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Electrochemical redox behaviour of Antimony (Sb) in Nitrilotriacetic acid (NTA) and Tartaric acid (TA) was investigated, owing to its relevance in nuclear, semiconducting and catalysis industry. A quasi-reversible to irreversible nature of Sb reduction was revealed from the type of chelant controlled the Sb availability as dissolved oxygenated species both in electrolyte and in the adsorbed form. Existence of two stable Sb-NTA complexes (denoted as Sb-NTA-a & Sb-NTA-b) was proposed based on the reduction peaks of Sb-NTA complexes observed in cyclic voltammetry. The stability constant of Sb-NTA-a is found to be lower when compared to Sb-TA. A more negative reduction potential compared to -0.3 V for Sb-NTA-b complex, indicates the formation of a stable species. Standard rate constant for Sb reduction evaluated by extended Nicholson method, supported the irreversible Sb reduction, and point towards the difficulty in dissolution of reduced Sb in presence of chelants. NTA acted as a habit modifier, where Sb2O3 acquired an octahedral shape with cubic crystal structure. The presence of NTA, thus modified both the kinetics of Sb reduction and the morphology of Sb deposits. These findings provide additional insights into the active role of chelants used for Sb mitigation both in nuclear and environmental applications.

In the field of peroxymonosulfate (PMS) activation technology, there is a pressing need to reduce PMS consumption and enhance its utilization rate. The present study demonstrates that the introduction of dissolved oxygen (DO) into the Mn(II)-nitrilotriacetic acid (NTA)-activated PMS system significantly enhances the degradation efficiency of sulfadiazine and increases the PMS utilization rate from approximately 15.0 to 41.3 %. Mechanistic analysis reveals that the Mn(II)-NTA/PMS system generates sulfate radicals as well as intermediate valent manganese species in the absence of DO; while in the presence of DO, Mn(II) is oxidized to Mn(III) by dioxygen to form superoxide anions and Mn(III), which can be further oxidized by PMS to higher valence states such as Mn(V) and Mn(VII). Consequently, the production of free radicals decreases while intermediate valent manganese species become more abundant. Additionally, O 2 •- can also reduce both Mn(VII) and Mn(IV) back to their lower oxidation state (Mn(II)). The cooperative interactions between these active species enhance the efficiency of catalytic cycles of manganese species. Moreover, the influence of multiple factors, the degradation products, and their associated toxicity assessment were investigated. Overall, this research provides valuable insights into the design of highly efficient PMS and DO activation systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nitrilotriacetic acid functionalized Adansonia digitata (NFAD) biosorbent has been synthesized using a simple and novel method. NFAD was characterized by X-ray Diffraction analysis technique (XRD), Scanning Electron Microscopy (SEM), Brunauer-Emmett-Teller (BET) surface area analyzer, Fourier Transform Infrared spectrometer (FTIR), particle size dispersion, zeta potential, elemental analysis (CHNS/O analyzer), thermogravimetric analysis (TGA), differential thermal analysis (DTA), derivative thermogravimetric analysis (DTG) and energy dispersive spectroscopy (EDS). The ability of NFAD as biosorbent was evaluated for the removal of Pb (II) and Cu (II) ions from aqueous solutions. The particle distribution of NFAD was found to be monomodal while SEM revealed the surface to be heterogeneous. The adsorption capacity of NFAD toward Pb (II) ions was 54.417 mg/g while that of Cu (II) ions was found to be 9.349 mg/g. The adsorption of these metals was found to be monolayer, second-order-kinetic, and controlled by both intra-particle diffusion and liquid film diffusion. The results of this study were compared better than some reported biosorbents in the literature. The current study has revealed NFAD to be an effective biosorbent for the removal of Pb (II) and Cu (II) from aqueous solution.

