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Myoglobin (Mb) and horseradish peroxidase (HRP) were both reconstituted with a meso-unsubstituted iron corrole and their electronic configurations and peroxidase activities were investigated. The appearance of the 540 nm band upon incorporation of the iron corrole into apoMb indicates axial coordination by the proximal histidine imidazole in the Mb heme pocket. Based on (1)H NMR measurements using the Evans method, the total magnetic susceptibility of the iron corrole reconstituted Mb was evaluated to be S = 3/2. In contrast, although a band does not appear in the vicinity of 540 nm during reconstitution of the iron corrole into the matrix of HRP, a spectrum similar to that of the iron corrole reconstituted Mb is observed upon the addition of dithionite. This observation suggests that the oxidation state of the corrole iron in the reconstituted HRP can be assigned as +4. The catalytic activities of both proteins toward guaiacol oxidation are quite different; the iron corrole reconstituted HRP decelerates H(2)O(2)-dependent oxidation of guaiacol, while the same reaction catalyzed by iron corrole reconstituted Mb has the opposite effect and accelerates the reaction. This finding can be attributed to the difference in the oxidation states of the corrole iron when these proteins are in the resting state.

Sequential detections of different proteins on Western blot save time and precious samples. The main problem concerning reprobing is that stripping buffers can unbind both the antibody and the tested antigen. An original reprobing method has been set up based on horseradish peroxidase (HRP) inhibition after enhanced chemiluminescence detection. Instead of removing previously fixed antibodies as common stripping buffers do, the HRP activity linked to the secondary antibody is irreversibly inhibited by excess of hydrogen peroxide. A 15-min incubation allows one to perform at least five different sequential detections without losing significant amounts of blotted proteins.

This article describes the employment of a novel p-phenol derivative, 4-(1,2,4-triazol-1-yl)phenol (TRP), as a highly potent signal enhancer of the luminol-hydrogen peroxide (H(2)O(2))-horseradish peroxidase (HRP) chemiluminescence (CL) system. The CL reaction conditions were optimized, and the enhancement characteristics of TRP were compared with those of p-iodophenol (PIP). TRP produced a strong enhancement of the CL with the effect of prolonging the light emission. The developed system was then applied to the determination of H(2)O(2) with immobilized HRP using magnetic beads as a solid support. The linear range for H(2)O(2) was 2.0x10(-6) to 1.0x10(-3) M. The detection limit for H(2)O(2) was 2.0x10(-6) M. The proposed sensor was applied successfully to the determination of H(2)O(2) in rainwater.

Cytochrome c (cyt c), a mitochondrial intermembrane electron shuttle between complexes III and IV, can, upon binding with an anionic phospholipid, cardiolipin (CL), act as a peroxidase that catalyzes cardiolipin oxidation. H(2)O(2) was considered as a source of oxidative equivalents for this reaction, which is essential for programmed cell death. Here we report that peroxidase cyt c/CL complexes can utilize free fatty acid hydroperoxides (FFA-OOH) at exceptionally high rates that are approximately 3 orders of magnitude higher than for H(2)O(2). Similarly, peroxidase activity of murine liver mitochondria was high with FFA-OOH. Using EPR spin trapping and LC-MS techniques, we have demonstrated that cyt c/CL complexes split FFA-OOH predominantly via a heterolytic mechanism, yielding hydroxy-fatty acids, whereas H(2)O(2) (and tert-butyl hydroperoxide, t-BuOOH) undergo homolytic cleavage. Computer simulations have revealed that Arg(38) and His(33) are important for the heterolytic mechanism at potential FFA-OOH binding sites of cyt c (but not for H(2)O(2) or t-BuOOH). Regulation of FFA-OOH metabolism may be an important function of cyt c that is associated with elimination of toxic FFA-OOH and synthesis of physiologically active hydroxy-fatty acids in mitochondria.

In living organisms, heme is an essential molecule for various biological functions. Recent studies also suggest that heme functions as organelle-derived signal that regulates fundamental cell processes. Furthermore, estimation of heme is widely used for studying various blood disorders. In this regard, development of a rapid, sensitive, and high throughput heme assay has been sought. The most frequently used method of measuring heme by pyridine hemochrome is time, labor, and material intensive, and therefore limiting in its utility for large scale, high throughput analysis. Recently, we reported alternative method that is sensitive and specific to heme, which is based on the ability of horseradish peroxidase (HRP) apo-enzyme to reconstitute with heme to form an active holo-enzyme. Here, we developed high throughput heme assay by performing reactions on multi-well plate with highly sensitive chemiluminescence detection reagents. Detection of chemiluminescence in charged coupled device (CCD)-based gel doc apparatus enables simultaneous measurement of multiple samples. Furthermore, the high sensitivity of this assay allowed a direct measurement of heme in solvent extracts after dilution. This assay is sensitive, quick, provides a large dynamic range, and is well suited for large-scale analysis of heme extracted from minute amount of samples.

