Presence Of EDTA Research Articles

Pseudothrombocytopenia — problems and ways to solve diagnostic problems

EDTA-dependent pseudothrombocytopenia is the laboratory phenomenon of a spurious low platelet count which appears in vitro while using EDTA as anticoagulant. The problem of EDTA-dependent pseudothrombocytopenia has significant relevance due to the widespread using of automated hematology analyzers which EDTA stabilized blood is most recommended for. The mechanism of this phenomenon is based on the production of the specificantibodies causing the platelet aggregation in the presence of EDTA. The aim of this study was to evaluate MgSO4 as an in vitro anticoagulant for platelet count, MPV and correction WBC. Early determination of the patients with EDTA-dependent pseudothrombocytopenia is an important diagnostic criterion necessary to exclude blood diseases and prevent errors in treatment.

α-Amylase purified and characterized from fenugreek (Trigonella foenum-graecum) showed substantial anti-biofilm activity against Staphylococcus aureus MTCC740

Starch hydrolyzing α-amylase from germinated fenugreek (Trigonella foenum-graecum) has been purified 104-fold to apparent electrophoretic homogeneity with a final specific activity of 297.5 units/mg. SDS-PAGE of the final preparation revealed a single protein band of 47.5 kDa, supported by LC/MS analysis and size-exclusion chromatography on the Superdex 200 (ÄKTA-FPLC). α-Amylase exhibited maximum activity at pH 5.5. An activation energy (Ea) of 9.12 kcal/mol was found to exist in the temperature range of 20 to 90 °C. When substrate concentrations were evaluated between 0.5 and 10 mg/mL, the Km and Vmax values for starch were observed to be 1.12 mg/mL and 384.14 μmol/min/mg, respectively. The major substrate starch exhibited high specificity for fenugreek α-amylase. In the presence of EDTA (5 mM), the activity was lost, however, it could be largely reversed with the addition of calcium. Furthermore, an effort was made to assess the ability of fenugreek seed-derived partially purified (DEAE-cellulose enzyme) and purified α-amylase to disperse inside 48 h-old biofilms of Staphylococcus aureus MTCC740. The outcomes clearly demonstrated that the purified and partially purified α-amylase both exhibited strong biofilm dispersion activity.

Experimental and modeling study of the diffusion path of Ce(III)-EDTA in compacted bentonite

The impact of the diffuse double-layer on the diffusion of radionuclide anions remains unclear, impeding the application of Archie's law for adsorptive anions. Through-diffusion experiments were carried out to measure the effective diffusion coefficient and distribution coefficient of Ce(III) in the presence of EDTA. A multi-porosity model, in combination with Archie's law, was used to estimate the effective diffusion coefficient. The predominant species, CeEDTA−, exhibited an effective diffusion coefficient of (0.88–1.92) × 10−11 m2/s and the distribution coefficient of (0.81–1.18) × 10−3 m3/kg at the compacted dry density of 1300–1800 kg/m3. The accessible porosity, the diffuse double-layer porosity, and the interlayer porosity were analyzed using the multi-porosity model, based on the characterization of bentonite via Scanning electron microscopy with energy dispersive spectrometry, X-ray diffractometer, and Nitrogen-Brunner-Emmett-Teller method. Due to the low external surface area of Zhisin bentonite (27.7–35.3 m2/g), the impact of the diffuse double-layer was insignificant on the total and accessible porosities. Additionally, the diffuse double-layer porosity had insignificant impact on the effective diffusion coefficient of CeEDTA− in compacted bentonite, as its value was one order magnitude lower than that of the interlayer porosity and the accessible porosity.

Purification and characterization of a high molecular weight serine protease from Microbacterium paraoxydans sp. SKS10.

