Enzymatic determination of serum-free fatty acids: a colorimetric method

A simple and sensitive enzymatic method is described for the determination of serumfree fatty acids. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The reaction is followed as the production of hydrogen peroxide by using acyl-CoA oxidase/peroxidase/4-aminoantipyrine/phenol system. Results on human sera correlate well with those obtained by our previously described enzymatic method (Shimizu et al. (1979) Anal. Biochem.98, 341–345) and the chemical colorimetric method.     

A model of peroxidase activity with inhibition by hydrogen peroxide

The rate of color formation in an activity assay consisting of phenol and hydrogen peroxide as substrates and 4-aminoantipyrine as chromogen is significantly influenced by hydrogen peroxide concentration due to its inhibitory effect on catalytic activity. A steady-state kinetic model describing the dependence of peroxidase activity on hydrogen peroxide concentration is presented. The model was tested for its application to soybean peroxidase (SBP) and horseradish peroxidase (HRP) reactions based on experimental data which were measured using simple spectrophotometric techniques. The model successfully describes the dependence of enzyme activity for SBP and HRP over a wide range of hydrogen peroxide concentrations. Model parameters may be used to compare the rate of substrate utilization for different peroxidases as well as their susceptibility to compound III formation. The model indicates that SBP tends to form more compound III and is catalytically slower than HRP during the oxidation of phenol.     

Sensitive and Simple Methods for Determination of l-Lysine with l-Lysine α-Oxidase

l-Lysine could be determined satisfactorily with a new fungal enzyme, l-Iysine α-oxidase (EC 1.4.3). The method consists of the oxidative deamination of l-Iysine with l-lysine α-oxidase and the spectrophotometric determination of one of the reaction products: α-keto-ε-aminocaproate, its intramolecular dehydrated form, Δ¹-piperideine-2-carboxylate or hydrogen peroxide. The method on the basis of the color reaction of hydrogen peroxide formed from l-lysine with 4-aminoantipyrine and phenol in the presence of peroxidase was most sensitive and simple. The method could be used for the direct assay of l-lysine levels in serums from several animals without pretreatments.     

Effect of temperature and pH on phenol removal using purified Coprinus cinereus peroxidase

The removal of phenol by peroxidase-catalysed polymerization was examined using purified Coprinus cinereus peroxidase. The phenol removal efficiency increased with a decrease in the reaction temperature over the range of 0–70 °C, though only a trace of enzyme activity with 4-aminoantipyrine (4-AAP), phenol and hydrogen peroxide was found at 0 °C. The optimum pH value for phenol removal was 9.0, while the enzyme expressed maximum activity at pH 7.5 in the presence of 4-AAP, phenol and hydrogen peroxide. By measuring residual enzyme activity in the polymerizing reaction mixture, it was shown that enzyme inactivation by free radicals was more suppressed at 0 °C than at 40 °C and that the adsorption of the enzyme on the polymerized precipitate was more suppressed at pH 9.0 than that at pH 7.5.     

Determination of each constituent in mixtures of catechol and protocatechuic acid,quinol and gentisic acid,phenol and p-hydroxybenzoic acid,and pyrogallol and gallic acid

Simple, rapid spectrophotofluorometric methods were developed for determining each constitutent in the mixtures of catechol and protocatechuic acid and in mixtures of quinol and gentisic acid. A colorimetric method involving the use of 4-aminoantipyrine and extraction with chloroform was proposed for determining each constituent in mixtures of phenol and p-hydroxybenzoic acid. Two simple and rapid colorimetric methods were used in conjunction to determine each constituent in mixtures of pyrogallol and gallic acid. The accuracy of all methods was within ±5%.     

Susceptibility ofParacoccidioides brasiliensis conidia to products of oxidative metabolism

The toxic effect of components of the peroxide-peroxidase-halide system onParacoccidioides brasiliensis conidia was investigated. By itself, hydrogen peroxide was lethal at a concentration of 0.5M. The addition of peroxidase (14 U/ml) and KI (5 × 10⁻⁴M) markedly reduced the amount of hydrogen peroxide (from 5 × 10⁻¹ to 5 × 10⁻⁶M) required to kill 99% of the conidia. The lethal effect of the system suggested that it may play a role in host defense againstP. brasiliensis.     

