Chelator Concentration Research Articles

BioF is a novel B2 metallo-β-lactamase from Pseudomonas sp. isolated from an on-farm biopurification system.

Antimicrobial resistance represents a major global health concern and environmental bacteria are considered a source of resistance genes. Carbapenems are often used as the last antibiotic option to treat multidrug-resistant bacteria. Metallo-β-lactamases (MBLs) are able to render resistance to almost all β-lactam antibiotics, including carbapenems. Unfortunately, there are no inhibitors against MBLs for clinical use. Subclass B2 MBLs are the only enzymes working as strict carbapenemases, under-represented, encoded in chromosome genes and only functional as mono-zinc enzymes. Despite current efforts in MBLs inhibitor development, B2 carbapenemase activity is especially difficult to suppress, even in vitro. In this study we characterized BioF, a novel subclass B2 MBL identified in a new environmental Pseudomonas sp. strain isolated from an on-farm biopurification system (BPS). Although blaBioF is most likely a chromosomal gene, it is found in a genomic island and may represent a step previous to the horizontal transmission of B2 genes. The new B2 MBL is active as a mono-zinc enzyme and is a potent carbapenemase with incipient activity against some cephalosporins. BioF activity is not affected by excess zinc and is only inhibited at high metal chelator concentrations. The discovery and characterization of B2 MBL BioF as a potent carbapenemase in a BPS bacterial isolate emphasizes the importance of exploring antibiotic resistances existing in the environmental microbiota under the influence of human activities before they could emerge clinically.

Isolation and Chelation Activity of Plant Phenolic Root Exudates As Potential Clinical Iron Chelators

Introduction:Hemoglobinopathies such as β-Thalassemia (Thal) and other genetic disorders such as neurodegeneration with brain iron accumulation have complications of iron overload, which can impair vital processes such as cardiac and neurological function. In Thal, ineffective erythropoiesis leads to elevated gastrointestinal iron absorption and in severe cases may necessitate blood transfusions exacerbating the condition. Iron chelating agents such as deferoxamine (DFO), deferiprone, and deferasirox must be used, but can have unwanted side effects. While effective in treating iron overload, the increased risk of side effects at higher doses highlights the importance of finding new potent iron-chelating agents, either as replacements or as adjuvants. Plant based treatments may be better tolerated by patients, offering an alternative therapeutic approach. Plant secondary metabolites secreted from roots under iron limiting conditions may provide a novel source for this bioactivity. Plants rich in phenolic compounds and those tolerating alkaline soils, which necessitate efficient mobilization of soil-precipitated iron, are our focus here. The aim of this project is to functionally and analytically characterize naturally occurring plant root exudates as possible clinical iron chelators.Methods : We cultivated Populus trichocarpa ( Poplar), Thuja plicata (Western red-cedar), and Lavendula X intermedia (Lavender) cuttings under iron normal and iron limiting conditions. From their roots, exudates were isolated to characterize and determine their effectiveness as iron chelators. Total phenolic concentrations of exudates were evaluated using Folin-Ciocalteu reagent. The ferrozine assay was used as a competition assay to characterize total iron binding ability of root exudates and standard chelators, which was sensitive enough to differentiate chelation effectiveness between DFO, EDTA, and isolated root exudates at 800 nM. Furthermore, known plant iron chelators chlorogenic acid (CA), a phenolic, and the more lipophilic β-thujaplicin (hinokitiol) were compared to DFO, then used tomodel root exudates in a cell culture bioassay. The bioassay was developed using monocytic THP-1 cells (as an RE system model) to quantify intra- and extracellular iron, comparing iron uptake in cells exposed to varying concentrations of iron chelators as a way of evaluating chelation strengths of crude root exudates, β-thujaplicin, and CA in comparison to DFO.Results:Total phenolic production in P. trichocarpa root exudates increased significantly (p < 0.05) in response to iron depletion after 12 hrs. Iron binding ability increased almost four-fold upon iron depletion. In the ferrozine competition assay, iron depleted root exudates inhibited iron binding by 15% whereas iron normal exudates inhibited by only 4%. Iron binding of root exudates was similar to EDTA (13% inhibition) and about half that of DFO (30% inhibition). In the THP-1 cell bioassay, DFO and CA showed comparable trends of chelation activity. Cells exposed to either 50 or 100 μM CA or DFO reduced intracellular iron by at least 47% or 89%, respectively. However, at 50 and 100 µM β-thujaplicin increased iron uptake by 82 to 86% respectively. Further testing of plant root exudates is ongoing.Conclusions: Exposing P. trichocarpa to an iron deficient environment resulted in plants that secreted root exudates with significantly higher level of phenolics and increased iron chelation ability. This was shown in the ferrozine competition assay where iron depleted root exudates showed higher binding affinity for iron when compared to the iron normal exudates resulting in an iron chelation ability similar to the known iron chelator EDTA. In the bioassay using THP-1 monocytic cells, testing the plant phenolic CA, there was reduction of intracellular iron comparable to DFO, whereas the lipophilic chelator β-thujaplicin increased cellular iron. Consequently, chelators such as CA exclude iron from being taken up by the cells, while more lipophilic chelators, like β-thujaplicin enable transport through membranes, and thus have the capacity to transport iron both into and out of cells. Thus, we have shown that plant root exudates can yield potential iron chelators and that in general plant phenolics have iron chelation capacity comparable to the classic iron chelator DFO. DisclosuresWalter:Apopharma: Research Funding.