Alkaline proteases from microbial sources have been found suitable for diverse industrial applications, with serine proteases being the most common enzymes used in the detergent industry. In the present study, we have purified and characterized an extracellular alkaline serine protease from Microbacterium paraoxydans sp. SKS10. The protease was purified using ammonium sulfate precipitation followed by different chromatography techniques (fold purification 6.919). Km and Vmax for the protease were determined to be 0.183mg/mL and 4.904U/mL, respectively. This enzyme is a thermostable high molecular weight (∼109.4kDa) protease which has maximal activity at 60°C, and above pH 10. Inhibitor assays revealed the enzyme to be a serine protease whose activity increased by 2.5-fold in the presence of EDTA. This enzyme remained active in the presence of various metal salts and organic solvents and was compatible with commercially available laundry detergents highlighting its potential for use in the detergent industry.

Statistical optimization of enzyme cocktail production using Jew's mallow stalks residues by a new isolate Aspergillus flavus B2 via statistical strategy and enzymes characterization.

This study investigates the production of the enzyme cocktail by the isolated fungi Aspergillus flavus B2 (GenBank accession number OL655454) using agricultural and industrial (AI) residues as the sole substrate. Of all the AI residues tested, Jew's mallow stalk was the best inducer substrate for enzyme cocktail production without adding any nutrients. Statistical optimization using Response Surface Methodology enhanced the production by 5.45, 5.20, and 3.34-fold, respectively for pectinase, xylanase, and CMCase. Optimum temperature, activation energy (Ea), and activation energy for denaturation (Ed) were determined. Michaelis constant (Km) for CMCase, xylanase, and pectinase enzyme was 1.82, 1.23, and 1.05mg/mL, respectively. Maximum reaction rate (Vmax) was 4.67, 5.29, and 17.13U/mL, respectively for CMCase, xylanase, and pectinase. Thermal stability revealed that pectinase, CMCase, and xylanase enzymes retained 64.7%, 61.8%, and 53.2% residual activities after incubation for 1h at 50°C. Half-life time (t0.5) of pectinase, CMCase, and xylanase at 50°C were 189.38, 129.8, and 127.89min, respectively. Thermodynamics of the produced enzymes enthalpy (ΔH*d), free energy (ΔG*d), and entropy (ΔS*d) were determined at 40, 50, and 60°C. In the presence of EDTA (5mM), CMCase, xylanase, and pectinase retained 69.5%, 66.2%, and 41.2%, respectively of their activity. This work is significant for the valorization of AI residues and the production of value-added products.

Facile Synthesis of ZnO/g-C3N4 with Enhanced Photocatalytic Performance for the Reduction of Cr(VI) in Presence of EDTA Under Visible Light Irradiation

Facile Synthesis of ZnO/g-C3N4 with Enhanced Photocatalytic Performance for the Reduction of Cr(VI) in Presence of EDTA Under Visible Light Irradiation

Matrix metalloproteinase sensing in wound fluids: Are graphene-based field effect transistors a viable alternative?

Wound dressings based on graphene sheets are expected to play a crucial role in wound treatment. The good conductivity and charge mobility of graphene as an electronic material makes graphene-based field effect transistors well-adapted for sensing of wound biomarkers. In this work, an aptamer-based field effect transistor using graphene as sensing channel material was developed and evaluated for the selective and sensitive monitoring of matrix metalloproteinase 9 (MMP-9), a wound biomarker upregulated in non-healing wounds. Concentration-dependent measurements performed with wound fluid spiked with MMP-9 on GFET-MMP/PEG sensors exhibited real-time detection of MMP-9 with a detection limit of 97 pM in 1x MOPS/1 mM CaCl2. The sensor was tested on swab samples from non-healing wounds and compared to results of antibody-based ELISA analysis. In contrast to ELISA, the GFET sensor was only sensing the active MMP-9 form and not the mixture of active and inactive forms, as evidenced by the analysis performed in the presence of EDTA as inactivation medium.

