A more sensitive modification of the catalase assay with the Clark oxygen electrode: Application to the kinetic study of the pea leaf enzyme

A modification of the method of catalase determination by means of the Clark oxygen electrode is described. The assay is based on measurement of the initial rate at which oxygen is released by catalase in an oxygen-free buffer. Displacement of oxygen was brought about by flushing with nitrogen, and the substrate used was hydrogen peroxide at a 33.5 mm final concentration. The method is rapid and can be used with crude catalase preparations. Its sensitivity is at least 20 times higher than that of previous methods; it has an interval of measurable activity of about 0.01–8.4 μmol of O₂/min and, therefore, is applicable to an 840-fold range of catalase concentrations. This modification was applied to the kinetic study of crude extracts of pea leaf catalase. An apparent K_m of 0.190 m was calculated.     

Dioxygen uptake by isolated thylakoids from lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.): simultaneous measurements of dioxygen uptake,pH change of the medium and chlorophyll fluorescence parameters

The setup has been elaborated for the simultaneous measurements of dioxygen uptake, pH changes, and chlorophyll a fluorescence parameters of an isolated thylakoid suspension. Using this equipment we have found at least three kinetically distinguishable components in the response of dioxygen uptake and pH increase to light intensity in the range of 0–1600 μE m⁻² s⁻¹. The pH changes were not observed in the presence of uncouplers (2 μM valinomycin plus 2 μM nigericin) while O₂ uptake increased by about 10% and F _v /F _m ratio appeared to be unaffected by this treatment. Treatment with DNP-INT, an inhibitor of plastoquinol oxidation, led to a significant reduction of pH increase and O₂ consumption whereas F _v /F _m was impaired only to 71% of the control. Incubation with catalase (580 U/ml) caused a total inhibition of oxygen uptake, while the pH increased and the F _v /F _m ratio decreased to about 60% and 85% of the control, respectively. The addition of catalase after the irradiation period led to an evolution of the same amount of dioxygen as was consumed during the light period. These results show that hydrogen peroxide was formed in the investigated system and accumulated during illumination. On the basis of the obtained data, three sites of dioxygen reduction within isolated thylakoid membranes and the dependence of dioxygen uptake on the photosystem activities were discussed.     

Escherichia coli Hmp, an “oxygen-binding flavohaemoprotein”, produces superoxide anion and self-destructs

Escherichia coli Hmp is a homologue of Ralstonia eutropha FHP, the first reported bacterial flavohaemoglobin, and functions in NO detoxification. Photolysis of CO-ligated Hmp in the presence of oxygen gave a photodissociable oxy species with k_on 2.82×10⁷ M^–1 s^–1 and k_off 4.49×10³ s^–1. The dissociation constant of the primary O₂ compound was 160 M (25°C, pH 7.0). In order to detect superoxide formation, ferric horseradish peroxidase was used. Hmp formed the oxy compound within milliseconds, followed by formation of compound III, arising from superoxide formation. The rate of superoxide formation was independent of oxygen concentration between 0.05 and 0.7 mM oxygen, suggesting a K_m <0.05 mM. During prolonged oxidation of NADH, the spectral signals of Hmp decayed and iron was released in a process prevented by superoxide dismutase or catalase. NADH oxidation by purified Hmp was characterised by progressive slowing of oxygen uptake. Inclusion of NO, superoxide dismutase or catalase during NADH oxidation partially protected oxygen uptake, consistent with the formation, in the absence of NO, of reactive oxygen species that inhibit Hmp function. The results are discussed in relation to the tight control exerted on Hmp synthesis in vivo.This paper is dedicated to Professor Dr Hans G. Schlegel, on the occasion of his 80th birthday.     

Kinetic Characterization of Extracellular Catalases fromPenicillium piceum F-648 and Its Hydrogen Peroxide-Adapted Variants

A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H₂O₂ was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H₂O₂ degradation (V _max), V _max/K _M ratio, and the catalase inactivation rate constant in the enzymatic reaction (k _in, s^–1) were estimated in phosphate buffer (pH 7.4) at 30°C. The effective constant representing the rate of catalase thermal inactivation (k _in ^*, s^–1) was determined at 45°C. In all samples, the specific activity and K _M for catalase were maximum at a protein concentration in culture liquid filtrates of (2.5–3.5) × 10^–4 mg/ml. The effective constants describing the rate of H₂O₂ degradation (k, s^–1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H₂O₂. Catalases from variants 5 and 3 with high and low affinities for H₂O₂, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H₂O₂ can be used to obtain fungal variants producing extracellular catalases with improved properties.     