Silicon nutrition potentiates the antioxidant metabolism of rice plants under iron toxicity

Iron toxicity reduces growth of rice plants in acidic lowlands. Silicon nutrition may alleviate many stresses including heavy metal toxicity in plants. In the present study, the ameliorating effects of silicon nutrition on rice (Oryza sativa L.) plants under toxic Fe levels were investigated. Plants were cultivated in greenhouse in hydroponics under different Fe treatments including 10, 50, 100, and 250 mg L^?1 as Fe-EDTA and silicon nutrition including 0 and 1.5 mM sodium silicate. Iron toxicity imposed significant reduction in plant fresh weight, tiller, and leaf number. The activity of catalase, cell wall, and soluble peroxidases, and polyphenol oxidase in shoots decreased due to moderate Fe toxicity (50 and 100 mg L^?1), but increased at greater Fe concentration. Ascorbate peroxidase activity increased in both roots and shoots of Fe-stressed plants. Iron toxicity led to increased tissue hydrogen peroxide and lipid peroxidation. Silicon nutrition improved plant growth under all Fe treatments and alleviated Fe toxicity symptoms, probably due to lower Fe concentration of Si-treated plants. Silicon application could improve the activity of antioxidant enzymes such as catalase, ascorbate peroxidase, and soluble peroxidase under moderate Fe toxicity, which resulted in greater hydrogen peroxide detoxification and declined lipid peroxidation. Thus, silicon nutrition could ameliorate harmful effects of Fe toxicity possibly through reduction of plant Fe concentration and improvement of antioxidant enzyme activity.     

Cell-free one-pot conversion of (+)-valencene to (+)-nootkatone by a unique dye-decolorizing peroxidase combined with a laccase from <Emphasis Type="Italic">Funalia trogii</Emphasis>

A combined system of a unique dye-decolorizing peroxidase (Ftr-DyP) and a laccase obtained from the basidiomycete Funalia trogii converted the precursor (+)-valencene completely to the high-value grapefruit flavour constituent (+)-nootkatone, reaching a concentration maximum of 1100 mg/L. In the presence of 1 mM Mn²⁺ and 2.5 mM p-coumaric acid, (+)-nootkatone was the predominating volatile product, and only traces of substrate and the nootkatols were detectable after 24 h. Hence, the two-enzyme-system reproduced the oxidizing activity observed before for the crude culture supernatant. The newly discovered Ftr-DyP was purified, sequenced and further characterized as a thermostable, non-glycosylated protein with a pH-optimum in the acidic range and a calculated mass of 52.3 kDa. Besides the typical activity of DyPs towards anthraquinone dyes, Ftr-DyP also oxidized Mn²⁺ and showed activity in the absence of hydrogen peroxide. Neither the DyP from Mycetinis scorodonius nor the manganese peroxidase from Nematoloma frowardii were able to replace Ftr-DyP in this reaction. A hypothetical reaction mechanism is presented.     

Assessment of plant secondary metabolites in oil palm seedlings after being treated with calcium,copper ions and salicylic acid

The effect of calcium, copper ions and salicylic acid (SA) amendment on the incidence of basal stem rot and activity of secondary metabolites in oil palm seedlings were investigated in glasshouse study. Disease incidence (DI) in positive control (T8) was 75% at nine months after inoculation (9 MAI). However, weekly pre-immunisation with Ca^2+?+^?Cu^2+?+^?SA prior to inoculation significantly suppressed DI and delayed disease onset as noted in T7. In the present study, the lowest %DI was observed in T7 (15%) followed by T1, T5, T6, T3, T4 and T2. The Ca²⁺, Cu²⁺ and SA amendments were resulted in earlier and higher accumulation of plant secondary metabolites as noted in leaves, stems and root tissues in response to invasion by Ganoderma boninense. High total phenolic content concentration was detected in T7 (leaf: 233.38 ± 0.12 mg/g; stem: 132.78 ± 0.04 mg/g and root: 86.98 ± 0.28 mg/g). Similar trend was obtained in peroxidase activity, total lignin content and hydrogen peroxide scavenging activity. These results suggested that it could be due to the accumulation of phenolics, peroxidase activities, lignin content and hydrogen peroxide scavenging activities in oil palm seedling tissues which might have collectively contributed to induce resistance against G. boninense.     

