Nitrilotriacetic Acid Research Articles

Exploring the promoting effect of nitrilotriacetic acid on hydroxyl radical and humification during magnetite-amended composting of sewage sludge

The OH production by adding magnetite (MGT) alone has been reported in composting. However, the potential of nitrilotriacetic acid (NTA) addition for magnetite-amended sludge composting remained unclear. Three treatments with different addition [control check (CK); T1: 5 % MGT; T2: 5 % MGT + 5 % NTA] were investigated to characterize hydroxyl radical, humification and bacterial community response. The NTA addition manifested the best performance, with the peak OH content increase by 52 % through facilitating the cycle of Fe(Ⅱ)/Fe(Ⅲ). It led to the highest organic matters degradation (22.3 %) and humic acids content (36.1 g/kg). Furthermore, NTA addition altered bacterial community response, promoting relative abundances of iron-redox related genera, and amino acid metabolism but decreasing carbohydrate metabolism. Structural equation model indicated that temperature and Streptomyces were the primary factors affecting OH content. The study suggests that utilizing chelators is a promising strategy to strengthen humification in sewage sludge composting with adding iron-containing minerals.

Siderite with chelator as high-efficiency Fenton reagent to degrade naphthalene via ferrous liberation and carbon dioxide radical anion-mediated iron redox cycle

Siderite (FeCO3) has been verified to be an effective iron material in Fenton-like process, but large dose of FeCO3 and oxidants limits its application. Here, chelators were successfully used to enhance the degradation of naphthalene (NAP). When 1.0 mM of hydrogen peroxide (H2O2) and 1.0 g L−1 of FeCO3 were applied, in the presence of 0.2 mM oxalic acid (OA), 0.1 mM nitrilotriacetic acid (NTA), or 0.1 mM DL-tartaric acid (DL-TA), 99.3 %, 100 %, or 96.7 % of NAP was degraded within 24 h, respectively. The different roles of chelators were confirmed. For OA and DL-TA, due to weak complexation with iron ions, acid effect and generation of carbon dioxide radical anion (CO2−) contributed to high concentration of Fe2+, while for NTA, chelation and CO2− were two major factors. In addition, the excellent potential application of H2O2/FeCO3/chelator was determined because of (1) lower environmental risk of chemicals; (2) strong tolerance to water matrixes; (3) the excellent stability of FeCO3 confirmed by surface characterizations and cyclic experiment, as NAP was completely degraded after the 6th cycle. At last, H2O2/FeCO3/chelator had superiority over other reduced iron minerals due to lower running cost in lab study, better reusability, and environmental friendliness. Therefore, it is expected that H2O2/FeCO3/chelator technique can provide an effective alternative to Fenton-like process in remediation of NAP contaminated sites.

Overcoming Fe0/Cr(VI) redox-induced low electron transfer efficiency under neutral pH by iron-based dual active sites-mediated hydrogen atom activation

Efficiently removing hexavalent chromium (Cr(VI)) using iron-based adsorbents under neutral pH conditions continues to pose a challenge. In this study, we constructed nano zero-valent iron (nZVI) supported by carboxylated multi-walled carbon nanotubes (OCNT) composites with iron-based dual active sites through the nitrilotriacetic acid modification (FOC-NA). The acid dissolution experiment, high-resolution transmission electron microscopy (HRTEM), thermogravimetric analyzer (TGA), X-ray photoelectron spectroscopy (XPS), and Raman analysis proved these active sites consisted of iron-carboxylate complex (−COOFe(II)) and OCNT-confined nZVI (Fe0-in-OCNT). The existence of iron-based dual active sites enhanced electron transfer efficiency in Cr(VI) removal under neutral pH conditions. This leaded to a superior adsorption capacity for Cr(VI), as evidenced by a maximum adsorption capacity of 141.29 mg·g−1 at a pH of 6.5. Meanwhile, the formation of a Cr-containing product (−COOFe-O-Cr) on the surface of FOC-NA substantially hindered the re-release of chromium into the environment. Furthermore, this study proposed the significant involvement of active hydrogen atoms (H*) in the reduction of Cr(VI), as confirmed by quenching experiments and analyses using electron spin resonance (EPR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). The formation pathway and energy barrier of H* and the −COOFe-O-Cr complex were analyzed by density functional theory (DFT) calculations. This study provided insights for the rational design of iron-based adsorbents with dual active sites to facilitate the activation of H* for Cr(VI) reduction under neutral pH conditions.

