Protein purification via consecutive histidine-polyphosphate interaction.

While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.

Preparation and characterization of iron(III) nitrilotriacetate complex in aqueous solutions for quantitative protein binding experiments.

Various preparations of iron(III) nitrilotriacetate (FeNTA) solution reported in the literature lack a comprehensive method for accurate determination of FeNTA concentration and often result in unstable solutions. A detailed procedure for the preparation of FeNTA solution is presented that includes the standardization of both components of the chelate. The standardization of the components allowed the accurate determination of the molar absorption coefficients for the calculation of the FeNTA concentration in two different buffers at pH 5.6 and 7.4. The variation of pH in this range or ionic strength in the range from 0 M to 3 M (KCl) has little effect on the value of the molar absorption coefficient. The precise concentrations of all species involved in the equilibria between Fe and NTA were determined in the pH range 2-12 using the Jenkins-Traub algorithm to solve the 5th-order polynomial in Microsoft Excel. In view of the experimental observations and the calculated distribution of species, the stability of FeNTA solutions may be affected by the Fe : NTA ratio and the total concentrations, with dilute solutions and those with an excess of NTA over Fe showing higher stability.

Strategies for Mitigating Commercial Sensor Chip Variability with Experimental Design Controls.

Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni2+-nitrilotriacetic acid (NTA) functional group. While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.

Biodegradable chelant-metal complexes enhance cadmium phytoextraction efficiency of Solanum americanum.

Toxic contaminants (metals and metal-containing compounds) are accumulating in the environment at an astonishing rate and jeopardize human health. Remarkable industrial revolution and the spectacular economic growth are the prime causes for the release of such toxic contaminants in the environment. Cadmium (Cd) is ranked the 7th most toxic compound by the Agency for Toxic Substances and Disease Registry (USA), owing to its high carcinogenicity and non-biodegradability even at miniscule concentration. The present study assessed the efficiency of four biodegradable chelants [nitrilotriacetic acid (NTA), ethylenediamine disuccinate (EDDS), ethylene glycol tetraacetic acid (EGTA), and citric acid (CA)] and their dose (5 mM and 10 mM) in enhancing metal accumulation in Solanum americanum Mill. (grown under 24 mg Cd kg-1 soil) through morpho-physiological and metal extraction parameters. Significant variations were observed for most of the studied parameters in response to chelants and their doses. However, ratio of root and shoot length, and plant height stress tolerance index differed non-significantly. The potential of chelants to enhance Cd removal efficiency was in the order - EGTA (7.44%) > EDDS (6.05%) > NTA (4.12%) > CA (2.75%). EGTA and EDDS exhibited dose-dependent behavior for Cd extraction with 10 mM dose being more efficient than 5 mM dose. Structural equation model (SEM) depicted strong positive interaction of metal extraction parameters with chelants (Z-value = 11.61, p = 0.001). This study provides insights into the importance of selecting appropriate dose of biodegradable chelants for Cd extraction, as high chelant concentration might also result in phytotoxicity. In the future, phytoextraction potential of these chelants needs to be examined through field studies under natural environmental conditions.

Using peptides to promote delivery and improve anti-tumour efficacy of liposomal drug.

Liposomal drugs exhibit advantages for cancer therapy, but efficacy is often limited by their rapid clearance from the blood by the reticuloendothelial system, and an inability to target and penetrate tumours. Interestingly, a 21-amino acid SIRP-α- (signal regulatory protein-α) interacting 'self' peptide is reported to inhibit uptake by phagocytes. Also, 'iRGD' a 9-amino acid cyclic peptide that binds αvß3 integrins and neuropilin-1 (NRP-1), promotes targeting and penetration of the drug into tumours. Here we explore the potential of nitrilotriacetic acid-ditetra-decylamine (NTA3-DTDA)-containing liposomes (NTA-liposomes) engrafted with His-tagged forms of 'self' peptide (pCD47) to prolong circulation time in blood after iv administration, and of iRGD peptide (piRGD) to enhance treatment efficacy of doxorubicin-containing liposomes (Caelyx). Our results show that pre-incubation of murine phagocytic DC2.4 and RAW246.7 cells with pCD47 inhibits the uptake of NTA-liposomes in vitro, but engraftment of pCD47 surprisingly reduces liposome lifetime in blood. Engraftment of piRGD promoted binding of NTA-liposomes to murine B16 melanoma and CT26 colorectal carcinoma cells in vitro. Importantly, iv administration of piRGD-engrafted Caelyx was found to significantly inhibit tumour growth and prolong survival in both B16 and CT26 murine tumour models. Our results show that engraftment of piRGD onto Caelyx is a convenient strategy to enhance treatment efficacy.