In this work, both red and white winery wastewaters (WW) and olive mill wastewater (OMW) were submitted to a treatment by Fenton-based processes (FBPs). The main aim was to evaluate the most efficient and economic process. Initial tests, resorting to a batch reactor, demonstrated that UV-C/Fenton (λ = 254 nm) was the most effective process. Operational conditions such as pH, H 2 O 2 and Fe 2+ concentrations revealed to have a superior influence within dissolved organic carbon (DOC) removal as well as regarding the reactor's energy consumption. As a means to prevent iron precipitation, the addition of nitrilotriacetic acid (NTA) was tested. With experimental conditions pH = 3.0, [H 2 O 2 ] = 194 mM, [Fe 2+ ] = 1.0 mM, [NTA] = 1.0 mM, radiation UV-C (254 nm), time = 240 min, the kinetic rate related with DOC removal showed a k red WW  = 0.0128 min -1  > k OMW  = 0.0124 min -1  > k white WW  = 0.0104 min -1 and both the WW and OMW achieved the Portuguese legal limit values for wastewater discharge. Furthermore, comparative experiments were performed in a semi-continuous reactor, being that the results put in evidence that the concentration of H 2 O 2 added and the flow rate of reagents' addition (F) had a significant effect on the efficiency of the reactor. Under an optimum experimental procedure pH = 3.0, [H 2 O 2 ] = 97 mM, [Fe 2+ ] = 1.0 mM, [NTA] = 1.0 mM, radiation UV-C (254 nm), F = 1 mL min -1 , time = 240 min, there were observed higher DOC removal kinetic rates (k OMW  = 15.20 × 10 -3 min -1  > k red WW  = 11.64 × 10 -3 min -1  > k white WW  = 11.57 × 10 -3 min -1 ) and a cost ranging between 0.0402 and 0.0419 €/g.DOC. These results showed that semi-continuous reactors have the potential to be applied to large scale treatments, with low reagents consumption and reduced energy requirements., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This work investigated the activation of peroxymonosulfate by electrochemical (EC) system assisted with Fe(III)-nitrilotriacetic acid (NTA) complex for degradation of persistent naphthenic acids (NAs) under neutral initial pH conditions. As NAs are a complicated mixture, 1-adamantanecarboxylic acid (ACA) was selected as the model NA compound for degradation experiment. The addition of NTA is to chelate with Fe(III), gaining stability under neutral pH condition to facilitate the circulation of Fe(II)/Fe(III) by the electrochemical process to activate PMS. The EC/Fe(III)-NTA/PMS system was explored with applicable pH range of 3-9 and an optimized molar ratio 1: 2 for Fe: NTA. Results of quenching and chemical probe experiment together with results of electron paramagnetic resonance (EPR) analysis revealed the main reactive species of the system, including • OH, SO 4 •- , 1 O 2 and possibly Fe(IV). With the addition of NTA, the yields of • OH, SO 4 •- , 1 O 2 were enhanced. Results of mass spectrometry analysis and DFT calculations indicated the formation of 9 degradation byproducts of ACA via three primary degradation pathways such as hydroxyl substitution, carbonyl substitution, and decarboxylation. Furthermore, the EC/Fe(III)-NTA/PMS system could achieve excellent removal efficiency of ACA with different anions such as Cl - , HCO 3 - , NO 3 - and H 2 PO 4 - in the background. The practical applicability of the system was also verified with the high removal of commercial NAs mixture standard. Overall results have indicated the EC/Fe(III)-NTA/PMS system could be utilized for efficient reclamation of authentic oil and gas industrial wastewater under natural pH conditions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In recent years, visible light assisted advanced oxidation processes (AOPs) are appealing in the elimination of pollutants. Herein, an innovative and eco-friendly visible light enhanced Fe 3+ -nitrilotriacetic acid system for the activation of peroxydisulfate (Vis/Fe 3+ -NTA/PDS) was proposed for the removal of bisphenol A (BPA). Fe 3+ -NTA could be dissociated through ligand-to-metal charge transfer (LMCT) to realize the generation of Fe 2+ for the continuous activation of PDS to remove BPA. The use of 0.10 mM Fe 3+ , 0.10 mM NTA and 1.00 mM PDS led to 97.5% decay of 0.05 mM BPA and 66.3% of TOC removal in 30 min with the illumination of visible light at initial pH 3.0. The sulfate and hydroxyl radicals were proved to be the dominant species leading to BPA removal by means of radical scavenging experiments, radical probes and electron paramagnetic resonance (EPR) technique. The effects of various operating parameters, natural water constituents as well as different water matrices on BPA abatement were explored. The intermediate products of BPA degradation were identified and a