Protective effect of seminal plasma proteins on the degradation of ascorbic acid

The objectives of this study were to determine ascorbic acid stability and its effect on antiproteinase activity of seminal plasma in the presence of an oxidant. Effect of seminal plasma, and additives: glutathione, albumin, hydrogen peroxide and Tris buffer, on ascorbic acid degradation was investigated by UV absorbance. Antiproteinase against trypsin amidase activity was measured spectrophotometrically using N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) as substrate. Ascorbic acid was destroyed much more rapidly with the addition of hydrogen peroxide than in Tris buffer at pH 8.2 alone. Seminal plasma protected ascorbic acid more efficiently than glutathione and albumin alone. The protective effect of seminal plasma on ascorbic acid degradation may closely relate to the function of ascorbic acid in reproductive system of scurvy-prone animals including teleost fish. Within the range of 1–8 mM concentrations, ascorbic acid had a pro-oxidant action on seminal plasma antiproteinase activityin vitro when they were incubated with hydrogen peroxide.Abbreviations AA Ascorbic acid - BAPNA N-benzoyl-DL-arginine-p-nitroanilide - DMSO dimethyl sulfoxide - GSH glutathione - H₂O₂ hydrogen peroxide     

A new polyaniline–catalase–glutaraldehyde-modified biosensor for hydrogen peroxide detection

A new biosensor based on catalase enzyme immobilized on electrochemically constructed polyaniline (PANI) film modified with glutaraldehyde has been developed for the determination of hydrogen peroxide (H₂O₂) in milk samples. Assembly processes of polyaniline and immobilization of the enzyme were monitored with the help of electrochemical impedance spectroscopy. Amperometric measurements have been performed at cathodic peak (?0.3?V vs. Ag/AgCI) which was attributed to reduction of PANI. Hydrogen peroxide was determined by using amperometric method at ?0.3?V. The biosensor responses were correlated linearly with the hydrogen peroxide concentrations between 5.0?×?10^?6 and 1.0?×?10^?4?M by amperometric method. Detection limit of the biosensor is 2.18?×?10^?6?M for H₂O₂. In the optimization studies of the biosensor, some parameters such as optimum pH, temperature, concentration of aniline, amount of enzyme, and number of scans during electropolymerization were investigated.     

THE DECOMPOSITION OF HYDROGEN PEROXIDE BY LIVER CATALASE

1. The velocity of decomposition of hydrogen peroxide by catalase as a function of (a) concentration of catalase, (b) concentration of hydrogen peroxide, (c) hydrogen ion concentration, (d) temperature has been studied in an attempt to correlate these variables as far as possible. It is concluded that the reaction involves primarily adsorption of hydrogen peroxide at the catalase surface. 2. The decomposition of hydrogen peroxide by catalase is regarded as involving two reactions, namely, the catalytic decomposition of hydrogen peroxide, which is a maximum at the optimum pH 6.8 to 7.0, and the "induced inactivation" of catalase by the "nascent" oxygen produced by the hydrogen peroxide and still adhering to the catalase surface. This differs from the more generally accepted view, namely that the induced inactivation is due to the H₂O₂ itself. On the basis of the above view, a new interpretation is given to the equation of Yamasaki and the connection between the equations of Yamasaki and of Northrop is pointed out. It is shown that the velocity of induced inactivation is a minimum at the pH which is optimal for the decomposition of hydrogen peroxide. 3. The critical increment of the catalytic decomposition of hydrogen peroxide by catalase is of the order 3000 calories. The critical increment of induced inactivation is low in dilute hydrogen peroxide solutions but increases to a value of 30,000 calories in concentrated solutions of peroxide.     

A kinetic study of the effects of hydrogen peroxide and pH on ascorbate oxidase.

