Horseradish Peroxidase Intermediates Research Articles

Carbon-felt-based Bioelectrocatalytic Flow-detectors: Role of Ultrasound Irradiation during the Adsorption of Horseradish Peroxidase and Thionine for a Highly Sensitive Amperometric Determination of H<sub>2</sub>O<sub>2</sub>

This study reports on the first example, to our knowledge, of the usefulness of an ultrasound (US)-irradiation during an enzyme adsorption step, for enhancing the performance of a redox-enzyme-based amperometric biosensor. Horseradish peroxidase (HRP) and thionine (Th) were coadsorbed from a mixed aqueous solution of HRP and Th onto a carbon-felt (CF) under US-irradiation for 5 min with an ultransonic bath operating at 40 kHz frequency and 55 W of electric power output. The resulting HRP and Th-coadsorbed CF (HRP/Th-CF) was successfully used as a working electrode unit of a bioelectrocatalytic flow-detector for hydrogen peroxide (H(2)O(2)), which detects the cathodic peak currents based on the direct (unmediated) reduction of oxidized HRP intermediates at 0 V vs. Ag/AgCl. Compared with ordinary adsorption without US-irradiation, US-irradiation during the HRP adsorption step was effective to obtain highly sensitive peak current responses to H(2)O(2). The measurements of electrochemical impedance spectroscopy and cyclic voltammetry suggested that the adsorption of HRP and Th under the US-irradiation provides a suitable interfacial microenvironment for a favorable orientation and conformation of an enzyme with active site available for both substrates and the electrode, which results in larger bioelectrocatalytic activity. The peak currents for H(2)O(2) increased up to 3 × 10(-6) M (sensitivity, 4.72 µA/µM) with a lower detection limit of 2 × 10(-8) M (S/N = 3; current noise level, 0.03 µA).

Photooxidation of cytochrome P450-BM3

High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a Ru(II) photosensitizer to a nonnative cysteine near the heme (RuIIK97C-FeIIIP450), in order to promote electron transfer from the Fe(III) porphyrin to photogenerated Ru(III). The conjugate was structurally characterized by X-ray crystallography (2.4 Å resolution; Ru-Fe distance, 24 Å). Flash-quench oxidation of the ferric-aquo heme produces an Fe(IV)-hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.

Mechanism of horseradish peroxidase-catalyzed heme oxidation and polymerization (β-hematin formation)

Horseradish peroxidase (HRP) catalyzes the polymerization of free heme (beta-hematin formation) through its oxidation. Heme when added to HRP compound II (FeIV=O) causes spectral shift from 417 nm (Compound II) to 402 nm (native, FeIII) indicating that heme may be oxidized via one-electron transfer. Direct evidence for one-electron oxidation of heme by HRP intermediates is provided by the appearance of an E.s.r signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-heme radical adduct (a1H=14.75 G, a2H=4.0 G) in E.s.r studies. Heme-polymerization by HRP is inhibited by spin trap indicating that one-electron oxidation product of heme ultimately leads to the formation of heme-polymer. HRP, when incubated with diethyl pyrocarbonate (DEPC), a histidine specific reagent, shows concentration dependent loss of heme-polymerization indicating the role of histidine residues in the process. We suggest that HRP catalyzes the formation of heme-polymer through one-electron oxidation of free heme.

Oxidative activation of indole-3-acetic acids to cytotoxic species— a potential new role for plant auxins in cancer therapy

