Inhibition mechanism of Tb(III) on horseradish peroxidase 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.     

Molecular and cellular mechanism of the effect of La(III) on horseradish peroxidase

Horseradish is an important economic crop. It contains horseradish peroxidase (HRP) and lots of nutrients, and has specific pungency. Lanthanum is one of the heavy metals in the environment. It can transfer through the food chain to humans. In this paper, the molecular and cellular mechanism of the toxic effects of La(III) on HRP in vivo was investigated with an optimized combination of biophysical, biochemical, and cytobiological methods. It was found that La(III) could interact with O and/or N atoms in the backbone/side chains of the HRP molecule in the cell membrane of horseradish treated with 80 μM La(III), leading to the formation of a new complex of La and HRP (La–HRP). The formation of the La–HRP complex causes the redistribution of the electron densities of atoms in the HRP molecule, especially the decrease in the electron density of the active center, Fe(III), in the heme group of the La–HRP molecule compared with the native HRP molecule in vivo. Therefore, the electron transfer and the activity of HRP in horseradish treated with 80 μM La(III) are obviously decreased compared with those of the native HRP in vivo. This is a possible molecular and cellular mechanism for the toxic effect of La(III) on HRP in vivo. It is suggested that the accumulation of La in the environment, especially the formation of the La–HRP complex in vivo, is harmful to organisms.     

Tb(III)-ion-binding-induced conformational changes in platelet factor XIII.

Ca(II) ions are crucial during proteolytic conversion of Factor XIII zymogen into the active enzyme Factor XIIIa. Factor XIII proteolyzed by thrombin or trypsin in the presence of 5 mM-EDTA resulted in rapid inactivation of transglutaminase activity. Factor XIIIa formed by thrombin or trypsin in the presence of 40 microM-Tb(III) ions, however, was indistinguishable from Factor XIIIa formed in the presence of 2-5 mM-Ca(II) ions with respect to molecular mass and transglutaminase activity. Thrombin treatment of Factor XIII in the presence of 1-5 microM-Tb(III) ions resulted in three fragments (76 kDa, 51 kDa and 19 kDa) with simultaneous loss of transglutaminase activity. Tb(III) ions at concentrations greater than 40 microM made platelet Factor XIII resistant to proteolysis by either thrombin or trypsin. Other lanthanide(III) ions [Ln(III) ions] tested [Ce(III), La(III) and Gd(III) ions] functioned similarly to Tb(III) ions during proteolytic activation of Factor XIII. Ln(III) ions (10-100 microM) were unable to replace the Ca(II) ions required for transglutaminase activity of Factor XIIIa. Tb(III) ions also inhibited in a non-competitive manner the transglutaminase activity of Factor XIIIa (Ki 71 microM) even when measured in the presence of 200-fold molar excess of Ca(II) ions. Factor XIII selectively bound to a Tb(III)-chelate affinity column, and could not be eluted by 100 mM-CaCl2. Binding of Tb(III) ions to Factor XIII was demonstrated by fluorescence emission due to Forster energy transfer. A 10(4)-fold molar excess of CaCl2, but not NaCl, partially quenched Tb(III) fluorescence. Low concentrations (5-20 microM) of Tb(III) ions also inhibited the binding of Factor XIII to des-A-fibrinogen by about 43%, whereas higher concentrations (40-100 microM) promoted binding. Conformational changes in Factor XIII consequent to the binding of Tb(III) ions could be responsible for the observed effects on protein structure and function.     

Spectroscopic studies on calcium depleted horseradish peroxidase: Observation of tryptophan sensitized bound terbium(III) flourescence

Fluorescence, circular dichroism (CD), and UV-visible spectroscopic studies on horseradish peroxidase (HRP) and its calcium depleted derivative (CaD-HRP) are reported. CaD-HRP with its emission maximum at 338 nm is found to be six times as fluorescent as native HRP. The red shift relative to HRP emission observed at 328 nm indicates conformation change around trp towards more hydrophilic environment. CD spectrum of HRP in the 250–700 nm range shows that CD bands of HRP in the Soret region (403 nm) and in the aromatic region (280 nm) also undergo red shift on removal of endogenous calcium, indicating a change in conformation in the vicinity of both heme as well as aromatic residues. Comparison of CD of low spin HRP cyanide in the same region shows that CaD-HRP has some intermediate-spin character. CaD-HRP reconstituted with Tb³⁺ ion showed recovery of the enzyme activity by 89% of the original. Fluorescence of trp sensitized Tb³⁺ ion bound to apo-CaD-HRP was observed in the 450–700 nm wavelength region. trp-heme distance in CaD-HRP, calculated using the Förster theory of resonance energy transfer, was found to be 29.5 Å as opposed to 20.1 Å in HRP. The trp-Tb³⁺ distance was similarly estimated to be 7.1 Å.     