Addition of hydrogen peroxide to ascorbate oxidase results in formation of a complex which has been analyzed kinetically. Since reduction of molecular oxygen to water by this enzyme occurs in more than one step, the peroxide complex may be a mimic for a catalytic intermediate. The properties of the complex are similar to those reported for a peroxide complex with laccase. Changes in the activity of ascorbate oxidase as a function of pH indicate the presence of a group in the enzyme with an apparent pK of 7.8 which must be protonated in order for the enzyme to function. The ascorbate K_m is known to be insensitive to pH in this region and the present work indicates the same to be true for the oxygen K_m. It would appear that the rate determining step in the catalytic mechanism involves protonation of an intermediate.     

Nitration of Tyrosine by Hydrogen Peroxide and Nitrite

Peroxynitrite anion is a powerful oxidant which can initiate nitration and hydroxylation of aromatic rings. Peroxynitrite can be formed in several ways, e.g. from the reaction of nitric oxide with superoxide or from hydrogen peroxide and nitrite at acidic pH. We investigated pH dependent nitration and hydroxylation resulting from the reaction of hydrogen peroxide and nitrite to determine if this reaction proceeds at pH values which are known to occur in vivo. Nitration and hydroxylation products of tyrosine and salicylic acid were separated with an HPLC column and measured using ultraviolet and electrochemical detectors. These studies revealed that this reaction favored hydroxylation between pH 2 and pH4, while nitration was predominant between pH 5 and pH 6. Peroxynitrite is presumed to be an intermediate in this reaction as the hydroxylation and nitration profiles of authentic peroxynitrite showed similar pH dependence. These findings indicate that hydrogen peroxide and nitrite interact at hydrogen ion concentrations present under some physiologic conditions. This interaction can initiate nitration and hydroxylation of aromatic molecules such as tyrosine residues and may thereby contribute to the biochemical and toxic effects of the molecules.     

Borate buffer inhibition of peroxidatic intermediate formation in the deutero-ferriheme-hydrogen peroxide system

The rate of formation of peroxidatically active reaction intermediate(s) via oxidation of the iron(III)-porphyrin complex, deuteroferriheme, with hydrogen peroxide decreases with increasing borate content of mixed borate-carbonate buffer solutions. Studies at pH = 9.25 in 0.035 M borate buffer and 0.035 M carbonate buffer suggest borate to function as an uncompetitive inhibitor. A comparison of slopes and intercepts of double reciprocal plots for inhibited and uninhbited reactions allows calculation of selected parameters for the deuteroferriheme-H₂O₂ reaction at pH = 9.25 in terms of a typical enzymatic stoichiometric mechanism for heme activity. This includes the Michaelis constant (K_m = 8.1 × 10^?5 M) and the first-order rate constant for conversion of heme-substrate complex to intermediate(s) (k₃ = 7.4 sec^?1). A tentative mechanistic model involving reversible interaction of borate inhibitor with heme-substrate complex is considered, and pseudo-first-order rate constants calculated on the basis of this scheme are in reasonable agreement with those obtained experimentally. It is suggested that comparable inhibitory action may be responsible for some previously reported cases of decreased catalase enzyme activity in borate buffer solutions     

Hydrogen peroxide production in wheat leaves infected with the fungus Septoria nodorum Berk. Strains with different virulence

The effect of two strains of the phytopathogenic fungus Septoria nodorum Berk. of different virulence on the intensity of local generation of hydrogen peroxide in common wheat leaves and the role of oxidoreductases in this process was studied. Differences in the pattern of hydrogen peroxide production in wheat plants infected with high- and low-virulence pathogen strains have been found. The low-virulent S. nodorum strain caused a long-term hydrogen peroxide (H₂O₂) generation in the infection zone, whereas the inoculation of leaves with the highly virulent strain resulted in a transient short-term increase in the H₂O₂ concentration at the initial moment of contact between the plant and the fungus. It was shown that the low level of H₂O₂ production by plant cells at the initial stages of pathogenesis facilitates S. nodorum growth and development. The decrease in the H₂O₂ concentration induced by the highly virulent S. nodorum strain is determined by inhibition of the oxalate oxidase activity in plant tissues and by the ability of the fungus to actively synthesize an extracellular catalase. The pattern of hydrogen peroxide generation at the initial stages of septoriosis may serve as an index of virulence of S. nodorum population.     