Synthesis and Application of Acylhydrazone Probe with High Selectivity and Rapid Detection of Mercury Ion

AbstractA Schiff base acylhydrazone fluorescent probe (DPTYMH) was designed and synthesized using 3‐bromo‐N,N‐diphenylaniline, 5‐formyl‐2‐thiopheneboronic acid, and 2‐hydroxybenzohydrazide. The structure was then verified by 1H NMR, 13C NMR and HR‐MS. DPTYMH exhibited positive solvent photochromic effect based on the mechanism of intra‐molecular charge transfer (ICT), and demonstrated effectiveness, high selectivity, and specificity to capture mercury ion (Hg2+). The response time and the detection limit of DPTYMH on Hg2+ was under 10 s and as low as 1.01×10−8 M, respectively. Density Functional Theory (DFT) calculation and 1H NMR were used to investigate the potential coordination mechanism. The Hg2+ level of several water samples had been effectively analyzed using DPTYMH. The molecular logic gate was also built using Hg2+ and EDTA as chemical inputs by taking use of DPTYMH′s reversibility in the presence of EDTA. Moreover, the DPTYMH was produced as test strips, which has the enormous potential to detect Hg2+ ions in the environment.

Bacterial DNase activity as a putative inductor of sperm DNA fragmentation in infected bull frozen-thawed semen samples

The aim of this study was to investigate the relationship between DNase activity associated with bacterial contamination of incubated bovine frozen-thawed spermatozoa and elevated sperm DNA fragmentation. Electrophoresis analysis of plasmid PBR322 incubated for 30 min at 37 °C with the supernatant of the diluent of frozen-thawed centrifuged bovine semen straws infected with bacteria showed clear evidence of DNase activity when compared to plasmid incubated in similarly prepared non-infected bovine diluent supernatant (Experiment 1). This DNase activity was subsequently found to be time dependent (0–60 min) and its activity prevented in the presence of EDTA (10 and 20 mM; Experiment 2). Semen straws infected (n = 10) and not infected (n = 10) with bacteria where incubated at 37 °C for up to 48h post-thaw. Semen infected with bacteria showed an exponential increase in bacterial growth and a corresponding increase in sperm DNA fragmentation. Non-infected semen samples showed no change in the incidence of sperm DNA fragmentation over the same period of incubation (Experiment 3). Our experiments reinforce the idea that exogenous DNases present in the semen should be considered as one of the primary contributing causes of sperm DNA fragmentation post ejaculation. In the case of the bull, post-thaw incubation of commercial straws contaminated with bacteria, resulted in increased levels of sperm DNA fragmentation, most likely associated with DNase activity (potentially restriction endonucleases) derived from the bacteria. Such adverse changes in sperm DNA fragmentation, as described here in vitro, may be also operative after insemination in the female reproductive tract (in vivo) and highlight the importance of implementing high levels of hygiene practice during semen processing, especially in light of future trends of bacterial resistance to the common antibiotics used in semen diluents.

Americium Sorption by Microplastics in Aqueous Solutions

The interaction of americium by polyamide (PN6) and polyethylene (PE) microplastics (MPs) has been investigated in de-ionized and seawater samples traced with the Am-241 isotope. The effect of the pH and composition of the aqueous solution (seawater) on the sorption efficiency (Kd) have been studied as function of time. In addition, the americium sorption using radionuclide mixtures (e.g., Am-241 and U-232) and its desorption in the presence of EDTA was investigated by means of bath-type experiments. The experimental data (e.g., Kd values) revealed that the sorption efficiency depends on the MP type and the pH that governs the Am(III) speciation in the solution, and the PN6 surface charge determines the sorption efficiency. Moreover, the desorption of Am(III) from MPs is a time-depended process, and the presence of complexing agents (e.g., EDTA) enhances the desorption efficiency. The Kd values in the studied water samples were relatively close to one another and similar to corresponding values in soil systems, indicating the significant role of microplastics on the behavior of americium in natural aquatic systems.

Phytoremediation of CdS/Te quantum dots by Ocimum basilicum in the presence of EDTA.