Raman evidence that the lyoprotectant poly(ethylene glycol) does not restore nativity to the heme active site of horseradish peroxidase suspended in organic solvents

Resonance Raman spectroscopy was used to interrogate the heme active site of horseradish peroxidase (HRP) lyophilized in the presence and absence of the lyoprotectant poly(ethylene glycol) (PEG; FW 5000; 0-80% w/w) suspended in acetone, chloroform, or acetonitrile. In aqueous solution, Fe(3+)HRP is characterized by a five-coordinate high-spin (5-c HS) heme system. The structure of the heme-active site of HRP in all solvents is perturbed by co-lyophilization of HRP with PEG. Heme active site structural changes are consistent with coordination of water in the distal axial coordination site of the ferric heme iron and disruption of the hydrogen-bond network when the protein is lyophilized in the presence of PEG (>or=60% w/w) in all of the solvent systems studied. Similar active site structural changes were previously observed for HRP in benzene and attributed to a change in the reaction mechanism for HRP in benzene. (Mabrouk, P. A.; Spiro, T. G. J. Am. Chem. Soc. 1998, 120, 10303-10309.) Thus, PEG is proposed to increase the catalytic activity of HRP in nonaqueous media by locking the heme active site into a structure that functions through an alternative catalytic pathway in nonaqueous media.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

Expression of a synthetic gene for horseradish peroxidase C in Escherichia coli and folding and activation of the recombinant enzyme with Ca2+ and heme

A synthetic gene encoding horseradish peroxidase isoenzyme C (HRP C) has been synthesized and expressed in Escherichia coli. The nonglycosylated recombinant enzyme (HRP C*) was produced in inclusion bodies in an insoluble inactive form containing only traces of heme. HRP C* was solubilized and conditions under which it folded to give active enzyme were determined. Folding was shown to be critically dependent upon the concentrations of urea, Ca2+, and heme and on oxidation by oxidized glutathione. Purification of active HRP C* from the folding mixture gave a peroxidase, with about half the activity of HRP C. Glycosylation is thus not essential for correct folding and activity. The C-terminal and N-terminal extensions to HRP identified previously in cloned cDNA sequences are also not required for correct folding. However, Ca2+ appears to play a key role in folding to give the active enzyme. The overall yield of purified active enzyme was 2-3%, but this could be increased by reprocessing material that precipitated during folding.     

Tb(III) as a fluorescent probe for the structure of bovine serum albumin

Tb(III) was used as a fluorescent probe in the study of the calcium-binding sites on Bovine Serum Albumin (BSA). The fluorescence of Tb(III) is enhanced markedly when bound to BSA and nonradiative energy transfer between two fluorescent tryptophan(Trp) residues and Tb(III) bound to calcium-binding sites on BSA occurred. Experimental results show that the major groups in BSA bound to metal ion are the carboxyl side groups of glutamic acid (Glu) and aspartic acid (Asp). The average distance between the bound Tb(III) and the two tryptophan residues in BSA calculated by a F?ster dipole-dipole nonradiative energy transfer mechanism is 1.48 nm.     

Interaction between lanthanum ion and horseradish peroxidase in vitro

The interaction between lanthanum ion (La³⁺) and horseradish peroxidase (HRP) in vitro was investigated using a combination of biophysical and biochemical methods. When the molar ratio of La³⁺ and HRP is low, it was found that the interaction between La³⁺ and HRP mainly depends on the electrostatic attraction, van der waals force and hydrogen bond etc. Thus, the interaction is weak and the La–HRP complex cannot be formed in vitro. As expected, the interaction can change the conformation of HRP molecule, leading to the increase in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure degree of the active center, Fe(III) of the porphyrin ring of HRP molecule. Therefore, the catalytic activity of HRP for the H₂O₂ reduction is improved. When the molar ratio of La³⁺ and HRP is high, La³⁺ can strongly coordinate with O and/or N in the amide group of the polypeptide chain of HRP molecule, forming the La–HRP complex. The formation of the La–HRP complex causes the change in the conformation of HRP molecule, leading to the decrease in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure degree of the active center, Fe(III) of the porphyrin ring of HRP molecule. Thus, the catalytic activity of HRP for the H₂O₂ reduction is decreased comparing with that of HRP in the absence of La³⁺. The results can provide some references for understanding the interaction mechanism between trace elements ions and peroxidase in living organisms.     

Heterogeneous inhibition of horseradish peroxidase activity by cadmium

Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd(2+) for various periods of time. The H(2)O(2)-mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd(2+) was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd(2+) concentration. Furthermore, for the same inhibition type, K(i) values dropped as incubation time increased. These results suggested that Cd(2+) would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd(2+) altered the heme Soret absorption band on binding HRP and exhibited a K(d) which decreased as the incubation time of HRP with Cd(2+) increased. Hill plots suggested a cooperative binding of up to three Cd(2+) ions per molecule of HRP. Thus, Cd(2+) binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd(2+) ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them.     

