Forms Of Horseradish Peroxidase Research Articles

Probing nitrite coordination in horseradish peroxidase by resonance Raman spectroscopy: Detection of two binding sites

Nitrite is a powerful oxidant that affects the activity of peroxidases towards various substrates and leads to heme macrocycle modifications in members of the peroxidase family, such as the horseradish peroxidase (HRP). We have applied resonance Raman spectroscopy to investigate the structural properties of the species formed in the reaction of NO2− with the ferric form of HRP. Our data demonstrate that the heme nitrovinyl group is partially formed at near neutral pH, without coordination of NO2− to the heme Fe. Nitrite coordinates to the heme Fe at acidic pH in the nitro binding mode, characterized by the detection of the ν(Fe-NO2) at 563cm−1, δ(FeNO2) at 822cm−1 and νsym(NO2) at 1272cm−1. The sensitivity of the vibrations of the heme Fe-nitro complex to H/D exchange indicates H-bonding interaction of the heme-bound ligand with the distal environment that determines the NO2− binding mode. A model describing the different modes of NO2− binding in HRP is presented.

Chemically glycosylation improves the stability of an amperometric horseradish peroxidase biosensor

We constructed a biosensor by electrodeposition of gold nano-particles (AuNPs) on glassy carbon (GC) and subsequent formation of a 4-mercaptobenzoic acid self-assembled monolayer (SAM). The enzyme horseradish peroxidase (HRP) was then covalently immobilized onto the SAM. Two forms of HRP were employed: non-modified and chemically glycosylated with lactose. Circular dichroism (CD) spectra showed that chemical glycosylation did neither change the tertiary structure of HRP nor the heme environment. The highest sensitivity of the biosensor to hydroquinone was obtained for the biosensor with HRP-lactose (414nAμM−1) compared to 378nAμM−1 for the one employing non-modified HRP. The chemically glycosylated form of the enzyme catalyzed the reduction of hydroquinone more rapidly than the native form of the enzyme. The sensor employing lactose-modified HRP also had a lower limit of detection (74μM) than the HRP biosensor (83μM). However, most importantly, chemically glycosylation improved the long-term stability of the biosensor, which retained 60% of its activity over a four-month storage period compared to only 10% for HRP. These results highlight improvements by an innovative stabilization method when compared to previously reported enzyme-based biosensors.

A Streptavidin-SOG Chimera for All-Optical Immunoassays

Immunological detection has been developed into a sensitive and versatile technique and is based on two requisite elements: targeting antibodies and an indicator, usually in the form of horseradish peroxidase or alkaline phosphatase. The specificity and turnover rate of these enzymes provide an efficient means of signal amplification, but both require a stopping agent to prevent overdevelopment, which limits scalability to mechanized fluidic systems. As an alternative, we present a fully optical detection system based on a streptavidin-singlet oxygen generating chimeric protein that produces singlet oxygen from blue light. When used with trans-1-(2'-methoxyvinyl)pyrene, the photosynthetic streptavidin combines indicator development and reporting in sufficiently distinct visible wavelengths while retaining the sensitivity and scale of enzymatic systems that use horseradish peroxidase. By combining photosensitive development and detection into one system, we can enable future, highly parallel immunological testing to be controlled with the spatial and temporal precision of light.

Long-range electron transfer in recombinant peroxidases anisotropically orientated on gold electrodes

Long-range electron transfer (ET) in horseradish peroxidase (HRP) was studied with a wild-type recombinant form of HRP, rHRP, and recombinant forms containing histidine and cysteine tags at Gln1, Asn57, Asn189, or Ser309 amino acid residues of the protein. Chemisorption of the enzyme onto the Au electrodes through the tags introduced in different positions of the protein surface provided anisotropic orientations of the rHRPs on the Au surface, which allowed a restricted "rotation" of the rHRP molecules on the electrodes. Atomic force microscopy (AFM) studies revealed the monolayer coverage of the enzyme on gold surfaces and the specific orientations of different forms of rHRP, which may be characterized by different distances between the heme active site of rHRP and the gold electrode. The efficiency of long-range ET between the electrode and the heme of rHRP was estimated from direct non-catalytic electrochemistry of rHRPs differently orientated on Au and compared with the theoretically calculated values from the protein ET model (C. C. Page, C. C. Moser, X. Chen, P. L. Dutton, Nature, 1999, 402, 47-51), under the assumption that ET occurs within the protein structure between the heme and the tag-modified amino acid residue of the protein. Comparative analysis of the long-range ET through the rHRP showed that the highest ET rates, obtained for the rHRP forms containing the tags at C- or N-termini of the enzyme, did not correlate with the shortest ET distance, but were instead consistent with the directional ET along the most favourable ET pathway within the protein matrix.