Reactive Extrusion of Nonmigratory Antioxidant Poly(lactic acid) Packaging.

Reactive extrusion of bio-derived active packaging offers a new approach to address converging concerns over environmental contamination and food waste. Herein, metal-chelating nitrilotriacetic acid (NTA) ligands were grafted onto poly(lactic acid) (PLA) by reactive extrusion to produce metal-chelating PLA (PLA-g-NTA). Radical grafting was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy with the introduction of secondary alkyl stretches (2919 and 2860 cm-1) and by X-ray photoelectron spectroscopy (XPS) with an increase in the atomic percentage of nitrogen. Compared to films prepared from native, granular PLA (gPLA), PLA-g-NTA films had lower contact angles and hysteresis values (86.35° ± 2.49 and 31.89° ± 2.27 to 79.91° ± 1.58 and 21.79° ± 1.72, respectively), supporting the surface orientation of the NTA ligands. The PLA-g-NTA films exhibited a significant antioxidant character with a radical scavenging capacity of 0.675 ± 0.026 nmol Trolox(eq)/cm2 and an iron chelation capacity of 54.09 ± 9.36 nmol/cm2. PLA-g-NTA films delayed ascorbic acid degradation, retaining ∼45% ascorbic acid over the 9-day study compared to <20% for control PLA. This research makes significant advances in translating active packaging technologies to bio-derived materials using scalable, commercially translatable synthesis methods.

Probing Protein-Protein Interactions with Label-Free Mass Spectrometry Quantification in Combination with Affinity Purification by Spin-Tip Affinity Columns.

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.

Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand.

Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc) is a hollow cylindrical decameric protein complex with an inner space 110 Šin diameter and 160 Šin height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246) in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA) chelated to a Ni2+ ion (Ni2+-NTA) to TpHc. We found that His-tagged green fluorescent protein (GFP) was encapsulated into the Ni2+-NTA-conjugated TpHc via the interaction between the His-tag and the Ni2+-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering.

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.

Folding and Assembly of Vanilloid Receptor Secondary-Structure Peptide with Hexahistidine Linker at Nickel-Nitrilotriacetic Acid Monolayer for Capsaicin Recognition.

Herein, we report the self-assembly of a synthetic vanilloid receptor (VR) peptide that selectively binds capsaicin. We synthesized a 26-mer peptide-YSEILFFVQS-HHHHHH-LAMGWTNMLY (S3HS4)-comprising two chemoreceptor domains of transient receptor potential channel (TRPV1) linked by a hexahistidine sequence. High-speed atomic force microscopy (AFM) imaging in water revealed that the peptide structures alternated rapidly between wedge shape and linear forms. Circular dichroism spectroscopy showed that 65% of the amide units in the peptide chain adopted an α-helix structure, which was ascribed to the chemoreceptor domains. S3HS4 developed well-packed monolayers at the Ni-treated thiolated nitrilotriacetic acid self-assembled monolayers by chelation of the hexahistidine segment, as characterized by infrared spectroscopy and AFM, which exhibited statistically constant specific height. Therefore, S3HS4 was expected to fold spontaneously upon chelation, and the resulting helix-turn-helix conformers developed films while uniformly oriented: the tilt angle was 69° from the surface normal to the substrate. According to microgravimetric analysis using a quartz crystal microbalance (QCM), the adsorption was 84 ± 47 pmol cm-2 ( n = 3), which was almost consistent with the saturation adsorption of an α-helix unit. We also used a QCM to investigate the host-guest reactions of S3HS4 and found that the S3HS4-attached QCM-chip-bound capsaicin with an apparent binding constant of (4.2 ± 3.6) × 104 M-1 ( n = 4), whereas there was no evidence of binding to vanillin or acetophenone. Two controls-a blank chip without S3HS4 and a chip modified with a single helical peptide (LAMGWTNMLY-HHHHHH)-produced no capsaicin response. To the best of our knowledge, S3HS4 is the first example of a synthetic VR mimic peptide. We believe that the present surface-directed structure-based design can be used to exploit the α-helix bundle in hexahistidine-linked bishelical peptides.

