[Cytochemical and cytoenzymatic leukocytic disorders in chronic post-viral hepatitis].
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Hepatitis↗
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Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue.
We developed a new, highly sensitive enzymatic method for quantifying creatine in erythrocytes, which comprises creatine amidinohydrolase, sarcosine oxidase, and peroxidase. In the present method, an N-methylcarbamoyl derivative of methylene blue, 10-N-methylcarbamoyl-3,7-bis(dimethylamino)phenothiazine (MCDP), was used as a sensitive chromogenic compound. Potassium ferrocyanide was used to prevent nonspecific oxidation of MCDP. The enzymatic method exhibited good analytical performance: precision, within-run CVs <1.0% and between-day CVs <2.0%; average analytical recovery, 99.3% +/- 1.8%; detection limit, 1.0 micromol/L in hemolysate; and linearity, at least up to 500 micromol/L as creatine concentration in hemolysate. Excellent agreement was observed between the present method (y) and HPLC (x), y = 1.029x - 0.002 micromol/g hemoglobin, r = 0.9998, S(y/x) = 0.053 micromol/g hemoglobin (n = 110). No significant interference was produced by various compounds, including guanidino compounds, amino acids, and reducing materials. The reference intervals (mean +/- 2 SD) for erythrocyte creatine obtained from 60 males and 60 females were (in micromol/g hemoglobin) 1.18 +/- 0.52 (0.66-1.70) for males and 1.35 +/- 0.49 (0.86-1.84) for females. Using this method, we documented changes in erythrocyte creatine in patients with various hemolytic conditions, including hemolytic anemia, liver cirrhosis, renal insufficiency, and chronic renal failure treated with hemodialysis with or without the administration of erythropoietin. We conclude that the use of MCDP allows sensitive measurement of erythrocyte creatine and that MCDP with potassium ferrocyanide can improve the sensitivity of assays that use peroxidase for detection of H2O2.
Adult↗
Histochemical localization of trehalase activity in dorsal flight muscle of the flesh fly Sarcophaga bullata with light and electron microscopy.
Trehalase activity in flight muscle of the flesh fly Sacrophaga bullata is detected histochemically at light- and electron-microscopic levels by using diaminobenzidine, glucose oxidase and peroxidase in the incubation medium. The association of trehalase activity with the inner mitochondrial membrane is confirmed. Biochemical assay shows that about 50% of the initial total trehalase activity is lost from the tissue during the histochemical processing and about 50% remains for histochemical detection.
Animals↗
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.
Apoenzymes↗
Ferrocyanide-peroxidase activity of cytochrome c oxidase
Redox interaction of mitochondrial cytochrome c oxidase (COX) with ferrocyanide/ferricyanide couple is greatly accelerated by polycations, such as poly-l-lysine [Musatov et al. (1991) Biological Membranes 8, 229-234]. This has allowed us to study ferrocyanide oxidation by COX at very high redox potentials of the ferrocyanide/ferricyanide couple either following spectrophotometrically ferricyanide accumulation or measuring proton uptake associated with water formation in the reaction. At low [ferrocyanide]/[ferricyanide] ratios (Eh values around 500 mV) and ambient oxygen concentration, the ferrocyanide-oxidase activity of COX becomes negligibly small as compared to the reaction rate observed with pure ferrocyanide. Oxidation of ferrocyanide under these conditions, is greatly stimulated by H2O2 or ethylhydroperoxide indicating peroxidatic reaction involved. The ferrocyanide-peroxidase activity of COX is strictly polylysine-dependent and is inhibited by heme a3 ligands such as KCN and NaN3. Apparently the reaction involves normal electron pathway, i.e. electron donation through CuA and oxidation via heme a3. The peroxidase reaction shows a pH-dependence similar to that of the cytochrome c oxidase activity of COX. When COX is preequilibrated with excess H2O2, addition of ferrocyanide shifts the initial steady-state concentrations of the Ferryl-Oxo and Peroxy compounds towards approximately 2:1 ratio of the two intermediates. It is suggested that in the peroxidase cycleferrocyanide donates electrons to both P and F intermediates with a comparable efficiency. Isolation of a partial redox activity of COX opens a possibility to study separately proton translocation coupled to the peroxidase half-reaction of the COX reaction cycle. Copyright 1998
Journal Article↗
Peroxidase activities of hemoproteins. V. Hematin complexes with heterogeneous liganes.
