Disodium P-nitrophenyl Phosphate Research Articles

Enhanced peroxymonosulfate activation by (NH4)2Mo3S13 for organic pollutant removal: Crucial roles of adsorption and singlet oxygen

Though sulfide materials have attracted much attentions in sulfate radicals (SO4•–) based advanced oxidation processes, the catalytic performance and inherent mechanism of ammonium thiomolybdate ((NH4)2Mo3S13) activating peroxymonosulfate (PMS) are less studied. In this work, (NH4)2Mo3S13 materials were prepared by the hydrothermal method and applied as the PMS activator to degrade various organic pollutants. The (NH4)2Mo3S13/PMS system significantly decomposed tetracycline, oxytetracycline, methylene blue, and congo red. However, the degradation of carbamazepine, disodium p-nitrophenyl phosphate, norfloxacin, and sulfamethoxazole was relatively weaker. This selective catalytic performance was attributed to the adsorption affinities of (NH4)2Mo3S13 for organic pollutants. Besides, the exposed Mo(IV) and S on the surface of (NH4)2Mo3S13 were found to be the main active sites for activation of O2 and PMS. Among various active species, singlet oxygen was the crucial active species, while sulfate and hydroxyl radicals played limited roles for the removal of methylene blue in (NH4)2Mo3S13/PMS system. The findings of this work proved (NH4)2Mo3S13 materials are promising catalyst for organic pollutants removal in advanced oxidation processes.

Isolation and selection of salt-tolerant bacterial strains capable of solubilizing phosphorus and synthesizing phosphatase enzyme from rice-shrimp soil in Mekong River Delta, Vietnam

The aim of this study was to isolate and select salt tolerant bacteria having both functions in phosphorus solubilization and phosphatase synthesis from rice-shrimp farming soil in saline areas in Mekong River Delta of Vietnam. Phosphorus solubilizing bacteria were isolated on National Botanical Research Institute's Phosphate (NBRIP) agar medium containing 1% NaCl and the activity of phosphatase enzyme was determined by disodium p-nitrophenyl phosphate reagent method at a wavelength of 420 nm. The result showed that from 15 saline soil samples, a total of 95 strains of phosphorus solubilizing bacteria were isolated and 19 of them showed their good phosphorus solubilization. The results about phosphatase activities of these 19 strains illustrated that TBT5-3 bacterial strain was the highest phosphatase producing strain with an amount of 0.377 U/mL after 10 days of incubation. This strain showed its best phosphatase producing capacity when cultured in the liquid culture medium containing pH 5, 1% NaCl, glucose and urea under the shaking speed of 120 rpm. Based on 16S rRNA gene sequence analysis this phosphatase synthesizing bacterial strain was genetically identified as species of Bacillus sp. TBT5-3 since 100% of this train sequence is affiliated with Bacillus megaterium.

Isolation and Purification of Organophosphorus Hydrolases Secreted from Acetone-acclimated Phosphorus Accumulating Organisms and Study of Their Properties for Hydrophobic Organophosphorus Sensor.

The present work studied an acclimation method for phosphorus accumulating organisms (PAOs) with a high content of acetone in culture solutions to develop microbial-based enzyme sensors for highly hydrophobic organophosphorus (OP) pesticides. Through three steps of cultivation and acclimation, only rod-shaped bacteria survived among the various PAOs. The extracellular enzymes released from the acclimated PAOs were salted out by using ammonium sulfate, then purified by a dialysis membrane and a DEAE-Sepharose FF anion exchange column. Two enzyme components were successfully separated-both of which showed hydrolase activity on disodium p-nitrophenyl phosphate (enzyme I, 1.57 μmol/(min·μg); enzyme II, 0.88 μmol/(min·μg) at 45°C). Further, SDS-PAGE gel electrophoresis results showed that the molecular weights of enzymes I and II were about 15.11 and 11.98 kDa, respectively. On this basis, the applicability of the enzyme in hydrophobic OP biosensors was demonstrated.