Heterologous Expression,Purification and Characterization of a Peroxidase Isolated from <Emphasis Type="Italic">Lepidium draba</Emphasis>

Peroxidase is one of the most widely used enzymes in biotechnology and medicine. In the current study, cDNA encoding peroxidase from Lepidium draba (LDP) was cloned and expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies (IBs). To achieve purified active enzyme, IBs were solubilized before being purified and refolded. The deduced amino acid sequence (308) of the LDP gene (924 bp) revealed 88.96% identity to horseradish peroxidase C1A (HRP C1A). The results of basic local alignment search tool (BLAST) and phylogenetic analysis of the protein sequence showed that this enzyme belongs to the neutral group of class III plant peroxidases. According to sequence analysis and structural modeling, critical amino acids in heme and calcium binding domain as well as cysteine residues were conserved as HRP C1A except for calcium binding domain where valine228 was replaced with isoleucine. The far-UV circular dichroism (CD) results were confirmed by homology modeling data showing the enzyme consists mainly of α-helices as other plant peroxidases. Overall, according to the results of catalytic activity and refolding yield, LDP can be introduced as a novel peroxidase for medical and biotechnology applications.     

Photoluminescence profiles and fast/slow annealing effects of Eu(III)/Tb(III)‐codoped silica phosphor materials

A silica (SiO₂) nanoparticle matrix was codoped with luminescent Eu(III) and Tb(III) ions using a modified Stöber method. The effects of fast and slow thermal annealing on photoluminescence profile imaging were examined. Slow annealing treatment suppressed more quenching sites than fast thermal annealing to further increase the photoluminescence signals. The photoluminescence signals observed between 450 and 720 nm were assigned to the ⁵D₀ → ⁷F_J (J = 0,1,2,3,4) of Eu(III) and the ⁵D₄ → ⁷F_J (J = 6,5,4,3) transitions of Tb(III). Photoluminescence was largely sensitized by indirect excitation and was much stronger than that generated by direct excitation. The Eu(III) and Tb(III) ions were doped at lower symmetry sites in the silica matrix. Copyright © 2015 John Wiley & Sons, Ltd.     

Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical

Previous studies established that the cyanyl radical ((*)CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.     

Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth

 It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 μg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immuno-reactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by M_r-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth. Received: 22 July 1999 / Accepted: 11 October 1999     

Extensive labelling of injured insect neurons with seven different heme peptides

Seven different heme peptides were used in neuronal uptake and labelling experiments in flies. The peptides were: catalase, lactoperoxidase, hemoglobin, horseradish peroxidase (HRP), myoglobin, cytochrome c and microperoxidase. All of these peroxidase active peptides were taken up by lesioned neurons and the markers spread throughout the entire cells resulting in a detailed labelling of their processes and cell bodies. Only HRP was taken up by intact neurons. Attempts were made to block axonal transport of HRP with colchicine, vinblastine and 2,4-dinitrophenol. These attempts were unsuccessful and it is proposed that HRP and the other six heme peptides testes are non-selectively diffusing through lesioned or damaged nerve cells in flies.     

Redox properties of the Fe3+/Fe2+ couple in Arthromyces ramosus class II peroxidase and its cyanide adduct

The thermodynamics of the one-electron reduction of the ferric heme in free and cyanide-bound Arthromyces ramosus peroxidase (ARP), a class II plant peroxidase, were determined through spectro-electrochemical experiments. The data were compared with those for class III horseradish peroxidase C (HRP) and its cyanide adduct, and were interpreted in terms of ligand binding features, electrostatic effects and solvent accessible surface area of the heme group and of catalytically relevant residues in the heme distal site. The values for free and cyanide-bound ARP (−0.183 and −0.390 V, respectively, at 25 °C and pH 7) are higher than those for HRP and HRP-CN. ARP features an enthalpic stabilization of the ferrous state and a remarkably negative reduction entropy, which are both unprecedented for heme peroxidases. Once the compensatory contributions of solvent reorganization are partitioned from the measured reduction enthalpy, the resulting protein-based value for ARP turns out to be less positive than that for HRP by +10 kJ mol⁻¹. The smaller stabilization of the oxidized heme in ARP most probably results from the less pronounced anionic character of the proximal histidine, and the decreased polarity in the heme distal site as compared with HRP, as indicated by the X-ray structures. The surprisingly negative value for ARP is the result of peculiar reduction-induced solvent reorganization effects.     