Negative air ions as a source of superoxide

The physico-chemical characteristics and possible formation mechanisms of negative air ions are considered. It was found that the products of oxygen and nitrogen negative ionization reduce ferricytochromec and nitroblue tetrazolium, and that these reactions were inhibited by superoxide dismutase. The interaction of negatively ionized oxygen with water led to hydrogen peroxide accumulation, which was inhibited by tetranitromethane or catalase. Nitrogen ionization under these conditions caused the formation of the hydrated electron e _aq ^— and the superoxide anion O ₂ ^— . The data obtained indicate that the biological activity of negative air ions may be dependent on superoxide. The generation of reactive oxygen ions in the gas phase and also at a gas/water interface is described. A scheme for superoxide production under oxygen and nitrogen ionization is proposed.     

Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells

Although it is understood that hydrogen peroxide (H₂O₂) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H₂O₂ in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H₂O₂. The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0  ±  4.7 h versus 28.6  ±  0.8 h, p < 0.05), consistent with a diminished growth rate and H₂O₂ release. Incubation with aminotriazole, a catalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G₀/G₁ phase in hCatTg MAECs compared to the wild-type cells (25.0  ±  0.9 h versus 15.9  ±  1.4 h, p  <  0.05). The hCatTg MAECs also exhibited decreased activities of the cyclin-dependent kinase (Cdk) complexes responsible for G₀/G₁- to S-phase transition in the cell cycle, including the cyclin D–Cdk4 and cyclin E–Cdk2 complexes. Moreover, the reduction in cyclin–Cdk activities in hCatTg MAECs was accompanied by increased protein levels of two Cdk inhibitors, p21 and p27, which inhibit the Cdk activity required for the G₀/G₁- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H₂O₂ scavenger, suggest that endogenously produced H₂O₂ mediates MAEC proliferation by fostering the transition from G₀/G₁ to S phase.     

Energy metabolism of Chironomus anthracinus (Diptera: Chironomidae) from the profundal zone of Lake Esrom,Denmark, as a function of body size,temperature and oxygen concentration

The profundal zone of Lake Esrom, Denmark has a dense population of Chironomus anthracinus, which survives 2–4 months of oxygen depletion each summer during stratification. The metabolism of 3rd and 4th instar larvae was examined in regard to variation in biomass and temperature. Respiration at air saturation was described by a curvilinear multiple regression relating oxygen consumption to individual AFDW and temperature. At 10 °C and varying oxygen regimes the O₂ consumption and CO₂ production of 4th instar larvae were almost unaltered from saturation to about 3 mg O₂ l^–1, but decreased steeply below this level. The respiratory quotient increased from 0.82 at saturation to about 3.4 at oxygen concentrations near 0.5 mg O₂ l^–1. This implied a shift from aerobic to partially anaerobic metabolism. At 0.5 mg O₂ l^–1 the total energy production equalled 20% of the rate at saturation of which more than one third was accounted for by anaerobic degradation of glycogen. This corresponded to a daily loss of 12 µg mg AFDW^–1 or approximately 5% of the body reserves. At unchanged metabolic rate the glycogen store would last three weeks, but long term oxygen deficiency causes a further suppression of the energy metabolism in C. anthracinus.     

Physiological and antioxidant response by Beauveria bassiana Bals (Vuill.) to different oxygen concentrations

The effect of three levels of oxygen (normal atmosphere (21% O₂), low oxygen (16% O₂) and enriched oxygen (26% O₂)) on the production and germination of conidia by Beauveria bassiana was evaluated using rice as a substrate. The maximum yield of conidia was achieved under hypoxia (16% O₂) after 8 days of culture (1.51 × 10⁹ conidia per gram of initial dry substrate), representing an increase of 32% compared to the normal atmosphere. However, germination was reduced by at least 27% due to atmospheric modifications. Comparison of antioxidant enzyme activity (superoxide dismutases and catalases) with the oxidation profiles of biomolecules (proteins and lipids) showed that a decrease in catalase activity in the final days of culture coincided with an increase in the amount of oxidized lipids, showing that oxidative stress was a consequence of pulses of different concentrations of O₂. This is the first study describing oxidative stress induction by atmospheric modification, with practical implications for conidia production.     