Development of a Chromatographic Method for the Determination of Alkaline Phosphatase Activity in Pasteurized Milk

Alkaline phosphatase (ALP) is an important native enzyme of milk which is an established indicator for testing the adequacy of milk pasteurization. In the present study, a HPLC-UV method was standardized for the determination of ALP activity in pasteurized milk in terms of p-nitrophenol concentration liberated from the enzyme substrate di-sodium p-nitrophenyl phosphate. p-Nitrophenol was extracted by clarification, centrifugation, evaporation, and solvent exchange. The analyte was separated by a reversed-phase C18 column using an isocratic mobile phase composed of distilled water and acetonitrile (1:1) in a low-pressure gradient elution mode with a total run time of 12 min. The UV detection was achieved at a wavelength of 319 nm. Values obtained for different validation parameters confirmed that the method was fit to serve the purpose because of good specificity, linearity demonstrated under various experimental conditions (r2 > 0.9), very low limit of detection (LOD, 0.024 mg/L) and limit of quantitation (LOQ, 0.072 mg/L), accuracy (>86 %), and precision (percent relative standard deviation (% RSD) < 6.152). Validation studies also clearly demonstrated the ability of the HPLC method to detect as low as 0.1 mg/L of p-nitrophenol and 0.0006 % of raw milk. Laboratory evaluation of the HPLC method in comparison with an international reference method (ISO method) showed that the HPLC method has advantages over the ISO method due to lower LOD and LOQ, ability to detect very low levels of raw milk contamination, and comparatively high accuracy and precision.

A Novel NF-κB Pathway Involving IKKβ and p65/RelA Ser-536 Phosphorylation Results in p53 Inhibition in the Absence of NF-κB Transcriptional Activity

Nuclear factor kappaB (NF-kappaB) plays an important role in regulating cellular transformation and apoptosis. The human T-cell lymphotropic virus type I protein, Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NF-kappaB. We have demonstrated previously that Tax inhibits p53 activity through the p65/RelA subunit of NF-kappaB. We now present evidence that suggests that the upstream kinase IKKbeta plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblast (MEF) IKKbeta-/-cells did not support Tax-mediated p53 inhibition, whereas MEFs lacking IKKalpha allowed Tax inhibition of p53. Second, transfection of IKKbeta wild type (WT), but not a kinase-dead mutant, into IKKbeta-/-cells rescued p53 inhibition by Tax. Third, the IKKbeta-specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA-/-cells transfected with p65/RelA WT or mutant plasmids. Moreover, Tax induced p65/RelA Ser-536 phosphorylation in WT or IKKalpha-/- cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKKbeta-/-cells, suggesting a link between IKKbeta and p65/RelA phosphorylation. Consistent with this observation, blocking IKKbeta kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus type I-transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NF-kappaB transcription activation pathway. Our work, therefore, describes a novel Tax-NF-kappaB p65/RelA pathway that functions to inhibit p53 but does not require NF-kappaB transcription activity.

Modelling the kinetics of enzyme-catalysed reactions in frozen systems: the alkaline phosphatase catalysed hydrolysis of di-sodium- p-nitrophenyl phosphate

The applicability of the William, Landel and Ferry (WLF) equation with a modification to take into account the effect of melt-dilution and an empirical log-logistic equation were evaluated to model the kinetics of diffusion-controlled reactions in frozen systems. Kinetic data for the alkaline phosphatase catalysed hydrolysis of disodium- p-nitrophenyl phosphate (DNPP) in four model systems with different glass transition temperatures at maximal freeze-concentration ( T g′) comprising sucrose ( T g′=−34.2 °C), maltodextrin ( T g′=−14.4 °C), carboxymethylcellulose (CMC), ( T g′=−12.6 °C) and CMC–lactose ( T g′=−23.1 °C) in a temperature range of −28 to 0 °C were used. The modified WLF equation was used with a concentration-dependent glass transition temperature ( T g) as well as T g′ as reference temperatures. For both cases, the equation described well the reaction kinetics in all the systems studied. The log-logistic equation also described the kinetics in all model systems except in the vicinity of the melting temperature of ice. The effect of melt-dilution on reactant concentration was found to be significant only in the dilute model systems near the melting temperature of ice.

