Effect of strophanthin G on peroxidase oxidation kinetics of slowly oxidizable peroxidase substrates

Steady-state kinetics of thioproperazine, triftazine, aminazine, and o-dianisidine oxidation with hydrogen peroxide catalyzed by horseradish peroxidase were studied in the presence of strophanthin G. Values of the inhibition and activation constants (Ki, Ka) were determined in the pH range 5.0-7.5. At acidic pH, strophanthin G activated peroxidase during the oxidation of thioproperazine by the uncompetitive mechanism, and when triftazine was oxidized, the inhibition was noncompetitive. At pH > 6.0, the patterns of activation and inhibition changed to mixed-type during the peroxidase oxidation of thioproperazine and triftazine and to competitive inhibition of peroxidase with strophanthin G during the oxidation of aminazine. These effects are suggested to be due to an ionizable enzyme group of pK approximately 6.0. Strophanthin G inhibited free-radical oxidation of o-dianisidine via binding to the enzyme-substrate complex, preventing the generation of a stable semi-oxidized product of o-dianisidine, and thus inhibiting the enzyme by the anticompetitive mechanism. Mechanisms of oxidation of slowly and rapidly oxidizable substrates of peroxidase in the presence of strophanthin G are suggested.     

Chromogenic substrates for horseradish peroxidase

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4-chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate.     

New pteridine substrates for dihydropteridine reductase and horseradish peroxidase.

The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported.     

Stereospecificity of horseradish peroxidase

We report here on the stereospecificity observed in the action of horseradish peroxidase (HRPC) on monophenol and diphenol substrates. Several enantiomers of monophenols and o-diphenols were assayed: L-tyrosinol, D-tyrosinol, L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-alpha-methyldopa, DL-alpha-methyldopa, DL-adrenaline, D-adrenaline, L-isoproterenol, DL-isoproterenol and D-isoproterenol. The electronic density at the carbon atoms in the C-1 and C-2 positions of the benzene ring were determined by NMR assays (delta1 and delta2). This value is related to the nucleophilic power of the oxygen atom of the hydroxyl groups and to its oxidation-reduction capacity. The spatial orientation of the ring substituents resulted in lower Km values for L- than for D-isomers. The kcat values for substrates capable of saturating the enzyme were lower for D- than for L-isomers, although both have the same delta1 and delta2 NMR values for carbons C-1 and C-2, and therefore the same oxidation-reduction potential. In the case of substrates that cannot saturate the enzyme, the values of the binding constant for compound II (an intermediate in the catalytic cycle) followed the order: L-isomer>DL-isomer>D-isomer. Therefore, horseradish peroxidase showed stereospecificity in its affinity toward its substrates (K m) and in their transformation reaction rates (k cat).     

New fluorogenic substrates for horseradish peroxidase: Rapid and sensitive assays for hydrogen peroxide and the peroxidase

p-Hydroxyphenyl compounds [3-(p-hydroxyphenyl)propionic acid, p-hydroxyphenethyl alcohol, hordenine, p-ethylphenol, 3-(p-hydroxyphenyl)-1-propanol, p-n-propylphenol, and p-hydroxyphenyllactic acid] were recently found to be excellent fluorogenic substrates for the horseradish peroxidase-mediated reaction with hydrogen peroxide. A very rapid and sensitive method for the fluorometric assays of hydrogen peroxide and the peroxidase was established by using 3-(p-hydroxyphenyl)propionic acid as the best of these substrates; hydrogen peroxide can be assayed precisely in amounts as small as 0.1 nmol, with peroxidase activity as low as 7.8 μU.     

Cobalt-substituted horseradish peroxidase.

Horseradish peroxidase can be reconstituted with cobalt porphyrin to give a cobaltic holoenzyme having physicochemical properties quite similar to those of the native ferric protein. The cobaltic protein (Co3+HRP) can be reduced to the cobaltous form (CoHRP), the analogue of ferroperoxidase and the reduced cobalt protein can bind O2 to form an analogue of oxyferroperoxidase (Compound III). Since both the CoHRP and oxy-CoHRP are EPR-visible, the cobalt has been used to probe the nature of the heme crevice in these two protein forms. The occurrence of a three-line 14N superhyperfine pattern in the spectrum of the former unambiguously shows that in the divalent state of the protein the proximal axial ligand is a nitrogenous base. The spectrum of the latter shows a uniquely large Aparallel(59Co) = 23.2 G. Although we confirm the reported failure of the Co3+HRP to catalyze peroxide-dependent oxidations of classical peroxidase substrates (Gjessing, E.C., and Sumner, J.B. (1942) Arch. Biochem. 1, 1), the oxy-CoHRP does undergo oxidation-reduction reactions analogous to those exhibited in the cytochrome P-450 catalytic cycle.     

Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase.

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl- or sn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM(-1) min(-1)) were k(1) = 1-3 x 10(5) and k(2) = 4-11 x 10(4). In the presence of 0.36 mM GSH, apparent K(m) was 120-130 microM and apparent V(max) was 1.5-1.6 micromol/min/mg of protein. Assays with H(2)O(2) and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K(i) 4 miroM. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.     