The use of nanomaterials like quantum dots (QDs) with optical properties has increased in the last decade because of their electronics, medicine, and environmental applications. The lack of recycling and appropriate disposal causes these materials to be considered new emerging contaminants. In this research, the extraction and translocation (phytoextraction) of cadmium as QDs by Ocimum basilicum "basil" in the presence of EDTA (chelating agent) was studied. For edible plants are essential to know where the contaminants are located to minimize human consumption. In this work, the phytoextraction, distribution, and translocation of cadmium (under ionic solution-CdIS and CdS/Te QDs) at 25 and 50mg/kg-soil and in the presence of 1,000mg/kg-soil EDTA was studied in O. basilicum (a commercialized culinary herb) for three and six weeks. Basil seedlings were grown in an environmentally controlled chamber at 24-28o C and 12/12 hours periods of light and darkness. The highest cadmium concentration was found in the roots from where it is translocated to stems and leaves. CdS/Te QDs at low concentration (25mg/kg and 3 weeks of exposure) had the highest translocation factor, indicating that cadmium's nanocrystal (QDs) forms can affect the phytoextraction mechanism. The highest bioconcentration of cadmium was reached at a high contaminant concentration. The bioconcentration factors confirm that O. basilicum phytoextracts cadmium from soils contaminated with CdS/Te QDs and CdIS. The bioconcentration factors of cadmium (under both species CdS/Te QDs and CdIS) increase in the presence of EDTA. The bioconcentration and translocation factors suggest that consuming "basil" from contaminated soils with CdS/Te QDs at low concentrations increases the exposition to this metal.

Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12

Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg−1 towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes.

Amine Oxidation by Electrochemically Generated Peroxodicarbonate

AbstractThe N-oxidation of tertiary amines was achieved by using electrochemically generated peroxodicarbonate solutions as sustainable oxidizers. The presence of EDTA and 2,2,2-trifluoroacetophenone as a mediator was found to be crucial for converting water-insoluble substrates. Various tertiary amines were converted into their corresponding N-oxides in yields of up to 98%. The scope includes economically important surfactants and potential platform oxidizers.

Reprint of: Generation of Superoxide Radical during Autoxidation of Hydroxylamine and an Assay for Superoxide Dismutase

Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of autoxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, indicating the participation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide dismutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.

Synthesis, Characterization of Nano Zinc Sulphide and Evaluation of DNA Binding Interactions

In recent years Bio-Nanotechnology is a new multidisciplinary field of research and development that has offered wide spectrum of potential applications in the medical field. Several hyperproliferative diseases such as cancer are treated by inhibiting DNA synthesis. For the innovative design and implementation of DNA applications in biological systems, it is extremely important to understand how DNA interacts with nanoparticles or small molecules. The ZnS NPs have been synthesized by solvothermal approach by refluxing an aqueous solution of zinc nitrate and Thiourea in presence of EDTA as chelating agent. The interactions of zinc sulfide nanoparticles with calf thymus DNA were investigated and the structural characterization of ZnS nanoparticles was performed using XRD, FTIR, FE-SEM. The average grain size of ZnS nanoparticles were determined from XRD was found to be nearly 3 nm. Binding interaction studies of nanoparticles with CT DNA were carried out using UV–Visible and fluorescence spectroscopy. The binding efficiency was evaluated and the binding constant (Kb) of ZnS was observed to be 1.6x 105M-1 indicating ZnS NPs binds to DNA. The fluorescence quenching investigation revealed that the fluorescence of ZnS Nps was quenched by CT DNA via a static quenching mechanism. The binding studies research on Zns NPs and CT DNA interactions provide new horizons in biomedical applications which may be beneficial for drug designing.