Kinetic study of peroxidase-catalyzed oxidation of 1-hydroxypyrene. Development of a nanomolar hydrogen peroxide detection system

Kinetics of 1-hydroxypyrene (1-HP) oxidation catalyzed with recombinant Coprinus cinereus (rCiP) and horseradish (HRP) peroxidases was investigated with a special emphasis for developing a nanomolar hydrogen peroxide (H₂O₂) detection system. At pH 8.0 the bimolecular constants of 1-HP oxidation with the ferryl compounds of rCiP and HRP were equal to (1.0 ± 0.3) × 10⁸ M⁻¹ s⁻¹ and (0.6 ± 0.2) × 10⁸ M⁻¹ s⁻¹, respectively. High bimolecular constants and fluorescence quantum yield of 1-HP (0.66) permitted detection as low as 21 nM of H₂O₂. To optimize the detection system 1-HP oxidation was modeled at steady-state conditions in the range pH 5.0 to pH 8.0. The 1-HP based detection system was compared with the Amplex Red system. The peroxidase-catalyzed 1-HP oxidation system was used for determination of ozone in the air.     

Alkaline peroxide pretreatment of corn stover: effects of biomass, peroxide, and enzyme loading and composition on yields of glucose and xylose

Background

Pretreatment is a critical step in the conversion of lignocellulose to fermentable sugars. Although many pretreatment processes are currently under investigation, none of them are entirely satisfactory in regard to effectiveness, cost, or environmental impact. The use of hydrogen peroxide at pH 11.5 (alkaline hydrogen peroxide (AHP)) was shown by Gould and coworkers to be an effective pretreatment of grass stovers and other plant materials in the context of animal nutrition and ethanol production. Our earlier experiments indicated that AHP performed well when compared against two other alkaline pretreatments. Here, we explored several key parameters to test the potential of AHP for further improvement relevant to lignocellulosic ethanol production.

Results

The effects of biomass loading, hydrogen peroxide loading, residence time, and pH control were tested in combination with subsequent digestion with a commercial enzyme preparation, optimized mixtures of four commercial enzymes, or optimized synthetic mixtures of pure enzymes. AHP pretreatment was performed at room temperature (23°C) and atmospheric pressure, and after AHP pretreatment the biomass was neutralized with HCl but not washed before enzyme digestion. Standard enzyme digestion conditions were 0.2% glucan loading, 15 mg protein/g glucan, and 48 h digestion at 50°C. Higher pretreatment biomass loadings (10% to 20%) gave higher monomeric glucose (Glc) and xylose (Xyl) yields than the 2% loading used in earlier studies. An H₂O₂ loading of 0.25 g/g biomass was almost as effective as 0.5 g/g, but 0.125 g/g was significantly less effective. Optimized mixtures of four commercial enzymes substantially increased post-AHP-pretreatment enzymatic hydrolysis yields at all H₂O₂ concentrations compared to any single commercial enzyme. At a pretreatment biomass loading of 10% and an H₂O₂ loading of 0.5 g/g biomass, an optimized commercial mixture at total protein loadings of 8 or 15 mg/g glucan gave monomeric Glc yields of 83% or 95%, respectively. Yields of Glc and Xyl after pretreatment at a low hydrogen peroxide loading (0.125 g H₂O₂/g biomass) could be improved by extending the pretreatment residence time to 48 h and readjusting the pH to 11.5 every 6 h during the pretreatment. A Glc yield of 77% was obtained using a pretreatment of 15% biomass loading, 0.125 g H₂O₂/g biomass, and 48 h with pH adjustment, followed by digestion with an optimized commercial enzyme mixture at an enzyme loading of 15 mg protein/g glucan.