A kinetic study on the copper-albumin catalyzed oxidation of ascorbate

Serum albumin can specifically bind one Cu(II)-ion, and is proposed to function as a copper transport protein in vivo. Cu(II)-albumin is rapidly reduced by ascorbate. A second order rate constant of 0.54 mM^–1 min^–1 was estimated for the reaction. The oxidation process is catalytic, the Cu(I)-albumin molecule being reoxidized by molecular oxygen. The reaction was found to follow Michaelis-Menten kinetics, characterized by an apparent K_m-value of 0.89 mM, and a catalytic constant of 0.066 M O₂/min. An apparent inhibition of oxygen uptake was obtained with catalase (but not with superoxide dismutase), suggesting the formation of H₂O₂ in the system. Wilson's disease patients usually have increased amounts of non-ceruloplasmin copper in plasma. The low level of plasma ascorbate observed in such patients could possibly be due, at least in part, to an oxidation by Cu(II)-albumin.     

An inhibitor of catalase induced by cold in chilling-sensitive plants

An inhibitor of catalase accumulated when leaves of chilling-sensitive species were stored in the dark at 0°C. The inhibitor could be removed from crude extracts by passing them through a column of Sephadex G-25. After this treatment, the catalase activity of extracts of chilled tissues was found to be equal to that of extracts from unchilled leaves. When chilled tissues were incubated at 20°C, the inhibitor of catalase was lost, unless the tissues had been irreversibly damaged. It specifically inhibited plant catalase, and had no effect on mammalian catalase, plant malic dehydrogenase, or plant superoxide dismutase.

Despite the presence of catalase inhibitor in extracts of chilled plants, no increase in the level of H₂O₂ in chilled tissues was found, suggesting either that the inhibitor is compartmentalized and not in contact with catalase in vivo, or that the level of H₂O₂ is controlled by means other than through catalase activity. Plant tissues normally contain H₂O₂ which is destroyed by catalase when they are damaged. After chilling, H₂O₂ leaking from already injured cells would not be so readily removed by the inhibited catalase, and could contribute to further injury by acting as a source of free radical oxidants.

     

Microbodies und Diaminobenzidin-Reaktion in den Acetat-Flagellaten Polytomella caeca und Chlorogonium elongatum

Summary In two forms of acetate flagellates, the colourless Volvocale Polytomella caeca and the green Volvocale Chlorogonium elongatum, cell organelles can be demonstrated which are ultrastructurally similar to microbodies of higher organisms. The organelles do not have a close association with the endoplasmic reticulum and are located in the peripheral cytoplasm between the elongated mitochondria. In Polytomella they exhibit more or less spherical profiles in section and have a maximum diameter of approximately 0.2–0.25 . In Chlorogonium the organelles occasionally have an elongated shape and are larger than in Polytomella. Employing the electron microscopic cytochemical reagent diaminobenzidine (DAB)/H₂O₂ to localize the microbodial marker enzyme catalase in these organelles, it was found that no accumulation of the electron-opaque product occurs in the microbodies either at alkaline or neutral pH or at room temperature or 37° C. Only the cristae of mitochondria are stained with the DAB reaction caused by cytochrome oxidase and possibly by a cytochrome peroxidase.Organelles of Polytomella caeca containing catalase or cytochrome oxidase can be separated by rate centrifugation of a crude particulate fraction on a sucrose gradient (Gerhardt, 1971). The particles isolated from the peak of catalase activity show the same fine structural characteristics as the microbodies in situ do. But again, there is no detectable staining of these organelles by the DAB/H₂O₂ reaction.The identity of the microbody-like particles in Polytomella caeca and Chlorogonium elongatum with microbodies in general is deduced despite the negative results in cytochemical localization of catalase in these organelles.     

Immobilization of catalase via adsorption onto -histidine grafted functional pHEMA based membrane

Poly(2-hydroxyethylmethacrylate) (pHEMA) based flat sheet membrane was prepared by UV-initiated photopolymerization technique. The membrane was then grafted with -histidine. Catalase immobilization onto the membrane from aqueous solutions containing different amounts of catalase at different pH was investigated in a batch system. The maximum catalase immobilization capacity of the pHEMA–histidine membrane was 86 μg cm⁻². The activity yield was decreased with the increase of the enzyme loading. It was observed that there was a significant change between V_max value of the free catalase and V_max value of the adsorbed catalase on the pHEMA–histidine membrane. The K_m value of the immobilized enzyme was higher 1.5 times than that of the free enzyme. Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. It was observed that enzyme could be repeatedly adsorbed and desorbed without loss of adsorption capacity or enzyme activity.     