Microcontaminants degradation in tertiary effluent by modified solar photo-Fenton process at neutral pH using organic iron complexes: Influence of the peroxide source and matrix composition

Peroxymonosulfate (HSO5–) and persulfate (S2O82–), both sources of sulfate radicals (SO4*–), were evaluated for the first time in modified solar photo-Fenton using Fe-chelating agents (nitriloacetic acid (NTA), ethylene diaminetetraacetic acid (EDTA), ethylenediamine-N,N′-disuccinic acid (EDDS) and citrate (Cit)) in comparison with hydrogen peroxide (H2O2) to degrade benzophenone-3 (BP-3), fipronil (FIP) and propylparaben (PPB) (100 µg L–1 each) in municipal tertiary effluent. Increasing HSO5– concentrations from 1.5 to 5.9 mmol L–1 reached ≥ 96 % removal for all iron complexes, while treatment efficiencies for all tested S2O82– concentrations (1.5–5.9 mmol L–1) were limited to 20 % to 55 %. This difference can be attributed to a pH decrease in the HSO5– system. The influence of pH and matrix composition upon treatment performance were also assessed in the FeNTA/S2O82– system, which was more efficient at pH < 7.0 due to higher availability of soluble iron, and partial removal of bicarbonate and humic acids caused by acidification. Matrix composition significantly influenced the process as higher degradation rates were achieved after isolated or combined removals of chloride, bicarbonate and sulfate. The comparison between naproxen (a high electron density compound) degradation and that of BP-3, FIP, and PPB by SO4*– - based advanced oxidation processes (SR-AOPs) was used to evaluate the influence of target compound structures upon treatment performance. S2O82− and HSO5– were ineffective for treatment at pH 7.0. Thus, H2O2 was the best peroxide source for target compounds degradation at neutral pH as this is the natural pH of municipal tertiary effluent and results indicated that the assessment and choice for SR-AOPs should consider the chemical structure of target compounds.

Experimental analysis and prediction of radionuclide solubility using machine learning models: Effects of organic complexing agents

Radioactive wastes contain organic complexing agents that can form complexes with radionuclides and enhance the solubility of these radionuclides, increasing the mobility of radionuclides over great distances from a radioactive waste repository. In this study, four radionuclides (cobalt, strontium, iodine, and uranium) and three organic complexing agents (ethylenediaminetetraacetic acid, nitrilotriacetic acid, and iso-saccharic acid) were selected, and the solubility of these radionuclides was assessed under realistic environmental conditions such as different pHs (7, 9, 11, and 13), temperatures (10 °C, 20 °C, and 40 °C), and organic complexing agent concentrations (10−5–10−2 M). A total of 720 datasets were generated from solubility batch experiments. Four supervised machine learning models such as the Gaussian process regression (GPR), ensemble-boosted trees, artificial neural networks, and support vector machine were developed for predicting the radionuclide solubility. Each ML model was optimized using Bayesian optimization algorithm. The GPR evolved as a robust model that provided accurate predictions within the underlying solubility patterns by capturing the intricate relationships of the independent parameters of the dataset. At an uncertainty level of 95%, both the experimental results and GPR simulated estimations were closely correlated, confirming the suitability of the GPR model for future explorations.

Fe2+-NTA synergized UV254 photolytic defluorination of perfluorooctane sulfonate (PFOS): Enhancing through intramolecular electron density perturbation via electron acquisition

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant posing a risk in environmental persistence, bioaccumulation and biotoxicity. This study was to reach a comprehensive and deeper understanding of PFOS elimination in a UV254 photolytic treatment with the co-presence of Fe2+ and nitrilotriacetic acid trisodium salt (NTA). PFOS defluorination was noticeably enhanced in the UV/Fe2+-NTA treatment compared with UV/NTA, UV/Fe2+ and our previously studied UV/Fe3+ treatments. UV–vis, FTIR, and UPLC/MS-MS results indicated the formation of PFOS-Fe2+-NTA complex in PFOS, Fe2+ and NTA mixture. The transition energy gap of PFOS-Fe2+-NTA decreased below the excitation energy supplied by UV254 irradiation, corresponding with red shift appearing in UV–vis scanning spectrum. This favored intramolecular electron transfer from Fe2+-NTA to PFOS under UV254 irradiation to form electron-accepting PFOS. Molecular electrostatic potential and atom charge distribution analyses suggested electron density rearrangement and perturbation in the perfluorinated carbon chain of electron-accepting PFOS, leading to the decrease in bond dissociation energies. Intermediate products detection suggested the parallel defluorination pathways of PFOS desulfonation, middle carbon chain scission and direct C-F cleavage. NTA exhibited crucial functions in the UV/Fe2+-NTA treatment by holding Fe2+/Fe3+ in soluble form as a chelant and favoring water activation to generate hydrated electrons (eaq−) under UV irradiation as a photosensitizer. Fe2+ acting as the conduit for electron transfer and the bridge of PFOS anion and NTA was thought functioning best at 200 µM in this study. The degree of UV/Fe2+-NTA -synergized PFOS defluorination also depended on eaq− yield and UV254 photon flux. The structure dependence on the electron transfer process of PFOS and PFOA was explored incorporating molecular structure descriptors. Because of possessing greater potential to acquire electrons or less likeliness to donate its electrons than PFOA, PFOS exhibited faster defluorination kinetics in the published “reduction treatments” than “oxidation” ones. Whereas, PFOA defluorination kinetics were at similar level in both “reduction” and “oxidation” treatments.