Conclusions

Alkaline peroxide is an effective pretreatment for corn stover. Particular advantages are the use of reagents with low environmental impact and avoidance of special reaction chambers. Reasonable yields of monomeric Glc can be obtained at an H₂O₂ concentration one-quarter of that used in previous AHP research. Additional improvements in the AHP process, such as peroxide stabilization, peroxide recycling, and improved pH control, could lead to further improvements in AHP pretreatment.     

An Automated System for Monitoring and Regulating the pH of Bicarbonate Buffers

The bicarbonate buffer is considered as the most biorelevant buffer system for the simulation of intestinal conditions. However, its use in dissolution testing of solid oral dosage forms is very limited. The reason for this is the thermodynamic instability of the solution containing hydrogen carbonate ions and carbonic acid. The spontaneous loss of carbon dioxide (CO₂) from the solution results in an uncontrolled increase of the pH. In order to maintain the pH on the desired level, either a CO₂ loss must be completely avoided or the escaped CO₂ has to be replaced by quantitative substitution, i.e. feeding the solution with the respective amount of gas, which re-acidifies the buffer after dissociation. The present work aimed at the development of a device enabling an automatic pH monitoring and regulation of hydrogen carbonate buffers during dissolution tests.     

Stoichiometric cooperation of NADPH and hexobarbital in hepatic microsomes during the catalysis of hydrogen peroxide formation

The interaction of NADPH and hexobarbital during catalysis of microsomal mixed function oxidase-dependent hydrogen peroxide formation has been investigated in hepatic microsomes from phenobarbital-treated rabbits. The application of Job's method (25) of continuous variation revealed optimal conditions for the rate and extent of hydrogen peroxide formation when hexobarbital and NADPH were in equimolar amounts. The formation of a complex of 1 mol NADPH with cytochrome c-reductase and 1 mol hexobarbital with cytochrome P-450 seems to be responsible for limitation of hydrogen peroxide formation. Rate and extent of hydrogen peroxide formation are directly proportional to the amount of hexobarbital and NADPH present and are governed by the mass action equation in a manner similar to that reported for interaction of purified enzymes (G. T. Miwa, S. B. West, M. T. Huang, and A. H. Lu, 1979,J. Biol. Chem.254, 5695–5700). Depending on either the NADPH concentration maintained by a generating system or the hexobarbital concentration, the extent of hydrogen peroxide formation could be shown to be a function of either compound alone, as long as the other one is in excess. The question whether the formation of hydrogen peroxide depends on the availability of two independent one-electron transfer reactions forming O₂^? or of one simultaneous two-electron transfer forming O₂^2? might thus become rather a matter of association of substrate and cosubstrate to a catalytically active complex in which the substrate augments the availability of reducing equivalents.     

Response of Plant-Colonizing Pseudomonads to Hydrogen Peroxide

Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H₂O₂) at concentrations up to 10 mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1 mM were killed. Extracts of logarithmic-phase cells displayed only band A catalase activity. Protection against 5 mM H₂O₂ was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentrations (30 to 300 μM) of H₂O₂. Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.     

Oxidation of Methanol and Formaldehyde by a System Containing Alcohol Oxidase and Catalase Purified from Candida sp. N-16

The oxidation of methanol and formaldehyde was investigated by using some combination systems of alcohol oxidase, catalase, which were purified from Candida N-16, and hydrogen peroxide. The activity of alcohol oxidase was irreversibly inhibited when the enzyme was incubated with 2.5 mm hydrogen peroxide for 15 min. However, the oxidation of methanol to formaldehyde by alcohol oxidase in the presence of catalase was extremely promoted by the addition of 30 mm hydrogen peroxide. Alcohol oxidase could oxidize not only methanol but also formaldehyde as follows: HCHO + 0₂ + H₂O→HCOOH + H₂O₂. The formaldehyde oxidizing activity was inhibited by hydrogen peroxide. The system containing alcohol oxidase and catalase appears to be the entity of the oxygen-dependent oxidation system of formaldehyde previously found in the cell-free extract of the yeast.     

