A hydrogen peroxide electrode assay to measure thiol peroxidase activity for organoselenium and organotellurium drugs

2012 ◽  
Vol 429 (2) ◽  
pp. 103-107 ◽  
Author(s):  
Niroshini M. Giles ◽  
Sweta Kumari ◽  
Rosemary A. Stamm ◽  
Siddharth Patel ◽  
Gregory I. Giles
Keyword(s):  
Hydrogen Peroxide ◽  
Peroxidase Activity ◽  
Thiol Peroxidase

scholarly journals Removal of hydrogen peroxide by the 29 kDa protein of Entamoeba histolytica

1997 ◽  
Vol 326 (3) ◽  
pp. 785-789 ◽  
Author(s):  
Iris BRUCHHAUS ◽  
Symi RICHTER ◽  
Egbert TANNICH
Keyword(s):  
Hydrogen Peroxide ◽  
Entamoeba Histolytica ◽  
Recombinant Proteins ◽  
Peroxidase Activity ◽  
Cumene Hydroperoxide ◽  
High Sensitivity ◽  
Coli Strain ◽  
Amino Acid Residues ◽  
Genes Encoding ◽  
Thiol Peroxidase

The 29 kDa protein of Entamoeba histolytica (Eh29), as well as a truncated variant of this protein, which lacks a cysteine-rich N-terminal region of 40 amino acid residues (Eh29mut), were recombinantly expressed in Escherichia coli and purified to homogeneity. Both recombinant proteins (recEh29, recEh29mut) were found to have hydrogen peroxide (H2O2)-removing activity, but recEh29 was twice as active as recEh29mut. For the consumption of exogenous H2O2, activity was dependent on the presence of reducing equivalents, such as dithiothreitol (DTT), indicating that Eh29 constitutes a thiol-dependent peroxidase. DTT was not required to remove H2O2 by recEh29 or recEh29mut when H2O2 was generated enzymically by the E. histolytica NADPH:flavin oxidoreductase. This enzyme produces H2O2 under aerobic conditions and simultaneously serves as a hydrogen donor for Eh29. Peroxidase activity of the recombinant proteins was further supported by complementation of an E. coli strain that lacks the entire alkyl hydroperoxide reductase locus. The high sensitivity of these bacteria against cumene hydroperoxide was significantly reduced by the introduction of the genes encoding recEh29 or recEh29mut. Using antisera raised against the recombinant proteins, native Eh29 was localized within the cytoplasm of the amoebae. In addition, the antisera reacted with proteins of E. histolytica lysates with apparent molecular masses of 35 kDa and 160–300 kDa. All of them exhibited thiol-peroxidase activity.

scholarly journals Homocysteine peroxidase activity in rat blood plasma: stoichiometry and enzymatic character of the reaction

2013 ◽  
Vol 59 (6) ◽  
pp. 636-643 ◽  
Author(s):  
A.V. Razygraev
Keyword(s):  
Hydrogen Peroxide ◽  
Glutathione Peroxidase ◽  
Blood Plasma ◽  
Peroxidase Activity ◽  
Protein Fraction ◽  
Specific Activity ◽  
Rat Blood ◽  
Significant Acceleration ◽  
Thiol Peroxidase ◽  
Reaction Stoichiometry

Recently it was shown that the presence of rat blood plasma (as well as of erythrocyte hemolysate) in the reaction mixture containing 43 mM Tris-HCl-buffer (pH 8.5), 0.29 mM EDTA, 19.2 mM sodium azide, 1 mM DL-homocysteine (Hcy), and 198 mM hydrogen peroxide (incubation at 37°C) results in a significant acceleration of the decrease in Hcy concentration caused by addition of H O . In this paper, we present data indicating that the observed activity is the homocysteine:H O -oxidoreductase (homocysteine peroxidase) activity. It has been found that the level of H O -dependent Hcy decrease observed in the presence of blood plasma corresponds to homocysteine:H O -oxidoreductase reaction stoichiometry of 2:1 (mole ratio). The activity observed belongs to the protein fraction isolated by saturation with ammonium sulfate to 50%; the specific activity in this protein fraction is significantly higher than that in the whole plasma. The results confirm the hypothesis that the reaction between Hcy and H O at the presence of plasma is catalyzed by the protein component of plasma and this is the homocysteine peroxidase reaction. This activity is not associated with serum albumin, which is known to function as thiol peroxidase, and probably belongs to extracellular glutathione peroxidase (Gpx3).