Association Between Iron and Cholesterol in Neuroblastomas.

Neuroblastoma is the most common childhood extracranial solid malignancy. Although cancer cells need iron and lipids for active cell division, possible links between iron and lipid metabolism in neuroblastomas have not been studied. We evaluated the levels and association between iron and cholesterol on in vitro neuroblastoma cancer models. We found that the levels of iron and cholesterol are diverse among neuroblastoma cell lines. There is a bi-directional association between iron and cholesterol in drug-resistant neuroblastoma SK-N-AS cells. In drug-resistant neuroblastoma cells, low concentration of an iron chelator did not have an impact on iron levels, but on cellular cholesterol levels. Furthermore, a cholesterol decreasing agent, simvastatin, influenced both iron and cholesterol levels in drug-resistant neuroblastoma cells. Cholesterol decreasing agents may be more effective than iron chelators for drug-resistant neuroblastoma treatment.

Development of the L-type CaV / BK Complex Simulator (II): estimation of the distance between the channels

The presence, in the cell membrane, of high-conductance K+ channels and voltage-gated Ca2+ channels (CaV) forming complexes has been reported. These complexes have important functions in excitable cells. The [Ca2+]i at the mouth of the CaV channel decreases with distance and with the concentration of chelators. For the BK channel to be activated with internal Ca2, a concentration of the order of M is necessary and this implies a closeness between the BK-CaV channels. A simulator of the decay of Ca2+ in the presence of BAPTA to estimate the distance between the channels was developed. The mathematical models were implemented in Visual Basic® 6.0 and were solved numerically. The results indicate the coexistence of L-type CaV channel and BK grouped in nanodomains with a distance between channels of ~30 nm.

Zinc import mediated by AdcABC is critical for colonization of the dental biofilm by Streptococcus mutans in an animal model.

Trace metals are essential to all domains of life but toxic when found at high concentrations. Although the importance of iron in host-pathogen interactions is firmly established, contemporary studies indicate that other trace metals, including manganese and zinc, are also critical to the infectious process. In this study, we sought to identify and characterize the zinc uptake system(s) of Streptococcus mutans, a keystone pathogen in dental caries and a causative agent of bacterial endocarditis. Different than other pathogenic bacteria, including several streptococci, that encode multiple zinc import systems, bioinformatic analysis indicated that the S. mutans core genome encodes a single, highly conserved, zinc importer commonly known as AdcABC. Inactivation of the genes coding for the metal-binding AdcA (ΔadcA) or both AdcC ATPase and AdcB permease (ΔadcCB) severely impaired the ability of S. mutans to grow under zinc-depleted conditions. Intracellular metal quantifications revealed that both mutants accumulated less zinc when grown in the presence of a subinhibitory concentration of a zinc-specific chelator. Notably, the ΔadcCB strain displayed a severe colonization defect in a rat oral infection model. Both Δadc strains were hypersensitive to high concentrations of manganese, showed reduced peroxide tolerance, and formed less biofilm in sucrose-containing media when cultivated in the presence of the lowest amount of zinc that support their growth, but not when zinc was supplied in excess. Collectively, this study identifies AdcABC as the major high affinity zinc importer of S. mutans and provides preliminary evidence that zinc is a growth-limiting factor within the dental biofilm.

