A kinetic study of the reaction of horseradish peroxidase with hydrogen peroxide.

A kinetic study has been carried out over the pH range of 2.63-9.37 for the reaction of horseradish peroxidase with hydrogen peroxide to form compound I of th;e enzyme. Analysis of the results, indicates that there are two kinetic influencing, ionizable groups on the enzyme with pKa values of 3.2 and 3.9. Protonation of these groups results in a decrease in the rate of reaction of the enzyme with H2O2. A previous study of the kinetics of cyanide binding to horseradish peroxidase (Ellis, W.D. & Dunford, H.B.: Biochemistry 7, 2054-2062 (1968)) has been extended to down to pH 2.55, and analysis of these results also indicates the presence of two kinetically important ionizable groups on the enzyme with pKa values of 2.9 and 3.9.     

The oxidation of phenol and its reaction product by horseradish peroxidase and hydrogen peroxide

Phenol and its oxidized products are shown to be substrates in the HRP, H₂O₂ enzyme system. The homogeneous nature of the product of phenol oxidation suggests that the radical generated remains enzyme-bound until coupling occurs. Kinetics of the reaction was investigated and was suggestive of a three substrate ping-pong mechanism.     

Inactivation of creatine kinase during the interaction of mefenamic acid with horseradish peroxidase and hydrogen peroxide: participation by the mefenamic acid radical

Creatine kinase (CK) was used as a marker molecule to examine the side effects of damage to tissues by mefenamic acid, an effective drug to treat rheumatic and arthritic diseases, with horseradish peroxidase and hydrogen peroxide (HRP-H(2)O(2)). Mefenamic acid inactivated CK during its interaction with HRP-H(2)O(2). Also, diphenylamine and flufenamic acid caused a loss of CK activity, indicating the imino group, not substituent groups, in the phenyl rings have a crucial role in CK inactivation. Rapid change in mefenamic acid spectra was detected, suggesting that mefenamic acid is efficiently oxidized by HRP-H(2)O(2). Peroxidases oxidize xenobiotics to free radicals by a one-electron transfer. However, direct detection of mefenamic acid radicals by electron spin resonance (ESR) was unsuccessful. Reduced glutathione and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) in the reaction mixture containing mefenamic acid with HRP-H(2)O(2) produced ESR signals consistent with a DMPO-glutathionyl radical adduct. These results suggest that inactivation of CK is probably caused through formation of mefenamic acid radicals. Sulfhydryl groups and tryptophan residues of CK were diminished by mefenamic acid with HRP-H(2)O(2). Other SH enzymes, including alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, were very sensitive to mefenamic acid with HRP-H(2)O(2). Inactivation of SH enzymes may explain some deleterious actions of mefenamic acid.     

Interaction of thiocyanate with horseradish peroxidase. 1H and 15N nuclear magnetic resonance studies

Interaction of thiocyanate with horseradish peroxidase (HRP) was investigated by relaxation rate measurements (at 50.68 MHz) of the 15N resonance of thiocyanate nitrogen and by following the hyperfine shifted ring methyl proton resonances (at 500 MHz) of the heme group of SCN-.HRP solutions. At pH 4.0, the apparent dissociation constant (KD) for thiocyanate binding to HRP was deduced to be 158 mM from the relaxation rate measurements. Chemical shift changes of 1- and 8-ring methyl proton resonances in the presence of various amounts of thiocyanate at pH 4.0 yielded KD values of 166 and 136 mM, respectively. From the pH dependence of KD and the 15N resonance line width, it was observed that thiocyanate binds to HRP only under acidic conditions (pH less than 6). The binding was found to be facilitated by protonation of an acid group on the enzyme with pKa 4.0. The pH dependence of the 15N line width as well as the apparent dissociation constant were quantitatively analyzed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. The KD for thiocyanate binding to HRP was also evaluated in the presence of an excess of exogenous substrates such as resorcinol, cyanide, and iodide ions. It was found that the presence of cyanide (which binds to heme iron at the sixth coordination position) and resorcinol did not have any effect on the binding of thiocyanate, indicating that the binding site of the thiocyanate ion is located away from the ferric center as well as from the aromatic donor binding site. The KD in the presence of iodide, however, showed that iodide competes with thiocyanate for binding at the same site. The distance of the bound thiocyanate ion from the ferric center was deduced from the 15N relaxation time measurements and was found to be a 6.8 A. From the distance as well as the change in the chemical shifts and line width of 1- and 8-methyl proton resonances, it is suggested that the binding site of thiocyanate may be located near heme, placed symmetrically with respect to 1- and 8-methyl groups of the heme of HRP. Similarity in the modes of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H2O2 may also proceed via the two-electron transfer pathway under acidic conditions, as is the case for iodide.     