ALKALINE CELLULASE FREE XYLANASE FROM BACILLUS SP. ASX42: PROPERTIES, PURIFICATION AND ITS EFFECT ON SEED GERMINATION

Bacillus sp. ASX42 isolated from soil samples of Lake Van shores, Turkey, and the strain producing cellulose-free xylanase enzyme with an optimum pH 9.0, at 60ºC. The molecular weight of the enzyme was estimated as 66 kDa on SDS PAGE analysis. Thermal stability of enzyme was detected average 68.7% at 4-60ºC and 60% at 65-95ºC while pH stability was observed about 90% between pH 3.5-13.0 for 15 min. As HgCl2 presented a strong inhibitor activity, Cobalt (132%) and Manganese (130%) showed a stimulatory effect on xylanase activity. The remaining activity was found to be 77%, 89%, and 92% in the presence of EDTA, 1,10-Phenanthroline monohydrate and β- mercaptoethanol, respectively. In this study, some known purification materials were compared for effectiveness. According to the results, cellulase-free xylanase ASX42 has shown a stimulatory effect on germination of Anagyris foedita and Ceratonia siliqua seeds. It has also produced a whitening effect on wastepaper pulp.

Exploring Agaricomycetes from the Paranaense rainforest (Misiones, Argentina) as an unconventional source of fibrinolytic enzymes

ABSTRACT Fungal fibrinolytic enzymes, secreted by some Agaricomycetes, are recognized as important thrombolytic agents due to their ability to rapidly dissolve thromboembolic clots. The present work evaluated fibrinolytic and proteolytic secretion abilities of 35 Agaricomycetes isolates from the Paranaense rainforest (Misiones, Argentina). We detected proteolytic activity in 40% of the strains while nine strains showed fibrinolytic activity. Schizophyllum commune LBM 026, Schizophyllum commune LBM 223, and Hornodermoporus martius LBM 224 exhibited the highest levels of fibrinolytic activity. Fibrin zymography from S. commune LBM 026 and LBM 223 showed an enzyme of 27.5 kDa, while H. martius LBM 224 presented an enzyme of 29 kDa. The evaluation of the enzymatic stability of culture supernatant of these strains revealed that the fibrinolytic activity was highly stable over a wide temperature and pH range. Long-term stability of fibrinolytic activity at physiological conditions evidenced that the strains had a half­life of at least 72 h. Fibrinolytic enzymes produced by S. commune LBM 026 and LBM 223 were inhibited in the presence of EDTA indicating that they are metalloproteases. This work reveals the potential of S. commune LBM 026, S. commune LBM 223, and H. martius LBM 224 as an unconventional source of thrombolytic agents.

Tuning the sorption properties of amidoxime-functionalized algal/polyethyleneimine beads for La(III) and Dy(III) using EDTA: Impact of metal speciation on selective separation

Amidoximated algal/polyethyleneimine beads are successfully applied for the sorption of two rare earths elements (REEs, Ln(III)): La (light) and Dy (heavy). The sorption properties are characterized by FTIR and SEM-EDX analyses. The pH effect on metal sorption is investigated with a double objective: optimizing metal recovery and separation. In binary solutions, Dy(III) shows slightly higher affinity for the sorbent than La(III); without significant differences in the selectivity. The difficult separation of REEs can be modulated using EDTA. Playing with the pH and the concentration of the ligand, La(III) can be separated from Dy(III). Selectivity coefficient, SCLa/Dy, may reach up to 40–50. Speciation diagrams show that the preferential formation of Dy-EDTA complexes limits Dy(III) sorption while La(III) remains predominant and highly sorbed (reduced effect of Dy-competition). Optimum conditions for Ln(III) separation can be predicted by the calculation of speciation diagrams. The concept is verified for the separation of La(III) from Er(III) (heavy REE). In the presence of EDTA, sorption isotherms (mono-component solutions) present sigmoidal shapes due to the effect of metal complexation. Converting the total concentration to the concentration of sorbable species (non-complexed by EDTA) allows plotting favorable curves (Langmuir equation). The fits of experimental profiles with the modified Langmuir equation confirm the preference of the sorbent for non-complexed species. The kinetic profiles fitted by the Crank equation highlight the contribution of the resistance to intraparticle diffusion in the control of uptake kinetic; this effect is reinforced by the presence of EDTA. Metal desorption using CaCl2 (50 mM, pH 2) or EDTA (0.5 mM, pH 6) is used for La(III) and Dy(III) desorption (yield: 80–95%), while sorption capacity progressively decreases over 3 cycles.