An ESR study of the peroxidase reaction catalyzed by human methaemoglobin and methaemoglobin-haptoglobin complex

1991 ◽  
Vol 56 (4) ◽  
pp. 923-932
Author(s):  
Jana Stejskalová ◽  
Pavel Stopka ◽  
Zdeněk Pavlíček
Keyword(s):  
Hydrogen Peroxide ◽  
Ascorbic Acid ◽  
Amino Acid ◽  
Peroxidase Activity ◽  
Amino Acid Analysis ◽  
Superoxide Radical ◽  
Esr Spectra ◽  
The Other ◽  
Delocalized Electron

The ESR spectra of peroxidase systems of methaemoglobin-ascorbic acid-hydrogen peroxide and methaemoglobin-haptoglobin complex-ascorbic acid-hydrogen peroxide have been measured in the acetate buffer of pH 4.5. For the system with methaemoglobin an asymmetrical signal with g ~ 2 has been observed which is interpreted as the perpendicular region of anisotropic spectrum of superoxide radical. On the other hand, for the system with methaemoglobin-haptoglobin complex the observed signal with g ~ 2 is symmetrical and is interpreted as a signal of delocalized electron. After realization of three repeatedly induced peroxidase processes the ESR signal of the perpendicular part of anisotropic spectrum of superoxide radical is distinctly diminished, whereas the signal of delocalized electron remains practically unchanged. An amino acid analysis of methaemoglobin along with results of the ESR measurements make it possible to derive a hypothesis about the role of haptoglobin in increasing of the peroxidase activity of methaemoglobin.

scholarly journals Experience of measuring glutathione peroxidase activity in surgically induced endometrial-like lesions in rats

2021 ◽  
Vol 70 (2) ◽  
pp. 55-61
Author(s):  
Aleksey V. Razygraev ◽  
Elena V. Baziyan ◽  
Lyudmila S. Polyanskikh ◽  
Mariya A. Petrosyan
Keyword(s):  
Hydrogen Peroxide ◽  
Antioxidant Enzymes ◽  
Glutathione Peroxidase ◽  
Rat Model ◽  
Peroxidase Activity ◽  
Specific Activity ◽  
Reduced Glutathione ◽  
Glutathione Peroxidase Activity ◽  
Nitrobenzoic Acid ◽  
Surgically Induced

BACKGROUND: Endometriosis is known to be linked with altered activities of antioxidant enzymes and with their gene polymorphisms. Progestins are known to induce glutathione peroxidase activity in the endometrium and promote reduction of endometrial lesions. It could be useful to estimate the correlation between the activity of glutathione peroxidase within endometrial lesions and their degree of reduction. AIM: The present study was aimed at estimating glutathione peroxidase activity in surgically induced endometrial-like lesions of different degree of reduction in rat model of endometriosis. MATERIALS AND METHODS: The method for determining glutathione peroxidase activity using hydrogen peroxide as a substrate and 5,5-dithiobis(2-nitrobenzoic acid) for estimation of residual reduced glutathione was applied for quantitative analysis of the enzyme activity in endometriotic foci, surgically induced in female Wistar rats. An assay of glutathione peroxidase activity in tissue homogenates was performed at 37C in a reaction medium containing Tris-HCl buffer supplemented with tetrasodium ethylenediaminetetraacetate and sodium azide (pH 8.5) in the presence of 0.55 mM reduced glutathione and 0.192 mM hydrogen peroxide. Before adding trichloroacetic acid, 40-second incubation was used. The correlation between the specific activity of the enzyme and protein amount in endometriotic foci was estimated. RESULTS: In a rat model of endometriosis, there was a high, well-determined glutathione peroxidase activity in endometriotic foci. For the same endometriotic tissue sample, the enzymatic activity was proportional to the amount of protein in the reaction mixture. The range of specific glutathione peroxidase activity was 2.436.45 micromoles of consumed glutathione per minute per milligram of protein (n = 7). In most reduced endometriotic foci (with the minimum amount of endometriotic tissue), the highest specific activity of glutathione peroxidase was found (the Spearmans rho of 0.93 with p = 0.0067). CONCLUSIONS: The method for determining glutathione peroxidase activity using hydrogen peroxide and 5,5-dithiobis(2-nitrobenzoic acid) is convenient for working with the endometriotic tissue in a rat model of endometriosis. We can accept, with p 0.01, that weight of endometriotic foci is negatively linked with specific glutathione peroxidase activity within their tissue. The results are analogous to the previously obtained data on catalase activity and suggest the involvement of both antioxidant enzymes in reduction of endometrial lesions.