Manganese Peroxidase-Dependent Oxidation of Glyoxylic and Oxalic Acids Synthesized by Ceriporiopsis subvermispora Produces Extracellular Hydrogen Peroxide

The ligninolytic system of the basidiomycete Ceriporiopsis subvermispora is composed of manganese peroxidase (MnP) and laccase. In this work, the source of extracellular hydrogen peroxide required for MnP activity was investigated. Our attention was focused on the possibility that hydrogen peroxide might be generated by MnP itself through the oxidation of organic acids secreted by the fungus. Both oxalate and glyoxylate were found in the extracellular fluid of C. subvermispora cultures grown in chemically defined media, where MnP is also secreted. The in vivo oxidation of oxalate was measured; ¹⁴CO₂ evolution was monitored after addition of exogenous [¹⁴C]oxalate to cultures at constant specific activity. In standard cultures, evolution of CO₂ from oxalate was maximal at day 6, although the MnP titers were highest at day 12, the oxalate concentration was maximal (2.5 mM) at day 10, and the glyoxylate concentration was maximal (0.24 mM) at day 5. However, in cultures containing low nitrogen levels, in which the pH is more stable, a better correlation between MnP titers and mineralization of oxalate was observed. Both MnP activity and oxidation of [¹⁴C]oxalate were negligible in cultures lacking Mn(II). In vitro assays confirmed that Mn(II)-dependent oxidation of [¹⁴C]oxalate by MnP occurs and that this reaction is stimulated by glyoxylate at the concentrations found in cultures. In addition, both organic acids supported phenol red oxidation by MnP without added hydrogen peroxide, and glyoxylate was more reactive than oxalate in this reaction. Based on these results, a model is proposed for the extracellular production of hydrogen peroxide by C. subvermispora.     

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.     

Lipopolysaccharide challenge of the mammary gland in cows induces nitrosative stress that impairs milk oxidative stability

The aim of this work was to study the effects of mastitis induced by intramammary lipopolysaccharide (LPS) challenge on milk oxidative stability, as well as to understand the underlying biochemical processes that cause such changes. LPS challenge was associated with nitric oxide burst from the surrounding mammary epithelial cells and consequently induced nitrosative stress that was induced by the formation of NO₂• from nitrite by lactoperoxidase. This response was associated with an ∼3-fold increased formation of hazardous compounds: nitrotyrosines, carbonyls and lipid peroxides. We sustained the involvement of xanthine oxidase as a major source of hydrogen peroxide. In consistent with previous findings, catalase has been shown to play a major role in modulating the nitrosative stress by oxidizing nitrite to nitrate. The current hygienic quality criteria cannot detect mixing of low-quality milk, such as milk with high somatic cells, and nitrite with high-quality milk. Thus, development of an improved quality control methodology may be important for the production of high-quality milk.     

Terephthalic acid: A dosimeter for the detection of hydroxyl radicals in vitro

Hydroxylation reactions of aromatic compounds have been used to detect hydroxyl radicals produced by gamma irradiation and ultrasound. The present study investigated the suitability of terephthalic acid (THA) as a hydroxyl radical dosimeter for general use in biologically relevant reactions. Hydroxyl radicals were generated by: (1) irradiating, THA with a 254 nm ultraviolet; (2) irradiating with gamma rays from a cesium source; and (3) generating hydroxyl radicals with 1 mM H₂O₂ and 10 μM Cu⁺². In each of the three experiments, a fluorescent product was generated which exhibited identical fluorescent excitation and emission spectra. THA is non-fluorescent, eliminating the problem of a high initial background. Because THA has four ring hydrogens, only one mon-hydroxylated isomer was formed. The hydrogen peroxide reaction was dependent on the presence of a metal and cupric ions were effective in enhancing the reaction. With a Cu⁺² concentration of 10 μM, the reation was linear between 0–30 mM H₂O₂. Catalase abolished the reaction at a concentration of 100 μg/ml and the effects could still be observed at 10 ng/ml, consistent with the very high rate at which catalase destroys hydrogen peroxide. Tertbutyl- hydroperoxide did not generate any fluorescence in this system which makes THA a very specific detector of hydroxyl radicals.     