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Acetates↗
Effect of petroleum-containing wastewater irrigation on bacterial diversities and enzymatic activities in a paddy soil irrigation area.
Effects of petroleum contamination on bacterial diversities and enzymatic activities in paddy soils were investigated in the Shenfu irrigation area, the largest area irrigated by oil-containing wastewater for more than 50 yr in northeastern China. Bacterial diversities were determined by conventional colony morphology typing techniques and 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Dehydrogenase, hydrogen peroxidase, polyphenol oxidase, urease, and substrate-induced respiration (SIR) were measured to evaluate the effects of petroleum-containing wastewater irrigation on soil biochemical characteristics. Results showed that paddy soil total petroleum hydrocarbon (TPH) concentration in the irrigation area varied from 277.11 to 5213.37 mg kg(-1) dry soil. Soil TPH concentration declined along the gradient of the irrigation channel from up- to downstream. At the current pollution level, the paddy soil TPH concentration was positively correlated with the colony forming units (CFU) of aerobic heterotrophic bacteria (AHB) (r = 0.928, p < 0.001) and the genetic diversity based on DGGE profiles (r = 0.655, p < 0.05). The bacterial diversities in the soils based on colony morphotypes of AHB also increased with TPH concentration (r = 0.598), but not significant statistically (p = 0.052). Analysis of soil enzyme activities indicated a significant positive correlation between soil TPH concentration and activities of dehydrogenases (r = 0.974, p < 0.001), hydrogen peroxidases (r = 0.957, p < 0.001), polyphenol oxidases (r = 0.886, p < 0.001), and SIR (r = 0.916, p < 0.001). On the contrary, the urease activity showed a negative correlation with paddy soil TPH concentration (r = -0.814, p = 0.002), and could be used as a sensitive indicator of petroleum contamination.
Agriculture↗
Preliminary studies on differential defense responses induced during plant communication.
We compared the expression patterns of three representative genes in undamaged tomato and tobacco plants in response to exposure to either tomato or tobacco fed on by Helicoverpa armigera (cotton bollworm). When tomato and tobacco, two species of one family, were incubated in the chambers with the tomato plants damaged by the cotton bollworm, the expression of the PR1, BGL2, and PAL genes was up-regulated in leaves of both plants. However, the levels of gene expression were significantly higher in the tomato than that in the tobacco. In addition, the activities of enzymes, peroxidase, polyphenol oxidase, and lipoxygenase were found to be higher in the tomato than those in the tobacco. Similar results were obtained when the damaged plants were replaced by the tobacco.
Animals↗
Immunoaffinity layering of enzymes. Stabilization and use in flow injection analysis of glucose and hydrogen peroxide.
A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae.
Animals↗
Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris.
The effect of regurgitant from Leptinotarsa decemlineata Say larvae on wound-induced responses was studied using two plant species, Solanum tuberosum L. and Phaseolus vulgaris L. Wounding of one leaf of intact S. tuberosum plants differentially affected ethylene production and activities of peroxidase and polyphenol oxidase. Only polyphenol oxidase activity was stimulated by wounding in both wounded and systemic leaves. Peroxidase activity was not affected by wounding. Wounding caused only a transient increase of ethylene production from wounded leaves. The application of regurgitant to wound surfaces stimulated ethylene production as well as activities of peroxidase and polyphenol oxidase in both wounded and systemic leaves. Wounding significantly enhanced ethylene production and polyphenol oxidase activity in wounded and systemic leaves of P. vulgaris. The application of regurgitant caused an amplification of ethylene production, peroxidase activity, and polyphenol oxidase activity, in both wounded and systemic leaves of bean plants. Several substances were tested for their role as possible endogenous signals in P. vulgaris. Hydrogen peroxide and methyl jasmonate appeared as potential local and systemic signals of ethylene formation in wounded bean plants. Local ethylene production in leaf discs was differentially affected by the regurgitant application in potato versus bean plants. While all tested concentrations of regurgitant caused stimulation of ethylene formation from potato leaf discs, ethylene production was completely inhibited by increasing concentrations of the regurgitant in bean leaf discs. Our data present evidence that ethylene may play an important role in the interaction between plants and herbivores at the level of recognition of a particular herbivore leading to specific induction of signalling cascades.