Tomato spotted wilt tospovirus, Impatiens necrotic spot tospovirus and Watermelon silver mottle tospovirus

<i>Tomato spotted wilt tospovirus</i>, <i>Impatiens necrotic spot tospovirus</i> and <i>Watermelon silver mottle tospovirus</i>

Copper(II) complexes of novel N-alkylated derivatives of cis,cis-1,3,5-triaminocyclohexane. 2. Metal-promoted phosphate diester hydrolysis.

Aqueous copper(II) N,N',N' '-trimethyl-cis,cis-1,3,5-triaminocyclohexane (Cu(tach-Me(3))(2+)(aq)) promotes the hydrolysis of activated phosphate diesters in aqueous medium at pH 7.2. This complex is selective for cleavage of the phosphate diester sodium bis(p-nitrophenyl) phosphate (BNPP), the rate of hydrolysis of the monoester disodium p-nitrophenyl phosphate being 1000 times slower. The observed rate acceleration of BNPP hydrolysis is slightly greater than that observed for other Cu(II) complexes, such as [Cu([9]aneN(3))Cl(2)] ([9]aneN(3) identical with 1,4,7-triazacyclononane). The rate of hydrolysis is first-order in phosphate ester at low ester concentration and second-order in [Cu(tach-Me(3))](2+)(aq), suggesting the involvement of two metal complexes in the mechanism of substrate hydrolysis. The reaction exhibits saturation kinetics with respect to BNPP concentration according to a modified Michaelis-Menten mechanism: 2CuL + S <==> LCu-S-CuL --> 2CuL + products (K(M) = 12.3 +/- 1.8 mM(2), k(cat) = (4.0 +/- 0.4) x 10(-)(4) s(-1), 50 degrees C) where CuL (triple bond) [Cu(tach-Me(3))](2+), S (triple bond) BNPP, and LCu-S-CuL is a substrate-bridged dinuclear complex. EPR data indicate that the dicopper complex is formed only in the presence of BNPP; the active LCu-S-CuL intermediate species then slowly decays to products, regenerating monomeric CuL.

Reaction rate modeling in cryoconcentrated solutions: alkaline phosphatase catalyzed DNPP hydrolysis.

The hydrolysis of disodium p-nitrophenyl phosphate catalyzed by alkaline phosphatase was chosen as a model to study the kinetics of changes in frozen food products. The initial reaction rate was determined in concentrated sucrose solutions down to -24 degrees C, and the enzymatic characteristics K(M) and V(max) were calculated. The experimental data were compared to the kinetics predicted by assuming that the reaction was viscosity dependent. Indeed, an analysis of the enzymatic reaction demonstrated that both the diffusion of the substrate and the flexibility of the enzyme segments were controlled by the high viscosity of the media. When the temperature was too low for the viscosity to be measured simply, the Williams-Landel-Ferry equation was used to predict the viscosity, taking, as reference temperature, the glass transition temperature (T(g)) corresponding to the concentration of the freeze-concentrated phase at the test temperature. Predicted values of the reaction rate were very close to the experimental ones in the studied temperature range.

On the solubility of disodium p-nitrophenyl phosphate in supercritical carbon dioxide

The title compound is shown to be insoluble in supercritical carbon dioxide, and an earlier report of its enzymatic hydrolysis in supercritical carbon dioxide is shown to be erroneous.

A rapid, sensitive enzyme-linked immunoassay for human thyrotropin.

In this enzyme-linked immunoassay for human thyrotropin (TSH) in unextracted serum we use 96-well immunoenzymometric assay plates, first coated with polyclonal antibody to TSH, then incubated with the serum samples and reacted with mouse monoclonal antibody to human TSH. After incubation with alkaline phosphatase-labeled antibody against mouse IgG, disodium p-nitrophenyl phosphate is added and the color change is measured spectrophotometrically. Assay sensitivity is 0.1 milli-int. unit/L. Cross reactivity with lutropin, follitropin, or choriogonadotropin was negligible. TSH concentrations ranged from 0.4 to 4.1 milli-int. units/L in 43 normal subjects (mean 2.0, SD 1.0), and were uniformly less than 0.3 milli-int. unit/L in 23 patients with hyperthyroidism. Features which make this assay advantageous to the clinical laboratory include ease of set-up, ability to assay many samples at a time, high sensitivity, rapid turnaround time (8 h), and absence of requirements for radioactive materials.