Effects of pressure and temperature on the reactions of horseradish peroxidase with hydrogen cyanide and hydrogen peroxide.

Reactions of ferric horseradish peroxidase with hydrogen cyanide and hydrogen peroxide were studied as a function of pressure. Activation volumes are small and differ in sign (delta V = 1.7 +/- 0.5 ml/mol for peroxidase + HCN and -1.5 +/- 0.5 ml/mol for peroxidase + H2O2). The temperature dependence of cyanide binding to horseradish peroxidase was also determined. A comparison is made of relevant parameters for cyanide binding and compound I formation.     

High-valent intermediates in the reaction of N alpha-acetyl microperoxidase-8 with hydrogen peroxide: models for compounds 0, I and II of horseradish peroxidase.

N-acetyl microperoxidase-8 (Ac-MP-8) is a water soluble, ferric heme model for the peroxidases. The reaction of Ac-MP-8 with H2O2 in 10 mM potassium phosphate over the pH range of 7-11 gives rise sequentially to relatively stable green and red species with properties that closely mimic those of HRP compounds I and II, respectively. Low-temperature stopped-flow studies of this reaction carried out in 50% v/v methanol/10 mM potassium phosphate, pH* 9.1 at -25.8 degrees C indicate that the pseudo-first-order rate constant, kobs, that describes the formation of the green intermediate exhibits saturation kinetics as a function of [H2O2] with kmaxobs = 95 s-1 and KM = 87 mM. Rapid-scan studies carried out with [H2O2] = 200 mM at -38.0 degrees C show that a compound 0 species with a characteristic band near 340 nm is formed whose conversion to the green species is rate limiting. Thus, Ac-MP-8 has high-valent forms that are models for all three known intermediates in the peroxidase cycle of horseradish peroxidase.     

Oxidation of 2-nitropropane by horseradish peroxidase. Involvement of hydrogen peroxide and of superoxide in the reaction mechanism

Incubation of aqueous solutions of 2-nitropropane in air causes a slow oxidation reaction that generates H(2)O(2). Purified horseradish peroxidase catalyses the oxidation of such preincubated 2-nitropropane solutions according to the equation: [Formula: see text] The pH optimum is 4.5 and K(m) for 2-nitropropane is 16mm. Other nitroalkanes or nitro-aromatics tested are not oxidized at significant rates by peroxidase. H(2)O(2) or 2,4-dichlorophenol increases the rate of 2-nitropropane oxidation by peroxidase. Catalase inhibits the reaction completely. Superoxide dismutase or mannitol, a scavenger of the hydroxyl radical, OH(.), each inhibits partially. Aniline and guaiacol are also powerful inhibitors of 2-nitropropane oxidation. It is suggested that peroxidase uses the traces of H(2)O(2) generated during preincubation of 2-nitropropane to catalyse oxidation of this substrate into a radical species that can reduce O(2) to the superoxide ion, O(2) (-.).O(2) (-.), or OH(.) derived from it, then appears to react with more nitropropane, generating further radicals and H(2)O(2) to continue the oxidation. Inhibition by aniline and guaiacol seems to be due to a competition for H(2)O(2).     

Inactivation of creatine kinase during the interaction of indomethacin with horseradish peroxidase and hydrogen peroxide: involvement of indomethacin radicals

Creatine kinase (CK) was used as a marker molecule to examine the side effect of damage to tissues by indomethacin (IM), an effective drug to treat rheumatoid arthritis and gout, with horseradish peroxidase and hydrogen peroxide (HRP-H2O2). IM inactivated CK during its interaction with HRP-H2O2. Under aerobic conditions, inactivation of CK significantly decreased. CK in rat heart homogenate was also inactivated by IM with HRP-H2O2. When IM was incubated with HRP-H2O2, the maximum absorption of IM at 280 nm rapidly decreased and a new peak at 410 nm occurred with isosbestic points at 260 and 312 nm. In contrast, under anaerobic conditions, the spectral change of IM was almost absent, indicating IM was oxidized to the yellow substance by HRP-H2O2. Adding catalase strongly inhibited the production of yellow substance. Sodium azide also blocked the formation of yellow substance and the inactivation of CK. Electron spin resonance signals of IM carbon-centered radical were detected using 2-methyl-2-nitrosopropane during the interaction of IM with HRP-H2O2 under anaerobic conditions. Oxygen was consumed during the interaction of IM with HRP-H2O2. These results suggest that IM carbon-centered radicals may rapidly react with O2 to generate the peroxyl radicals. Sulfhydryl groups and tryptophane residues of CK decreased during the interaction of IM with HRP-H2O2. Other sulfhydryl enzymes, including alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, were also readily inactivated during the interaction with HRP-H2O2. Sulfhydryl enzymes seem to be very sensitive to IM activated by HRP-H2O2.     