Wound-induced expression of horseradish peroxidase

Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the -glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.Abbreviations HRP horseradish peroxidase - prx gene for peroxidase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

Differences in the binding of aromatic substrates to horseradish peroxidase revealed by fluorescence line narrowing

The heme in horseradish peroxidase (HRP) isoenzyme C was replaced by mesoporphyrin (MP), and the binding effect of the aromatic substrates benzo-and naphthohydroxamic acid (BHA, NHA), resorcinol (RE), isomeric resorcylic acids (alpha-, beta-, gamma-RE), and hydroquinone (HQ) was studied at pH 5 by conventional and laser-excited fluorescence spectroscopy on the basis of the signal of the porphyrin. Under laser excitation at cryogenic temperatures site selection was demonstrated, and the fluorescence line narrowing data were used to characterize the HRP/substrate complexes by the inhomogeneous distribution function for the S0----S1 (0----0) transition energy and the vibrational energies in the S1 electronic state. A comparison with ground-state vibrational energies for MP in chloroform/ether showed a downward shift in vibrational energies for S1 by approximately 20 cm-1. The association characteristics of the substrates were in accordance with previous literature data indicating NHA to be of the strongest binding affinity. For BHA, spectral evidence was obtained for a second type of binding site where hydrophobic interactions with the porphyrin ring may be possible. The effect of the RE's was similar to each other, but only beta-RE showed saturation. Complexation in every case caused the strong reduction of the splitting in the 0----0 transition energy for the tautomeric forms of MP and an increase in the 0----0 energy by 100-200 cm-1 depending on the substrate. The substrate binding also affected the phonon coupling of vibronic transitions exciting into the delta v = 927- and 976-cm-1 modes; in the latter case, the vibrational energy was also increased to 983 cm-1 for beta-RE. In the same energy range, however, the transition into the delta nu = 958-960-cm-1 mode was not affected by binding. Both the magnitude of the energy shifting and the change in the strength of phonon coupling gave the same relation, BHA less than NHA less than HQ less than RE's, indicating a common conformational origin. A reduction of the fluctuational freedom of the protein chain at room temperature within the heme pocket was suggested on the basis of the reduction of the width of the inhomogeneous distribution of 0----0 energies (from 60-70 to approximately 30 cm-1 in case of HRP/HQ) upon substrate binding. Ways to relate the transition energy splitting and shifting effects to conformational changes are discussed by invoking the Jahn-Teller effect.     

Immobilization of horseradish peroxidase onto kaolin

Kaolin showed as a very perspective carrier for the enzyme immobilization and it was used for the adsorption of horseradish peroxidase (HRP). The effects of the enzyme concentration and pH on the immobilization efficiency were studied in the reaction with pyrogallol and anthraquinone dye C.I. Acid Violet 109 (AV 109). In addition, Fourier transform infrared spectroscopy, scanning electron microscopy and analysis by Brunauer–Emmett–Teller were performed for kaolin, thermally activated kaolin and the immobilized enzyme. It has been shown that 0.1 IU of HRP-kaolin decolorized 87 % of dye solution, under the optimal conditions (pH 5.0, temperature 24 °C, dye concentration 40 mg/L and 0.2 mM of H₂O₂) within 40 min. The immobilized HRP decolorization follows the Ping Pong Bi–Bi mechanism with dead-end inhibition by the dye. The biocatalyst retained 35 ± 0.9 % of the initial activity after seven cycles of reuse in the decolorization reaction of AV 109 under optimal conditions in a batch reactor. The obtained kinetic parameters and reusability study confirmed improvement in performances of k-HRP compared to free, indicating that k-HRP has a great potential for environmental purposes.     

Calcium-related properties of horseradish peroxidase.

Horseradish peroxidase has been shown to be a metalloprotein in which calcium contributes to the structural stability of the protein. Isoenzyme C and A contain 2.0 and 1.4 moles calcium/mole enzyme, respectively, which can be removed by treatment with guanidine hydrochloride and EDTA. Calcium-free isoenzyme C, but not isoenzyme A, reconstitutes upon addition of calcium and regains enzymatic activity. Free calcium readily exchanges with isoenzyme C, but only to a small extent with isoenzyme A. In addition the role of calcium in maintaining molecular conformation is evidenced by the effects of calcium removal from the isoenzyme C on the thermal stability of the protein.     

Chemical stabilization of recombinant horseradish peroxidase

Summary Unglycosylated recombinant horseradish peroxidase (HRP C^*) had a half life of 21 minutes at 65°C compared with only 5 minutes for the plant enzyme (HRP). The half life of HRP C^* at 65°C increased by 5-fold following modification with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Tolerance to 60% 1,4-dioxan also increased whilst tolerance to 30% dimethylformamide was unchanged.     