Enzymatic catalysis in a supercritical fluid

The enzyme alkaline phosphatase, EC 3. 1. 3. 1, was found to be active in a supercritical carbo dioxide solvent system. A batch reaction of disodium p-nitrophenyl phosphate with a 0.1 vol. % water solution in supercritical CO2 at 100 atm and 35°C produced p-nitrophenol when catalyzed by alkaline phosphatase.

Some properties of acid and alkaline phosphatase in seminal fluid and isolated sperm.

Activities of acid and alkaline phosphatases were examined in spermatozoa isolated from 177 semen samples differing in sperm counts. Alkaline phosphatase was also determined in seminal fluid. The enzymes were assayed using disodium p-nitrophenyl phosphate as substrate and were studied with respect to susceptibility to various concentrations of tartrate (acid) and to heat (alkaline). Electrophoretic separation of alkaline phosphatase from seminal fluid was performed using an Helena apparatus. The results showed that acid phosphatase activity in spermatozoa decreased with increase in sperm densities and that elevation of tartrate from 0.028 to 0.17 M usually correlated an inhibition of the enzyme from 72% to 78% (mean values). Alkaline phosphatase was very low in sperm and generally below the sensitivity of the method used. Activity of alkaline phosphatase in seminal fluid showed a tendency to increase with the increase in sperm counts, but the significance of differences between groups was not statistically valid. Exposure of seminal fluid to 55 degrees C for 16 min resulted in enzyme inactivation of about 90% and in this respect the alkaline phosphatase resembles the enzyme of bone origin. The electrophoretic pattern, however, did not confirm this view and the type of alkaline phosphatase in seminal fluid is not clear.

The significance of inhibitor-resistant alkaline phosphatase in the cytochemical demonstration of transport adenosine triphosphatase.

The hydrolysis of disodium p-nitrophenyl phosphate at pH 9.0 by slices of formaldehydee-fixed rat renal cortex was investigated by colorimetric estimation of the nitrophenol liberated. It was found that three types of activity could be identified on the basis of their responses to inhibitors and cations: (a) alkaline phosphatase sensitive to inhibition by L-tetramisole; (b) potassium-dependent phosphatase, probably identifiable with the phosphatase component of sodium-potassium-dependent transport adenosine triphosphatase (?Na-K-ATPase); and (c) alkaline phosphatase insensitive to L-tetramisole. It was found that in the presence of strontium ions, as used in Na-K-ATPase cytochemistry, the activities of the second and third types of enzyme were approximately equal. The implications of these findings for the cytochemical demonstration of Na-K-ATPase are discussed.

Characteristics of acid phosphatase in Porcellio laevis Laterille (Porcellionidae, Isopoda)

1.1. Cellulose acetate electrophoretic pattern studies on the haemolymph acid phosphatase revealed the presence of two isozymes. The energy of activation for hydrolysis of disodium p-nitrophenyl phosphate amounted to 8200± 100 cal/mole.2.2. Inhibition by diisopropylfluorophosphate and sodium cyanide ranged from 50–79·5 per cent, while by trinitrobenzene sulphonic acid and sodium bisulphite inhibition was 25–43 per cent.3.3. Inhibition of the enzyme by EDTA, except in the presence of Mg2+ and Zn2+, gives it the status of metalloenzyme.4.4. l-Tyrosine effectively inhibits both the digestive gut and hepatopancreatic fractions, while l-Tryptophan inhibits the hepatopancreatic fraction only.5.5. l-Cysteine and glutathione (reduced) activate the acid phosphatase, while l-phenylalanine, d-phenylalanine, dl-phenylalanine, d-tyrosine, dl-tyrosine, d-tryptophan, dl-tryptophan and l-alanine have no effect. Parachloromercuribenzoate, “β-mercaptoethanol”, iodoacetic acid and l-cystine inhibit the enzyme to varying degrees.6.6. Hydrolysis of phenyl phosphate by the enzyme was inhibited to varying degrees in the presence of heavy metal salts. Inhibition by cadmium nitrate and nickel chrloride was of the order of 40·7 and 12·9 per cent, respectively, while in the case of lead nitrate, silver nitrate and ferric sulphate it was 55·0, 75·6 and 85·2 per cent, respectively.