Affinity Purification of Methyllysine Proteome by Site-Specific Covalent Conjugation.

A basic but critical step in targeted proteomics by mass spectrometry is the separation of the targeted proteins from the complex mixture of the whole proteome by affinity purification. The bait protein is usually immobilized on the surface of a solid support to enable affinity-based purification of the targeted proteome. Here, we developed a site-specific covalent immobilization of the bait protein through affinity-guided covalent coupling (AGCC) of a single cysteine residue of an SH2 domain (utilized as an affinity tag for the protein target) with an engineered ligand peptide. Site-specific covalent immobilization of a methyllysine-binding protein HP1ß chromodomain on the agarose resin was used to purify the methyllysine proteome from the whole-protein mixture. This new bait immobilization led to a notably low background in the affinity purification step, markedly outperforming the conventional (His)6 tag-nickel nitrilotriacetic acid (Ni-NTA) immobilization method. Subsequent analysis of the purified proteome identified 275 lysine methylated sites and 184 methylated proteins from 332 HP1ß CD-binding proteins, including 30 novel methylated proteins. This work demonstrates that a robust site-specific covalent protein immobilization method is well-suited for proteomic analysis of low-abundance proteins. This method also enables the identification of new methylated proteins and methylation sites in the methyllysine proteome.

Nitrilotriacetic Acid-Functionalized Glucose-Responsive Complex Micelles for the Efficient Encapsulation and Self-Regulated Release of Insulin.

Insulin plays a significant role in diabetes treatment. Although a huge number of insulin-loaded, glucose-responsive nanocarriers have been developed in past decades, most of them showed a lower loading capacity and efficiency due to the weak interaction between insulin and nanocarriers. In this work, a novel insulin-encapsulated glucose-responsive polymeric complex micelle (CM) is devised, showing (i) enhanced insulin-loading efficiency owing to the zinc ions' chelation by nitrilotriacetic acid (NTA) groups of NTA-functioned glycopolymer and the histidine imidazole of insulin, (ii) the glucose-triggered pulse release of insulin, and (iii) long stability under physiological conditions. This CM was fabricated by the self-assembly of block copolymer PEG- b-P(Asp- co-AspPBA) and glycopolymer P(Asp- co-AspGA- co-AspNTA), resulting in complex micelles with a PEG shell and a cross-linked core composed of phenylboronic acid (PBA)/glucose complexations. Notably, the modified nitrilotriacetic acid (NTA) groups of CM could specifically bind insulin via chelated zinc ions, thus enhancing the loading efficacy of insulin compared to that of nonmodified CM. The dynamic PBA/glucose complexation core of CM dissociates under the trigger of high glucose concentration (>2 g/L) while being quite stable in low glucose concentrations (<2 g/L), as demonstrated by the pulse release of insulin in vitro. Finally, in a murine model of type 1 diabetes, NTA-modified complex micelles loading an insulin (NTA-CM-INS) group exhibited a long hypoglycemic effect which is superior to that of free insulin in the PBS (PBS-INS) group and insulin-loaded complex micelles without an NTA modification (CM-INS) group. This long-term effect benefited from Zn(II) chelation by NTA-modified complex micelles and could avoid hypoglycemia caused by the burst release of insulin. Taken together, this constitutes a highly effective way to encapsulate insulin and release insulin via an on-demand manner for blood glucose control in diabetes.

Effect of Bacillus subtilis and NTA-APG on pyrene dissipation in phytoremediation of nickel co-contaminated wetlands by Scirpus triqueter.

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.

Development of a Small Hybrid Molecule That Mediates Degradation of His-Tag Fused Proteins.

In recent years, the induction of target-protein degradation via the ubiquitin-proteasome system (UPS) mediated by small molecules has attracted attention, and this approach has applications in pharmaceutical development. However, this technique requires a ligand for the target protein that can be incorporated into tailor-made molecules, and there are many proteins for which such ligands have not been found. In this study, we developed a protein-knockdown method that recognizes a His-tag fused to a protein of interest. This strategy theoretically allows comprehensive targeting of proteins of interest by a particular molecule recognizing the tag. As expected, our hybrid molecule 10 [SNIPER(CH6)] efficiently degraded His-tagged CRABP-II and Smad2 in cells. This system provides an easy method to determine the susceptibility of proteins of interest to UPS-mediated degradation. Furthermore, we hope that this method will become an efficient tool to analyze the function of the UPS.