Indole-3-acetic acid (IAA) and some derivatives can be oxidised by horseradish peroxidase (HRP) to cytotoxic species. Upon treatment with IAA/HRP, liposomes undergo lipid peroxidation, strand breaks and adducts are formed in supercoiled plasmid DNA, and mammalian cells in culture lose colony-forming ability. IAA is only toxic after oxidative decarboxylation; no effects are seen when IAA or HRP is incubated independently in these systems at equivalent concentrations. Toxicity is similar in both hamster fibroblasts and some human tumour cells. The effect of IAA/HRP is thought to be due in part to the formation of 3-methylene-2-oxindole, which may conjugate with DNA bases and protein thiols. Our hypothesis is that IAA/HRP could be used as the basis for targeted cancer therapy involving antibody-, polymer-, or gene-directed approaches. HRP can thus be targeted to a tumour allowing non-toxic IAA delivered systemically to be activated only in the tumour. Exposure to newly synthesised analogues of IAA shows a range of four orders of magnitude difference in cellular toxicity but no structure–activity relationships are apparent, in contrast to well-defined redox dependencies of oxidation by HRP intermediates or rates of decarboxylation of radical-cation intermediates.

Mechanism for the Decoloration of Orange II Catalyzed by Horseradish Peroxidase

Study was made to clarify the mechanism for decoloration of Orange II in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Thiol (RSH) was found to decrease the decoloration rate of Orange II. The time which takes for the decoloration rate of Orange II to start rising is equal to the time it takes for RSH to decrease and disappear. Decoloration reaction of Orange II thus occurs through formation of Orange II radicals. Next, the reaction time of HRP system was measured by the determination of the change on HRP oxidation states. Though the decoloration time of Orange II with HRP was a few seconds, the reaction time of HRP-H2O2 system was 60min. 1, 2-Naphthoquinone was separated from reaction compounds by HPLC. Therefore, it is evident that Orange II acts on HRP intermediates as a hydrogendonor to produce Orange II radicals in HRP-H2O2 reaction system and also that the reaction products of Orange II acts on HRP intermediates as a hydrogen donors.

Transient-state and steady-state kinetics of the oxidation of aliphatic and aromatic thiols by horseradish peroxidase

In the course of oxidation of thiols by peroxidases thiyl radicals are formed which are known to undergo several free-radical conjugative reactions, among others leading to hydrogen peroxide formation. The present paper for the first time presents a comparative transient-state and steady-state investigation of the reaction of 15 aliphatic and aromatic mono- and dithiols with horseradish peroxidase (HRP). Both sequential-stopped-flow spectrophotometric investigations of the reaction of HRP intermediates Compound I ( k 2) and Compound II ( k 3) with thiols and measurements of the overall thiol oxidation and the simultaneous oxygen consumption in the presence and absence of exogenously added hydrogen peroxide (10 μM) have been performed. With HRP as thiyl radical generator it was shown that three groups of thiols have to be distinguished: (i) Aromatic thiols (e.g. thiophenol, 2-mercaptopurine) were excellent electron donors of both Compounds ( k 2: 10 4–10 7 M −1 s −1 and k 3: 10 3–10 6 M −1 s −1); however, the overall reaction was shown to depend on addition of hydrogen peroxide, indicating insufficient peroxide regeneration by arylthiyl radicals. (ii) Aliphatic thiols which were extremely bad substrates ( k 3 <10 M −1 s −1) for HRP (e.g. homocysteine, glutathione) and/or have a p K a,SH >9.5 (e.g. N-acetylcysteine, α-lipoic acid) were also shown to depend on exogenously added H 2O 2 to maintain the peroxidasic reaction, whereas (iii) with those thiols with rates of k 3 between 11 and 1600 M −1 s −1 (e.g. cysteine, cysteamine, cysteine methyl ester, cysteine ethyl ester) and/or with a p K a,SH <8 (penicillamine) thiol oxidation was independent of exogenously added hydrogen peroxide, indicating sufficient hydrogen peroxide regeneration.