possible transformation pathway was proposed. Briefly, this research provides an attractive strategy for the remediation of refractory wastewater using NTA assisted with visible light in the homogeneous Fe 3+ /PDS system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This study presents, for the first time, the concurrent supply of the oxidants H 2 O 2 and NaOCl in solar-driven Fenton-like processes at neutral pH with ferric nitrilotriacetate (Fe 3+ -NTA) for wastewater reclamation. Simultaneous Escherichia coli (E. coli) inactivation and the removal of the antibiotic sulfamethoxazole (SMX) at 50 μg/L in municipal effluents were investigated in 5-cm deep raceway pond reactors. First, the individual effects of reagent concentrations (1.47, 2.94 and 4.41 mM for H 2 O 2 ; 0.134, 0.269 and 0.403 mM for NaOCl; 0.1 and 0.2 mM for Fe 3+ -NTA) on the economic efficiency (in terms of mass of SMX eliminated per Euro and per hour to attain complete E. coli inactivation and more than 50% of SMX removal) were considered. The highest economic efficiencies were 141 mg SMX /€·h with H 2 O 2 and 222 with NaOCl, the reaction times being 105 and 60 min, respectively. Second, a new strategy for solar photo-Fenton with the combination of the most cost-effective conditions (1.47 mM H 2 O 2 - 0.134 mM NaOCl - 0.1 mM Fe 3+ -NTA) was carried out in secondary effluents from two treatment plants with different technologies. Economic efficiency was substantially affected by wastewater composition, ranging from 178 to 1131 mg SMX /€·h with treatment times between 60 and 10 min, significantly improving the reported results for conventional solar photo-Fenton to date., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

A simplified sample pretreatment procedure was developed for quantitative measurement of nitrilotriacetic acid (NTA) in environmental water. On the basis of coordination capacity between NTA and metal ions, aluminum-based metal organic framework (MOF, MIL-53(Al)) was adopted for the adsorption of NTA, followed by stripping with copper sulfate as the eluent. The adsorbed NTA was converted into Cu-NTA during the desorption process, which facilitated the ensuing measurement by high performance liquid chromatography (HPLC). A linear range within 0.10 - 10 mg L -1 was achieved, along with a limit of detection (LOD, S/N=3, n=7) of 0.03 mg L -1 and an enrichment factor of 10.4. The developed method was validated by the analysis of sea water, influent of wastewater treatment plant and industrial wastewater, with satisfactory recoveries (90.2 - 91.1%) obtained., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ethylenediaminetetraacetic acid (EDTA) is one of the most effective chelating agents for enhancing lead (Pb) accumulation in various plant organs. However, it has a higher risk of causing secondary pollution than other chelating agents. To reduce such environmental risks and increase remediation efficiency, EDTA can be combined with degradable chelating agents for use in phytoremediation, but there are few reports on the combination of EDTA and nitrilotriacetic acid (NTA). This study evaluated the effects of combined EDTA and NTA application at different concentrations (900, 1200, or 1500 mg/kg) and with different methods (1 application or 3 applications) on dwarf bamboo (Sasa argenteostriata (Regel) E.G. Camus) growth and phytoremediation efficiency and on the soil environment in pot experiments with Pb-contaminated soil. Applying EDTA and NTA together resulted in lower soil water-soluble Pb concentrations than applying EDTA alone and therefore resulted in lower environmental risk. The increased availability of soil Pb produced a stress response in the dwarf bamboo plants, which increased their biomass significantly. Moreover, under the chelating treatments, the soil Pb availability increased, which promoted Pb translocation in plants. The Pb content in the aerial parts of the dwarf bamboo increased significantly in all treatments (translocation factors increased by 300~1500% compared with that in CK). The Pb content increase in the aerial parts caused high proline accumulation in dwarf bamboo leaves, to alleviate Pb toxicity. Maximum Pb accumulation was observed in the EN1500 treatment, which was significantly higher than that in the other treatments except the EN900 treatment. This study elucidates the choice of remediation techniques and the physiological characteristics of the plants used in such studies. In conclusion, the EN900 treatment resulted in the lowest environmental risk, greatest biomass production, and highest phytoremediation efficiency of all treatments, indicating that it has great potential for application in phytoremediation with dwarf bamboo in Pb-contaminated soil., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