Complexation of ferrous ions by ferrozine, 2,2′-bipyridine and 1,10-phenanthroline: Implication for the quantification of iron in biological systems

Iron is an essential nutrient for virtually all forms of life. Because of its redox properties and involvement in a wide range of biological processes, a number of qualitative and quantitative chemical tools have been developed to detect reduced (Fe2+) and oxidized (Fe3+) forms of iron in biomolecules. These types of measurements are not only important in detecting iron species in solution, but also in understanding iron distribution, accumulation, and role in physiological and pathological processes. Here, we use UV–vis spectrophotometry and three common chromogenic reagents, ferrozine, 2,2′-bipyridine, and 1,10-phenanthroline to detect and quantify the concentration of ferrous ions in aqueous solutions, owing to the unique absorption spectra, specific molar absorptivity, and characteristic colors of these Fe2+-chelator complexes. Our results show that the kinetics of the formation of the {Fe2+-(ferrozine)3} complex, but not the{Fe2+-(bipyridine)3} or the {Fe(II)-(phenanthroline)3} complexes depend on the concentration of the iron chelator, requiring up to 20 min to complete when close to stoichiometric ratios are employed. The molar absorptivity values of these complexes under excess chelator concentrations were ~ 10% to 15% higher than reported literature values (i.e. 31,500 ± 1500 M−1 cm−1 for ferrozine at 562 nm, 9950 ± 100 M−1 cm−1 for 2,2′-bipyridine at 522 nm, and 12,450 ± 370 M−1 cm−1 for 1,10-phenanthroline at 510 nm). Our results have important implications when quantifying iron in biological systems and reveal optimal experimental conditions that must be employed for the accurate measurements of ferrous ions, whether free in solution, or after reduction of protein-bound ferric ions.

Complexation of transition metals by chelators added during mashing and impact on beer stability

Beer inevitably changes due to an array of staling reactions. A major factor in beer ageing is the involvement of transition metals (iron, copper, manganese) in oxidative reactions. To tackle the flavour stability issue, metal chelation was investigated. Based on previous research, five primary chelators (tannic acid, gallic acid, EDTA, citric acid and phytic acid) were screened using experimental design for their capacity to reduce the content of wort transition metals. The chelating agents were added under varying conditions (mash out temperature, mash pH, grain bill, chelator concentration, addition time) during laboratory scale mashing to assess how they altered complexation and metal load. Fourteen alternative chelators (ferulic acid, tartaric acid, quercetin, chlorogenic acid and ten polyphenolic food extracts: green tea, pomegranate, grape seed, reishi, cinnamon, curcuma, milk thistle, ginkgo, grapefruit seed and raspberry) were also explored. Metal ions were analysed using inductively coupled plasma optical emission spectrometry and wort oxidative stability by electron spin resonance spectroscopy. Mash pH was the most decisive of all tested process variables: acidified mashing (pH 6 to 5) produced worts with more iron, manganese and zinc (230, 320 and 150%, respectively). Addition of effective chelators counteracted this undesirable effect for iron. Green tea extract, tannic acid and, particularly, pomegranate extract all resulted in lower wort iron. Conversely, addition of EDTA, caused iron, manganese and zinc to increase. Pomegranate extract (90% ellagic acid) was the best performing chelator and reduced radical generation in wort (80% reduction by 60 mg/L addition), making it a promising novel compound in the improvement of beer shelf life. © 2021 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling.