Longevity and antioxidant enzymes,non-enzymatic antioxidants and oxidative stress in the vertebrate lung: a comparative study

It has been proposed that antioxidants can be longevity determinants in animals. However, no comprehensive study has been conducted to try to relate free radicals with maximum life span. This study compares the lung tissue of various vertebrate species — amphibia, mammals and birds — showing very different and well known maximum life spans and life energy potentials. The lung antioxidant enzymes superoxide dismutase, catalase, Se-dependent and non-Se-dependent glutathione peroxidases, and glutathione reductase showed significantly negative correlations with maximum life span. The same was observed for the lung antioxidants, reduced glutathione and ascorbate. It is concluded that a generalized decrease in tissue antioxidant capacity is a characteristic of longevous species. It is suggested that a low rate of free radical recycling (free-radical generation and scavenging) can be an important factor involved in the evolution of high maximum animal longevities. A low free-radical production could be responsible for a low rate of damage at critical sites such as mitochondrial DNA.Abbreviations CAT catalase - COX cytochrome oxidase - GPx glutathione peroxidase - GR glutathione reductase - GSH reduced glutathione - GSSG oxidized glutathione - LEP life energy potential - MDA malondialdehyde - MLSP maximum life span - MR metabolic rate - MW molecular weight - PO₂ partial pressure of oxygen - SOD superoxide dismutase - VO₂ basal oxygen consumption     

Antagonism of Aeromonas hydrophila by propolis and its effect on the performance of Nile tilapia, Oreochromis niloticus

Propolis, a resinous substance collected by Apis mellifera bees from various plant sources and mixed with secreted beeswax, is a multifunctional material used by bees in the construction, maintenance, and protection of their hives. The collected propolis sample, from High Egypt, was dark-green with olive-odor. The minimal inhibition concentration (MIC) of propolis-ethanolic-extract, against Aeromonas hydrophila, was 80 μg Propolis-ethanolic-extract and crude propolis (1%) were added to artificial basal diet with (30% crude protein) to evaluate their efficacy on the fish growth-performance, immunostimulation and resistance to A. hydrophila. Two hundred and twenty-five Oreochromis niloticus (8 ± 0.45 g/fish) were divided into three equal treatments (T) of triplet replicates. The fish of T₁ were fed on basal diet (control). The fish of T₂ were given the basal diet, containing propolis-ethanolic-extract. The fish of T₃ were given the basal diet containing crude propolis for 28 day. The fish were intraperitoneally challenged by A. hydrophila (0.2 × 10⁷ cells ml⁻¹) at the end of the feeding period and kept for 15 more days.The best growth rate and feed conversion ratio were obtained with T_2. The increase in the average daily gain, specific growth rate and feed efficiency ratio were highly significances in T₂ followed by T₃ when compared with the control group. The HCT-level and monocyte-counts were increased (T₂). No significant change, in the large lymphocytic-count was found among the three treatments (28–27–28%), while the neutrophil-count was significantly decreased (7%) with T₂ and increased (13.11%) with the control. A significant increase in serum lysozyme and serum bactericidal activities was found with T₂. The RLP against A. hydrophila was high with T₂ and T₃.The propolis-ethanolic-extract enhanced the growth, immunity and resistance of O. niloticus against A. hydrophila more than the crude propolis.     

Thermo-alkali-stable catalases from newly isolated Bacillus sp. for the treatment and recycling of textile bleaching effluents

Three thermoalkaliphilic bacteria, which were grown at pH 9.3–10 and 60–65 °C were isolated out of a textile wastewater drain. The unknown micro-organisms were identified as thermoalkaliphilic Bacillus sp. Growth conditions were studied and catalase activities and stabilities compared. Catalases from Bacillus SF showed high stabilities at 60 °C and pH 9 (t_1/2=38 h) and thus this strain was chosen for further investigations, such as electron microscopy, immobilization of catalase and hydrogen peroxide degradation studies. Degradation of hydrogen peroxide with an immobilized catalase from Bacillus SF enabled the reuse of the water for the dyeing process. In contrast, application of the free enzyme for treatment of bleaching effluents, caused interaction between the denaturated protein and the dye, resulting in reduced dye uptake, and a higher color difference of 1.3 ΔE* of dyed fabrics compared to 0.9 ΔE* when using the immobilized enzyme.     