Characteristics of alkaline phosphatases from the terrestrial isopod, Porcellio laevis Latreille (Porcellionidae, Isopoda)

1. 1. The nature of alkaline phosphatases from the digestive gut, hepatopancreas and the haemolymph of a tenth instar Porcellio laevis adult has been examined by electrophoretic and biochemical means. 2. 2. Cellulose acetate electrophoretic pattern studies on the haemolymph alkaline phosphatase revealed the presence of two isozymes. The heat of activation for hydrolysis of disodium p-nitrophenyl phosphate was calculated to be 4900 ± 150 mcal/mole. 3. 3. Inhibition studies with EDTA gave these enzymes the status of metallo-enzymes, and denaturation studies with urea indicated the presence of enzyme subunits. 4. 4. Serine and dehydroalanine were essential for the enzyme catalytic activity, while amino and sulphydryl attacking reagents acted as inhibitors. 5. 5. l-Tyrosine effectively inhibited both digestive gut and hepatopancreatic fractions, while l-phenylalanine and l-tryptophan inhibited the digestive gut fraction only. 6. 6. Glutathione (reduced) and l-cystein activated the alkaline phosphatases, while d-alanine, dl-alanine, l-alanine, d-phenylalanine, dl-phenylalanine, d-tryptophan, dl-tryptophan, d-tyrosine and dl-tyrosine had no effect. 7. 7. Among heavy metals, lead and nickel were found to activate the enzyme, while iron, silver and cadmium acted as potent inhibitors.

Phosphomonoesterases of the digestive gut in Porcellio laevis latreille (Porcellionidae, isopoda)

1. 1. The biochemical characteristics of two phosphomonoesterases of the digestive canal and the hepatopancreas of a tenth instar Porcellio laevis have been studied. Enzyme activities are based on the liberation of p-nitrophenol from disodium p-nitrophenyl phosphate at 410 nm. 2. 2. Alkaline phosphatase was found to have a linear relationship between concentration and activity, maximum activity at pH 7.79–8.10, a Michaelis constant of 3.0 × 10 −4M and a temperature coefficient ( Q 10) of 2.25 between 20 and 30°C. 3. 3. Barium chloride, calcium chloride, magnesium acetate and magnesium chloride acted as activators for alkaline phosphatase, while bisodium hydrogen arsenate, bisodium hydrogen phosphate, sodium arsenate and cobalt chloride inhibited the action of the enzyme. 4. 4. Acid phosphatase has an optimum pH of 4.7, a Michaelis constant of 7.5 × 10 −4M, zero-order kinetics, an optimum temperature of 50°C for a 54 min incubation period and a temperature coefficient ( Q 10) of 2.55 between 20 and 30°C. 5. 5. Barium chloride, calcium chloride, magnesium acetate and magnesium chloride acted as activators for acid phosphatase, and sodium cyanide, mercuric chloride, cobalt chloride, bisodium hydrogen arsenate, bisodium hydrogen phosphate, sodium fluoride and cupric sulphate inhibited the action of the enzyme. 6. 6. Alkaline phosphatase required 0.08M concentration of magnesium ions for its maximum activation, while acid phosphatase required 0.01M magnesium ions to attain maximum activity peak.

Quantitative estimation of heat-stable alkaline phosphatase activity in dried blood stains and its application to the forensic diagnosis of pregnancy.

The placental isoenzyme of alkaline phosphatase is characterized by heat stability (56° C for 30 min). This paper describes a new technique for the forensic diagnosis of pregnancy from blood stains, based on the fact that in serum of pregnant women in the latter half of pregnancy the heat-stable alkaline phosphatase activity rises markedly. In this study the total and heat-stable alkaline phosphatase activities were quantitatively assayed in blood stains of various origins by the method of Bessey-Lowry-Brock with the use of disodium p-nitrophenyl phosphate as substrate. Our results indicate that the principle herein reported may be helpful in the diagnosis of pregnancy from blood stains.