Dynamics of Nitric Oxide Rebinding and Escape in Horseradish Peroxidase

Ultrafast kinetic measurements of NO rebinding to horseradish peroxidase (HRP) are reported for the first time. The geminate kinetics are found to be exponential for all HRP samples studied. The ferric forms of HRP have NO geminate recombination time constants in the range of 15-30 ps, while the ferrous form has a time constant of approximately 7 ps. The simple exponential NO geminate kinetics found for HRP demonstrate that heme relaxation is not the underlying source of the nonexponential NO rebinding in myoglobin (Mb). The NO ligand escape rates from HRP are also determined, and they are found to depend dramatically on the presence or absence of the competitive inhibitor benzohydroxamic acid (BHA). The kinetic results indicate that, in contrast to Mb, there is direct solvent access to the distal heme pocket of HRP.

Absence of a Detectable Intermediate in the Compound I Formation of Horseradish Peroxidase at Ambient Temperature*

A microsecond-resolved absorption spectrometer was developed to investigate the elementary steps in hydrogen peroxide (H(2)O(2)) activation reaction of horseradish peroxidase (HRP) at ambient temperature. The kinetic absorption spectra of HRP upon the mixing with various concentrations of H(2)O(2) (0.5-3 mm) were monitored in the time range from 50 to 300 mus. The time-resolved spectra in the Soret region possessed isosbestic points that were close to those between the resting state and compound I. The kinetic changes in the Soret absorbance could be well fitted by a single exponential function. Accordingly, no distinct spectrum of the putative intermediate between the resting state and compound I was identified. These results were consistent with the proposal that the O-O bond activation in heme peroxidases is promoted by the imidazolium form of the distal histidine that exists only transiently. It was estimated that the rate constant for the breakage of the O-O bond in H(2)O(2) by HRP is significantly faster than 1 x 10(4) s(-1).

Mechanistic and molecular investigations on stabilization of horseradish peroxidase C.

The enzyme horseradish peroxidase (HRP) shows a decreasing activity when the enzyme's substrate hydrogen peroxide is present with the degree of inactivation being dependent on the incubation time and the hydrogen peroxide concentration. Incubation times of some minutes do not inactivate the enzyme independent of the H2O2 concentration. After several hours, only 50% of the activity is found for a medium H2O2 excess, and a >100-fold excess of H2O2 completely inactivates the enzyme. Polymeric additives, in particular Gafquat, lead to higher residual activities, whereas stabilizers, such as aminopyrine, preserve the full activity. Circular dichroism (CD) measurements reveal that the enzyme structure remains more or less unchanged when hydrogen peroxide is added. Only when a 1000-fold excess of hydrogen peroxide is present are structural changes observed. UV spectra highlight that the heme group in the enzyme is affected by hydrogen peroxide in a first step. Without any prolonged incubation, a decrease of the Soret band to approximately 50% is found for low hydrogen peroxide concentrations (HRP/H2O2 from 1:1 to 1:100). Higher H2O2 concentrations lead to the formation of catalytically inactive HRP forms. Preincubation of Gafquat, which is a copolymer from vinylpyrrolidone and derivatized methyl methacrylate, with hydrogen peroxide shifts the influence of hydrogen peroxide to higher concentrations, the shift being dependent on the Gafquat concentration. This effect is not observed for other polymers, such as dextrans, but it is also found for the stabilizer aminopyrine. Extended incubation times (24 h) of HRP together with H2O2, however, lead to an at least partial recovery of the Soret band for lower H2O2 concentrations (H2O2/HRP from 1:1 to 1:100). When hydrogen peroxide is used in a >100 fold excess, the heme group is irreversibly destroyed, and even the characteristic band of cpd III is not found. Here, the presence of Gafquat only reduces the degree of destruction. Computer modeling of the interaction between the polymers and the enzyme shows no specific binding sites for the functional groups of the vinylpyrrolidone-methacrylate copolymer Gafquat or of DEAE-dextran on the enzyme, whereas for the only activating polymer, polyethylenimine clustering of binding sites is observed.