The effect of EDTA and citric acid on biochemical processes and changes in phenolic compounds profile of okra (Abelmoschus esculentus L.) under mercury stress

The present study aimed to explore the effect of synthetic and naturally occurring chelators, EDTA and citric acid (CA), respectively, on changes in physiological and biochemical factors including cell death, level of mercury ions accumulation, malondialdehyde (MDA) content, total phenol and total flavonoids, anthocyanins and DPPH free radical scavenging activity, in the leaves of okra (Abelmoschus esculentus L.) plants exposed to mercury stress. In addition, polyphenolic compounds profile was assessed by high-performance liquid chromatography. The okras were planted in completely controlled hydroponic conditions (Hoagland solution). After they reached the four-leaf stage, they were treated simultaneously with different concentrations of HgCl2, EDTA and CA chelators, and their combination for one month. At the stage of maturity, the physiological and biochemical factors of the plant leaves were measured. The results showed that with the application of higher concentration of HgCl2, cell death, level of shoot and root Hg2+ content and root MDA, total phenols and total flavonoids, anthocyanin content, and DPPH free radical scavenging activity were increased. Also, the results indicated that okra plants have high biomass and a high rate of Hg mobilization and accumulation in the shoot versus the roots (TF=2.152 for the plants treated with 60 mg L−1 Hg2+), hence, can be considered as Hg hyperaccumulator plant for the phytoremediation of Hg-polluted soils and waters. In the Hg-treated plants changes in their phenolic profile were induced, and the increase of chlorogenic acid, rosmaric acid, apigenin, quercetin and rutin content was observed. The application of EDTA and CA improved the toxic effects of Hg2+, by modifying phenolic compounds, chelating Hg2+, and its proper compartmentation, while EDTA outperformed CA in this respect. Based on the results, it could be concluded that due to the high biomass and growth of okra in the presence of Hg2+, this plant is suitable for phytoremediation of soil and water contaminated with mercury. In addition, EDTA and CA can play a significant role in removing this toxic metal through transferring it from the culture medium to the plant.

Development and validation of an economical uric acid-Fe3+/Fe2+-ferrozine-based colorimetric assay to estimate uric acid level of pure and biological samples.

This work presents the development and validation of a simple, rapid, and cost-effective spectrophotometric method for quantitative analysis of uric acid in biological samples. The method relies upon uric acid-led reduction of Fe(III) to Fe(II) of sample/standard solutions which stoichiometrically engages ferrozine to form a magenta-colored complex. Different parameters including pH, metal and chelator concentrations, temperature, etc., were optimized for the maximum intensity and stability of the complex. The uric acid concentrations of synthetic/plasma solutions were determined by comparing the color intensity of Fe(ferrozine)3 2+ complex produced by test solution with the standard curve formed by known uric acid concentrations. The method was validated in accordance with ICH guidelines and subjected to human plasma analysis. The results obtained were compared with a reference (enzymatic) method which revealed that there was no significant difference between the two methods at 95% confidence level. The method is highly specific, precise, linear, accurate, and robust.

Physicochemical properties of skim milk powder dispersions prepared with calcium-chelating sodium tripolyphosphate, trisodium citrate, and sodium hexametaphosphate

Due to health benefits of proteins, the demand for protein beverages has grown rapidly. Translucent protein drinks with neutral pH may have advantages over acidic beverages that may cause dental erosion, and skim milk powder (SMP) is an affordable protein ingredient. Dissociating casein micelles by calcium chelators is a well-known method to reduce SMP dispersion turbidity, but much is to be studied for physicochemical properties as affected by chelator type and concentration. The objective of the present study was to characterize physicochemical properties of dispersions with 5% (wt/vol) SMP after addition of 0 to 30 mM sodium tripolyphosphate, trisodium citrate, or sodium hexametaphosphate. The turbidity was decreased with increasing chelator concentration, with the lowest turbidity observed in the SMP dispersions with sodium hexametaphosphate. The smallest hydrodynamic diameter was observed at an intermediate chelator concentration, resulting from the balance of casein micelle dissociation and aggregation of dissociated caseins induced at an elevated ionic strength. Heating at 90°C for 5 min increased turbidity but lowered hydrodynamic diameter of SMP dispersions, with some exceptions. The morphology of SMP dispersions differed for each chelator and was also affected by chelator concentration and heating. Trisodium citrate was the most effective to demineralize colloidal calcium phosphate in casein micelles, but the amount of dissolved calcium was not directly correlated with the decreased turbidity, indicating different chelating mechanisms by each chelator. Analysis of serum calcium and phosphorus concentrations also suggested that the type and concentration of soluble and insoluble calcium phosphates and their partitioning in the serum and casein micelles were dynamically changed by the studied parameters to affect dispersion turbidity and structures of casein micelles. Findings from the present study may be used to formulate translucent beverages incorporating SMP and other casein micelle ingredients.