Evaluation of antioxidant and anticancer activities of chemical constituents of the Saururus chinensis root extracts

Evaluation of antioxidant and anticancer activities were screened by various Saururus chinensis root extracts. Four solvents (ethyl acetate, methanol, ethanol, and water) extracts were investigated for their total flavonoids, phenol contents and their antioxidant activity of DPPH (2,2-diphenyl-1-picrylhydrazyl), NO (nitric oxide), H₂O₂ (hydrogen peroxide), ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium assays, FRAP (ferric reducing ability of plasma) assays and anticancer activity. The total phenolic and flavonoid content of extracts were determined by using FC (Folin–Ciocalteu) and AlCl₃ colorimetric assay method. Total flavonoid content in these plants ranged from 24.7 to 72.1 mg g^?1 and amount of free phenolic compounds was between 11.2 and 67.1 mg g^?1 extract. The all extracts have significant levels of phenolics and flavonoids content. Anticancer activity was screened for MCF-7 breast cancer cell line. Ethanol extract shows significant of antioxidant activity and water extract shows significant of anticancer activity compared with standard (BHT) butylated hydroxy toluene. These ethanol and water extracts could be considered as a natural source for using antioxidant, and anticancer agents compared to commercial available synthetic drugs.     

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20–40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 ± 5 s⁻¹; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s⁻¹ can be estimated at 5°C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10⁻⁴ to 10⁻⁵-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:! covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.     

Nitrate nutrition enhances zinc hyperaccumulation in Noccaea caerulescens (Prayon)

Nitrate fertilization has been shown to increase Zn hyperaccumulation by Noccaea caerulescens (Prayon) (formerly Thlaspi caerulescens). However, it is unknown whether this increased hyperaccumulation is a direct result of NO₃ ^? nutrition or due to changes in rhizosphere pH as a result of NO₃ ^? uptake. This paper investigated the mechanism of NO₃ ^?-enhanced Zn hyperaccumulation in N. caerulescens by assessing the response of Zn uptake to N form and solution pH. Plants were grown in nutrient solution with 300 μM Zn and supplied with either (NH₄)₂SO₄, NH₄NO₃ or Ca(NO₃)₂. The solutions were buffered at either pH 4.5 or 6.5. The Zn concentration and content were much higher in shoots of NO₃ ^?-fed plants than in NH₄ ⁺-fed plants at pH 4.5 and 6.5. The Zn concentration in the shoots was mainly enhanced by NO₃ ^?, whereas the Zn concentration in the roots was mainly enhanced by pH 6.5. Nitrate increased Zn uptake in the roots at pH 6.5 and increased apoplastic Zn at pH 4.5. Zinc and Ca co-increased and was found co-localized in leaf cells of NO₃ ^?-fed plants. We conclude that NO₃ ^? directly enhanced Zn uptake and translocation from roots to shoots in N. caerulescens.     

Antioxidant responses and water status in Brassica seedlings subjected to boron stress

The effect of boron (B) on growth, water status and oxidative damage was investigated in the leaves and roots of 7-day-old seedlings of Brassica juncea var. Varuna. For this seedlings of Brassica were grown in solution culture with variable boron supply (0.033, 0.33, 3.3 and 33 mg B L^?1) under controlled conditions in green house. Photosynthetic pigments were found to be decreased more under excess (3.3 and 33 mg B L^–1) than deficient boron supply (0.033 mg B L^–1) when compared to control (0.33 mg B L^–1). Accumulation of hydrogen peroxide and thiobarbituric acid reactive substances content in both leaves and roots under deficient and excess boron supply suggested oxidative damage due to excessive production of reactive oxygen species. Increased activity of antioxidative enzymes: superoxide dismutase, catalase and peroxidase along with polyphenol oxidase was observed in leaves and roots under boron deficiency and excess than in control. Increased proline concentration, decreased total water content and water saturation deficit also indicated the water deficit condition in leaves and roots of boron-stressed Brassica seedlings.     