Material systems are needed that promote stabilization of entrained molecules, such as enzymes or therapeutic proteins, without destroying their activity. We demonstrate that the unique structure of silk fibroin protein, when assembled into the solid state, establishes an environment that is conducive to the stabilization of entrained proteins. Enzymes (glucose oxidase, lipase, and horseradish peroxidase) entrapped in these films over 10 months retained significant activity, even when stored at 37 degrees C, and in the case of glucose oxidase did not lose any activity. Further, the mode of processing of the silk protein into the films could be correlated to the stability of the enzymes. The relationship between processing and stability offers a large suite of conditions within which to optimize such stabilization processes. Overall, the techniques reported here result in materials that stabilize enzymes to an extent, without the need for cryoprotectants, emulsifiers, covalent immobilization, or other treatments. Further, these systems are amenable to optical applications and characterization, environmental distribution without refrigeration, are ingestible, and offer potential use in vivo, because silk materials are biocompatible and FDA approved, degradable with proteases, and currently used in biomedical devices.

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca(2+) ions. Earlier studies suggested a critical role of one of the bound Ca(2+) ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca(2+)-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca(2+)-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca(2+), perturbs a H-bonding network connecting this Ca(2+) to the heme crevice that involves the distal water. The measured effects of Ca(2)(+) depletion can be interpreted as supporting a structural role for the distal Ca(2+) and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.

The inhibition mechanism of Tb(III) on horseradish peroxidase (HRP) in vitro was discussed. The results from MALDI-TOF/MS and X-ray photoelectron spectroscopy (XPS) showed that Tb(III) mainly interacts with the O-containing groups of the amides in the polypeptide chains of the HRP molecules and forms the complex of Tb(III)-HRP, and, in the complex, the molar ratio Tb(III)/HRP is 2 : 1. The results from CD and atomic force microscopy (AFM) indicated that the coordination effect between Tb(III) and HRP can lead to the conformation change in the HRP molecule, in which the contents of alpha-helix and beta-sheet conformation in the peptide of the HRP molecules is decreased, and the content of the random coil conformation is increased. Meanwhile, the coordination effect also leads to the decrease in the content of inter- and intrapeptide-chain H-bonds in the HRP molecules, resulting in the HRP molecular looseness and/or aggregation. Thus, the conformation change in the HRP molecules can significantly decrease the electrochemical reaction of HRP and its electrocatalytic activity for the reduction of H2O2.

The spectroscopic properties of interactions involving horseradish peroxidase (HRP) and Tb3+ in the simulated physiological solution was investigated with some electrochemical and spectroscopic methods, such as cyclic voltammetry (CV), circular dichroism (CD), X-ray photoelectron spectroscopy (XPS) and synchronous fluorescence (SF). It was found that Tb3+ can coordinate with oxygen atoms in carbonyl groups in the peptide chain of HRP, form the complex of Tb3+ and HRP (Tb-HRP), and then lead to the conformation change of HRP. The increase in the random coil content of HRP can disturb the microstructure of the heme active center of HRP, in which the planarity of the porphyrin cycle in the heme group is increased and then the exposure extent of the electrochemical active center is decreased. Thus Tb3+ can inhibit the electrochemical reaction of HRP and its electrocatalytic activity for the reduction of H2O2 at the Au/Cys/GC electrode. The changes in the microstructure of HRP obstructed the electron transfer of Fe(III) in the porphyrin cycle of the heme group, thus HRP catalytic activity is inhibited. The inhibition effect of Tb3+ on HRP catalytic activity is increased with the increasing of Tb3+ concentration. This study would provide some references for better understanding the rare earth elements and heavy metals on peroxidase toxicity in living organisms.