Promoting crystallization of intrinsic membrane proteins with conjugated micelles

A new technique for promoting nucleation and growth of membrane protein (MP) crystals from micellar environments is reported. It relies on the conjugation of micelles that sequester MPs in protein detergent complexes (PDCs). Conjugation via amphiphilic [metal:chelator] complexes presumably takes place at the micelle/water interface, thereby bringing the PDCs into proximity, promoting crystal nucleation and growth. We have successfully applied this approach to two light-driven proton pumps: bacteriorhodopsin (bR) and the recently discovered King Sejong 1–2 (KS1–2), using the amphiphilic 4,4′-dinonyl-2,2′-dipyridyl (Dinonyl) (0.7 mM) chelator in combination with Zn2+, Fe2+, or Ni2+ (0.1 mM). Crystal growth in the presence of the [metal–chelator] complexes leads to purple, hexagonal crystals (50–75 µm in size) of bR or pink, rectangular/square crystals (5–15 µm) of KS1–2. The effects of divalent cation identity and concentration, chelator structure and concentration, ionic strength and pH on crystal size, morphology and process kinetics, are described.

225Ac-H4py4pa for Targeted Alpha Therapy.

Herein, we present the syntheses and characterization of a new undecadendate chelator, H4py4pa, and its bifunctional analog H4py4pa-phenyl-NCS, conjugated to the monoclonal antibody, Trastuzumab, which targets the HER2+ cancer. H4py4pa possesses excellent affinity for 225Ac (α, t1/2 = 9.92 d) for targeted alpha therapy (TAT), where quantitative radiolabeling yield was achieved at ambient temperature, pH = 7, in 30 min at 10-6 M chelator concentration, leading to a complex highly stable in mouse serum for at least 9 d. To investigate the chelation of H4py4pa with large metal ions, lanthanum (La3+), which is the largest nonradioactive metal of the lanthanide series, was adopted as a surrogate for 225Ac to enable a series of nonradioactive chemical studies. In line with the 1H NMR spectrum, the DFT (density functional theory)-calculated structure of the [La(py4pa)]- anion possessed a high degree of symmetry, and the La3+ ion was secured by two distinct pairs of picolinate arms. Furthermore, the [La(py4pa)]- complex also demonstrated a superb thermodynamic stability (log K[La(py4pa)]- ∼ 20.33, pLa = 21.0) compared to those of DOTA (log K[La(DOTA)]- ∼ 24.25, pLa = 19.2) or H2macropa (log K[La(macropa)]- = 14.99, pLa ∼ 8.5). Moreover, the functional versatility offered by the bifunctional py4pa precursor permits facile incorporation of various linkers for bioconjugation through direct nucleophilic substitution. In this work, a short phenyl-NCS linker was incorporated to tether H4py4pa to Trastuzumab. Radiolabeling studies, in vitro serum stability, and animal studies were performed in parallel with the DOTA-benzyl-Trastuzumab. Both displayed excellent in vivo stability and tumor specificity.

