again increased towards maturity. The maximum activities of DAO coincided with the maximum content of putrescine while the levels of PAO did not seem to be directly correlated with spermidine or spermine contents. Isoelectric focusing (IEF) of DAO and PAO activities revealed the presence of bands at 30 and 45 DPA. The possible involvement of DAO and PAO in the supply of H(2)O(2) to peroxidase-catalysed reactions in the chalazal cells during grain filling is discussed.