Isolation and characterization of iron chelators from turmeric (Curcuma longa): selective metal binding by curcuminoids.

Iron overload disorders may be treated by chelation therapy. This study describes a novel method for isolating iron chelators from complex mixtures including plant extracts. We demonstrate the one-step isolation of curcuminoids from turmeric, the medicinal food spice derived from Curcuma longa. The method uses iron-nitrilotriacetic acid (NTA)-agarose, to which curcumin binds rapidly, specifically, and reversibly. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin each bound iron-NTA-agarose with comparable affinities and a stoichiometry near 1. Analyses of binding efficiencies and purity demonstrated that curcuminoids comprise the primary iron binding compounds recovered from a crude turmeric extract. Competition of curcuminoid binding to the iron resin was used to characterize the metal binding site on curcumin and to detect iron binding by added chelators. Curcumin-Iron-NTA-agarose binding was inhibited by other metals with relative potency: (>90% inhibition) Cu2+ ~ Al3+ > Zn2+ ≥ Ca2+ ~ Mg2+ ~ Mn2+ (<20% inhibition). Binding was also inhibited by pharmaceutical iron chelators (desferoxamine or EDTA) or by higher concentrations of weak iron chelators (citrate or silibinin). Investigation of the physiological effects of iron binding by curcumin revealed that curcumin uptake by cultured cells was reduced >80% by addition of iron to the media; uptake was completely restored by desferoxamine. Ranking of metals by relative potencies for blocking curcumin uptake agreed with their relative potencies in blocking curcumin binding to iron-NTA-agarose. We conclude that curcumin can selectively bind toxic metals including iron in a physiological setting, and propose inhibition of curcumin binding to iron-NTA-agarose for iron chelator screening.

Controlled carbon nanotube layers for impedimetric immunosensors: High performance label free detection and quantification of anti-cholera toxin antibody.

An original impedimetric immunosensor was developed based on carbon nanotube (CNT) deposits with controlled thicknesses for enhanced electroactive surface areas leading to improved sensor performances. Cholera monitoring was chosen as the model immune system for this setup. These CNT deposits were characterized using confocal laser microscopy and electrochemical methods. To form the sensor device, the CNT deposits were functionalized via electrocoating of polypyrrole-nitrilotriacetic acid (poly(pyrrole-NTA)) followed by the formation of a Cu (II) complex with the NTA functions. The bioreceptor unit, cholera toxin B Subunit, modified with biotin, was then immobilized via coordination of the biotin groups with the NTA-Cu(II) complex. Each step of the formation of the immunosensor and the subsequent binding of the analyte antibody anti-cholera toxin were investigated with cyclic voltammetry and Electrochemical Impedance Spectroscopy. After optimization, the resulting impedimetric cholera sensor shows excellent reproducibility, increased sensitivities, a very satisfying detection limit of 10-13gmL-1 and an exceptional linear range for anti-cholera detection of 8 orders of magnitude (10-13-10-5gmL-1) and a sensitivity of 24.7 ± 0.4Ω per order of magnitude.

New insights into the antioxidant activity and cytotoxicity of arzanol and effect of methylation on its biological properties.

The heterodimeric phloroglucinyl pyrone arzanol (Arz) has raised considerable interest because of its antiviral, anti-inflammatory, and antioxidant activity. We have investigated the effect of methylation of the pyrone moiety on the antioxidant activity and cytotoxicity of Arz. This manoeuvre, that left the polyphenolic moiety unscathed, was nevertheless detrimental for antioxidant activity in both the cholesterol thermal degradation- and the Cu2+-induced liposome oxidation assays, providing evidence of structure-activity relationships that go beyond the preservation of the polyphenolic pharmacophore. The antioxidant activity of Arz was retained also in the Fe-NTA model of in vivo oxidative stress, with protective effect on the oxidative degradation of plasmatic lipids, unsaturated fatty acids and cholesterol. Both Arz and methylarzanol (Me-Arz) were devoid of toxic effect on colonic differentiated Caco-2 cells up to 100µM, but significantly reduced cancer Caco-2 cell viability at lower dosages. Arz could also selectively reduce viability of other cancer cell lines, [murine melanoma cells (B16F10 cells), human cervical carcinoma cells (HeLa cells)], suggesting that it can act as a selective modulator of cell processes typical of cancer cells. Taken together, our results qualify Arz as a lead structure for further in vivo investigation of its pharmacological potential.