Optimization of Degradation of Emulsified Oil by Microbial Missile Using Response Surface Methodology

Optimal conditions for microbial missile degradation of emulsified oil were predicted using the response surface method designed by Box-Behnken. The effects of the concentration of sodium alginate (SA), the concentration of calcium source (CaCO3), the calcium ion chelator (CaCl2), and the pH value on the degradation efficiency of oil pollution were investigated using calcium alginate gel as a matrix. The results show that the model fitting accuracy R2=0.9739, the concentration of SA, the concentration of calcium source, the concentration of calcium ion chelator and the pH value have a great influence on the response value of the degradation rate of emulsified oil. The model predicts that the optimal reaction conditions are SA concentration of 21.27 g/L, calcium source concentration of 29.71 g/L, calcium ion chelator concentration of 32.33 g/L, and pH is 8.56. The predicted degradation rate of emulsified oil reaches 71.50%. The model was further verified by experiments. The average value of the four experimental degradation rates under the optimal conditions was 71.61%, and the deviation was 0.11% compared with the predicted value. This proves that the method can be used to optimize the process parameters of microbial missiles to degrade the emulsified oil, provide optimized solutions for the treatment of actual oily wastewater.

A Wafer-Scale Material Removal Rate Model for Chemical Mechanical Planarization

In this work, a new wafer-scale material removal rate (MRR) model is proposed to investigate the underlying removal mechanism of wafer surface in the chemical mechanical planarization (CMP) process. Based on the governing equation of the plate theory, chemical reaction kinetics and wear theory, an analytical CMP model has been constructed to capture the influence of mechanical abrasion and chemical reactions on the removal rate with high efficiency. The effect of the elastic modulus of the pad, platen rotational speed, polishing temperature and reactant concentrations on the removal mechanism is investigated in detail to elucidate the intrinsic removal behavior. It is found that the material removal rate is sensitive to the elastic modulus of pad, temperature distribution, and chelator concentration, more sensitive to the oxidizer concentration, and most sensitive to the platen rotational speed. The magnitude of the removal rate can be adjusted significantly by properly selecting a good configuration of these influencing factors. The model predictions of the MRR profiles are consistent with the published experimental data at different polishing conditions. Therefore, the present CMP model can give insights into the wear mechanism of polishing process and may be utilized as a simulation tool for further optimization of removal rate and surface uniform control.

Conformational Stability Effect of Polymeric Iron Chelators.

SummaryThe design and synthesis of metal chelators with extraordinary metal affinities is a basic and challenging scientific problem of both fundamental and practical importance. Here, we demonstrate a “conformational stability effect” that can significantly enhance the metal affinity of ligands after conjugation to polymer chains with the ability to spontaneously adopt a specific conformation as an optimal “soft” scaffold to ensure maximum thermodynamic stability of the metal complexes. Using iron chelators as models, we show that simple conjugation of small molecule catechol ligands to a polyallylamine chain resulted in more than 8–9 orders of magnitude enhancement of the iron-binding affinity, which is comparable to that of enterobactin, the strongest iron chelator ever known. This study demonstrates that flexible polymer chelators may realize the highest possible metal affinities of the conjugated ligands owing to their ability to achieve an optimal conformation, which could advance the identification of strong metal chelators.

Effects of biodegradable chelator combination on potentially toxic metals leaching efficiency in agricultural soils

Soil washing with chelators, a viable method for treating soils contaminated with potentially toxic metals, has drawn increasing attentions. The objective of this study was to determine a new generation of mixed degradable chelating agents from N, N-bis (carboxymethyl) glutamic acid (GLDA), [S, S]-stereoisomer of ethyleneiaminedisucc--inic acid (EDDS), nitrilotriacetic acid (NTA), and citric acid (CA), and to evaluate its effectiveness and feasibility to reduce toxic metals contamination in two different agricultural soils. A comparative leaching test conducted on the four individual degradable chelating agents showed that the capacity of single chelator in mobilizing copper (Cu), zinc (Zn), cadmium (Cd), and lead (Pb) varied significantly. Using a combination of GLDA and NTA was more advantageous than using a single chelating agent in extracting potentially toxic metals. The removal efficiencies of Cu, Zn, Cd, and Pb reached 38.2, 9.8, 71.4, and 19.5% for soil 1, and 25.0, 5.2, 59.7, and 18.5% for soil 2, respectively, at mixed chelator (MC) concentrations of 3 mmol/L (GLDA) and 2 mmol/L (NTA), pH of 6.0, and a contact time of 4.0 h. The effects of washing conditions, chelator concentration, pH values, and contact time on the removal efficiencies of target toxic metals were investigated. The results showed that the combined chelating agent has a lower pH dependence, making it feasible for a wider range of applications. The effects of the chelating agents on the morphological distribution of potentially toxic metals and the soil enzyme activity before and after the treatments were also studied. After washing, the content of the water-soluble, acid-soluble, reducible, and oxidizable target metals showed a certain degree of decrease. Although the activities of catalase, urease, and invertase appeared to be inhibited during a short period of time, their activities were stimulated and later promoted with the degradation of the chelating agent. In general, the chelating agent combination has a great potential for toxic metals leaching.