A spectrophotometric method for the determination of glucose-6-phosphatase activity

A sensitive and rapid spectrophotometric method for determination of glucose-6-phosphatase activity is described. Glucose formed by the enzyme is oxidized by glucose oxidase to gluconolactone and hydrogen peroxide. The latter, phenol, and 4-aminoantipyrine are converted by peroxidase to quinoneimine. The formation of quinoneimine is followed directly on a spectrophotometer at 510 nm. The method described is as sensitive and accurate as conventional assays based on determination of phosphate released, and also works well in phosphate buffers.     

Efficacy of Acinetobacter sp. B9 for simultaneous removal of phenol and hexavalent chromium from co-contaminated system

The present study shows the feasibility of a newly isolated strain Acinetobacter sp. B9 for concurrent removal of phenol and Cr (VI) from wastewater. The experiments were conducted in a batch reactor under aerobic conditions. Initially, when mineral salt solution was used as the culture medium, the strain was found to utilize phenol as sole carbon and energy source while no Cr (VI) removal was observed. However, the addition of glucose as co-carbon source resulted in the removal of both toxicants. This co-removal efficiency of the strain was further improved with nutrient-rich media (NB). Optimum co-removal was determined at 188 mg L^?1 of phenol and 3.5 mg L^?1 of Cr (VI) concentrations at pH 7.0. Strain B9 followed the orthometabolic pathway for phenol degradation. Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR) studies showed sorption of chromium as one of the major mechanisms for Cr (VI) removal by B9 cells. Acinetobacter sp. B9 was later on checked for bioremediation of real tannery wastewater. After 96 h of batch treatment of tannery effluent containing an initial 47 mg L^?1 phenol and 16 mg L^?1 Cr (VI), complete removal of phenol and 87 % reduction of Cr (VI) were attained, showing high efficiency of the bacterial strain for potential application in industrial pollution control.     

Immobilization of d-glucose oxidase onto a hydrogen peroxide permselective membrane and application for an enzyme electrode

A hydrogen peroxide permselective membrane with asymmetric structure was prepared and d-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized d-glucose oxidase membrane was 0.34 units cm^?2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized d-glucose oxidase membrane was 1.6 × 10^?3 mol l^?1 and that of free enzyme was 4.8 × 10^?2 mol l^?1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized d-glucose oxidase membrane. The enzyme electrode responded linearly to d-glucose over the concentration 0–1000 mg dl^?1 within 10 s. When the enzyme electrode was applied to the determination of d-glucose in human serum, within day precision (CV) was 1.29% for d-glucose concentration with a mean value of 106.8 mg dl^?1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized d-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of d-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.     

Peroxidase and hydrogen peroxide detection by a bioenergized method

Peroxidase activity and hydrogen peroxide concentrations were measured by a chemiluminescent method based upon the reduction of peroxidase Compounds I and II by both eosin and EDTA. Eosin excess present in the reaction mixture was excited during the reaction to its fluorescent state. This bioenergized method allows calculation of peroxidase concentration in the range of 10^?12 to 10^?7m, and hydrogen peroxide concentration in the range of 10^?9 to 10^?5m. This approach has been applied to the estimation of peroxidase activity in human red cell membranes and hydrogen peroxide formation in the peroxidase-catalyzed oxidation of glutathione.     

