Determination Of Peroxidase Activity Research Articles

Quality changes during thermal processing of two mixed formulas of fruits and vegetables pulps

The present work aimed to evaluate, through thermal degradation kinetics (80 °C to 98 °C in time intervals of 0.5 to 25 min), the effects of different thermal treatments on the biochemical, physicochemical, sensory and microbiological parameters of two mixed fruit and vegetable pulps, a yellow and a red one. The evaluated fruit and vegetable pulps resulted from the mixture of different fruits and vegetables proportions (pineapple, beetroot, strawberry and lemon juice) added to a 50% (p/p) pear-based pulp to maximize their bioactivity, physicochemical stability and sensorial acceptance. Evaluated quality parameters included the determination of peroxidase activity (POD), pH, soluble solids content (SSC), total phenolic content (TPC), CIELab colour, sensory evaluation (colour, taste and aroma) and total mesophilic aerobic counts (TAPC). Regarding heat treatments optimization for both pulps with lower pH, it was concluded that higher temperature treatments (90 to 98 °C) applied over a shorter time (less than 5 min) were more effective to inactivate POD, to reduce the initial microbial load (>2 log10 cycles) and to maximize sensorial attributes. In both mix pulps, total phenolic content (TPC) was not significantly influenced by the different applied time-temperature binomials. From the degradation kinetic models and as an example, it was possible to conclude that POD followed a 1st order kinetic, where the temperature effect was well fitted to the Arrhenius equation. The results allowed to obtain optimized time-temperature binomials for each pulp to simultaneously achieve POD enzyme inactivation, microbial reduction, and maximization of quality parameters relatively to fresh pulps, 90 °C/5 min and 98 °C/2.5 min, for the yellow pulp and red pulp, respectively.

A fast and sensitive zymography method of peroxidase activity determination using sodium acetate buffer

A fast and sensitive zymography method of peroxidase activity determination using sodium acetate buffer

Determination of myeloperoxidase activity in nasal lavage and saliva in patients with allergic rhinitis

A technique for peroxidase activity determination in nasal lavage and saliva in patients with allergic rhinitis is described. An initially increased level of peroxidase activity in secrets (nasal lavage and saliva) in patients with allergic rhinitis in comparison with the control group was found, which can be used in the diagnosis of allergy. The method for peroxidase activity evaluation in nasal lavage and saliva can be used to diagnose allergic rhinitis. Diagnostic levels of peroxidase activity in nasal lavage and saliva (more than 1.011 and 1.061, respectively) may be criteria for neutrophil hypersensitivity.

Technological scoring of sugar beets which were infected by the pathogen of the vascular bacteriosis during vegetation period

Efficiency of the sugar production from sugar beets is close connected with quality of the raw material which uses for the processing. Quality of the raw material depends on complex impact of number of factors, like natural, climatic, agritechnical, and varietal. Currently, there are some negative factors, like decreasing of soil fertility, increasing of soil's infectious background, prevalence of the foreign hybrid sorts of sugar beets which are vulnerable for local pathogens. These factors cause the increase of the diseases of sugar beets' root system – fusarium decay and vascular bacteriosis. The explorations of sugar beets' crops for many years in different areas of Central-Chernozem region have revealed high damage for yield from these diseases. In some years the loss of yield was up to 50%. The determination of impact of bacterial infections on the processes of forming of the chemical composition of sugar beets' roots and the accumulation of sugar in sugar beets' roots is the very important task for science and production. The researches have been done in the laboratory of store and production raw materials, in the Laboratory of immunity, in the department of biotechnology of the FSTSO “All-Russian research institute for sugar beet and sugar named after A.L. Mazlumov”, and in the department of technology of fermentation and sugar productions of the Voronezh state university of engineering technologies. Was revealed, that increasing the level of the disease of sugar beets by vascular bacteriosis causes the rise amount of dry substances from 26.04% (healthy roots) up to 32.75% (damage of roots is 5 scores). And the sugar content decreases from 18.77% up to 16.83%. Proportion of sucrose in dry substances also decreases from 72.08% (healthy roots) up to 51.39% (damage of roots is 5 scores), because part of sucrose is utilized by bacteria in affected roots. Content of reducing substances increases by a factor of 1.2–4.9. Also was revealed that the pathogens of vascular bacteriosis make negative impact for quality of beets' juice and cleaned juice, and them purity decreases by 0.7–7.5% and 0.4-7.6%. The researches for determination of peroxidase activity in sugar beets' roots, which have different levels of damage by vascular bacteriosis, allowed to reveal enhancing common and specific activity of ferment if disease increases. Was determined that pathogens of vascular bacteriosis make negative impact on the factor of sugar exit which decreases by 1.0–5.5%.

