Self-assembling nitrilotriacetic acid nanofibers for tracking and enriching His-tagged proteins in living cells.

Specific and expeditious identification and enrichment of target proteins in living cells is often a challenging task. The hexahistidine (6His) tag is frequently used to label artificially engineered proteins produced in prokaryotic or eukaryotic cells. Utilizing the interaction between 6His-tag and nitrilotriacetic acid (NTA) mediated by divalent metal ions (Ni2+, Cu2+, Zn2+ or Co2+), we designed and synthesized a series of Nap-G/Biotin/ANA-FFpYGK-NTA probes that, assisted by alkaline phosphatase (ALP), self-assemble into nanofibers. The probe consists of an NTA group that specifically binds to 6His-tag, an FFpY group that promotes self-assembly facilitated by ALP, and a hydrophobic (Nap-G/ANA/Biotin) capping group for various applications. We demonstrate that the ANA-FFpYGK-NTA(Ni2+) nanofibers are fit for real-time tracking of His-tagged protein in living cells, and the Biotin-FFpYGK-NTA(Ni2+) nanofibers are for isolating His-tagged proteins and other proteins that they interact with.

Effect of EDTA and NTA on cadmium distribution and translocation in Pennisetum purpureum Schum cv. Mott.

The primary objective of this research was to investigate the cadmium (Cd) distribution in Pennisetum purpurem (Napier grass) in the presence of 30 mg/L of Cd and different types and concentrations of chelating agents (ethylenediaminetetraacetic acid disodium dihydrate (EDTA), nitrilotriacetic acid (NTA), and EDTA-NTA mixtures). Plant samples were collected every 15 d during a 105-d experimental period. Accumulation of Cd in each part of the plant was determined using atomic absorption spectrometer (AAS), and the distribution of Cd was determined by laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS) and synchrotron radiation micro X-ray fluorescence (SR-micro-XRF). The highest concentrations of Cd accumulation of 889 ± 53 mg kg-1 in the underground part (roots) and 265 ± 26 mg kg-1 in the aboveground part (stems and leaves) in the presence of 1:1 M ratio of Cd:EDTA after 30 d of exposure were observed. Plants grown in the presence of either NTA or EDTA-NTA mixtures showed significant lower Cd accumulation levels. The LA-ICP-MS analysis showed that Cd was primarily accumulated in the aboveground part (stems and leaves), especially in the xylem and intercalary meristem. In addition, translocation factor was very low. Thus, P. purpurem could be considered as a candidate plant for cadmium phytostabilization.

Determination of aminopolycarboxylic acids at ultra-trace levels by means of online coupling ion exchange chromatography and inductively coupled plasma-mass spectrometry with indirect detection via their Pd²âº-complexes.

A new indirect IC-ICP-MS method for the determination of aminopolycarboxylic acids in water samples is described. It is based on the addition of an excess of Pd(II) to water samples. The analytes are forced into very strong and negatively charged palladium complexes, separated by ion exchange chromatography and detected by their palladium content, utilizing an on-line coupled ICP-MS. This method is suitable to determine the concentration of 8 aminopolycarboxylic acids (nitrilotriacetic acid (NTA), (2-carboxyethyl) iminodiacetic acid (ß-ADA), methylglycinediacetic acid (MGDA), 2-hydroxyethyl) ethylenediamine triacetic acid (HEDTA), diethylene triamine pentaacetic acid (DTPA), ethylendiamine tetraacetic acid (EDTA), 1,3-diaminopropane tetraacetic acid (1,3-PDTA) and 1,2-diaminopropane tetraacetic acid (1,2-PDTA) at the ng kg(-1) level. The method is faster and easier than the established gas chromatography (GC)-method ISO 16588:2002 and up to two orders of magnitude more sensitive than the ion pair chromatography based method of DIN 38413-8. Analytic performance is superior to ISO 16588:2002 and the comparability is good.

Chemometric study on the electrochemical incineration of nitrilotriacetic acid using platinum and boron-doped diamond anode.

This study investigated the electrochemical incineration of nitrilotriacetic acid (NTA) at boron-doped diamond (BDD) and platinum (Pt) anodes. Trials were performed in the presence of sulfate electrolyte media under recirculation mode. The parameters that influence the degradation efficiency were investigated, including applied current density, flow rate, supporting electrolyte concentration and reaction time. To reduce the number of experiments, the system had been managed under chemometric technique named Doehlert matrix. As a consequence, the mineralization of NTA demonstrated similar behavior upon operating parameters on these two anodes. Further kinetic study indicated that the degradations followed pseudo-first-order reactions for both BDD and Pt anodes, and the reaction rate constant of the former was found to be higher than that of the latter. Such difference could be interpreted by results from fractal analysis. In addition, a reaction sequence for NTA mineralization considering all the detected intermediates was also proposed.