In this work, we found that the distribution of two specific atrazine (ATZ) oxidation products (desethyl-atrazine (DEA) and desisopropyl-atrazine (DIA)) was different in oxidation processes involving aqueous ferryl ion (Fe(IV)) species and • OH. Specifically, the molar ratio of produced DEA to DIA (i.e., [DEA]/[DIA]) increased from 7.5 to 13 with increasing pH from 3 to 6 when ATZ was oxidized by Fe(IV), while the treatment of ATZ by • OH led to the [DEA]/[DIA] value of 2 which was independent of pH. Moreover, ATZ showed high reactivity towards Fe(IV) over a wide pH range, especially at near-neutral pH, at which ATZ oxidation in Fe(II)/peroxydisulfate system was even much faster than another well-defined Fe(IV) scavenger, the sulfoxides. By using this approach, it was obtained that the [DEA]/[DIA] value remained at 2 during ATZ transformation by the nitrilotriacetic acid (NTA) assisted Fenton-like (Fe(III)/H 2 O 2 ) system, which was independent of solution pH and reactants dosage. This result clarified that • OH was the primary reactive intermediate formed in the NTA assisted Fe(III)/H 2 O 2 system. This study not only developed a novel sensitive diagnostic tool for distinguishing Fe(IV) from • OH, but also provided more credible evidence to the nature of reactive intermediate in a commonly controversial system., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The tris-nitrilotriacetic acid (tris-NTA) chip has been used for surface plasmon resonance (SPR) kinetic studies involving histidine (His)-tagged proteins. However, its full potential, especially for analyte quantification in complex biological media, has not been realized due to a lack of systematic studies on the factors governing ligand immobilization, surface regeneration, and data analysis. We demonstrate that the tris-NTA chip not only retains His-tagged proteins more strongly than its mono-NTA counterpart, but also orients them more uniformly than protein molecules coupled to carboxymethylated dextran films. We accurately and rapidly quantified immunoglobulin (IgG) molecules in sera by using the initial association phase of their conjugation with His-tagged protein G densely immobilized onto the tris-NTA chip, and established criteria for selecting the optimal time for constructing the calibration curve. The method is highly reproducible (less than 2% RSD) and three orders of magnitude more sensitive than immunoturbidimetry. In addition, we found that the amount of His-protein immobilized is highly dependent on the protein isoelectric point (pI). Reliable kinetic data in a multi-channel SPR instrument can also be rapidly obtained by using a low density of immobilized His-tagged protein. The experimental parameters and procedures outlined in this study help expand the range of SPR applications involving His-tagged proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Different degrees of protein purity have been observed in immobilized metal affinity chromatography ranging from extremely high purity to moderate and low purity. It has been hypothesized that the host cell protein composition and the metal ligands are factors governing the purity of a protein obtained after immobilized metal affinity chromatography (IMAC). Ni nitrilotriacetic acid (NTA) has become the first choice for facile His-tagged protein purification, but alternative ligands such as iminodiacetic acid (IDA) with other immobilized metal ions such as Zn, Cu and Co are valuable options when the expected purity or binding capacity is not reached. Especially Cu and Zn are very attractive, due to their reduced environmental and safety concerns compared to Ni. Co and Zn are more selective than Ni and Cu. This increased selectivity comes at the cost of weaker binding. In this work, the influence of ligand choice on protein purity after IMAC was evaluated by several methods, including peptide mapping. His-tagged GFP was used as model protein. We found that host cell protein (HCP) content varies drastically between ligands, as IDA eluates generally showing higher HCP concentrations than NTA. The relative content of the key amino acids His, Cys and Trp in the sequence of the co-eluted protein does not suffice to explain co-eluting propensity. The co-elution of HCPs is mostly influenced by metal binding clusters on the protein surface and not by total content or surface concentration of metal interacting amino acids. Prediction of co-elution is not dependent on these clusters alone, due to protein-protein interactions, indicted by a relative low metal binding cluster score but high co-elution propensity and in a lot