Journal Article↗
Biosensor for determination of glucose and sucrose in fruit juices by flow injection analysis.
Glucose and sucrose were measured with an amperometric method by using the flow injection analysis technique. A carbon paste electrode with a renewable surface containing glucose oxidase, horseradish peroxidase, and ferrocene was used in combination with the soluble enzymes invertase and mutarotase. The effect of invertase, mutarotase, and ascorbic acid on the electrode response was examined. Glucose and sucrose concentrations were determined with < 3% errors. The proposed method for glucose and sucrose measurements was validated in real samples of fruit juices. The results were also compared with those obtained with the ultraviolet method.
Ascorbic Acid↗
Development and application of an integrated system for monitoring ethanol content of fuels.
An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-amino-phenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05-1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91-mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05-1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.
Alcohol Oxidoreductases↗
Leishmania donovani: ultrastructural localization of diaminobenzidine reactivity in the amastigotes.
Intracellular amastigotes of Leishmania donovani obtained from spleens of infected hamsters were studied by means of the diaminobenzidine technique for the presence of cytochromes and the activities of cytochrome oxidase and peroxidase. In the absence of H2O2, the oxidation of DAB, evidenced by electron-dense deposits localized on the cristae, inclusions, and enveloping membranes of the mitochondria and kinetoplast, revealed the activity of the cytochrome oxidase and the presence of the cytochromes. The increased deposition of DAB oxidation especially on the enveloping membranes in the presence of H2O2 suggests the activity of a peroxidase, probably cytochrome c peroxidase.
3,3'-Diaminobenzidine↗
Biological energy from the igneous rock enhances cell growth and enzyme activity.
Some effects from natural resources might be ignored and unused by humans. Environmental hormesis could be a phenomena necessary to bio-organism existence on earth. Since 1919, radiation and some heavy metal hormesis from the environment were proved in various reports. In this study, igneous rock with very low radioactivity and high ferrous activity was measured by multichannel analyzer and inductively coupled plasma analyzer. The water treated by igneous rock, both directly soaked or indirectly in contact, induced increased activities of glucose oxidase, catalase, peroxidase, and superoxide dismutase. It also increased cell growth of SC-M1, HCT-15, Raji, and fibroblast cell lines. The water after treatment of igneous rock had no change in pH values, but displayed decreased conductivity values. We assume that the igneous rock could transfer energy to water to change the molecular structure or conformation of water cluster, or by radiation hormesis effect could then induce increased enzyme activity and cell growth. It is also possible that the energy from rock may combine radiation hormesis with other transferable biological energy forms to change water cluster conformation.
Animals↗
Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance.
The oxidative burst is an early response to pathogen attack leading to the production of reactive oxygen species (ROS) including hydrogen peroxide. Two major mechanisms involving either NADPH oxidases or peroxidases that may exist singly or in combination in different plant species have been proposed for the generation of ROS. We identified an Arabidopsis thaliana azide-sensitive but diphenylene iodonium-insensitive apoplastic oxidative burst that generates H(2)O(2) in response to a Fusarium oxysporum cell-wall preparation. Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a type III peroxidase, French bean peroxidase type 1 (FBP1) exhibited an impaired oxidative burst and were more susceptible than wild-type plants to both fungal and bacterial pathogens. Transcriptional profiling and RT-PCR analysis showed that the anti-sense (FBP1) transgenic plants had reduced levels of specific peroxidase-encoding mRNAs, including mRNAs corresponding to Arabidopsis genes At3g49120 (AtPCb) and At3g49110 (AtPCa) that encode two class III peroxidases with a high degree of homology to FBP1. These data indicate that peroxidases play a significant role in generating H(2)O(2) during the Arabidopsis defense response and in conferring resistance to a wide range of pathogens.
Arabidopsis↗
Role of alpha-tocopheroxyl radical in the initiation of lipid peroxidation in human low-density lipoprotein exposed to horse radish peroxidase.