Indirect ultra trace determination of aminopolycarboxylic acids in surface water using ion exchange chromatography coupled on-line to inductively coupled plasma mass spectrometry.

An indirect method for the determination of ultra trace amounts of aminopolycarboxylic acids in surface water with a minimum of sample preparation was developed. It is based on the addition of a metal ion to the water sample in order to transform the chelating reagents into negatively charged complexes. These are separated by means of ion exchange chromatography and the metal inside the complex is detected by on-line coupled ICP-MS. Iron and indium as two metal ions of the nominal charge of +3 were investigated, whereas In(3+) provided the best results in terms of sensitivity with detection limits in the lower ng/l range. The trivalent complexing agent NTA cannot be determined accurately because of the low retention of the complex. The indium and iron complexes of CDTA are strongly absorbed on anion exchangers and showed retention time of over 1 h. The use of column-switching reduced the retention time to less than 15 min. The method has been validated against a sample set of 62 samples which have previously been analysed by an external water lab according to DIN EN ISO 16588 using the GC­MS method.

Electrochemiluminescence from hydrophilic thin film Ru(bpy)3(2+)-modified electrode prepared using natural halloysite nanotubes and polyacrylamide gel.

Because Ru(bpy)(3)(2+)-modified electrodes are often used in aqueous condition, the development of a hydrophilic modified electrode is of critical importance. Herein a hydrophilic, thin film Ru(bpy)(3)(2+)-modified electrode is successfully developed using polyacrylamide gel to embed halloysite nanotubes on the electrode surface which is used to adsorb Ru(bpy)(3)(2+) by cation-exchange. X-ray photoelectron spectroscopy demonstrates the formation of the film on an electrode and the high adsorbing capacity of the halloysite nanotubes toward Ru(bpy)(3)(2+). The different electrochemiluminescence (ECL) behaviors of the electrode using nitrilotriacetic acid and tripropylamine, as co-reactants, illustrate the hydrophilic character of the modified electrode. Contrary to the previous works, the addition of carbon nanotubes into the modified electrode film leads to a decreased ECL emission, due to the reduction in the porosity of the film, which hinders the diffusion of the analyte.

Speciation of Zn-aminopolycarboxylic complexes by electrospray ionization mass spectrometry and ion chromatography with inductively coupled plasma mass spectrometry.

The speciation of Zn-aminopolycarboxylic complexes was investigated using both electrospray ionization mass spectrometry (ESI-MS) and ion chromatography (IC) with inductively coupled plasma mass spectrometry (ICP-MS). The resulting ESI mass spectra indicated that [Zn(HEDTA)](1-), [Zn(NTA)](1-), [Zn(EDTA)](2-) and [Zn(DTPA)](3-) were all simultaneously detected in solution; [Zn(NTA)](1-) exhibited the weakest intensity of all these Zn-aminopolycarboxylic complexes. IC/ICP-MS was also successfully used to separate Zn complexes by anion-exchange chromatography using a mobile phase containing 30 mM (NH(4))(2)HPO(4) at pH 7.5 giving reasonable resolution within 15 min. A weak peak attributable to the poor stability [Zn(NTA)](1-) ion was also observed using IC/ICP-MS. With the exception of [Zn(NTA)](1-), detection limits ranging from 0.5 to 1.0 microg/L were obtained and the proposed method was used for the determination of Zn aminopolycarboxylic complexes in soil solution.

Occurrence and fate of organic pollutants in combined sewer systems and possible impacts on receiving waters.

Selected organic pollutants are classified based on an intensive literature survey. Two wastewater parameters (COD and ammonium) and six selected organic pollutants (polycyclic aromatic hydrocarbons (PAH), diethylhexylphthalate (DEHP), estradiol (E2), ethinylestradiol (EE2), ethylenediamine tetraacetic acid (EDTA) and nitrilo triaceticacid (NTA)) are specified. As a result, for the first time representative concentrations in dry weather flow, surface runoff and effluent of wastewater treatment plants (WWTPs) in combined sewer systems (CSS) are stated. The second part of the paper presents a first estimation of main emission out of a combined sewer system and possible receiving water impacts in terms of (1) annual discharged loads calculated by pollution load simulations in a hypothetical catchment and (2) concentrations calculated in combined sewer overflows (CSO) discharges and resulting receiving water concentrations.