Influence of EDTA on the interaction between U(VI) and calcite

Uranium contamination is of great concern due to its long-term radioactivity and toxicity. The migration of uranium in environment is well known affected by its interaction with clay minerals and the presence of organic matters. The interaction of U(VI) with calcite in the presence of EDTA across weak to strong alkaline conditions was studied by batch sorption and coprecipitation experiments. Multiple spectroscopic methods and DFT calculation were applied to help analyze the experimental results, and the different roles EDTA plays in the interaction process between uranium and calcite were revealed. It is proposed that at hyperalkaline conditions (pH > 9.5), U(VI) favors to precipitate on calcite surface by forming uranium minerals, yet the presence of EDTA impedes the precipitation of uranium by increase the dissolution of calcite by complexing with Ca2+. In contrast, under weak alkaline conditions (pH 7.5–9.5), EDTA enhances the removal of uranium from solution by calcite, mainly through increasing the specific surface area and sorption sites of calcite due to the steps, vacancies and/or etch pits created by EDTA on calcite surface without modifying its lattice structure and surface functional groups. DFT calculations of Ca−UO2 −CO3 ternary complex interacted with calcite also support the theory that uranyl complexes prefer the sorption on the steps, vacancies and/or etch pits created by EDTA on calcite surface rather than on a flat calcite surface. These results unveil the details of interaction between uranium and calcite, including the presence of EDTA, will help to better understand the migration behavior of uranium in nature, especially in soil minerals.

Pu(iii) and Cm(iii) in the presence of EDTA: aqueous speciation, redox behavior, and the impact of Ca(ii).

The impact of calcium on the solubility, redox behavior, and speciation of the An(iii)–EDTA (An = Pu or Cm) system under reducing, anoxic conditions was investigated through batch solubility experiments, X-ray absorption spectroscopy (XAS), density functional theory (DFT), and time-resolved laser fluorescence spectroscopy (TRLFS). Batch solubility experiments were conducted from undersaturation using Pu(OH)3(am) as the solid phase in contact with 0.1 M NaCl–NaOH–HCl–EDTA–CaCl2 solutions at [EDTA] = 1 mM, pHm = 7.5–9.5, and [CaCl2] ≤20 mM. Additional samples targeted brine systems represented by 3.5 M CaCl2 and WIPP simulated brine. Solubility data in the absence of calcium were well-described by Pu(iii)–EDTA thermodynamic models, thus supporting the stabilization of Pu(iii)–EDTA complexes in solution. Cm(iii)–EDTA TRLFS data suggested the stepwise hydrolysis of An(iii)-EDTA complexes with increasing pH, and current Pu(iii)-EDTA solubility models were reassessed to evaluate the possibility of including Pu(iii)–OH–EDTA complexes and to calculate preliminary formation constants. Solubility data in the presence of calcium exhibited nearly constant log m(Pu)tot, as limited by total ligand concentration, with increasing [CaCl2]tot, which supports the formation of calcium-stabilized Pu(iii)–EDTA complexes in solution. XAS spectra without calcium showed partial oxidation of Pu(iii) to Pu(iv) in the aqueous phase, while calcium-containing experiments exhibited only Pu(iii), suggesting that Ca–Pu(iii)–EDTA complexes may stabilize Pu(iii) over short timeframes (t ≤45 days). DFT calculations on the Ca–Pu(iii)–EDTA system and TRLFS studies on the analogous Ca–Cm(iii)–EDTA system show that calcium likely stabilizes An(iii)–EDTA complexes but can also potentially stabilize An(iii)–OH–EDTA species in solution. This hints towards the possible existence of four major complex types within Ca–An(iii)–EDTA systems: An(iii)–EDTA, An(iii)–OH–EDTA, Ca–An(iii)–EDTA, and Ca–An(iii)–OH–EDTA. While the exact stoichiometry and degree of ligand protonation within these complexes remain undefined, their formation must be accounted for to properly assess the fate and transport of plutonium under conditions relevant to nuclear waste disposal.