Selective oxidation of enzyme extracts for improved quantification of peroxidase activity

Natural components endogenous to plant material extracts often interfere with traditional peroxidase assays by reducing the oxidized product generated as a result of the peroxidase-catalyzed reaction. This leads to an underestimation of peroxidase activity when the oxidized product provides the signal for enzyme activity quantification. This article describes a relatively simple way to alleviate complications arising due to the presence of such confounding compounds. The method is based on using 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the reducing substrate. The oxidized product of the reaction is ABTS+, the accumulation of which can be followed spectrophotometrically. It is shown here that one can selectively inactivate the endogenous compounds that confound the peroxidase assay by treating the enzyme preparation with the oxidized product itself, ABTS+, prior to initiating the quantification assay. This approach is selective for those compounds likely to interfere with peroxidase quantification. The presented method is shown to alleviate the complications associated with lag phases typical of plant extract peroxidase assays and, thus, to more accurately reflect total peroxidase activity. The presented assay is expected to be applicable to the wide range of biological systems for which the determination of peroxidase activity is desired.

Effects of Petroleum Products in Soil on α-Amylase, Starch Phosphorylase and Peroxidase Activities in Cowpea and Maize Seedlings

Aims: To determine the effect of petroleum products (kerosene, diesel, engine oil and petrol) contaminated soil at various concentrations on the activities of α-amylase, starch phosphorylase in the cotyledons of cowpea and maize seedlings as well as peroxidase activity in the leaves of both seedlings. Place and Duration of Study: This study was conducted in Delta State University, Abraka, Nigeria between April 2007 and August 2011. Methodology: Improved varieties of maize (Zea mays L) and Vigna unguiculata (L) Walp were planted in soil contaminated at different concentrations comprising six groups. Each group was replicated five times. Groups 1 to 5 contained 0.1%, 0.25%, 0.5%, 1.0% and 2.0% (v/w) respectively of each of the petroleum products while group six served as control (0.0%). Three Original Research Article Achuba and Okoh; AJEA, 6(2): 112-120, 2015; Article no.AJEA.2015.070 113 seeds were planted in each bag and watered daily. Four days after germination the activities of αamylase, starch phosphorylase in the cotyledons of the cowpea and maize seedlings were analysed. This was followed by the determination of peroxidase activity in the leaves of cowpea and maize seedlings four, eight and twelve days after germination. Results: The results showed that the petroleum products caused metabolic perturbations in the seedlings. This is indicated by the significant (P<0.05) decrease in the activities of starch degrading enzymes: α-amylase and phosphorylase as well as peroxidase activity compared to their respective control values. Conclusion: Kerosene decreased the activities of the enzymes more than the other petroleum products. The effect of petroleum products contaminated soil was more severe in cowpea seedlings relative to maize seedlings.