High-performance liquid chromatography-post-column chemiluminescence determination of aminopolycarboxylic acids at low concentration levels using tris(2,2'-bipyridyl)ruthenium(III).

A simple, selective and sensitive method for the determination of aminopolycarboxylic acids [diethylenetriaminepentaacetic acid (DTPA), S,S-ethylenediamine N,N'-disuccinic acid (EDDS), ethylenediaminetetraacetate (EDTA) and nitrilotriacetate (NTA)] has been developed using high-performance liquid chromatography with chemiluminescence detection. The aminopolycarboxylic acids were separated on a C18 reversed-phase column with an aqueous sulfuric acid mobile phase at a pH lower than 1.5. The eluate was mixed with tris(2,2'-bipyridyl)ruthenium(III), which was on-line generated by photooxidation of the ruthenium(II) complex in the presence of peroxydisulfate, and the generated chemiluminescence was detected. Calibration graphs, based on standard solutions, were linear over the range 8x10(-9) to 4x10(-5) M. The detection limits at a signal-to-noise ratio of 3 ranged from 9x10(-10) to 8x10(-8) M. The relative standard deviations of intra- and inter-day precision were below 1.3% and 2.1%, respectively. This HPLC system was successfully applied to the determination of aminopolycarboxylic acids in three different types of water samples. The low pH of the mobile phase limits interference from metal ions in natural waters. When such interference occurs, a cation-exchange column can be used to suppress it. The lowest amounts measurable were: 75 pg for NTA, 0.7 ng for EDDS, 0.8 ng for DTPA and 12ng for EDTA. The method was also applied for the EDTA assay in canned foods.

Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications.

High-affinity nitrilotriacetic acids (NTA) have great potential in the molecular manipulation of His-tagged proteins. We have developed a facile method to synthesize multivalent NTA and its conjugates. Starting with appropriately protected lysine, we synthesized the mono-NTA synthons functionalized with either an amino group or a carboxylic group. We then obtained tri-NTA through the condensation of the amino NTA and the carboxylic NTA. Using amino tri-NTA as the key intermediate, we synthesized a series of tri-NTA conjugates with a variety of functional units including biotin, dialkyl, fluorescein, and a hydroxybenzimidate moiety. The biotin-tri-NTA was employed to convert a Biacore streptavidin chip into a high-affinity tri-NTA chip. The equilibrium dissociation constants of tri-NTA/His-tagged protein complexes measured by surface plasmon resonance are in the 20 nM range. Histidine(6)-tagged yeast cytosine deaminase (His6-yCD) was incorporated onto the liposome surface by the lipid-tri-NTA conjugate without any activity loss. Fluorescein-tri-NTA formed a stable 1:1 complex with His6-yCD without significant fluorescence quenching. Specific tri-NTA derivatives for the radiolabeling and coupling of two His-tagged proteins to each other are described. Thus, we have added to the toolbox a number of high-affinity tri-NTA adaptors for the manipulation of His-tagged molecules.

Investigation of mechanisms of oxidation of EDTA and NTA by permanganate at high pH.

Permanganate has been used for oxidation of nuclear wastes containing chelating agents such as ethylenediaminetetraacetic and nitrilotriacetic acids (EDTA and NTA) to improve separation of radionuclides and heavy metals from the wastes, butthe mechanisms of degradation of these and related organic ligands at high pHs have not been studied. EDTA, NTA, and the model compound ethylenediamine (EN) were found to be readily oxidized by permanganate at pH 12-14. The reduction of permangante was accompanied by formation of unstable manganate and dispersed MnO2 particles, which constituted the final product of permanganate reduction. The yields and speciation of EDTA, NTA, and EN breakdown products were affected by the pH and permanganate dose. Iminodiacetic acid (IDA), oxalate, formate, and ammonia were the predominant EDTA and NTA oxidation products. Mineralization of EDTA, NTA, and EN to CO2 was more significant at pH 12. At pH 14 formation of oxalate and deamination to NH3 were the most important reactions. IDA was released upon the oxidation of both EDTA and NTA, but EDTA oxidation yielded no ethylenediaminediacetic acid (EDDA). The speciation of the reaction products indicated that the ethylene group in EDTA was the preferred attack site in oxidations by alkaline permanganate.

Simultaneous determination of DTPA, EDTA, and NTA by UV-visible spectrometry and HPLC.