The nature of the [Fe(IV)-O] center in hemoprotein Compounds II has recently received considerable attention, as several experimental and theoretical investigations have suggested that this group is not necessarily the traditionally assumed ferryl ion, [Fe(IV)=O]2+, but can be the protonated ferryl, [Fe(IV)-OH]3+. We show here that cryoreduction of the EPR-silent Compound II by gamma-irradiation at 77 K produces Fe(III) species retaining the structure of the precursor [Fe(IV)=O]2+ or [Fe(IV)-OH]3+, and that the properties of the cryogenerated species provide a report on structural features and the protonation state of the parent Compound II when studied by EPR and 1H and 14N ENDOR spectroscopies. To give the broadest view of the properties of Compounds II we have carried out such measurements on cryoreduced Compounds II of HRP, Mb, DHP and CPO and on CCP Compound ES. EPR and ENDOR spectra of cryoreduced HRP II, CPO II and CCP ES are characteristic of low-spin hydroxy-Fe(III) heme species. In contrast, cryoreduced "globins", Mb II, Hb II, and DHP II, show EPR spectra having lower rhombicity. In addition the cryogenerated ferric "globin" species display strongly coupled exchangeable (1)H ENDOR signals, with A max approximately 20 MHz and a iso approximately 14 MHz, both substantially greater than for hydroxide/water ligand protons. Upon annealing at T > 180 K the cryoreduced globin compounds II relax to the low-spin hydroxy-ferric form with a solvent kinetic isotope effect, KIE > 6. The results presented here together with published resonance Raman and Mossbauer data suggest that the high-valent iron center of globin and HRP compounds II, as well as of CCP ES, is [Fe(IV)=O]2+, and that its cryoreduction produces [Fe(III)-O]+. Instead, as proposed by Green and co-workers, CPO II contains [Fe(IV)-OH]3+ which forms [Fe(III)-OH]2+ upon radiolysis. The [Fe(III)-O]+ generated by cryoreduction of HRP II and CCP ES protonate at 77 K, presumably because the heme is linked to a distal-pocket hydrogen bonding/proton-delivery network through an H-bond to the "oxide" ligand. The data also indicate that Mb and HRP compounds II exist as two major conformational substates.

One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III) was investigated using some biophysical and biochemical methods. Firstly, it was found that a large amount of Tb(III) can be distributed on the cell wall, that some Tb(III) can enter into the horseradish cell, indicating that peroxidase was mainly distributed on cell wall, and thus that Tb(III) would interact with horseradish peroxidase (HRP) in the plant. In addition, peroxidase bioactivity was decreased in the presence of Tb(III). Secondly, a new peroxidase-containing Tb(III) complex (Tb-HRP) was obtained from horseradish after treatment with Tb(III); the molecular mass of Tb-HRP is near 44 kDa and the pI is about 8.80. Thirdly, the electrocatalytic activity of Tb-HRP is much lower than that of HRP obtained from horseradish without treatment with Tb(III). The decrease in the activity of Tb-HRP is due to the destruction (unfolding) of the conformation in Tb-HRP. The planarity of the heme active center in the Tb-HRP molecule was increased and the extent of exposure of Fe(III) in heme was decreased, leading to inhibition of the electron transfer. The microstructure change in Tb-HRP might be the result of the inhibition effect of Tb(III) on peroxidase activity in horseradish.

Chlorogenic acid is the major diphenol of many fruits, where it is oxidized enzymatically by polyphenol oxidase (PPO) or peroxidase (POD) to its o-quinone. In spectrophotometric studies of chlorogenic acid oxidation with a periodate ratio of [CGA]0/[IO4-]0 < 1 and [CGA]0/[IO4-]0 > 1, the o-quinone was characterized as follows: lambda(max) at 400 nm and epsilon = 2000 and 2200 M-1 cm-1 at pH 4.5 and 7.0, respectively. In studies of o-quinone generated by the oxidation of chlorogenic acid using a periodate at ratio of [CGA]0/[IO4-]0 > 1, a reaction with the remaining substrate was detected, showing rate constants of k = 2.73 +/- 0.17 M-1 s-1 and k' = 0.05 +/- 0.01 M-1 s-1 at the above pH values. A chronometric spectrophotometric method is proposed to kinetically characterize the action of the PPO or POD on the basis of measuring the time it takes for a given amount of ascorbic acid to be consumed in the reaction with the o-quinone. The kinetic constants of mushroom PPO and horseradish POD are determined.