Small-cell Lung Cancer (Human): Potentiation of Endocytic Membrane Activity by Voltage-gated Na<Superscript>+</Superscript> Channel Expression in Vitro

The possible functional role of voltage-gated Na⁺ channel (VGSC) expression in controlling endocytic membrane activity in human small-cell lung cancer (SCLC) cell lines (H69, H209, H510) was studied using uptake of horseradish peroxidase (HRP). The normal human airway epithelial (16HBE14o) cell line was used in a comparative approach. Uptake of HRP was vesicular, strongly temperature-sensitive and suppressed by cytoskeletal poisons (cytochalasin D and colchicine), consistent with endocytosis. Compared with the normal cells, HRP uptake into SCLC cells was kinetically more efficient, resulting in more than four-fold higher uptake under optimized conditions. Importantly, HRP uptake into SCLC cells was inhibited significantly by the specific VGSC blocker tetrodotoxin, as well as lidocaine and phenytoin. These effects were dose-dependent. None of these drugs had any effect on the uptake into the 16HBE14o cells. Uptake of HRP into SCLC cells was reduced by ∼66% in Na⁺-free medium and was partially (∼30%) dependent on extracellular Ca²⁺. The possibility that the endocytic activity in the H510 SCLC cells involved an endogenous cholinergic system was investigated by testing the effects of carbachol (a cholinergic receptor agonist) and eserine (an inhibitor of acetylcholinesterase). Both drugs inhibited HRP uptake, thereby suggesting that basal cholinergic activity occurred. It is concluded that VGSC upregulation could enhance metastatic cell behavior in SCLC by enhancing endocytic membrane activity.     

Kinetics of interconversion of ferrous enzymes, compound II and compound III, of wild-type synechocystis catalase-peroxidase and Y249F: proposal for the catalatic mechanism

With the exception of catalase-peroxidases, heme peroxidases show no significant ability to oxidize hydrogen peroxide and are trapped and inactivated in the compound III form by H2O2 in the absence of one-electron donors. Interestingly, some KatG variants, which lost the catalatic activity, form compound III easily. Here, we compared the kinetics of interconversion of ferrous enzymes, compound II and compound III of wild-type Synechocystis KatG, the variant Y249F, and horseradish peroxidase (HRP). It is shown that dioxygen binding to ferrous KatG and Y249F is reversible and monophasic with apparent bimolecular rate constants of (1.2 +/- 0.3) x 10(5) M(-1) s(-1) and (1.6 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C), similar to HRP. The dissociation constants (KD) of the ferrous-dioxygen were calculated to be 84 microm (wild-type KatG) and 129 microm (Y249F), higher than that in HRP (1.9 microm). Ferrous Y249F and HRP can also heterolytically cleave hydrogen peroxide, forming water and an oxoferryl-type compound II at similar rates ((2.4 +/- 0.3) x 10(5) M(-1) s(-1) and (1.1 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C)). Significant differences were observed in the H2O2-mediated conversion of compound II to compound III as well as in the spectral features of compound II. When compared with HRP and other heme peroxidases, in Y249F, this reaction is significantly faster ((1.2 +/- 0.2) x 10(4) M(-1) s(-1))). Ferrous wild-type KatG was also rapidly converted by hydrogen peroxide in a two-phasic reaction via compound II to compound III (approximately 2.0 x 10(5) M(-1) s(-1)), the latter being also efficiently transformed to ferric KatG. These findings are discussed with respect to a proposed mechanism for the catalatic activity.     

Specificity of a DNA-based (DNAzyme) peroxidative biocatalyst

A complex formation between hemin and a congruous oligonucleotide not only greatly enhances the former’s peroxidative activity but also results in a biocatalyst (DNAzyme) with a novel specificity. Herein substrate, regio-, enantiomeric, and diastereomeric selectivities of heme, the DNAzyme, and the enzyme horseradish peroxidase are comparatively examined.     

Activity,stability and conformational flexibility of seed coat soybean peroxidase

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase superfamily that includes the classical peroxidase, namely horseradish peroxidase (HRP). We have measured the catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(M)) of SBP and that of HRP-C for the oxidation of ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonate)] by hydrogen peroxide at 25 degrees C. We observed that the k(cat) and k(cat)/K(M) values for SBP are much higher than those for HRP-C at all pH values, rendering SBP a more potent peroxidase. This is attributed to the relatively more solvent exposed delta-meso heme edge in SBP. We observed that the maximum catalytic activity and conformational stability of SBP is at pH approximately 5.5. A pH maximum of 5.0 for the catalytic activity of SBP has recently been reported. Estimation of secondary structural elements at various pH values indicated that there is a maximal reduction of beta-strands and beta-turns at pH 5.5 causing the heme to be further exposed to the solvent and increasing the overall conformational flexibility of the protein.