Heme-containing (per)oxidases including horse radish peroxidase (HRP)/H2O2 have been shown to oxidatively modify isolated low-density lipoprotein (LDL) in vitro and oxidized LDL is implicated in the early events leading to atherosclerosis. The role of alpha-tocopherol (alpha-TOH) in the oxidation of LDL by HRP/H2O2 is unclear, although alpha-tocopheroxyl radical (alpha-TO.), which is formed during this process, can act as a chain transfer agent of lipid peroxidation in LDL. By combining HPLC and EPR spectroscopy, we hereby show that during HRP/H2O2-induced oxidation of human LDL: (i) the accumulation of cholesteryl linoleate hydroperoxides and hydroxides (CE-O(O)H) occurs concomitantly with the formation of alpha-TO. and consumption of alpha-TOH in the absence of other detectable organic (g approximately 2) radicals; (ii) the rates of alpha-TO. formation and subsequent decay reflect the rates of both alpha-TOH consumption and CE-O(O)H accumulation; (iii) CE-O(O)H accumulation is directly dependent on the level of endogenous alpha-TOH, and vitamin E supplementation results in increased lipid oxidizability; (iv) the inhibition of HRP activity by catalase plus urate results in a persistent alpha-TO. signal, the decay (t1/2 approximately 20 min) of which is accompanied by continued accumulation of CE-O(O)H, with complete cessation of lipid peroxidation upon loss of the chromanoxyl signal. These results demonstrate a direct correlation between alpha-TOH/alpha-TO. and the extent of HRP/H2O2-induced LDL lipid peroxidation, and that this type of oxidative modification can occur in the absence of g approximately 2 radicals other than alpha-TO.. Together, the results support a role for tocopherol-mediated peroxidation but not the involvement of a protein radical in the initiation of LDL lipid peroxidation induced by HRP/H2O2.
Free Radicals↗
Assay of nucleoside diphosphate kinase in microtiter plates using a peroxidase-coupled method.
A sensitive assay for nucleoside diphosphate kinase which utilizes microtiter plates is described. ATP, formed in the reaction between dGTP and ADP, is trapped by the glycerol kinase reaction. A colored compound is generated by glycerol-3-phosphate oxidase and peroxidase. This assay is useful for testing a large number of samples generated by chromatographic techniques or for screening purposes.
Adenosine Diphosphate↗
Effect of nitrogen and water treatment on leaf chemistry in horsenettle (Solanum carolinense), and relationship to herbivory by flea beetles (Epitrix spp.) and tobacco hornworm (Manduca sexta).
We studied the interaction between plants (horsenettle; Solanum carolinense) and herbivorous insects (flea beetles; Epitrix spp., and tobacco hornworm; Manduca sexta) by focusing on three questions: (1) Does variation in nitrogen availability affect leaf chemistry as predicted by the carbon-nutrient balance (CNB) hypothesis? (2) Does variation in plant treatment and leaf chemistry affect insect feeding? (3) Is there an interaction between the insect herbivores that is mediated by variation in leaf chemistry? For three successive years (1998-2001), we grew a set of clones of 10 maternal plants under two nitrogen treatments and two water treatments. For each plant in the summer of 2000, we assayed herbivory by hornworms in both indoor (detached leaf) and outdoor (attached leaf) assays, as well as ambient flea beetle damage. Estimates of leaf material consumed were made via analysis of digitized leaf images. We also assayed leaves for total protein, phenolic, and glycoalkaloid content, and for trypsin inhibitor, polyphenol oxidase, and peroxidase activity. Despite strong effects of nitrogen treatment on growth and reproduction, only total protein responded as predicted by CNB. Leaf phenolic levels were increased by nitrogen treatment, polyphenol oxidase activity was decreased, and other leaf parameters were unaffected. Neither hornworm nor flea beetle herbivory could be related to plant treatment or genotype or to variation in any of the six leaf chemical parameters. A negative relationship between flea beetle and hornworm herbivory was found, but was not apparently mediated by any of the measured leaf chemicals. Because leaf resistance was maintained in low nitrogen plants at the apparent expense of growth and reproduction, our results support the concept of a fitness cost of defense, as predicted by the optimal defense hypothesis.
Adaptation, Physiological↗