Aquatic surface respiration,an adaptive response to hypoxia in the guppy,Poecilia reticulata (Pisces,Poeciliidae)

Synopsis Aquatic respiration at the air-water interface, herein termed aquatic surface respiration (ASR), is used by the guppy,Poecilia reticulata (Poeciliidae) to meet oxygen demand in hypoxid water. A specific position in which the head contacts the surface and the jaws open just beneath the surface is adopted. ASR is initiated at a P0₂ of about 50 torr and the percent time spent rises in a steep, linear fashion as PO₂ decreases. Below 4 torr more than 90% of the animal's time is spent in ASR. Males spend less time in ASR than do females. The percent time in ASR increases with increasing size of female guppies, but decreases with increasing size of male guppies. At low oxygen (18 torr) laboratory-born guppies derived from stocks likely to experience deoxygenation spend less time in ASR than do guppies derived from stocks less likely to experience deoxygenation. The percent time in ASR increases with temperature when PO₂ is held constant. Acclimation to low oxygen decreases the percent time in ASR.Guppies not permitted ASR die in 6–41 h at 14–17 torr and 10–15 min at 1–4 torr. Guppies performing ASR survive the duration of experiments at 13–35 torr (13 days), 14–17 torr (96 h), and 1–4 torr (9.5 h). Activity, courtship, and dive duration in response to a shadow stimulus are all reduced by low oxygen. ASR is an effective alternative to aerial respiration as an adaptation to hypoxic waters, but is probably energetically and temporally more costly.     

Characterization and properties of catalase immobilized onto controlled pore glass and its application in batch and plug-flow type reactors

Bovine liver catalase was covalently immobilized onto controlled pore glass (CPG) beads modified with 3-aminopropyltriethoxysilane (3-APTES) followed by treatment with glutaraldehyde. Coupling of catalase onto CPG was optimized to improve the efficiency of the overall immobilization procedure. The optimum coupling conditions: pore diameter of CPG, pH, buffer concentration, temperature, coupling time and initial catalase amount per grams of carrier were determined as 70 nm, 6.0, 75 mM, 5 °C, 7 h and 6 mg catalase, respectively. Catalytic efficiencies (k_cat/K_m) and thermal inactivation rate constants (k_i) of ICPG1 were determined and compared with that of free catalase. Suitability of ICPG1 was also investigated by using it in batch and plug-flow type reactors. When the remaining activity of ICPG1 retained was about 50% of its initial activity the highest total productivity of ICPG1 was determined as 7.6 × 10⁶ U g immobilized catalase⁻¹ in plug-flow type reactor. However, the highest total productivity of ICPG1 was 6.2 × 10⁵ U g immobilized catalase⁻¹ in batch type reactor. ICPG1 may have great potentials as biocatalyst for the application in decomposition of hydrogen peroxide in plug-flow type reactor.     

Effects of aeration and of corn steep concentration on L-asparaginase production in Escherichia coli

Summary The production of L-asparaginase was investigated in Escherichia coli, growing under different conditions of aeration in a medium containing 2% or 6% corn steep. At both concentrations, excessive aeration decreased enzyme production. In the medium with 2% corn steep, L-asparaginase activity began to decline as soon as the oxygen absorption exceeded 0.22 mmol O₂ l^–1 min^–1, and when the oxygen absorption rate was 1.26 mmol O₂ l^–1 min^–1, enzyme activity reached only about 5% of maximum. In the medium with 6% corn steep, a decline of L-asperaginase activity did not appear until the oxygen absorption rate value exceeded 0.54 mmol O₂ l^–1 min^–1, at the oxygen absorption rate of 1.26 mmol O₂ l^–1 min^–1, the enzyme activity still reached about 50% of maximum.     

Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol

The chemiluminescence of luminol (3-aminophthalhydrazide) with H₂O₂ has been used to quantify endogenous amounts of H₂O₂ in plant tissues. The reaction is linear over at least three orders of magnitude between 10^?5 and 10^?2M H₂O₂. Interference by coloured compounds in the crude extract is calibrated by a purification step with Dowex AG 1-X8. The extract is calibrated with an internal H₂O₂ standard, and the specificity verified by H₂O₂ purging with catalase. The minimum delectability for H₂O₂ of this assay is at least 1 ng, corresponding to 0.1–1 g fresh material. Data are presented for the levels of H₂O₂ in potatoes after treatment with oxygen and ethylene, in tomatoes before and after ripening and in untreated germinating castor beans as well as in beans treated with aminotriazol to inhibit catalase activity. Though data using the titanium test are generally confirmed, the method presented here has the advantage of higher sensitivity and specificity.