Comparative study of horseradish mutant forms by radioenzymology

Radioenzymology was used to study the recombinant and mutant forms of horseradish peroxidase, namely, F221W, Q176E, Q176A, S35K, E64P, E64S, S35A, and S35KQ176E. Both removal of the Trp residue and introduction of an additional one result in a simpler dose response; the insertion of polar residues stabilizes the enzyme molecule through realization of a more closed conformation. The greatest oscillation changes were found for the replacement by Ala. It was assumed that the binding site of guaiacol as a substrate is located near the residue 64, which is structurally related to the residue 176 and the heme. A scheme of formation of the intermediate through rotation of the Trp aromatic ring was proposed.

Effect of pH on direct electron transfer in the system gold electrode–recombinant horseradish peroxidase

The effect of pH on the kinetics of the bioelectrocatalytic reduction of H 2O 2 catalysed by horseradish peroxidase (HRP) has been studied at –50 mV vs. Ag∣AgCl on HRP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Native HRP (nHRP) and a nonglycosylated recombinant form containing a six-histidine tag at the C-terminus, C HisrHRP, produced by genetic engineering of nonglycosylated recombinant HRP using an E. coli expression system, have been used for adsorptive modification of Au electrodes. A favourable adsorption of C HisrHRP on preoxidised Au from a protein solution at pH 6.0 provided a high and stable current response to H 2O 2 due to its bioelectrocatalytic reduction based on direct (mediatorless) electron transfer (ET) between Au and the active site of HRP. The heterogeneous ET rate constant, k s, calculated from experimental data on direct ET, on mediated ET in the presence of catechol as well as from microbalance data, increased more than 30 times when changing from nHRP to C HisrHRP. For both forms of HRP, the increasing efficiency of bioelectrocatalysis with increasing [H 3O +] was observed. The values of the apparent k s between C HisrHRP and Au changed from a value of 12±2 s −1 in PBS at pH 8.0 to a value of 434±62 s −1 at pH 6.0; a similar k s–pH dependence was also observed for nHRP, providing the possibility to consider the reaction mechanism involving the participation of a proton in the rate-determining step of the charge transfer.

Effect of interfering substances on current response of recombinant peroxidase and glucose oxidase-recombinant peroxidase modified graphite electrodes.

Graphite electrodes have been modified with different forms of horseradish peroxidase (HRP). These included native HRP, wild-type recombinant HRP, and two single-point recombinant HRP mutants, N70V and N70D. The mediator-less response of these electrodes to H2O2 was studied indicating that electrodes modified with recombinant HRP forms are more stable than those modified with native HRP. Various interfering compounds were investigated for their effect on the current response to H2O2. It was found that interferences such as acetaminophen and dopamine affected the response by mediating the electron transfer (ET) between graphite and peroxidases. The mediating behaviour manifested itself as an increased current of the electrode to H2O2. The interfering effect was less pronounced for the electrodes modified with recombinant HRPs possessing better electronic coupling with the graphite surface. The interfering behaviour of acetaminophen on the response for glucose with the bienzyme electrode containing co-immobilised glucose oxidase and HRP was mainly ascribed to mediation of ET between graphite and HRP. It was experimentally proven that a high efficiency of direct ET between graphite and recombinant HRP substantially reduces the interfering effect of acetaminophen.