Theoretical study of model compound I complexes of horseradish peroxidase and catalase

Theoretical studies of the electronic structure and spectra of models for the ferric resting state and Compound I intermediates of horseradish peroxidase (HRP-I) and catalase (CAT-I) have been performed using the INDO-RHF/CI method. The goals of these studies were twofold: i) to determine whether the axial ligand of HRP is best described as imidazole or imidazolate, and ii) to address the long-standing question of whether HRP-I and CAT-I are a1u and a2u tau cation radicals. Only the imidazolate HRP-I model led to a calculated electronic spectra consistent with the experimentally observed significant reduction in the intensity of the Soret band compared with the ferric resting state. These results provide compelling evidence for significant proton transfer to the conserved Asp residue by the proximal histidine. The origin of the observed reduction of the Soret band intensity in HRP-I and CAT-I spectra has been examined and found to be caused by the mixing of charge transfer transitions into the predominantly porphyrin tau-tau transitions. For both HRP-I and CAT-I, the a1u porphyrin tau cation state is the lowest energy, and it is further stabilized by both the anionic form of the ligand and the porphyrin ring substituents of protoporphyrin-IX. The calculated values of quadrupole-splitting observed in the Mossbauer resonance of HRP-I and CAT-I are similar for the a1u and a2u tau cation radicals. Electronic spectrum of the a1u tau cation radical of HRP-I are more similar to the observed spectra, whereas the spectra of both a1u tau and a2u tau cation radicals of CAT-I resemble the observed spectra. These results also indicate the limitations of using any one observable property to try to distinguish between these states. Taken together, comparison of calculated and observed properties indicate that there is no compelling reason to invoke the higher energy a2u tau cation radical as the favored state in HRP-I and CAT-I. Both ground-state properties and electronic spectra are consistent with the a1u tau cation radical.

Oscillatory Chemiluminescence during Peroxidation of Umbelliferone Catalyzed by Horseradish Peroxidase

Abstract The chemiluminescence (CL) which occurred during oxidation of umbelliferone catalyzed by horseradish peroxidase (HRP) oscillated, when a high concentration of H2O2 was employed. The oscillatory CL response reflected periodic changes in the concentration of HRP intermediates between active states, compound I and compound II, and an inactive state of compound III. When the reaction was initiated, the CL was generated by the action of compound I and compound II. Then, the concentration of compound III was gradually increased, because the formation of compound III from compound II and H2O2, the rate constant of which was very little, could not be ignored in a high H2O2 condition; thus the CL was decreased by degrees. Compound III was then decomposed into the active HRP intermediates and the CL was re-increased. The repetition of those processes causes the oscillation. A kinetic study on the mechanism of the oscillatory CL was performed with trial rate equations.

Mechanism of inhibition of horseradish peroxidase-catalysed iodide oxidation by EDTA.

EDTA inhibits horseradish peroxidase (HRP)-catalysed iodide oxidation in a concentration and pH-dependent manner. It is more effective at pH 6 than at lower pH values. A plot of log Kiapp. values as a function of pH yields a sigmoidal curve from which a pKa value of 5.4 can be calculated for an ionizable group on the catalytically active HRP for EDTA inhibition. Among the structural analogues of EDTA, tetramethylethylenediamine (TEMED) is 80% as effective as EDTA, whereas the EDTA-Zn2+ chelate and EGTA are ineffective. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation. Spectral studies show that EDTA can quickly reduce compound I to compound II, but reduction of preformed compound II to the native enzyme is relatively slow, as demonstrated by the time-dependent spectral shift from 417 nm to 402 nm through an isosbestic point at 408 nm. Under steady-state conditions, in a reaction mixture containing HRP, EDTA and H2O2, the enzyme remains in the compound-II form, with absorption maxima at 417, 527 and 556 nm. Direct evidence for one-electron oxidation of EDTA by HRP intermediates is provided by the appearance of an e.s.r. signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-EDTA radical adduct [aN (hyperfine splitting constant) = 1.5 mT] in e.s.r. studies. The signal intensity, however, decreases in the presence of iodide. The KD of the HRP-EDTA complex obtained from optical difference spectroscopy increases with an increase in iodide concentration, and the double-reciprocal plot for EDTA binding indicates that EDTA and iodide compete for the same binding site for oxidation. We suggest that EDTA inhibits iodide oxidation by acting as an electron donor.