A model of leprosy was used to study the therapeutic effect of horse-radish root (HRR) containing peroxidase in combination with rifampicin (RFP) and potassium iodide (PI) as compared to routine combined therapy with RFP and diaminodiphenylsulfonum. Therapy with HRR and iodide showed the best antimicrobial effect than the routine combined therapy. A combination of RFP, HRR, and PI increased the activity of neutrophilic myeliperoxidase produced an anti-inflammatory activity and caused no persistent anemia or toxic effect on the murine liver.

Vibrational changes in the catalytic site of horseradish peroxidase were investigated by FTIR (Fourier-transform infrared) spectroscopy in the 1000-2500 cm(-1) range. Difference spectra were generated by photolysis of the haemII-CO compound at different pH/pD values. The spectra report on the fine structure around the catalytic site and show vibrational changes of protein backbone, amino acid residues and cofactors. Assignments of the FTIR vibrations can be made based upon known crystal structures, comparisons with absorption frequencies and extinction coefficients of model amino acids and cofactors, effects of H2O/2H2O exchange and changes of pH/pD. Concomitant with the photolysis of the CO ligand are changes due to haem and protein vibrations, predominant among which are arginine and histidine residue vibrations.

The sulfoxidation of dimethyl sulfide (DMS), by two different heme-type enzyme models (without the protein), namely, horseradish peroxidase (HRP) and cytochrome P450 (P450), was studied using density functional theory. The models differ from each other by the axial ligand of the iron, which is imidazole in the case of HRP and thiolate in the case of P450. The computational results reveal a concerted oxygen atom transfer to sulfur, with spin-state selection dependent upon the identity of the proximal ligand. In the case of thiolate, the mechanism prefers the high-spin quartet pathway; whereas in the case of imidazole, the mechanism involves two-state reactivity (TSR), with competing quartet and doublet spin states. Furthermore, with thiolate the high-spin transition state, (4)TS(P450), has an upright conformation with a large Fe-O-S(DMS) angle of 147 degrees , whereas the low-spin species, (2)TS(P450), has a small angle and its Fe-O moiety makes an O-N(Por) bond with one of the nitrogen atoms of the porphine macrocycle. By contrast, when the proximal ligand is imidazole, both transition states possess a bent Fe-O bond and an O-N(Por) bond. These spin-state selection patterns obey simple orbital-selection rules, which are manifestations of the electronic nature of the ligand, i.e., the electron-releasing effect of the thiolate vis-a-vis the electron-withdrawal effect of imidazole. Other possible reactivity expressions of the spin-selection patterns are discussed [Dowers, T. S., Rock, D. A., Rock, D. A., Jones, J. P. (2004) J. Am. Chem. Soc. 126, 8868-8869]. Theory shows that intrinsically, HRP should be as reactive as P450 toward sulfoxidation.

Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.

Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the "hydrated" form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP-acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463-468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron-ligand distances of 2.3 and 1.8 A, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 A from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.

An anionic peroxidase RsPrx1 was purified (RZ=3.0) and characterized from roots of Chinese red radish (Raphanus sativus var. niger, Brassicaceae). The specific activity of RsPrx1 (micromol mg(-1) min(-1)) is 413.5 (ferulic acid); 258.7 (ABTS); 177.3 (caffeic acid) and 10.0 (guaiacol acid). The optimum pH is 4.0 (citrate buffer) using ABTS as substrate. RsPrx1 can utilise the red pigment present in the root, pelargonidin, as substrate and the specific activity is 93.6 micromol mg(-1) min(-1). The molecular mass of RsPrx1 is 45 kDa (denatured) and 46 kDa (native) as determined by SDS-PAGE and gel filtration, respectively. The isoelectric point (pI) determined by native IEF is 4.7 and by chromatofocusing (Mono P) is 5.1. Analysis of tryptic peptides by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) covered 27% of the RsPrx1 sequence and confirmed its identity. The gene encoding RsPrx1 was cloned by PCR and the amino acid sequence showed the highest identity (82%) to peroxidase AtPrx22 and AtPrx23 from Arabidopsis thaliana and to HRPC3 and HRPE5 from horseradish, respectively. Activity-stained IEF gels show that RsPrx1 is primarily expressed in the roots in agreement with the expression profile of the orthologous genes. These five orthologous peroxidases have three introns of variable length and sequence at conserved locations between the distal and proximal histidine. The results suggest that RsPrx1 orthologs are widespread in the Brassicaceae plant family with a 15-residue-long C-terminal propeptide in common. Based on the results, we propose that RsPrx1 and orthologs are targeted to the vacuoles to modify stored anthocyanins like pelargonidin.