Oxidation of Melatonin and Tryptophan by an HRP Cycle Involving Compound III

We recently described that horseradish peroxidase (HRP) and myeloperoxidase (MPO) catalyze the oxidation of melatonin, forming the respective indole ring-opening product N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) (Biochem. Biophys. Res. Commun. 279, 657–662, 2001). Although the classic peroxidatic enzyme cycle is expected to participate in the oxidation of melatonin, the requirement of a low HRP:H2O2 ratio suggested that other enzyme paths might also be operative. Here we followed the formation of AFMK under two experimental conditions: predominance of HRP compounds I and II or presence of compound III. Although the consumption of substrate is comparable under both conditions, AFMK is formed in significant amounts only when compound III predominates during the reaction. Using tryptophan as substrate, N- formyl-kynurenine is formed in the presence of compound III. Both, melatonin and tryptophan efficiently prevents the formation of p-670, the inactive form of HRP. Since superoxide dismutase (SOD) inhibits the production of AFMK, we proposed that compound III acts as a source of O−•2 or participates directly in the reaction, as in the case of enzyme indoleamine 2,3-dioxygenase.

Direct electron transfer in the system gold electrode–recombinant horseradish peroxidases

The kinetics of the bioelectrocatalytic reduction of hydrogen peroxide has been studied at gold electrodes modified with different forms of horseradish peroxidase (HRP). Native HRP, wild type recombinant HRP (rec-HRP) and its two mutant forms containing a six-histidine tag at the C- or N-terminus, C Hisrec-HRP and N Hisrec-HRP, respectively, have been used for an adsorptive modification of the gold electrodes. The histidine sequences, i.e. histidine tags, were introduced into the peroxidase structure by genetic engineering of non-glycosylated rec-HRP using an Escherichia coli expression system. Experiments with a gold rotating disc electrode demonstrated that electrodes with the adsorbed rec-HRP forms exhibited high and stable current response to H 2O 2 due to its bioelectrocatalytic reduction based on direct (mediatorless) ET between gold and the active site of HRP. The heterogeneous ET rate constants were evaluated to be in the order of 20 or 33 s −1 between rec-HRP or its histidine mutants and gold, respectively, in 0.01 M phosphate buffer (pH 7.4) containing 0.15 M NaCl. The increase in the heterogeneous ET rate found for C Hisrec-HRP and N Hisrec-HRP is probably due to the interaction of the histidine tag with the electrode surface. The kinetic data demonstrate that new possibilities for enhancing the catalytic activity of the enzyme at the electrode ∣ solution interface can be achieved by genetic engineering design of the enzyme molecules.

Mediatorless biosensor for H 2O 2 based on recombinant forms of horseradish peroxidase directly adsorbed on polycrystalline gold

Four forms of horseradish peroxidase (HRP) have been used to prepare peroxidase-modified gold electrodes for mediatorless detection of peroxide: native HRP, wild type recombinant HRP, and two recombinant forms containing six-His tag at the C-terminus and at the N-terminus, respectively. The adsorption of the enzyme molecules on gold was studied by direct mass measurements with electrochemical quartz crystal microbalance. All the forms of HRP formed a monolayer coverage of the enzyme on the gold surface. However, only gold electrodes with adsorbed recombinant HRP forms exhibited high and stable current response to H 2O 2 due to its bioelectrocatalytic reduction based on direct electron transfer between gold and HRP. The sensitivity of the gold electrodes modified with recombinant HRPs was in the range of 1.4–1.5 A M −1 cm −2 at −50 mV versus Ag∣AgCl. The response to H 2O 2 in the concentration range 0.1–40 μM was not dependent on the presence of a mediator (i.e. catechol) giving strong evidence that the electrode currents are diffusion limited. Lower detection limit for H 2O 2 detection was 10 nM at the electrodes modified with recombinant HRPs.

P-chip and P-chip bienzyme electrodes based on recombinant forms of horseradish peroxidase immobilized on gold electrodes.

Adsorption and bioelectrocatalytic activity of native horseradish peroxidase (HRP) and its recombinant forms on polycrystalline gold electrodes were studied. Recombinant forms of HRP were produced by a genetic engineering approach using an E. coli expression system. According to direct mass measurements with a quartz crystal microbalance, all the forms of HRP formed monolayer coverage of the enzyme on the gold surface. However, only gold electrodes modified with the recombinant HRP forms (non-glycosylated) exhibited high and stable current response to H2O2 due to its bioelectrocatalytic reduction based on direct electron transfer (ET) between gold and the active site of the enzyme. Introduction of a six-His tag either at the C-terminus or at the N-terminus of the enzyme molecule additionally increased the strength of the enzyme binding with the gold surface and the efficiency of direct ET. Immobilization of recombinant forms of HRP containing histidine functional groups on the surface of the gold electrode was used both for the development of a P-chip, a biosensor for hydrogen peroxide determination based on direct ET, and for the development of a bienzyme biosensor electrode for the determination of L-lysine based on co-immobilized recombinant forms of HRP and L-lysine-alpha-oxidase.