of cases these proteins are often part of complex such as ribosome and chaperones. The different co-eluting proteins were presented by a heatmap with a dendrogram. Ward's linkage method was used to calculate the distance between groups of co-eluting proteins. Clustering of co-eluting HCPs was observed according to ligand and by metal ions, with Zn and Co forming one cluster and Ni and Cu another. The co-elution of host cell proteins can be explained by clusters of metal interacting amino acids on the protein surface and by protein-protein interactions. While Ni NTA still appears to be highly advantageous, it might not be the cure-all for all applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Low pH requirement is one of the biggest limitations of the application of traditional Fenton and electro-Fenton (EF) process because Fe II /Fe III would precipitate at high pH. In this study, a flow-through EF system operated in batch recirculation mode was constructed. Nitrilotriacetic acid (NTA) was used as a chelating agent in the EF system (NTA/EF) to keep iron soluble at high pH values, producing OH by reaction of H 2 O 2 generated in situ with Fe II NTA that obtained by the reduction of Fe III NTA at the cathode. This flow-through NTA/EF system accelerated the mass transfer of target molecules to the electrode surface and showed high efficiency for phenol removal at pH 5-8 with rate constants (k) at around 0.26 min -1 , higher than that of the batch test (k = 0.15 min -1 ) and EF process without NTA (k = 0.16 min -1 ). The influences of aeration rate, current, flow rate, Fe dose, the ratio of NTA to Fe, pH, and initial phenol concentration on the phenol removal were investigated. The system could be used for at least 3 times for phenol removal without obvious efficiency decline. The flow-through NTA/EF system is promising for the removal of organic contaminants in a wide pH range., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Large amounts of insulin-loaded glucose-responsive micelles based on poly(amino acid)s have been developed for diabetes treatment over last decades, but most of them could not effectively protect insulin from enzymatic degradation in vivo because the micellar core was biodegradable and lacked protective structure for insulin, which would lower the efficacy of insulin to a large extent. In this study, we fabricated a new type of insulin-loaded glucose-responsive complex micelles (CMs), which were self-assembled by a phenylboronic acid (PBA)-modified block copolymer PEG-b-P(Asp-co-AspPBA) and a glucosamine (GA)/nitrilotriacetic acid (NTA)-functionalized block copolymer PNIPAM-b-P(Asp-co-AspGA-co-AspNTA), for self-regulated delivery of insulin with effective protection of insulin and enhanced hypoglycemic activity in vivo. The CMs possessed mixed shell of PEG/PNIPAM and cross-linked core of PBA/GA complex, which could be disintegrated under the condition of high glucose concentration (5 g/L) while maintaining stable at low glucose concentration (1 g/L). The NTA groups of CMs greatly improved the loading content of insulin by specifically bind insulin via the chelated zinc ions. More importantly, PNIPAM chains in the mixed shell would collapse under 37 °C and form hydrophobic domains around the micellar core, which could significantly protect the micellar core as well as the encapsulated insulin from attacking by external proteases. In a murine model of type 1 diabetes, the CMs with insulin chelated by NTA showed a long hypoglycemic effect, which is superior to insulin-loaded simple micelles without PNIPAM and insulin in PBS buffer (pH 7.4). Therefore, this kind of CMs could be a potential candidate for insulin delivery in diabetes therapy., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A complex mix of organic pollutants and heavy metal made the remediation of contaminated wetlands more difficult. Few research focus on the remediation for pyrene enhanced by chemical reagents and pyrene degrading bacteria in the nickel co-contaminated soil. In this paper, the effect of chemical reagents (nitrilotriacetic acid and alkyl polyglucoside) and Bacillus subtilis on pyrene dissipation in phytoremediation of nickel co-contaminated soil by Scirpus triqueter was investigated. Similar seedlings of Scirpus triqueter were moved to uncontaminated soil and pyrene-nickel co-contaminated soil. The pots (14.8 cm diameter and 8.8 cm height) were set up in greenhouse and treated in different ways. After 60 days, plant biomass, radial oxygen loss (ROL), soil dehydrogenase activity (DHA) and pyrene concentration in soil were determined. Results showed that ROL rate and DHA in different groups was positively correlated with pyrene