In this study, UV-visible spectrophotometry (UV-Vis) and high-performance liquid chromatography (HPLC) were used for simultaneous analysis of chelating agents diethylenetriamine pentaacetic acid (DTPA), ethylenediamine tetraacetic acid (EDTA), and nitrilotriacetic acid (NTA), as their metal chelates in dishwashing detergents, natural waters, and pulp mill water. The total amounts of the chelating agents in dishwashing detergents were verified by potentiometric titration with Fe(III) solution. Nickel(II) chelates were determined by UV-Vis and iron(III)chelates by HPLC and titration. Recoveries of DTPA, EDTA, and NTA from a standard mixture of analytes by UV-Vis were 107+/-7, 101+/-12 and 94+/-13%, respectively, and the recovery of the total amount of complexing agents was 99+/-4%. The limits of detection for DTPA, EDTA, and NTA were 667, 324, and 739 micromol L(-1), respectively. In HPLC measurements the optimized mobile phase contained 0.03 mol L(-1) sodium acetate, 0.002 mol L(-1) tetrabutylammonium bromide, and 5% methanol at pH 3.15 and the detection was by UV-Vis detection at 254 nm. All three complexing agents could be separated from each other in a simultaneous analysis in less than 5 min. The limits of detection were 0.34, 0.27, and 0.62 micromol L(-1) for DTPA, EDTA, and NTA, respectively. The total amounts of the analytes measured in the dishwashing detergents by the three techniques were found to be highly comparable (ANOVA: F=0.04, P=0.96). R(2) values were 0.99 for EDTA, 0.99 for NTA, and 0.99 for all the results when UV-Vis and HPLC determinations were compared using regression lines. The UV-Vis and HPLC methods were proved to be viable also for analyses of natural and pulp mill waters. The absence of matrix interferences was verified by the standard addition technique.

Prognosis of environmental concentrations by geo-referenced and generic models: a comparison of GREAT-ER and EUSES exposure simulations for some consumer-product ingredients in the Itter.

The aim of this study was the comparison between predicted environmental concentrations (PEC) derived using a generic aspacial model, European Union System for the Evaluation of Substances (EUSES), and a geo-referenced model, the Geo-referenced Regional Environmental Assessment Tool for European Rivers (GREAT-ER). The PECs of some consumer-product ingredients (boron, LAS) and professional uses (EDTA, NTA and Triclosan) were calculated for the river catchment of the Itter, a small tributary to the river Rhine. The PEClocal and PECregional for the water compartment generated by EUSES (default scenario) were subsequently refined with data that realistically reflects the region of North Rhine-Westphalia (NRW scenario) and the Itter catchment (Itter scenario). The results of the three scenarios were then compared with the PECinitial and PECcatchment calculated by GREAT-ER, that was designed as a higher-tiered exposure assessment tool, and with concrete concentrations in the Itter, measured as 24-h composite samples. While the PECregional of all scenarios was close to the lower end of the measured concentrations, the geo-referenced PECs described equally well the real spacial situation. The measured environmental concentrations confirmed the built-in conservatism of the PEClocal calculations by EUSES showing for all investigated chemicals an unrealistically high PEClocal (default). The refinement in the more realistic scenarios could not provide a straight forward general improvement of the PEClocal. In conclusion, when the EUSES prognosis is refined using more detailed substance and regional specific data, it may provide a fairly accurate modelling especially of substances that are not eliminated in the environment. However, in the case of eliminable substances, it does not match the accuracy of higher-tiered geo-referenced exposure models like GREAT-ER.

Environmental concentrations of boron, LAS, EDTA, NTA and Triclosan simulated with GREAT-ER in the river Itter.

A computer simulation of the environmental concentrations of some typical consumer-product ingredients was performed using the geo-referenced exposure model GREAT-ER (Geo-referenced Regional Environmental Assessment Tool for European Rivers) in the river Itter. Boron and LAS were chosen as typical detergent ingredients along with EDTA, NTA and Triclosan as examples of household and cosmetic product ingredients. The simulations were based on consumption figures of the respective chemical in consumer products in the year 2000. For EDTA, the consumption figure used for the calculation had to be extended to commercial products since the EDTA-use in domestic products could not account for the measured concentrations alone. The resulting PEC (Predicted Environmental Concentration) for all investigated compounds showed very good accordance to the measured concentrations in the Itter which were monitored in the same year. The concentrations did not deviate more than by a factor of 3. GREAT-ER's calculated 90th-percentile was never exceeded by the monitoring result thus reflecting a reasonable accuracy.

The control of nitrilotriacetic acid in edetic acid and its salts by liquid chromatography.