Influence of protein environment on magnetic circular dichroism spectral properties of ferric and ferrous ligand complexes of yeast cytochrome c peroxidase.

The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism (MCD) at 4 degrees C to determine the effect the protein environment may exercise on spectral properties. The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HRP) and myoglobin (Mb), two well-characterized histidine-ligated heme proteins. The ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an MCD spectrum very similar to that of ferric azide HRP. This complex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand and the distal arginine of CCP (Arg 48). For the ferric fluoride derivative all three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD characteristics. The MCD data for the cyanoferric complexes are similar for all three proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative. Similarly, the ferric NO complexes of the three proteins show MCD spectra similar in feature position and shape, but vary somewhat in intensity. Reduction of CCP at neutral pH yields a typical pentacoordinate high-spin complex with an MCD spectrum similar to that of deoxyferrous HRP. Formation of the NO and cyanide complexes of ferrous CCP gives derivatives with MCD spectra similar to the analogous forms of HRP and Mb in both feature position and shape. Addition of CO to deoxyferrous CCP results in a ferrous-CO complex with MCD spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD characteristics. Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that a pH dependent conformational change has occurred, leading to a coordination structure similar to that of ferrous cytochrome b5, a known bis-histidine complex. Exposure of this complex to CO further confirms that a conformational change has taken place in that the MCD spectral characteristics of the resulting complex are similar to those of ferrous-CO Mb but not ferrous-CO HRP.

A theoretical study of electronic factors affecting hydroxylation by model ferryl complexes of cytochrome P-450 and horseradish peroxidase

Density functional theory (DFT) is used to study model ferryl species of cytochrome P-450 and horseradish peroxidase (HRP), as well as of the product complex due to oxidation of H2 by the P-450 species (1–4 and 7). The ferryl species studied include neutral and cation radical states of the porphyrin, as well as high- and low-spin situations. A few issues are addressed concerning the mechanism of alkane hydroxylation, and theoretical support is provided for: (i) the contention that spin inversion occurs along the reaction path, (ii) that the cation radical state of the porphyrin is an essential feature required to accommodate an excess electron from the ferryl moiety and thereby stabilize the ground state of the hydroxylation product, and (iii) that the donor property of the proximal ligand has a significant influence on the energy of the ferryl-to-ring charge-transfer states which are essential to convert the reactant state to the hydroxylation product state. In this sense, our study sheds some light on the difference between the oxidized and reduced HRP forms, HRP(I) and HRP(II), and suggests that the combination of a cation radical porphyrin state and a good π-donor proximal ligand like thiolate, could be the underlying reason for the potent hydroxylation ability of the P-450 ferryl-complex.

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.