Rapid and reliable determination of the halogenating peroxidase activity in blood samples

By combining easy and fast leukocyte enrichment with aminophenyl-fluorescein (APF) staining we developed a method to quickly and specifically address the halogenating activity of the immunological relevant blood heme peroxidases myeloperoxidase and eosinophil peroxidase, respectively. For leukocyte enrichment a two-fold hypotonic lysis procedure of the blood with Millipore water was chosen which represents a cheap, fast and reliable method to diminish the amount of erythrocytes in the samples. This procedure is shown to be suitable both to human and murine blood micro-samples, making it also applicable to small animal experiments with recurring blood sampling. As all types of leukocytes are kept in the sample during the preparation, they can be analysed separately after discrimination during the flow cytometry analysis. This also holds for all heme peroxidase-containing cells, namely neutrophils, eosinophils and monocytes. Moreover additional parameters (e.g. antibody staining) can be combined with the heme peroxidase activity determination to gain additional information about the different immune cell types. Based on previous results we applied APF for specifically addressing the halogenating activity of leukocyte peroxidases in blood samples. This dye is selectively oxidized by the MPO and EPO halogenation products hypochlorous and hypobromous acid. This approach may provide a suitable tool to gain more insights into the immune-physiological role of the halogenating activity of heme peroxidases.

Tetra(p-tolyl)borate-Functionalized Solvent Polymeric Membrane: A Facile and Sensitive Sensing Platform for Peroxidase and Peroxidase Mimetics

The determination of peroxidase activities is the basis for enzyme-labeled bioaffinity assays, peroxidase-mimicking DNAzymes- and nanoparticles-based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost-effective solvent polymeric membrane-based peroxidase detection platform is described that utilizes reaction intermediates with different pKa values from those of substrates and final products. Several key but long-debated intermediates in the peroxidative oxidation of o-phenylenediamine (o-PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o-PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane-based methods. As an example of peroxidase mimetics, G-quadruplex DNAzymes were probed by the intermediate-sensitive membrane and a label-free thrombin detection protocol was developed based on the catalytic activity of the thrombin-binding G-quadruplex aptamer.

Colorimetric determination of peroxidase activity in the presence of a polyanion

A procedure was developed for the determination of peroxidase activity with respect to substrates whose oxidation products were insoluble or poorly soluble in the reaction medium. It was demonstrated that the introduction of water-soluble sulfonated polystyrene to a reaction medium containing aniline and hydrogen peroxide made it possible to transform insoluble polyaniline to the highly dispersed state, preventing the formation of a deposit at the walls of the cuvette in the course of analysis. The addition of sulfonated polystyrene to the reaction medium improves the sensitivity of the determination of horseradish peroxidase.

Effect of Ilex extracts and isolated compounds on peroxidase secretion of rat submandibulary glands

Free radicals are involved in diverse disorders such as tumoral, central nervous system alterations, immunological and inflammatory pathologies. Peroxidase is an oral enzyme involved in the defense of the oral cavity. Ilex species such as Ilex paraguariensis St. Hil. and the commercial product made with it “Yerba Mate” are used traditionally as antirheumatics and for the treatment of gastrointestinal diseases among others and also as a beverage with nutritional and stimulant properties. The presence of polyphenolic derivatives and flavonoids in the aqueous extract has been determined by HPLC analysis. In this study, the activity of aqueous extracts of I. paraguariensis and “Yerba Mate” on peroxidase secretion in female rat submandibular glands was investigated. The contribution to this pharmacological activity by some major hydrocynnamic acid derivatives present in the crude extracts, such as chlorogenic acid and caffeic acid and the most abundant methylxanthine, caffeine, was also evaluated. Spectrophotometrical determination of peroxidase activity showed that both extracts produced a significant increase in both secreted peroxidase and total peroxidase activity, though “Yerba Mate” showed a higher activity (EC 50 “Yerba Mate”: 148 ± 10 μg/ml; EC 50 I. paraguariensis: 841 ± 20 μg/ml). The HPLC/DAD analysis of the crude extracts was performed and chlorogenic acid, caffeic acid and caffeine were identified and quantified. The results (expressed as W/W percentage of dried material) were as follows: I. paraguariensis: chlorogenic acid: 2.80 ± 0.30, caffeic acid: 0.023 ± 0.004, caffeine: 1.06 ± 0.06; “Yerba Mate”: chlorogenic acid: 1.98 ± 0.37; caffeic acid: 0.020 ± 0.003, caffeine: 0.70 ± 0.06. Caffeine and chlorogenic acid were proved to play an important role in the induction of peroxidase secretion induced by the extracts.