scholarly journals Peroxidase Activity of Human Hemoproteins: Keeping the Fire under Control

Molecules ◽  
2018 ◽  
Vol 23 (10) ◽  
pp. 2561 ◽  
Author(s):  
Irina Vlasova
Keyword(s):  
Hydrogen Peroxide ◽  
Peroxidase Activity ◽  
Biological Effects ◽  
Reactive Intermediates ◽  
The Body ◽  
Main Function ◽  
Hypohalous Acids ◽  
Tyrosyl Radicals ◽  
External Conditions ◽  
Structure Properties

The heme in the active center of peroxidases reacts with hydrogen peroxide to form highly reactive intermediates, which then oxidize simple substances called peroxidase substrates. Human peroxidases can be divided into two groups: (1) True peroxidases are enzymes whose main function is to generate free radicals in the peroxidase cycle and (pseudo)hypohalous acids in the halogenation cycle. The major true peroxidases are myeloperoxidase, eosinophil peroxidase and lactoperoxidase. (2) Pseudo-peroxidases perform various important functions in the body, but under the influence of external conditions they can display peroxidase-like activity. As oxidative intermediates, these peroxidases produce not only active heme compounds, but also protein-based tyrosyl radicals. Hemoglobin, myoglobin, cytochrome c/cardiolipin complexes and cytoglobin are considered as pseudo-peroxidases. Рeroxidases play an important role in innate immunity and in a number of physiologically important processes like apoptosis and cell signaling. Unfavorable excessive peroxidase activity is implicated in oxidative damage of cells and tissues, thereby initiating the variety of human diseases. Hence, regulation of peroxidase activity is of considerable importance. Since peroxidases differ in structure, properties and location, the mechanisms controlling peroxidase activity and the biological effects of peroxidase products are specific for each hemoprotein. This review summarizes the knowledge about the properties, activities, regulations and biological effects of true and pseudo-peroxidases in order to better understand the mechanisms underlying beneficial and adverse effects of this class of enzymes.

Preparation of porphyrin modified CO9S8 nanocomposites and application for colorimetric biosensing of H2O2

2018 ◽  
Vol 22 (09n10) ◽  
pp. 935-943 ◽  
Author(s):  
Yan Gao ◽  
Chunqiao Jin ◽  
Miaomiao Chen ◽  
Xixi Zhu ◽  
Min Fu ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Catalytic Activity ◽  
Peroxidase Activity ◽  
Catalytic Mechanism ◽  
Color Change ◽  
Colorimetric Detection ◽  
Buffer Solution ◽  
Hydrogen Peroxide Detection ◽  
Steady State Kinetics ◽  
One Step

Hydrogen peroxide detection has been widely applied in the fields of biology, medicine, and chemistry. Colorimetric detection of hydrogen peroxide has proven to be a fast and convenient method. In this work, 5,10,15,20-tetrakis(4-chlorophenyl) porphyrin modified Co[Formula: see text]S[Formula: see text] nanocomposites (H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] were prepared via a facile one-step hydrothermal method. H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] nanocomposites were demonstrated to possess an enhanced mimetic peroxidase activity toward the substrate, 3,3[Formula: see text],5,5[Formula: see text]-tetramethylbenzidine (TMB), which can be oxidized to oxTMB (oxidized TMB) in a buffer solution of hydrogen peroxide with a color change from colorless to blue. The catalytic activity of H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] was further analyzed by steady-state kinetics, and H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] had high affinity towards both TMB and H[Formula: see text]O[Formula: see text]. Furthermore, fluorescence and ESR data revealed that the catalytic mechanism of the peroxidase activity of H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text] is due to hydroxyl radicals generated from decomposition of H[Formula: see text]O[Formula: see text]. Based on the catalytic activity of H[Formula: see text]TClPP-Co[Formula: see text]S[Formula: see text], a sensitive colorimetric sensor of H[Formula: see text]O[Formula: see text] with a detection limit of 6.803 [Formula: see text]M as well as a range of 7–100 [Formula: see text]M was designed.

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