Protein control of prosthetic heme reactivity. Reaction of substrates with the heme edge of horseradish peroxidase

Incubation of horseradish peroxidase with phenylhydrazine and H2O2 markedly depresses the catalytic activity and the intensity, but not position, of the Soret band. Approximately 11-13 mol of phenylhydrazine and 25 mol of H2O2 are required per mol of enzyme to minimize the chromophore intensity. The enzyme retains some activity after such treatment, but this activity is eliminated if the enzyme is isolated and reincubated with phenylhydrazine. The prosthetic heme of the enzyme does not react with phenylhydrazine to give a sigma-bonded phenyl-iron complex, as it does in other hemoproteins, but is converted instead to the delta-mesophenyl and 8-hydroxymethyl derivatives. The loss of activity is due more to protein than heme modification, however. The inactivated enzyme reacts with H2O2 to give a spectroscopically detectable Compound I. The results imply that substrates interact with the heme edge rather than with the activated oxygen of Compounds I and II and specifically identify the region around the delta-meso-carbon and 8-methyl group as the exposed sector of the heme. Horseradish peroxidase, in contrast to cytochrome P-450, generally does not catalyze oxygen-transfer reactions. The present results indicate that oxygen-transfer reactions do not occur because the activated oxygen and the substrate are physically separated by a protein-imposed barrier in horseradish peroxidase.     

Competition of glycerol with other oxidizable substrates in rat brain.

The rate of conversion of [1,3-14C]glycerol into 14CO2 was measured in the presence and absence of unlabelled alternative substrates in whole homogenates from the brains of young (4-6 and 18-20 days old) and adult rats. Unlabelled glucose decreased 14CO2 production from [1,3-14C]glycerol by about 40% at all ages studied. Unlabelled 3-hydroxybutyrate significantly decreased the 14CO2 production from both low (0.2 mM) and high (2.0 mM) concentrations of glycerol in 4-6- and 18-20-day-old rat pups. However, the addition of 3-hydroxybutyrate had no effect on the rate of 14CO2 production from 2.0 mM-glycerol in adult rats, suggesting that the interaction of 3-hydroxybutyrate with glycerol in adult rat brain is complex and may be related to the biphasic kinetics previously reported for glycerol oxidation. Unlabelled glutamine decreased the production of 14CO2 by brain homogenates from 18-20-day-old and adult rats, but not in 4-6-day-old rat pups. In the converse situation, the addition of unlabelled glycerol to whole brain homogenates had little effect on the rate of 14CO2 production from [6-14C]glucose, 3-hydroxy[3-14C]butyrate and [U-14C]glutamine, although some significant differences were noted. Collectively these results suggest that glycerol and these other substrates may be metabolized in separate subcellular compartments in brain such that the products of glucose, 3-hydroxybutyrate and glutamine metabolism can dilute the oxidation of glycerol, but the converse cannot occur. The data also demonstrate that there are complex age-related changes in the interaction of glycerol with 3-hydroxybutyrate and glutamine. The fact that glycerol oxidation was only partially suppressed by the addition of 1-5 mM-glucose, -3-hydroxybutyrate or -glutamine could also suggest that glycerol may be selectively utilized as an energy substrate in some discrete brain region.     

Immobilization of horseradish peroxidase on activated wool

This work is aimed to immobilize partially purified horseradish peroxidase (HRP) on wool activated by multifunctional reactive center, namely cyanuric chloride. The effect of cyanuric chloride concentration, pH and enzyme concentration on immobilization of HRP was studied. FT-IR and SEM analyses were detected for wool, activated wool and immobilized wool-HRP. The wool-HRP, prepared at 2% (w/v) cyanuric chloride and pH 5.0, retained 50% of initial activity after seven reuses. The wool-HRP showed broad optimum pH at 7.0 and 8.0, which was higher than that of the soluble HRP (pH 6.0). The soluble HRP had an optimum temperature of 30 °C, which was shifted to 40 °C for immobilized enzyme. The soluble and wool-HRP were stable up to 30 and 40 °C after incubation for 1 h, respectively. The apparent kinetic constant values (K_ms) of wool-HRP were 10 mM for guiacol and 2.5 mM for H₂O₂, which were higher than that of soluble HRP. The wool-HRP was remarkably more stable against proteolysis mediated by trypsin. The wool-HRP exhibited more resistance to heavy metal induced inhibition. The wool-HRP was more stable to the denaturation induced by urea, Triton X-100, isopropanol, butanol and dioxan. The wool-HRP was found to be the most stable under storage. In conclusion, the wool-HRP could be more suitable for several industrial and environmental purposes.     

The reaction of coumarins with horseradish peroxidase

The peroxidase catalyzed oxidation of indole-3-acetate is inhibited by naturally occurring coumarins such as scopoletin. This inhibition is due to the preferential reactivity of the coumarins with the peroxidase compounds I, II, and III. In view of the possible growth regulatory role of coumarins in plants, the mechanism of oxidation of scopoletin by horse-radish peroxidase has been investigated.