PHOSPHOMONOESTERASE ACTIVITY IN THE LIFE-CYCLE STAGES OFSCHISTOCERCA GREGARIA

AbstractThe acid and alkaline phosphatase activity was measured in the developing egg and in the alimentary canal of aging nymphs as well as adult males and females of different ages. Para-nitrophenol was used as colorimetric standard and disodiump-nitrophenyl phosphate as substrate. Activity was measured in terms of micromoles ofp-nitrophenol liberated from the substrate as a result of enzyme action.Acid phosphatase activity was noticed to increase with the embryonic development and was higher than in the case of alkaline phosphatase. The alkaline phosphatase activity was lowest in the freshly laid egg, but increased more sharply than acid phosphatase during embryonic development.The activity of both the acid and alkaline phosphatases was highest in the first instar and declined gradually to the fifth instar. The activity of acid phosphatase was higher than alkaline phosphatase in all stages except the first instar where it was almost equal. The activity of both the enzymes was higher during the intermoulting period and declined at each moult indicating a hormone–enzyme relationship.In adults, activity of both the enzymes increased up to the maturation period after which the activity gradually decreased. Acid phosphatase activity was generally higher in males whereas alkaline phosphatase activity was generally higher in females. In almost all cases, the acid phosphatase activity was found to be higher than the alkaline phosphatase.

Alkaline phosphatase of the developing down feather: Substrates, activators, and inhibitors

1. 1. The pattern of activity of alkaline phosphatase in the developing down feather and the specificity of the reaction were studied with respect to the following substrates: sodium glycerophosphate, disodium p-nitrophenyl phosphate, glucose-1-phosphate, glucose-6-phosphate, hexose diphosphate, fructose-6-phosphate, muscle and yeast adenylic acids. 2. 2. With the method of Gomori, the pattern of activity was the same, but of varying strength, with all substrates except yeast adenylic acid. Most substrates produced a strong reaction in the mesodermal pulp, with all parts of the cells being blackened by precipitate; the reaction in the epidermis consisted of a moderate blackening of the nucleus and cytoplasm of the cylinder cells lying next to the pulp, and of the nucleoli in more peripheral cells. With yeast adenylic acid there was a prominent reaction in the nucleoli of all cells and a reduction of the activity elsewhere. 3. 3. With the azo dye method, the cytoplasm of the cells of the mesodermal pulp was highly reactive. A weaker reaction was found in the epidermal cytoplasm. 4. 4. The possibility that several phosphatases were contributing to the Gomori reaction was tested by using Mg ++, Mn ++, Be ++, Zn ++, Fe +++, HAsO 4 −−, HAsO 3 −−, CN −, semicarbazide, iodoacetate, Versene, glycine, alanine, cysteine, histidine, methionine, proline, valine, serine, lysine, and arginine in conjunction with several of the substrates. 5. 5. Versene, iodoacetate, semicarbazide, Be ++, CN −, cysteine, histidine, glycine, alanine, arginine, lysine, and methionine in high concentrations inhibited phosphatase activity. Mn ++, Fe +++, and HAsO 3 −− had no apparent effect. In low concentrations, methionine, proline, and valine gave some indication of stimulating activity with sodium glycerophosphate. 6. 6. The reactions of the adenylic acids and fructose sugars to the inhibitors differed from those of other substrates. Mg ++ was necessary for activity with muscle adenylic acid, fructose-6-phosphate, and hexose diphosphate. At high concentrations Zn ++ inhibited activity with all substrates except fructose-6-phosphate, and in low concentration stimulated activity with yeast adenylic acid. There was strong nucleolar activity with yeast adenylic acid in concentrations of arsenate which inhibited activity with all other substrates. Glycine promoted activity with muscle adenylic acid. The reactions with fructose sugars were blocked by lower concentrations of inhibitors than with sodium glycerophosphate. 7. 7. Epidermal phosphatase was removed more readily by the inhibiting agents than the phosphatase in the pulp. 8. 8. These data suggest that the developing down feather contains more than one phosphatase capable of utilizing the various substrates at a pH of 9.0–9.4. They also suggest that there is a true nucleolar reaction for alkaline phosphatase.