Optimization of a Methodology for Quantification and Removal of Zinc Gives Insights Into the Effect of This Metal on the Stability and Function of the Zinc-Binding Co-chaperone Ydj1.

Ydj1, a class B J-protein (Hsp40) in yeast, has two zinc finger domains in each monomer and belongs to an important co-chaperone family that plays crucial roles in cells, such as recognizing and binding partially folded proteins and assisting the Hsp70 chaperone family in protein folding. Yeast cells with ydj1 deletion were less efficient at coping with zinc stress than wild-type cells, and site-directed mutagenesis studies that impair or delete the zinc finger region have confirmed the importance of this region to the function of Ydj1; however, little is known about whether the presence of zinc is critical for the function of the protein. To gain insights into the effect of zinc on the structure and function of Ydj1 without having to modify its primary structure, a method was developed and optimized to quantify and remove the zinc from the protein. Recombinant Ydj1 was produced and purified, and its zinc content was determined by ICP-MS. The result showed that two zinc atoms were bound per monomer of protein, a good indicator that all sites were saturated. To optimize the removal of the bound zinc, variations on chelating agent (EDTA, EGTA, 1,10-phenanthroline), chelator concentration, reaction time, pH, and temperature were tested. These procedures had no effect on the overall secondary structure of the protein, since no significant changes in the circular dichroism spectrum were observed. The most significant removal (91 ± 2%, n = 3) of zinc was achieved using 1,10-phenanthroline (1 × 10−3 mol L−1) at 37°C with a pH 8.5 for 24 h. Zinc removal affected the stability of the protein, as observed by a thermal-induced unfolding assay showing that the temperature at the middle of the transition (Tm) decreased from 63 ± 1°C to 60 ± 1°C after Zn extraction. In addition, the effect on the ability of Ydj1 to protect a model protein (luciferase) against aggregation was completely abolished after the Zn removal procedure. The main conclusion is that zinc plays an important role in the stability and activity of Ydj1. Additionally, the results highlight the medical importance of chaperones, as altered zinc homeostasis is implicated in many diseases, such as neurodegenerative disorders.

Hydrophilic Quantum Dots Functionalized with Gd(III)-DO3A Monoamide Chelates as Bright and Effective T1-weighted Bimodal Nanoprobes

Magnetic resonance imaging (MRI) is a powerful non-invasive diagnostic tool that enables distinguishing healthy from pathological tissues, with high anatomical detail. Nevertheless, MRI is quite limited in the investigation of molecular/cellular biochemical events, which can be reached by fluorescence-based techniques. Thus, we developed bimodal nanosystems consisting in hydrophilic quantum dots (QDs) directly conjugated to Gd(III)-DO3A monoamide chelates, a Gd(III)-DOTA derivative, allowing for the combination of the advantages of both MRI and fluorescence-based tools. These nanoparticulate systems can also improve MRI contrast, by increasing the local concentration of paramagnetic chelates. Transmetallation assays, optical characterization, and relaxometric analyses, showed that the developed bimodal nanoprobes have great chemical stability, bright fluorescence, and high relaxivities. Moreover, fluorescence correlation spectroscopy (FCS) analysis allowed us to distinguish nanosystems containing different amounts of chelates/QD. Also, inductively coupled plasma optical emission spectrometry (ICP – OES) indicated a conjugation yield higher than 75%. Our nanosystems showed effective longitudinal relaxivities per QD and per paramagnetic ion, at least 5 times [per Gd(III)] and 100 times (per QD) higher than the r1 for Gd(III)-DOTA chelates, suitable for T1-weighted imaging. Additionally, the bimodal nanoparticles presented negligible cytotoxicity, and efficiently labeled HeLa cells as shown by fluorescence. Thus, the developed nanosystems show potential as strategic probes for fluorescence analyses and MRI, being useful for investigating a variety of biological processes.