Fully developed horseradish (Armoracia rusticana Gaertn., Mey., & Scherb.) roots from 27 accessions and leaves from a subset of 9 accessions were evaluated for glucosinolates and myrosinase enzyme activity. Eight different glucosinolates were detected (based on HPLC retention times as desulfoglucosinolates) in both root and leaf tissues. The sum of these glucosinolates, referred to as total, ranged from 2 to 296 micromol g(-1) of dry weight (DW) in both tissues. Four glucosinolates (sinigrin, glucobrassicin, neoglucobrassicin, and gluconasturtiin) were detected in major quantities. In fully developed roots, sinigrin concentration represented approximately 83%, gluconasturtiin approximately 11%, and glucobrassicin approximately 1% of the total glucosinolates. Approximately the same proportions of individual glucosinolates appeared in fully developed leaves, except that glucobrassicin was substituted by neoglucobrassicin and gluconasturtiin concentration was significantly lower (<1%). At least four other glucosinolates were detected in very small quantities (<1%) in both roots and leaves. Myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) is the enzyme responsible for the hydrolysis of the parent glucosinolates into biologically active products. Very little is known about myrosinase activity and the correlation of its activity to total and individual glucosinolates in plant tissues. Significant differences in myrosinase activity were detected between the roots and leaves, ranging from 1.2 to 57.1 units g(-1) of DW. Data showed no correlation between myrosinase activity and total and/or individual glucosinolates in the roots. However, in the leaves, significant correlations were found between myrosinase activity and total glucosinolates (0.78 at P = 0.01) and between myrosinase activity and sinigrin (0.80 at P = 0.01). Glucosinolates content and myrosinase activity were also correlated in young and fully developed roots and leaves and during tissue crushing. Glucobrassicin concentration in the roots and neoglucobrassicin concentration in the leaves were significantly higher in young than in fully developed tissue. Crushing of the tissue resulted in rapid hydrolysis of sinigrin and glucobrassicin, as expected, from the presence of myrosinase. Likewise, myrosinase activity declined rapidly after crushing, perhaps due to inactivation by the reaction products and/or the depletion of its substrates.

Glutathione S-transferase (GST) and peroxidase (POX) activities have a direct relation to the effect of stress on plant metabolism. Changes in the activities of the enzymes were therefore studied. Horseradish hairy roots were treated by selected bivalent ions of heavy metals (HMs) and nitroaromatic compounds (NACs). We have shown differences in GST activity when assayed with substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB). The conjugation of DCNB catalysed by GST was inhibited in all roots treated with HMs as compared to non-treated roots, whereas NACs caused induction of the activity in dependence on the exposition time and concentration of compounds. The conjugation of CDNB by GST was not affected to the same extent. The increase of GST activity was determined in cultures treated by nickel (0.1 mM) and diaminonitrotoluenes (DANTs, 0.1 mM) for 6 h, whereas the roots treated by 2,4,6-trinitrotoluene (TNT), 4-amino-2,6-dinitrotoluene (ADNT) and dinitrotoluene (DNT, 1.0 mM) needed 27 h treatment to induce the activity. The POX activity of cultures treated by HMs was inhibited to 17-35% in comparison to non-treated cultures. The POX activity of roots treated by TNT (0.1 and 1.0 mM) for 6 and 27 h and by ADNT (0.1 and 1.0 mM) for 6 h was inhibited. A partial increase of POX activity was measured in roots treated by all NACs for 27 h. The content of oxidized glutathione (GSSG) and reduced glutathione (GSH) in the roots differed significantly. It was followed as a symptom of the stress reaction of the plant metabolism to the effect of NACs and HMs.