A Rapid Microassay for Determination of Peroxidase in Wheat and Flour

ABSTRACTA rapid sensitive microplate assay for the determination of peroxidase activity in wheat, flour, or individual wheat kernels has been developed using a commercially prepared 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) diammonium salt (ABTS) solution. The assay derives a six‐point calibration curve based on commercially available horseradish peroxidase (r2 &gt; 0.990), while simultaneously analyzing eight extracts in triplicate. The coefficients of variation (CV) of triplicate assays of a single extract from any of the three sources investigated were generally &lt;2.0%. Multiple extracts (n = 5) of either whole meal or flour yielded assays with an average CV &lt; 5.0%. Preparation of the calibration standards with commercially available peroxidase stabilizing buffer allowed the standards to be used for five days without any deterioration in the assay's reproducibility. This assay is ideally suited for high‐throughput operations such as millstream analysis or plant breeder screening evaluations.

Analysing the freshness of intact rice grains by colour determination of peroxidase activity

AbstractFreshness is an important palatability characteristic of rice grains. However, this characteristic deteriorates rapidly during dry storage. In the present study we have developed an indirect method to examine the freshness of rice grains harvested in different crop seasons or stored under conditions known to accelerate deterioration. Bulk samples of intact brown rice grains were soaked in dianisidine solution containing guaiacol and H2O2, and the optical absorbance of the incubation solution was determined colorimetrically. Alternatively, intact brown rice grains were incubated in a similar solution in the wells of a 96‐well microplate, with one grain per well, and the optical absorbance of the incubation solution was measured by a microplate reader. The results indicated that cultivar, storage duration and temperature significantly affected the activities of grain peroxidase. Marked differences in the development of purple colour in the incubation solutions and in vitro peroxidase activity were also found among the brown rice grains sampled in different seasons. The changes in colour of the incubation solutions measured by either of the above methods paralleled the changes in grain activity of peroxidase assayed using an in vitro method. The proposed method can differentiate brown rice grains harvested in different seasons. Moreover, this method, if combined with suitable statistical clustering procedures, can detect the blending status of fresh/old brown grains in a sample. The advantages of this test method using A385 over the conventional enzyme activity assay are the rapidity and simplicity with which A385 can estimate the in vitro determination of peroxidase activity of tested rice grains. Copyright © 2003 Society of Chemical Industry

Dual method for the determination of peroxidase activity and total peroxides-iodide leads to a significant increase of peroxidase activity in human sera

Peroxidases are very important enzymes, e.g., as preventive antioxidants by removing noxious peroxides from the blood. For this reason we evaluated a colorimetric method which detects the activity of endogenous peroxidases by their reaction with hydrogen peroxide, using tetramethylbenzidine as the chromogenic substrate. This assay design can be easily reversed by change of the variable compound to measure also total peroxides in plasma or serum. An increased total antioxidant status was reported previously by the addition of iodide to human serum. In this study iodide activated the endogenous peroxidases significantly in comparison to control sera and isomolar NaCl as well as horseradish peroxidase. Corresponding to the increased peroxidase activity a concomitant decrease of total peroxides occurred in the same samples. This exchangeable assay design is a beneficial opportunity to screen total peroxide levels as well as peroxidase activity in human sera without time-consuming preparations. The method proved to be simple and is favorable due to its specificity, reproducibility, and low costs. Moreover, we were able to find an explanation for the increased total antioxidant status in the presence of iodide, which is presumably an indirect protective effect via an enhanced activity of enzymatic antioxidants, thereby reducing endogenous peroxides.

Electrochemical characterization of screen-printed carbonaceous electrodes for the determination of peroxidase activity in novel screen-printed flow-through modules.