5-Aminolevulinic acid enhances photosynthetic gas exchange, chlorophyll fluorescence and antioxidant system in oilseed rape under drought stress

This study evaluates the role of exogenous foliar application of 5-aminolevulinic acid (ALA) on water relations, gas exchange, chlorophyll fluorescence, and the activities and gene expression patterns of antioxidant enzymes in leaves of oilseed rape under drought stress and recovery conditions. Seedlings at four-leaf stage were imposed to well-watered condition (80 % of water-holding capacity) or drought stress (40 % of water-holding capacity) and subsequently foliar sprayed with water or ALA (30 mg l^?1). Drought suppressed the accumulation of plant biomass and decreased chlorophyll content and leaf water status (relative water content and water potential). The actual quantum yield of photosystem II and electron transport rates were hampered in parallel to net photosynthetic rate. However, drought stress induced the accumulation of malondialdehyde (MDA) and hydrogen peroxide, enhanced the activities of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and superoxide dismutase and up-regulated the expression of APX and GR. After rehydration for 4 days, the growth of drought-treated seedlings was restored to normal level for most of the physiological parameters. Foliar application of ALA maintained relatively higher leaf water status and enhanced chlorophyll content, net photosynthetic rate, actual quantum yield of photosystem II, photochemical quenching, non-photochemical quenching and electron transport rates in stressed leaves. Exogenous ALA also alleviated the accumulation of MDA and hydrogen peroxide, increased the activities of antioxidant enzymes and enhanced the expression of CAT and POD in drought-treated plants. These results indicate that ALA may effectively protect rapeseed seedlings from damage induced by drought stress.     

Reactive oxygen species generated in the presence of fine pyrite particles and its implication in thermophilic mineral bioleaching

In the tank bioleaching process, maximising solid loading and mineral availability, the latter through decreasing particle size, are key to maximising metal extraction. In this study, the effect of particle size distribution on bioleaching performance and microbial growth was studied through applying knowledge based on medical geology research to understand the adverse effects of suspended fine pyrite particles. Small-scale leaching studies, using pyrite concentrate fractions (106–75, 75–25, ?25 μm fines), were used to confirm decreasing performance with decreasing particle size (D ₅₀ <40 μm). Under equivalent experimental conditions, the generation of the reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals from pyrite was illustrated. ROS generation measured from the different pyrite fractions was found to increase with increasing pyrite surface area loading (1.79–74.01 m² L^?1) and Fe²⁺ concentration (0.1–2.8 g?L^?1) in solution. The highest concentration of ROS was measured from the finest fraction of pyrite (0.85 mM) and from the largest concentration of Fe²⁺ (0.78 mM). No ROS was detected from solutions containing only Fe³⁺ under the same conditions tested. The potential of ROS to inhibit microbial performance under bioleaching conditions was demonstrated. Pyrite-free Sulfolobus metallicus cultures challenged with hydrogen peroxide (0.5–2.5 mM) showed significant decrease in both cell growth and Fe²⁺ oxidation rates within the concentration range 1.5–2.5 mM. In combination, the results from this study suggest that conditions of large pyrite surface area loading, coupled with high concentrations of dissolved Fe²⁺, can lead to the generation of ROS, resulting in oxidative stress of the microorganisms.     

Enhancement of acid resistance of Scenedesmus dimorphus by acid adaptation

When using flue gas as carbon source for microalgae cultivation, the resulting acidic environment caused by SO _X and NO _X can inhibit microalgal growth. In this study, Scenedesmus dimorphus acquired increased acid resistance by prior exposure to sublethal acid stress; a process defined as acid adaptation. Among the five algal species tested, S. dimorphus showed the highest level of acid tolerance to extreme acid challenge (exposure to pH 3.0). Non-adapted and acid-adapted exponential algal cells were used as inocula for tubular photobioreactors aerated with 2 % CO₂. Previously adapted at pH 4.0 for 1 h, S. dimorphus developed highest growth rate under extreme acidic condition, and the maximum biomass concentration and specific growth rate at pH 3.0 (3.638?±?0.074 g?L^?1 and 1.037?±?0.008 d^?1, respectively) were respectively 14.22 and 10.79 % higher than those of non-adapted cells. Moreover, acid-adapted cells could tolerate lower pH of 2.5, at which the growth of non-adapted cells was totally inhibited. All the results indicated that acid adaptation was an effective approach for the acid resistance enhancement of microalgae.