dissipation from soil. In the process of remediation, chemical reagents might have an indirect slight effect on pyrene dissipation (pyrene dissipation increased 21%) by affecting DHA firstly and redistributing pyrene fractions in the presence of pyrene degrading bacteria. Pyrene degrading bacteria were likely to affect pyrene dissipation by impacting ROL rate and DHA and played a more vital role in contributing to pyrene dissipation (pyrene dissipation increased 45%) from wetland. This study demonstrated that phytoremediation for pyrene in nickel co-contaminated soil by Scirpus triqueter can be enhanced by the application of NTA-APG and pyrene degrading bacteria and they could be reasonably restore the ecological environment of PAH-contaminated wetlands., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Poly(1-vinylimidazole)-grafted Fe3O4@SiO2 magnetic nanoparticles (FSPV) were developed to remove both free Cd(II) and nitrilotriacetic acid (NTA) complexed Cd(II) from water. The adsorption capacity of NTA-complexed Cd(II) (42.1 mg/g) was larger than that of free Cd(II) (24.8 mg/g). Free Cd(II) and NTA-complexed Cd(II) could be reduced from 50 μg/L to below 5 μg/L within 10 and 20 min, respectively. FSPV could effectively remove trace free and NTA-complexed Cd(II) over broad pH ranges of 5-10 and 5-11, respectively. Moreover, the presence of seven coexisting ions (up to 100 mM) including four cations (Na(+), K(+), Ca(2+), and Mg(2+)) and three anions (Cl(-), NO3(-), and SO4(2-)) all slightly promoted the removal of both free and NTA-complexed Cd(II) onto FSPV. The removal of free Cd(II) or NTA-complexed Cd(II) was not obviously affected by the presence of Ni(II) and Co(II) (50 μg/L), humic acid (up to 3 mg/L as TOC) or alginate (up to 8 mg/L as TOC). In addition, FSPV could be regenerated with 10 mM HCl in 10 min, and the removal of both types of Cd(II) maintained above 95% in five consecutive adsorption/regeneration cycles. Therefore, FSPV could be utilized as promising adsorbent for both free and NTA-complexed Cd(II)., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In all cells, ATP-dependent proteases play central roles in the controlled degradation of short-lived regulatory or misfolded proteins. A hallmark of these enzymes is that proteolytic active sites are sequestered within a compartmentalized space, which is accessible to substrates only when they are fed into the cavity by protein-unfolding ATPases. HslVU is a prototype of such enzymes, consisting of the hexameric HslU ATPase and the dodecameric HslV protease. HslV forms a barrel-shaped proteolytic chamber with two constricted axial pores. Here, we report that structural alterations of HslV's pore motif dramatically affect the proteolytic activities of both HslV and HslVU complexes. Mutations of a conserved pore residue in HslV (Leu88 to Ala, Gly, or Ser) led to a tighter binding between HslV and HslU and a dramatic stimulation of both the proteolytic and ATPase activities. Furthermore, the HslV mutants alone showed a marked increase of basal hydrolytic activities toward small peptides and unstructured proteins. A synthetic peptide of the HslU C-terminal tail further stimulated the proteolytic activities of these mutants, even allowing degradation of certain folded proteins in the absence of HslU. Moreover, expression of the L88A mutant in Escherichia coli inhibited cell growth, suggesting that HslV pore mutations dysregulate the protease through relaxing the pore constriction, which normally prevents essential cellular proteins from random degradation. Consistent with these observations, an X-ray crystal structure shows that the pore loop of L88A-HslV is largely disordered. Collectively, these results suggest that substrate degradation by HslV is controlled by gating of its pores., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The use of surface plasmon resonance (SPR), for the comparison of metal binding properties of polyhistidine tags, was evaluated. Six different tags containing various number of histidines, either none (tags n and t), three (tags H3A3 and HA2HA2H) or six (tags H6 and His6), were genetically fused to the N-terminal of lactate dehydrogenase (LDH). The binding ability of these constructs to nickel ions, immobilised with nitrilotriacetic acid (NTA), was tested both by conventional immobilised metal ion affinity chromatography (IMAC) and SPR. The relative binding strengths of the tags to nickel were identical using both methods (n approximately t < HA2HA2H < H3A3 < His6 < H6), confirming the value of the SPR technique for investigating metal-protein interactions. Protein modelling has also proved to be useful in supporting the experimental results.