A novel totally screen-printed flow-through cell for immunoanalysis is presented. It contained screen-printed carbonaceous electrodes, which allowed the determination of peroxidase activity through the electrochemical reduction of p-benzoquinone. As different electrode materials differ strongly in their electrochemical properties, electrodes resulting from various screen-printable carbonaceous pastes were characterized using the hydroquinone/ p-benzoquinone redox couple. For most of the electrodes, cyclic voltammogram peak separations of between 550 and 670 mV were observed indicating only quasi-reversible electrochemical behavior. This was confirmed by variation of the peak separation with scan rate. Heterogeneous electron transfer rates of ca. 0.5 - 1 x 10(-3) cm s(-1) and electrochemical activation energies of ca. 20 kJ mol(-1) were found. These flow-through cells were not only applied to electrochemical peroxidase activity determinations but also, in combination with a separate detector, as affinity reactors. After biotinylation of screen-printed layers, streptavidin and then biotinylated peroxidase could be bound. However, as signals were only 10-20% of those obtained with a column filled with biotinylated glass beads, only the screen-printed electrochemical detector was applied to the detection of antibodies against the African Swine Fever Virus.

Changes in chalazal cell walls and in the peroxidase enzymes of the crease region during grain development in barley.

In an investigation of the role of peroxidase enzymes in the differentiation of the tissues of the crease region of barley, plants of winter barley cv. Halcyon were grown from anthesis onwards in controlled conditions at a constant temperature of 16 degrees C. Four ears were harvested at 2-d intervals from 6 d after anthesis (daa) until 50 daa. Grains from mid-ear were used for (i) fresh and dry weight determinations, (ii) extraction of crease tissue for the determination of peroxidase activity and for the separation of isozymes of peroxidase by isoelectric focusing (IEF) and (iii) detection of lignin and suberin in the tissues of the crease using autofluorescence and cytochemistry. Peroxidase activity was located histochemically in the crease tissue of cv. Chariot. Scanning electron microscopy studies were carried out on developing grains of cv. Blenheim. Maximum grain water content was achieved at 14 daa. Lignin and suberin were detected in the walls of the chalazal cells from 18 daa onwards. No changes in the staining of chalazal cell walls were detected at the end of grain filling (32 daa), but loss of autofluorescence and staining were observed at 42 daa, just prior to the final, rapid phase of grain dehydration. Peroxidase activity per fresh weight of crease tissue was high at 6 daa and low at 22 daa. It was also low between 32 and 40 daa, but it rose again from 42 daa onwards. IEF demonstrated that both anionic and cationic isozymes of peroxidase were present in crease tissue, the pattern of bands showing some marked changes during the course of grain development.

Correlation Between Glutathione Peroxidase Activity and The Quantity of Selenium in The Whole Blood of Beef Calves

The aim of the study was to establish concentration of selenium and the pertinent activity of glutathione peroxidase (GSH-Px) in whole blood that could be used as reference values in future research. The first part of study was carried out on clinical healthy beef calves (n = 35), fed a basal diet and a ready-made fodder mix that contained 0.1 mg.kg(-1) selenium. In the second part of our research, we investigated the GSH-Px activity in a group of calves (n = 47) that had not received supplements added to the basal diet. The research was carried out in the north-west Croatia, in the region known to be poorer in selenium. We determined GSH-Px activity in whole blood by the Ransel method adapted to a Technicon RA-1000 at 37 degrees C. Determination of Se concentration was done with a modification of the method given by Perkin-Elmer HGGS. The mean value of the Se concentration in the whole blood of the first group of calves was 200.22 +/- 45.2 mu g.l(-1) and pertinent average GSH-Px activity was 764.6 +/- 197.8 mu kat.l(-1). From the results obtained, a correlation between Se and GSH-Px was calculated (r = 0.82 ; P < 0.001). The mean values of the GSH-Px activity in herd that did not receive a supplement were 435.3 +/- 155.76 mu kat.l(-1). Out of 47 animals of the second herd, four animals (8.51%) had lower values than those recommended as sufficient. This study confirmed that after the calculation of the correlation between Se and GSH-Px, glutathione peroxidase activity determination can be used as a rapid and simple proxy for the determination of selenium concentration in whole blood.