The effects of trace metal impurities on Ga-68-radiolabelling with a tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelator.

GMP-grade 68Ge/68Ga generators provide access to positron-emitting 68Ga, enabling preparation of Positron Emission Tomography (PET) tracers and PET imaging at sites that do not have access to cyclotron-produced radionuclides. Radiotracers based on tris(3-hydroxy-1,6-dimethylpyridin-4-one) (THP) chelators enable simple one-step preparations of 68Ga PET radiopharmaceuticals from pre-fabricated kits without pre-processing of generator eluate or post-purification. However, trace metal impurities eluted along with 68Ga could compete for THP and reduce radiochemical yields (RCY). We have quantified trace metal impurities in 68Ga eluate from an Eckert & Ziegler (E&Z) generator using ICP-MS. The metals Al, Fe, natGa, Pb, Ti and natZn were present in generator eluate in significantly higher concentrations than in the starting eluent solution. Concentrations of Fe and natGa in eluate were in the range of 0.01–0.1 μM, Al, Zn and Pb in the range of 0.1–1 μM, and Ti in the range of 0.9–1.5 μM. To assess the ability of THP to chelate 68Ga in the presence of such metal ions, radiolabelling reactions were undertaken in which selected metal ions were added to make them equimolar with THP, or higher. Al3+, Fe3+, natGa3+ and Ti4+ reduced RCY at concentrations equimolar with THP and higher, but at lower concentrations they did not affect RCY. Pb2+, Zn2+, Ni2+ and Cr3+ had no effect on RCY (even under conditions in which each metal ion was present in 100-fold molar excess over THP). The multi-sample ICP-MS analysis reported here is (to date) the most comprehensive and robust quantification of metal impurities in the widely used E&Z 68Ga generator. 68Ga from an E&Z generator enables near-quantitative radiolabelling of THP at chelator concentrations as low as 5 μM (lower than other common gallium chelators) without pre-processing. The combination of Al3+, Fe3+, natGa3+ and Ti4+ in unprocessed 68Ga eluate is likely to decrease RCY of 68Ga radiolabelling if a lower amount of THP chelator is used, and future kit design should take this into account. To increase specific activities by using even lower THP concentrations, purification of 68Ga from trace metal ions will likely be required.

Enhanced azo dye removal from wastewater by coupling sulfidated zero-valent iron with a chelator

Recent research on zero-valent iron (ZVI) has evolved from a proof-of-concept stage to an application-focus stage with the goal of enhancing the applicability of ZVI for water decontamination. Sulfidation represents one of the most prominent ways to enhance the reactivity and efficiency of ZVI. In this study, sulfidated ZVI (S-ZVI) coupled with ethylenediamine tetraacetic acid (EDTA) was used to remove acid red 73 (AR 73) from wastewater and the results showed that the coupling of chelator and sulfidation significantly enhanced the removal of AR 73. The first-order kinetic constant (kobs) followed a bimodal pattern with increasing S/Fe ratio, chelator concentration, and mixing rate, but a unimodal pattern with changes in initial AR 73 concentration. Under the optimal conditions, the removal rate of AR 73 obtained in the EDTA + S-ZVI system was about 6.5 times faster than that in the ZVI system. Further analysis by gas chromatography-mass spectrometry (GC-MS) confirmed that reduction played a major role in the removal of AR 73. X-ray photoelectron spectroscopy (XPS) energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectrometry (FTIR) were used to investigate the surface chemical compositions of the solids before and after reaction. The results showed that the surface of freshly prepared S-ZVI was covered with FeSx, and EDTA on the surface of S-ZVI could complex with the Fe(II) to form an FeII-EDTA complex. The FeII-EDTA complex can inhibit the oxidation of Fe(II) and reduce the redox potential of the solution, which together enhance the removal of AR 73.