Simultaneous determination of thermodynamic and kinetic parameters of aminopolycarbonate complexes of cobalt(II) and nickel(II) based on isothermal titration calorimetry data.

The influence of the different side chain residues on the thermodynamic and kinetic parameters for complexation reactions of the Co2+ and Ni2+ ions has been investigated by using the isothermal titration calorimetry (ITC) technique supported by potentiometric titration data. The study was concerned with the 2 common tripodal aminocarboxylate ligands, namely, nitrilotriacetic acid and N-(2-hydroxyethyl) iminodiacetic acid. Calorimetric measurements (ITC) were run in the 2-(N-morpholino)ethanesulfonic acid hydrate (2-(N-morpholino) ethanesulfonic acid), piperazine-N,N'-bis(2-ethanesulfonic acid), and dimethylarsenic acid buffers (0.1 mol L-1 , pH 6) at 298.15 K. The quantification of the metal-buffer interactions and their incorporation into the ITC data analysis enabled to obtain the pH-independent and buffer-independent thermodynamic parameters (K, ΔG, ΔH, and ΔS) for the reactions under study. Furthermore, the kinITC method was applied to obtain kinetic information on complexation reactions from the ITC data. Correlations, based on kinetic and thermodynamic data, between the kinetics of formation of Co2+ and Ni2+ complexes and their thermodynamic stabilities are discussed.

Effects of [S,S]-ethylenediaminedisuccinic acid and nitrilotriacetic acid on the efficiency of Pb phytostabilization by Athyrium wardii (Hook.) grown in Pb-contaminated soils.

Chelate-assisted phytoextraction with biodegradable chelants has been demonstrated as an efficient method to enhance heavy metal remediation efficiency by plants, while there is little available information on phytostabilization. A pot experiment was conducted to investigate the effects of biodegradable [S,S]-ethylenediaminedisuccinic acid (EDDS) and nitrilotriacetic acid (NTA) on plant growth and Pb accumulation of Pb phytostabilizer Athyrium wardii (Hook.) grown in Pb contaminated soils and to explore the feasibility of chelate-assisted phytostabilization. Greater adverse effects on plant biomass under high EDDS treatments were observed than NTA treatments. Significant increase of shoot Pb concentrations of A. wardii was noticed with increasing NTA and EDDS dosages, while EDDS induced higher shoot Pb concentrations than NTA. Moreover, root Pb concentrations of A. wardii under NTA treatments were 1.18-1.28-time higher than EDDS treatments, and a peak value of root Pb concentrations was observed at 2 mmol kg(-1) of NTA. Shoot Pb accumulations significantly increased with increasing dosages, and EDDS treatments caused a 1.44-1.6-time increase of shoot Pb accumulation than NTA. Root Pb accumulations under NTA treatments were 1.18-1.28-time higher than EDDS treatments. Maximum root Pb accumulation (155.5 mg plant(-1)) was found at 2 mmol kg(-1) of NTA on the 14th day. Higher BCF values and lower TF values were found under NTA treatments as compared to EDDS treatments. Available Pb concentrations in soil significantly increased on the 7th day with increasing NTA and EDDS dosages, then gradually decreased on the 14th day. Soil pH slightly decreased with increasing NTA and EDDS dosages. Therefore, chelate-assisted phytostabilization could be a feasible way to enhance the efficiency of Pb phytostabilization by A. wardii.

Self-assembly of Ni-NTA-modified ß-annulus peptides into artificial viral capsids and encapsulation of His-tagged proteins.

ß-Annulus peptides bearing Cys at the N-terminal from tomato bushy stunt virus were synthesised using a standard Fmoc-protected solid-phase method, and the peptide was modified with Ni-NTA at the N-terminal. The Ni-NTA-modified ß-annulus peptide self-assembled into virus-like nanocapsules of approximately 40 nm in diameter. The critical aggregation concentration of these nanocapsules in 10 mM Tris-HCl buffer (pH 7.3) at 25 °C was 0.053 µM, which is 470 times lower than that of unmodified ß-annulus peptides. Moreover, size exclusion chromatography of the peptide assembly indicated encapsulation of His-tagged green fluorescent protein in the Ni-NTA-modified artificial viral capsid.