Flow cytometric evaluation of bone marrow differentials in rats with pharmacologically induced hematologic abnormalities.

Previously, flow cytometric determination of peroxidase activity, cell size, and reactivity to lymphocyte antibodies were used to produce bone marrow differentials in untreated rats. In the present study, abnormal hematologic profiles were induced with erythropoietin (EPO), recombinant murine stem cell factor (rm-SCF), granulocyte-macrophage stimulating factor (GM-CSF), and cyclophosphamide (CP). Manual and flow cytometric data showed comparable levels of erythroid and myeloid hyperplasia in EPO- and rm-SCF/GM-CSF-treated animals, respectively. In CP-treated animals, flow cytometric data revealed significant decreases in cellularity at concentrations of CP > or = 5 mg/kg. In contrast, 20 mg/kg CP were necessary to induce microscopically apparent hypoplasia in histologic bone sections, showing that the automated methodology was a more sensitive indicator of bone marrow hypocellularity than was the more conventional manual method. Megakaryocyte counts were consistently higher by flow cytometer than by manual counts performed on cytocentrifuge preparations made from the same cell suspensions but were similar to megakaryocyte counts performed on histologic sections of femur, indicating that the automated methodology produced a more accurate reflection of true megakaryocyte numbers. Induction of hematologic abnormalities in the present study showed that manual bone marrow differentials can be replaced with the more efficient and reliable flow cytometric method in most preclinical toxicology studies.

Colorimetric assay for lignin peroxidase activity determination using methylene blue as substrate

A colorimetric method for determination of lignin peroxidase activity has been developed which is based on the demethylation of methylene blue. The use of this dye shows the practical advantage of using a wavelength in the visible region of spectrum. The method can be efficiently used for the enzyme quantification as its sensitivity is close to the veratryl alcohol assay.

A disposable amperometric immunosensor for 2,4-dichlorophenoxyacetic acid

A disposable immunochemical biosensor for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was developed using a screen-printed electrode system as an amperometric transducer and monoclonal antibodies against 2,4-D as a biospecific part. At first, the electrochemical system for determination of peroxidase activity was studied. For measurements, the peroxidase substrates were hydrogen peroxide together with either hydroquinone, 1,2-phenylenediamine or pyrocatechol as electron donors, which were amperometrically detected using platinum, gold and graphite composite layers as working electrode materials. The optimal concentrations of reaction components were found, the highest signals being achieved for hydroquinone with gold or graphite electrodes. The immunochemical system makes use of the competitive assay principle with 2,4-D covalently immobilized either directly on the surface of the gold electrode or on a separate nylon membrane. The peroxidase-antibody conjugate was synthesized and used as a tracing reagent. By using 2,4-D immobilized through its carboxylic group directly to the silanized surface of the gold working electrode, a detection limit close to 0.1 μg/1 of free 2,4-D was achieved. One analysis was completed in 1 h (30 min for the immunochemical reaction, washing, 25 min incubation with substrate, measurement).

Peroxidase activities in bull spermatozoa.

The present study was carried out to determine the localization of peroxidase activity in bull spermatozoa. 3,3'-Diaminobenzidine (DAB) was used as a substrate for revealing peroxidase activity, and light and electron microscopic analysis of the results obtained was performed. Peroxidase activity was detected in the mitochondria of the middle piece and the outer acrosomal membrane. Catalase was excluded as an enzyme, catalyzing the detected peroxidase activity. Concerning the biochemical properties of bull sperm peroxidases, peroxidase activity was found to be manifested in a large pH range, 4-10.5. Bull sperm peroxidase activity appeared to be temperature sensitive and azide sensitive and could be readily inhibited by phenylhydrazine. Electrophoretic analysis of the proteins from bull sperm extracts separated in a Davis-Ornstein system of 7% polyacrylamide gel, followed by the determination of peroxidase activity on the polyacrylamide gels, revealed that all 14 sperm protein fractions available on the gel possessed peroxidase when benzidine was used as a substrate. The possible reasons for the electrophoretic heterogeneity of bull sperm peroxidases are discussed.