Fluorescence labeling of proteins has become increasingly important since fluorescent techniques like FRET and fluorescence polarization are now commonly used in protein binding studies, proteomics, and for high-throughput screening in drug discovery. In our efforts to study the binding of the beta(')-subunit from Escherichia coli RNA polymerase (RNAP) to sigma70, we synthesized a fluorescent-labeled beta(')-fragment (residues 100-309) in a very convenient way, that could be used as a general protocol for hexahistidine-tagged proteins. By performing all the following steps, purification, reduction, derivatization with IC5-maleimide, and free dye removal while the protein was bound to the column, we were able to reduce the procedure time significantly and at the same time achieve better labeling efficiency and quality. The beta(')-fragment with a N-terminal His(6)-tag was purified from inclusion bodies and could be refolded prior to or after binding to a Ni-NTA affinity column. Reduction prior to labeling was achieved with TCEP that does not interfere with Ni-NTA chemistry. The labeled beta(')-fragment was tested with sigma70 that was labeled with an europium-based fluorophore for binding in a electrophoretic mobility-shift assay. The sigma-to-core protein interaction in bacterial RNA polymerase offers a potentially specific target for drug discovery, since it is highly conserved among the eubacteria, but differs significantly from eukaryotes.

Non-transferrin-bound iron (NTBI) has been reported to be associated with several clinical states such as thalassemia, hemochromatosis, and in patients receiving chemotherapy. We have investigated a number of ligands as potential alternatives to nitrilotriacetic acid (NTA) to capture NTBI without chelating transferrin- or ferritin-bound iron in plasma. We have established, however, that NTA is the optimal ligand to chelate the different forms of NTBI present in sera and can be adopted for utilization in the NTBI assay. NTA (80 mM) removes all forms of NTBI, while only mobilizing a small fraction of the iron bound to both transferrin and ferritin. We have compared three different detection systems for the quantification of NTA-chelated NTBI: the established HPLC-based method, a simple colorimetric method, and a method based on inductive conductiometric plasma spectroscopy. The sensitivity and reproductibility of the colorimetric method were acceptable compared with the other two methods and would be more convenient as a routine laboratory screening assay for NTBI. However, the limitations of this method are such that it can only be utilized in situations where desferrioxamine is not used and when transferrin saturation levels are close to 100%. Only the HPLC-based method is applicable for patients receiving (desferrioxamine) chelation therapy. In some diseases such as hemochromatosis, transferrin may be incompletely saturated. In such cases, to avoid in vitro donation of iron onto the vacant sites of transferrin, sodium-tris-carbonatocobaltate(III) can be added to block the free iron binding sites on transferrin. If this step is not taken, there may be an underestimation of NTBI values., (Copyright 1999 Academic Press.)

While BIACORE instruments are routinely used for kinetic measurements and for the determination of binding constants, the immobilization of a ligand onto the sensor chip surface has to be individually optimized for every system. We show here that the histidine (His) tag, routinely used in protein purification and in detection is an ideal tag for immobilization, despite the intrinsically low affinity between an immobilized metal ion and the His tag. This is due to strong rebinding effects caused by the high surface density of immobilized Ni2+-nitrilotriacetic acid (NTA) on the chips used here. The immobilization of the ligand can be adjusted to a low level using the same chip, such that mass transport limitation and rebinding of the analyte to the immobilized ligand is minimal. Nine different proteins with different numbers of His tags were tested for stable binding to the Ni2+-NTA surface. Most proteins with one His tag dissociate very rapidly from the Ni2+-NTA surface, and the KD for the interaction between His tag and Ni2+-NTA was estimated to about 10(-6) m at neutral pH. In contrast, two His tags are usually found to be sufficient for stable binding. The kinetics of the chaperonin system of Escherichia coli GroEL and GroES were analyzed as a model using this system and found to be very similar to those obtained with covalently immobilized ligands. The sensor chip can be reused many times, because of the powerful regeneration methods. The ligand can be freshly immobilized after each cycle, thus eliminating potential denaturation upon regeneration as a source of error., (Copyright 1997 Academic Press.)

Using a one-step reaction, a bifunctional compound was synthesized for detecting histidine-tagged proteins immobilized on nitrocellulose. This compound has a biotin as one functional group and a nitrilotriacetic acid as the other. The nitrilotriacetic acid is used to chelate a Ni(II) ion at four of its six coordination sites. The remaining two sites are available for binding to a histidine tag. The biotin functional group can then be detected using a streptavidin-horseradish peroxidase conjugate and chemiluminescence. Using this biotinylated nitrilotriacetic acid, it is possible to detect less than 0.11 pmol of histidine-tagged Escherichia coli RNA polymerase sigma70 subunit. This reagent is also able to specifically detect His-tagged sigma70 from a whole cell lysate following SDS-PAGE and transfer to nitrocellulose. The reagent can be dissociated from the His-tagged protein at pH 4.8 and the blot can be reprobed with a monoclonal antibody for detection of different proteins on the same blot.