Abatement of Sorbed Crude Oil by Heterogeneous Fenton Process Using A Contaminated Soil Pre-Impregnated with Dissolved Fe(II) and Humic Acid

Dissolved Fe(II) and humic acid (HA) were pre-impregnated into contaminated soil to catalyze hydrogen peroxide to remove crude oil (CO). The effects of parameters such as initial Fe(II), HA and H₂O₂ concentrations on the oxidation of total petroleum hydrocarbon (TPH) were investigated using response surface methodology based on Box–Behnken design. The rate of hydrogen peroxide decomposition is decreased by pre-impregnating with dissolved Fe(II) + HA compared with only pre-impregnated Fe(II) and modified Fenton (MF). Oxygen evolution is the predominant route of hydrogen peroxide decomposition at natural pH. Unlike O₂ evolution, the kinetics of hydroxyl radical (OH^?) production are clearly uncoupled from H₂O₂ decay in these systems. The steady-state hydroxyl radical production rate is higher in the systems with pre-impregnated dissolved Fe(II) and HA, and more significance is the decrease in detectable TPH (70.84% removal efficiency) when soil is pre-impregnated with dissolved 25 mM Fe(II) + 0.7 mg/mL HA, and with the application of 700 mM H₂O₂, possibly due to hydrogen peroxide catalyzed by the iron of this complex (CO-HA–Fe(II)) producing hydroxyl radical in close proximity to the CO. Meanwhile, the removal efficiency of C₂₁–C₃₀ is up to 65.69%, which is 2.6 times higher than that of the MF (25.52%).     

Urea Hydrogen Peroxide Reduces the Numbers of Lactobacilli, Nourishes Yeast, and Leaves No Residues in the Ethanol Fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from ~10⁷ to ~10² CFU/ml in a 2-h preincubation at 30°C of normal-gravity wheat mash at ~21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at ~10⁷ CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at ~10⁷ CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30°C whether the bactericidal agent was added as H₂O₂ or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H₂O₂) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H₂O₂. H₂O₂-resistant mutants were not expected or found when lethal levels of H₂O₂ or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

The novel non-heme vanadium bromoperoxidase from marine algae: Phosphate inactivation

Summary Vanadium bromoperoxidase is a naturally occurring vanadium-containing enzyme isolated from marine algae. V-BrPO catalyzes the oxidation of halides by hydrogen peroxide which can result in the halogenation of organic substrates. Bromoperoxidase activity is measured by the halogenation of monochlorodimedone (2-chloro-5,5-dimethyl-1,3-dimedone, MCD). In the absence of an organic substrate, V-BrPO catalyzes the halide-assisted disproportionation of hydrogen peroxide yielding dioxygen. The dioxygen formed is in the singlet excited state (¹O₂). V-BrPO is quite stable to thermal denaturation and denaturation by certain organic solvents which makes V-BrPO an excellent candidate for industrial applications. The stability of V-BrPO in the presence of strong oxidants and in the presence of phosphate is reported. Incubation of V-BrPO in phosphate buffer (1–100 mM at pH 6; 2–10 mM at pH 5) inactivates the enzyme. The inactivity can be fully restored by the addition of vanadate if excess phosphate is removed. The inactivation of V-BrPO by phosphate can be prevented by the presence of H₂O₂ (4–40 mM). We are currently investigating the mechanism of V-BrPO inactivation by phosphate. V-BrPO was not inactivated by HOCl (1 mM) nor H₂O₂. In addition V-BrPO was not inactivated under turnover conditions of 1 mM H₂O₂ with 0.1–1 M Cl^– at pH 5 nor 2 mM H₂O₂ with 0.1 M Br^–.