Organic phase enzyme electrodes: kinetics and analytical applications

The suitability of organic media for enzymatic reactions has led to tremendous advancement in biosynthesis and, more recently, biosensing. Organic phase enzyme-based biosensors (OPEE), i.e. biosensors that operate in organic media, combine the high selectivity and specificity of enzymes with the ability to control their reactivity by altering some physiochemical properties of the reaction media, such as solvent polarity and degree of hydration. Classical enzymology predicts that hydrophobic organic solvents, rather than hydrophilic solvents, are suitable for enzyme activity, on account of the latter's ability to strip the enzyme of essential water of hydration necessary for the flexibility and polarity of the active site micro-environment. We have demonstrated with solvents, such as acetonitrile, acetone, butan-2-ol, chloroform, hexane and tetrahydrofuran, that organic-phase amperometric biosensors can be developed to exhibit excellent reactivities in both hydrophilic and hydrophobic organic solvents by controlling the degree of hydration of the biosensing environment. Amperometric biosensors containing tyrosinase, glucose oxidase (GOx), horseradish peroxidase (HRP), and more recently chemically modified HRP, have been constructed and successfully applied to assays in organic solvents. Several enzyme immobilization matrices have been used in our laboratory for the fabrication of sensors. Poly(estersulphonic acid)-entrapped tyrosinase and HRP electrodes have been found to be very effective biosensors in a variety of organic solvents for determining phenols, organic peroxides and pesticide compounds. The Os-polymers, [Os(bpy) 2(PVP) nCl]Cl [bpy = 2,2′-bipyridyl, PVP = poly(4-vinylpyridine); n = 10, 20 or 25] and [Os(byp) 2(PVI) 10Cl]Cl [PVI = poly(4-vinylimidazole)] have been used with bifunctional cross-linking agents, such as glutaraldehyde or polyethylene glycol, to form reagentless biosensors with GOx, tyrosinase and HRP (native and modified forms of HRP). These biosensors, based on the electrostatic complexation of the enzyme and the positively charged Os-polymer, have been found to be very stable in organic phase. GOx sensors that employ soluble mediators, such as, ferrocenemonocarboxylic acid, exhibited better catalytic efficiency, k cat ′ K M ′ in the organic phase than in the aqueous phase. In our studies we have demonstrated that the reactivities of amperometric organic phase biosensors follow the Michaelis-Menten kinetic paradigm. However, kinetic analyses have shown that the values of the sensor biocatalytic parameters, including I max, k cat′ and K M′ depend on solvent media. Under certain conditions, k cat ′ K M ′ is the second order rate constant through ΔG =/ −RT ln[ k cat ′ K M ′ )hlk B T], where T, h, and k B are the absolute temperature, Planck's constant and Boltzmann's constant, respectively. Thus, k cat ′ K M ′ is a measure of the activation of the biosensor redox-catalytic reaction in different solvents. This parameter has been evaluated at different temperatures in both aqueous and organic media. The ΔG =/ values (the difference in Gibbs energy between transition and ground state of sensor reaction for a given solvent and substrate) in organic and aqueous media have been used to evaluate the role of the solvent on the stabilization of the transition state on the electrocatalytic reaction. Organic phase HRP biosensors have also been applied in our laboratory for the determination of thiourea, ethylenethiourea and other thio-compounds (which are the parent compounds for some pesticides). The thio-compounds act as inhibitors. Their determination is based on the change in the electrocatalytic current of peroxide reduction by the biosensor, which accompanies the addition of the organic sulphides. Tyrosinase electrodes have been applied as detectors in reversed-phase HPLC for the detection of the eight phenols that are normally found in cigarette filters. Native HRP and bis-succinimide-modified-HRP electrodes have also been applied in the amperometric determination of organic peroxides, the aim being to produce a more sensitive, reproducible and stable organic phase HRP electrode.

Horseradish peroxidase—catalyzed hydroxylation of phenol: I. Thermodynamic analysis

We analyzed the horseradish peroxidase (HRP)—catalyzed hydroxylation of phenol in the presence of dihydroxy-fumaric acid and oxygen. All of the intermediate forms of the enzyme are reviewed. The last step of hydroxylation, consisting of the production of OH • radicals that further react on phenol, is emphasized. Possible OH • radicals production reactions were compiled and analyzed with respect to the available thermodynamic data. Some results of electrochemical experiments were also used to choose the correct set of reactions. At the end of analysis only two reactions for producing OH • seemed to be consistent with the thermodynamic and experimental data. Neither of these reactions involved compound III or any other intermediate form of HRP. The last step of hydroxylation was thus totally independent of the pure catalytic cycle of the enzyme. As a consequence, HRP cannot be used as an hydroxylation enzyme in place of the P 450 cytochrome, as is sometimes suggested.

Hydroxylation of benzene by horseradish peroxidase and immobilized horseradish peroxidase in an organic solvent

Horseradish peroxidase (HRP) catalyzed hydroxylation of benzene with an oxidant when benzene was used as the reaction solvent. HRP immobilized on poly(γ-methyl-L-glutamate) also catalyzed the reaction with higher activity than that of free HRP. The results of the reaction using [ 18O]hydrogen peroxide show that free and immobilized HRP incorporated the oxygen atom of the reactive species into the product, indicating that both forms of HRP can catalyze hydroxylation of benzene in an analogous manner to that of cytochrome P450.