Two liquid chromatographic methods have been developed and validated for the determination of nitrilotriacetic acid (NTA) in edetic acid (EDTA) and its salts. In one method NTA and EDTA are separated on a polymer column without pretreatment, and are detected amperometrically using a glassy-carbon electrode. In the other method NTA and EDTA are complexed with ferric ions and the complexes separated on a porous graphite carbon stationary phase with ultraviolet detection at 215 nm. Both methods were sufficiently selected and sensitive to allow the control of NTA (0.1% m/m) in sample of EDTA and its salts.

Gas chromatographic and mass spectrometric analysis of nitrilotriacetic acid in environmental aqueous samples.

This study describes a fast and accurate method for the sample preparation, identification, and quantitation of nitrilotriacetic (NTA) acid in environmental aqueous samples at a concentration of ppb level. The method is sensitive, specific, and free from the interferences of fatty and amino acids. The tri-n-propyl- and tri-n-butyl-NTA acid esters were prepared by the reaction of n-propyl-HCl and n-butyl-HCl solutions and NTA acid, respectively. The derivatives were analyzed by a gas chromatograph equipped with a mass spectrometric detector. The method detection limit, 0.006 mg/L of each NTA ester, was determined and validated by an analysis of a fortified water sample. The overall recoveries were 103-115%, n = 8. The method was applied to a real sample and a 0.90 mg/L concentration of NTA acid was found. Mass fragmentation patterns of the derivatives are also reported.

[Nitrilotriacetic acid (NTA)--properties and environmental behavior. II. Ecotoxicity and biodegradability of NTA in the environment]. / Kwas nitrylotrioctowy (NTA)--wlasciwosci i zachowanie w srodowisku. CZ. II. Ekotoksycznosc i biodegradacja NTA w srodowisku.

The results are described of studies on the effects of NTA on aqueous organisms and crops. The results show that NTA at concentrations determined in natural water systems causes no disturbances of the equilibrium in ecosystems. The conditions of the NTA biodegradation process in communal sewage and in surface waters and the effects of the processes taking place in water treatment systems destined for providing drinking water on NTA degradation are described. In a summary of the results of monitoring studies conducted in countries where NTA has been accepted for widespread use in washing powders the problems are stressed which will require solving before the decision is made of permission of NTA use in Poland. As of now, there are no full data making possible permission of using NTA in products of household chemistry.

Determination of free nitrilotriacetic acid in environmental water samples by ion chromatography with potentiometric and amperometric detection with a copper electrode.

An ion-chromatographic method is described for the analysis of free nitrilotriacetic acid in water samples. Separations are achieved on a polymer-based anion-exchange column with 6 mM nitric acid as eluent. Both potentiometric and amperometric detection have been applied using metallic copper as the indicator electrode. Detection limits are at about 500 ng injected in the potentiometric mode and 100 ng in the amperometric mode. On-line sample preconcentration is possible for volumes up to 2 ml of river water samples. The response of the detector to other aminopolycarboxylic and aminopolyphosphonic acids has been investigated.

Characterization of antibody-chelator conjugates: determination of chelator content by terbium fluorescence titration.

Fluorescence titrations were performed by adding varying mole ratios of terbium(III) to antibody conjugates formed by benzyl isothiocyanate derivatives of three different polyaminopolycarboxylate chelators (NTA, EDTA, and DTPA) and the results compared to values for average chelator content obtained by cobalt-57 binding assays. For two different murine monoclonal antibodies, the average chelator content obtained by terbium fluorescence titration correlated closely with that measured by the cobalt-57 binding assay. It is concluded that lanthanide fluorescence titrations provide a useful alternative to radiometal binding assays for the determination of chelator content in protein-chelator conjugates.

Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 26. Mixed ligand complexes of cadmium, nitrilotriacetic acid, glutathione, and related ligands.

The complexation of glutathione and related ligands by the nitrilotriacetic acid complex of Cd2+ (Cd(NTA)-) has been investigated by 1H NMR as a model for the coordination chemistry of Cd2+ and GSH in biological systems. Related ligands included glycine, glutamic acid, cysteine, N-acetylcysteine, penicillamine, N-acetylpenicillamine, mercaptosuccinic acid, and the S-methyl derivative of glutathione. The nature of the complexes formed was deduced from 1H NMR spectra of Cd(NTA)- and the ligands. Mixed ligand complexes (Cd(NTA)L) and single ligand complexes (CdLx) are formed with the thiol ligands, whereas only mixed ligand complexes form with glycine, glutamic acid and S-methylglutathione. Formation constants of the mixed and the single ligand complexes were determined from NMR data. The results indicate that formation constants for binding of a thiolate donor group by Cd2+, either as the free ion or in a coordinately unsaturated complex, are in the range 10(5)-10(6).