Horseradish peroxidase C (HRPC) is a class III peroxidase whose structure is stabilized by the presence of two endogenous calcium atoms. Calcium removal has been shown to decrease the enzymatic activity of the enzyme. The spin state of the iron, a mixture of high spin (HS) and mixed quantum spin state (QS) consisting of intermediate spin (IS) 3/2 + (HS) 5/2, is also significantly affected by calcium removal, going from a predominant QS component to a predominant HS component upon removal of one calcium. Removal of both calcium ions, however, results in the appearance of a significant LS contribution, easily monitored in the charge transfer (CT) band region by low-T absorption. Normal structural decomposition (NSD) calculations of the in-plane (ip) modes of the heme extracted from HRPC native and Ca(2+)-depleted models show that removal of the proximal calcium is associated with perturbed E(u) and increased A(1g) ip distortions of the heme. The effect of complete or distal calcium removal on the heme also results in increased A(1g) ip distortions, but in significantly decreased E(u) distortions. The overall effect is to decrease the nonplanarity of the heme: the total ip distortion of the native HRPC heme is 0.200 and 0.134 A for the Ca(2+)-depleted species. Our NSD results corroborate the role proposed for the protein matrix, namely to fine-tune the active site by inducing subtle changes in heme planarity and spin state of the iron., (Copyright 2004 Wiley Periodicals, Inc. Biopolymers, 2004)

In biotransformations carried out under similar conditions enzymatic systems from carrot (Daucus carota L.), celeriac (Apium graveolens L. var. rapaceum) and horse-radish (Armoracia lapathifolia Gilib.) hydrolyzed the ester bonds of acetates of phenols or alicyclic alcohols. Nevertheless, methyl esters of aromatic acids did not undergo hydrolysis. Alcohols were oxidized to ketones in a reversible reaction.

Background and Aims: Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence., Methods: Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques., Key Results: Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels., Conclusions: There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity. These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.

Many products of interest are synthesized in organized tissues, but not formed in suspension or callus culture. Therefore, most attention has been focused on root cultures. The transgenic plant,"hairy root", has brought us to dramatic improvements in growth rate and high content of desirable products. Since the roots are quite different from callus in morphology, the culture manner should be explored independently. By providing a growth environment, an elite hairy root can be a more attractive plant. Both of strain selection to generate more competent plants in breeding and engineering development are necessary to overcome various limitations. In this chapter the engineering issues involved in using hairy root culture are discussed, as follows. 1. Measurement of cell concentration on line, and a designing bioreactors for hairy root in liquid culture. 2. High cell density culture and its kinetic parameters. 3. Secretion of target products. 4. The micropropagation of the regenerated hairy root by means of artificial seed system. In some cases where callus and suspension culture show negligible productivity, organ culture will be necessary to achieve good formation. This study on hairy root culture indicates one of the best attempts to the recovery of products from the organ culture in plant biotechnology.

A panel of eight monoclonal antibodies raised against horseradish root peroxidase has been assembled and characterized. Affinity constants were determined for all antibodies, and their specificity for various structural forms of the enzyme (native peroxidase, apoperoxidase, and denatured peroxidase) were assessed by competitive enzyme immunoassay. The effects of the antibodies on the process of refolding of peroxidase after its denaturing with 6.5 M guanidine hydrochloride were studied spectrophotometrically, by the restoration of the enzymatic activity in the reaction of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate). The yield of the active enzyme in the course of the refolding was increased 1.5 to 1.7 times in the presence of antibody H1. Effects of the antibodies constituting the panel on the activity of native peroxidase and the stability of its dilute solutions were analyzed.

Hairy root cultures of Armoracia lapathifolia established by infection with Agrobacterium rhizogenes LBA 9402 present a level and isoenzyme pattern of peroxidases (POD) comparable to nontransformed roots. Elicitation with chitosan (10, 50, and 100 mg/L) was used in order to improve POD production. Total POD activity increased about 170% after 48 h of treatment with chitosan 100 mg/L. Elicitation effect on soluble and ionically cell-wall-bound POD fractions of A. lapathifolia hairy roots was analyzed. POD activity of the ionically cell-wall-bound protein fraction increased in the presence of chitosan in a dose-response manner. No effect on soluble POD fractions was observed, but the isoenzyme pattern analyzed by isoelectrofocusing showed an increase of an acidic isoenzyme (pI = 3.4) after the elicitation treatment. The ionically cell-wall-bound protein fraction showed only basic isoenzymes, with an increase of an isoenzyme of pI = 8.7, after the elicitation treatment.

The dynamics of structural and functional changes proceeding in a peroxidase molecule under the effect of temperature was studied. It was shown that peroxidase thermoinactivation proceeds in two consequent stages. Based on the analysis of enzyme peroxidase and oxidase activity and peroxidase spectral and buffer characteristics, it was established that at temperatures from 20 to 55 degrees C reversible conformation there occur changes of the hemoprotein molecule related to consequent unfolding and folding of the protein globule. The influence of temperature of 60 degrees C and above induces the protein globule unfolding and loosing of peroxidase activity.