Resonance Raman Spectroscopy of Horseradish Peroxidase Derivatives and Intermediates with Excitation in the Near Ultraviolet

Resonance Raman enhancement of derivatives and intermediates of horseradish peroxidase in the near ultraviolet (N-band excitation) results in intensity and enhancement patterns that are different from those normally observed within the porphyrin Soret (B-band) and alpha-beta (Q-band) absorptions. In particular it allows the resolution of resonance Raman spectra of horseradish peroxidase compound I. The bands above 1300 cm-1 can be assigned to porphyrin vibrational modes that are characteristically shifted in frequency due to removal of an electron from the porphyrin ring. The resonance Raman frequency shifts follow normal mode compositions. Relative to resonance Raman spectra of compound II, the v4 frequency (primarily Ca-N) exhibits a 20 cm-1 downshift. The v2, v11, and v37 vibrational frequencies whose mode compositions are primarily porphyrin Cb-Cb, exhibit 10-20 cm-1 upshifts. The v3, v10, and v28 frequencies, whose mode compositions are primarily Ca-Cm, exhibit downshifts. The downshifts for v3 and v10 are small, 3-5 cm-1; however, the downshift for v28 is 14 cm-1. These frequency shifts are consistent with those of previously published resonance Raman studies of model compounds. In contrast to reports from other laboratories, the data presented here for horseradish peroxidase compound I can be attributed unambiguously to resonance Raman scattering from a porphyrin pi-cation radical.

Polarized x-ray absorption near-edge structure of highly oxidized chromium porphyrins

Polarized X-ray absorption near-edge spectra have been measured for Cr/sup IV/(TTP)O and Cr/sup V/(TTP)N (TTP is the dianion of 5,10,15,20-tetra-p-tolylporphyrin). These spectra, which are very similar for the two molecules, show an intense preedge absorption feature polarized perpendicular to the porphyrin plane. The absorption edges are relatively featureless when the polarization is parallel to the porphyrin plane. On the basis of multiple-scattered-wave X..cap alpha.. calculations, the intense preedge feature is interpreted as a bound ..-->.. bound transition with significant metal character in the excited state. This transition, which has been observed in many metal-oxo species, was not observed in the putative ferryl (Fe=o) intermediates of horseradish peroxidase. The reasons for this absence in Fe=O species and its relationship to the reactivity of metal-oxo porphyrin complexes are discussed in light of the present results.

Role of Phenolic Inhibitors in Peroxidase-mediated Degradation of Indole-3-acetic Acid

7-Hydroxy-2,3-dihydrobenzofuran derivatives, metabolites of a carbamate insecticide carbofuran, and five other phenolic inhibitors of indoleacetic acid (IAA) oxidase interfered with IAA-induced spectral change in the Soret band of horseradish peroxidase (HRP). The onset of IAA degradation required transformed HRP intermediates. The inhibitors, when added before IAA, protected HRP from reacting with IAA, thus preventing formation of highly reactive enzyme intermediates, and consequently, IAA degradation. When added after IAA, the inhibitors quickly reversed the IAA-induced spectral change of HRP and inhibited further IAA degradation.The phenolic inhibitors differed in stability and reactivity. 7-Hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran, 3,7-dihydroxy-2,2-dimethyl-2,3-dihydrobenzofuran, catechol, protocatechuic acid, caffeic acid, ferulic acid, and scopoletin belonged to one group which produced only a temporary inhibition to IAA-induced spectral change of HRP and IAA degradation since the inhibitors were metabolized in the reaction. The length of the lag was dependent on the IAA, inhibitor, and enzyme concentrations. 3-Keto-7-hydroxy-2,2-dimethyl-2,3-dihydrobenzofuran and 3-keto-carbofuran belonged to the other group which produced a persistent inhibition.Degradation of IAA required both the heme group and apoprotein of HRP. Reconstituted enzyme from bovine hemin and apoprotein or HRP after unfolding by urea or guanidine treatment were inhibited by the inhibitors in a way similar to the native HRP. The inhibition was reversible by higher concentrations of IAA, but the plot of 1/v versus 1/s and 1/v versus i were curvilinear, reflecting the complex nature of a competitive inhibition.