A small fraction of affinity-purified MHC class II molecules are known to bind antigenic peptides in vitro. No simple method with acceptable recovery exists for separation of complexes of a known antigenic epitope and MHC class II from empty MHC class II and complexes of MHC class II and endogenously bound peptide. Here we describe an one step metal chelate affinity chromatography method to purify complexes of MHC class II and antigenic peptide of known composition. Complexes of human HLA-DR2 (DRB1*1501/DRB5*0101) and a peptide analog from human myelin basic protein MBP(84-102) containing a 6 histidine tag (6 x His) and a tyrosine residue at the N-terminus end [6 x His-MBP(83-102)Y83] were prepared and purified. The absence of residual free 6 x His-MBP peptide in the complex preparations were confirmed by gel filtration and TLC analyses. The purified complexes were applied onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose affinity support and 6 x His-tagged peptide class II complexes were selectively eluted with imidazole-containing buffer. The quantitation of bound peptide in the eluted complexes showed 100% occupancy of HLA-DR2 (DRB1*1501/DRB5*0101) with [6 x His-MBP(83-102)Y83] peptide with a recovery of 50-75%. The presence of a single peptide entity in the eluted complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid-extracted supernatant and by amino acid sequencing analyses. As expected, no endogenous polypeptide was detected in the Ni2+.NTA eluted complexes when analyzed by two-dimensional IEF gel electrophoresis. Finally, we demonstrate that both MBP(84-102) and [6 x His-MBP(83-102)Y83] peptides were equally capable of stimulating restricted T cell line in the presence of autologous antigen presenting cells (APCs). These results demonstrate that metal chelate affinity chromatography can be used to prepare MHC class II-peptide complexes containing single peptide. Such complexes of class II molecules containing known peptide have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may provide better understanding of the trimolecular interaction between MHC class II, antigenic peptide and T cell receptor (TCR).

Pre-column derivatization with o-phthaldialdehyde is a rapid and sensitive method for the quantitation of amino acids in biological fluids. This method uses acetonitrile as a deproteinizing reagent which gives improved recovery of tryptophan compared with 5-sulfosalicylic acid and permits the measurement of aspartic acid which coelutes with 5-sulfosalicylic acid. The method is automated to increase reproducibility and convenience. Mean coefficients of variation for peak areas relative to internal standard were 3.2 and 5.2% for amino acid standards and plasma samples, respectively. The presence of nitrilotriacetic acid stabilized the o-phthaldialdehyde reagent which is important in an automated system. The method is suitable for the analysis of large numbers of plasma samples where total tryptophan and aspartic acid are of interest.

The etiology of diabetes in some conditions of iron overload is not known. We studied growth, glucose tolerance, and pancreatic islet cell morphology and cytochemistry in rats administered parenteral FeNTA. These rats developed glucosuria, slowing of growth with eventual weight loss, polyuria, polydipsia, and death. They had normal fasting plasma glucose levels but decreased glucose tolerance and insulin response to glucose. Although no Prussian blue staining of iron was observed in pancreatic islets by light microscopy, at the ultrastructural level insulin-secreting beta-cells showed ferrin iron deposits localized to the plasmalemma and the cytoplasmic surface of secretory granule membranes. Prussian blue staining was also observed in parenchymal cells of the liver, heart, and kidney, in order of decreasing intensity. Animals treated with an equivalent dose of NTA, saline, or iron-dextran in saline had normal growth and response to glucose and did not exhibit pancreatic iron deposits at the light or ultrastructural level. These results support the hypothesis that iron affects pancreatic islet cell function and may be an etiologic agent of diabetes mellitus in hemochromatosis.

A novel nitrilotriacetic acid adsorbent has been prepared for metal chelate affinity chromatography. The new resin is a quadridentate chelate former and specially suitable for metal ions with coordination numbers of six, since two valencies remain for the reversible binding of biopolymers. It has been found to chelate Cu2+ and Ni2+ strongly and to be superior to the known iminodiacetic acid adsorbent. Charged with Ni2+, it was evaluated for the ability to bind peptides and proteins containing neighbouring histidine residues. The remarkable specificity found makes it an attractive addition to the range of adsorbents for metal chelate affinity chromatography.