Oxidation of acetylacetone catalyzed by horseradish peroxidase in the absence of hydrogen peroxide

Horseradish peroxidase (HRP) is a plant enzyme widely used in biotechnology, including antibody-directed enzyme prodrug therapy (ADEPT). Here, we showed that HRP is able to catalyze the autoxidation of acetylacetone in the absence of hydrogen peroxide. This autoxidation led to generation of methylglyoxal and reactive oxygen species. The production of superoxide anion was evidenced by the effect of superoxide dismutase and by the generation of oxyperoxidase during the enzyme turnover. The HRP has a high specificity for acetylacetone, since the similar beta-dicarbonyls dimedon and acetoacetate were not oxidized. As this enzyme prodrug combination was highly cytotoxic for neutrophils and only requires the presence of a non-human peroxidase and acetylacetone, it might immediately be applied to research on the ADEPT techniques. The acetylacetone could be a starting point for the design of new drugs applied in HRP-related ADEPT techniques.     

Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide

Horseradish peroxidase (HRP) is a commonly used enzyme in many biotechnological fields. Improvement of HRP stability would further increase its potential application range. In the present study, 13 single- and three double-mutants of solvent exposed, proximal lysine and glutamic acid residues were analysed for enhanced H(2)O(2) stability. Additionally, five single- and one pentuple-consensus mutants were investigated. Most mutants displayed little or no alteration in H(2)O(2) stability; however, three (K232N, K241F and T110V) exhibited significantly increased H(2)O(2) tolerances of 25- (T110V), 18- (K232N), and 12-fold (K241F). This improved stability may be due to an altered enzyme-H(2)O(2) catalysis pathway or to removal of potentially oxidisable residues.     

Oxidative destruction of estradiol after treatment with hydrogen peroxide catalyzed by horseradish peroxidase and methemoglobin]

It is shown that estradiol in the presence of horse radish peroxidase interacts with hydrogen peroxide, which is evidenced by an increase in its optical density at 280 nm. The photometering of samples containing estradiol and horse radish peroxidase upon their titration with hydrogen peroxide indicated that the increase in optical density stops after introducing hydrogen peroxide equimolar in concentration to estradiol. The stoichiometric ratio of estradiol consumed during oxidative destruction to hydrogen peroxide was 1:1. In the presence of ascorbate, the oxidative destruction of estradiol by the action of hydrogen peroxide, catalyzed by horse radish peroxidase, was observed only after a latent period and showed the same regularities as in the absence of ascorbate. It was found by calorimetry that, during the latent period, estradiol catalyzes the degradation of hydrogen peroxide and ascorbate without undergoing oxidative destruction. The substrates of the peroxidase reaction benzidine, 1-naphthol, and phenol interact with hydrogen peroxide in the presence of ascorbate and horse radish peroxidase in a similar way. Presumably, upon interaction with hydrogen peroxide in the presence of horse radish peroxidase, estradiol, like other substrates of this reaction, undergoes oxidative destruction by the mechanism of peroxidase reaction. It is shown that oxidative destruction of estradiol by the action of hydrogen peroxide can also be catalyzed by methemoglobin by the same mechanism. These data are important for understanding the role of estradiol in the organism and the pathways of its metabolic conversions.     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.     

Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes

The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD = 5.0 pmol/assay and LLQ = 30 pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09 nmol hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31 nmol of hydrogen peroxide/min/100 Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.     

Kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase]

A kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase within the pH range of 3.7-9.0 has been carried out. It was shown that the reaction of o-dianisidine peroxidase oxidation obeys the Michaelis--Menten kinetics; the kcat and Km values within the pH range used were determined. The optimum of peroxidase catalytic activity during o-dianisidine oxidation was observed at pH 5.0-6.0. The kinetic pattern of the reaction is discussed. It was demonstrated that deprotonation of the group at pK 6.5 decreases the kcat value 60 times. At pH greater than 8.0 an additional ionogenic group controls the enzyme activity.     

Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H₂O₂ is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H₂O₂.

Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H₂O₂ and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL.

These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.     

Radical mechanisms of the decomposition of peroxynitrite and the peroxynitrite-CO(2) adduct and of reactions with L-tyrosine and related compounds as studied by (15)N chemically induced dynamic nuclear polarization.

Enhanced absorption is observed in the (15)N NMR spectra of (15)NO(-)(3) during decomposition of peroxynitrite and the peroxynitrite-CO(2) adduct at pH 5.25, indicating the formation of (15)NO(-)(3) in radical pairs [(15)NO(*)(2), HO(*)] and [(15)NO(*)(2), CO(*-)(3)]. During the reaction of peroxynitrite and the peroxynitrite-CO(2) adduct with L-tyrosine, the (15)N NMR signal of the nitration product 3-nitrotyrosine exhibits emission showing a radical pathway of its formation. The nuclear polarization is built up in radical pairs [(15)NO(*)(2), tyr(*)] generated by free radical encounters of nitrogen dioxide and tyrosinyl radicals. The (15)N NMR signal of (15)NO(-)(2) formed during reaction of peroxynitrite with L-tyrosine appears in emission. It is concluded that tyrosinyl radicals are generated by reaction of nitrogen dioxide with L-tyrosine. In contrast to this, (15)NO(-)(2) does not show (15)N chemically induced dynamic nuclear polarization (CIDNP) during reaction of the peroxynitrite-CO(2) adduct with L-tyrosine, indicating a different reaction mechanism, which is assumed to be a hydrogen transfer between CO(*-)(3) and L-tyrosine. Emission is also observed in the (15)N NMR signals of 2-nitro-4-fluorophenol, 3-nitro-4-hydroxyphenylacetic acid, 2-nitrophenol, and 4-nitrophenol during reaction of 4-fluorophenol, 4-hydroxyphenylacetic acid, and phenol with peroxynitrite and the peroxynitrite-CO(2) adduct. 3-Nitro-4-hydroxyphenylacetic acid is also observed in emission during reaction of phenylacetic acid with peroxynitrite, but is not formed with the peroxynitrite-CO(2) adduct. The magnitude of the (15)N CIDNP effect during reaction of peroxynitrite with 4-fluorophenol and of the peroxynitrite-CO(2) adduct with 4-fluorophenol and phenol is determined. It excludes the occurrence of nonradical reactions. Only weak emission signals are observed during the reaction of peroxynitrite with phenol in (15)NO(-)(2), 2-nitrophenol, and 4-nitrophenol. 2-Nitrophenol is only formed in traces, and 4-nitrophenol is only formed in higher yields. The latter might be generated in part via a nonradical pathway.     

Mechanistic insight into the peroxidase catalyzed nitration of tyrosine derivatives by nitrite and hydrogen peroxide.

Peroxidases perform the nitration of tyrosine and tyrosyl residues in proteins, in the presence of nitrite and hydrogen peroxide. The nitrating species is still unknown but it is usually assumed to be nitrogen dioxide. In the present investigation, the nitration of phenolic compounds derived from tyrosine by lactoperoxidase and horseradish peroxidase was studied, with the aim of elucidating the mechanism of the reaction. The results indicate that nitrogen dioxide cannot be the only nitrating species and suggest the presence of two simultaneously operative pathways, one proceeding through enzyme-generated nitrogen dioxide and another through a more reactive species, assumed to be complexed peroxynitrite, which is generated by reaction of hydrogen peroxide with the enzyme-nitrite complex. The importance of the two pathways depends on peroxide and nitrite concentrations. With lactoperoxidase, nitration through the highly reactive intermediate is preferred except at very low nitrite concentration, while with horseradish peroxidase, the nitrogen dioxide driven mechanism is preferred except at very high nitrite concentration. The preferred mechanism for the two enzymes is that operative in the physiological nitrite concentration range.     

Application of chemically induced dynamic nuclear polarization to the nitration of N-acetyltyrosine and to some reactions of peroxynitrite.

By the observation of chemically induced dynamic nuclear polarization in (15)N NMR spectroscopy it has been shown that nitration of N-acetyltyrosine, even under acidic conditions, is largely a radical process. In the alkaline reaction of tyrosine with peroxynitrite the main products are nitrite and nitrate, both produced by a radical pathway, and tyrosine nitration is a minor reaction. It is suggested that tyrosine catalyzes the production of NO(*)(2) and HO(*) from peroxynitrite.