Dielectric barrier discharge combined with Fe(III)-NTA activated persulfate for efficient degradation of enrofloxacin in water

ABSTRACT Enrofloxacin (ENR) is widely used in aquaculture due to its broad-spectrum activity against a large number of pathogenic bacteria. However, ENR exhibits strong adsorption and a low biodegradation rate in the environment. This study proposes a new method for degrading ENR, which combines dielectric barrier discharge (DBD) with advanced oxidation of peroxymonosulfate (PMS, HSO5 −) and the use of nitrilotriacetic acid (NTA) combined with Fe(III) activator to improve reaction efficiency. The effects on the degradation efficiency were experimentally investigated by adjusting the internal process parameters and adding coexisting substances. The ESR and free radical scavenging experiments showed that both hydroxyl and sulfate radicals contributed to the degradation of the pollutant ENR. Based on the identified intermediates, and three possible degradation pathways were suggested. And the intermediates were less harmful. In conclusion, the Fe(III)-NTA/PMS/DBD system may be an efficient, environmentally friendly and new technology with development potential.

The effect of chelating agents on the Zn-phytoextraction potential of hemp and soil microbial activity

BackgroundHemp (Cannabis sativa) is a crop with a wide range of uses, from the production of fiber and seeds to the secondary metabolites for medicinal purposes. In addition, it is characterized by high biomass yield and the ability to accumulate heavy metals, which makes this plant convenient for phytoremediation purposes. In this study, the effect of applying exogenous biodegradable chelating agents, citric acid (CA) and nitrilotriacetic acid (NTA), to zinc-contaminated soil on zinc (Zn) uptake by two industrial hemp varieties ‘Felina 32’ and ‘Monoica’ was studied. The effect of CA and NTA on available Zn in soils was investigated using an ‘in pot’ experiment under controlled conditions. The effect of both tested compounds on soil microbial activity was simultaneously evaluated.ResultsAfter the application of NTA at a concentration of 5 mmol L−1, a > threefold increased accumulation of Zn in the above-ground parts was recorded in the ‘Felina 32’ variety. In the ‘Monoica’ variety, the levels of Zn in the above-ground parts were increased > twofold. NTA affected the soil microbiome negatively, causing decreased enzyme activity (in ‘Monoica’ planted soil) and induced respiration (in ‘Monoica’ and especially in ‘Felina 32’ planted soil). On the other hand, CA application did not lead to significantly increased Zn levels in any of the studied hemp varieties. Together with CA’s negative effects on some soil enzymes, CA enhanced urease activity, dehydrogenase and several respiration types for the ‘Felina 32’ variety and exerted less detrimental effect on the soil microbiome. No toxic effects from increased Zn uptake and accumulation in experimental plants were detected, accounting for the unchanged physiological stress markers (levels of photosynthetic pigments and proline in leaves, chlorophyll fluorescence parameters) and selected growth traits of the above-ground organs and root system.ConclusionsFrom the studied varieties, ‘Felina 32’ seems to be more suitable for Zn-phytoextraction because of its higher tolerance to increased Zn levels, higher biomass production and Zn accumulation capacity. Our results indicate the potential of using the ‘Felina 32’ variety in NTA-assisted Zn phytoextraction from contaminated soils.Graphical

Flotation Separation of Fluorite from Calcite using an Efficient Depressant Nitrilotriacetic Acid in the NaOL System.

Fluorite and calcite were separated with nitrilotriacetic acid (NTA) as a depressant. The single mineral flotation experiment confirmed that with 40 mg/L NaOL and 80 mg/L NTA, the fluorite recovery and calcite recovery were 24.37 and 94.13%, respectively, at pH 9. Meanwhile, in the fluorite-calcite binary mixed ore flotation experiment, the calcite recovery and fluorite recovery were 75.50 and 26.84%, respectively, and the CaCO3 and CaF2 grade in concentrate was 74.32 and 25.61%, respectively. The results confirmed that NTA could be used as a depressant to selectively inhibit fluorite flotation. The mechanism study illustrated that NTA was selectively reacted with fluorite by chemical interaction between O of NTA and Ca of fluorite. The adsorption of NTA on fluorite will impede the interaction between fluorite and NaOL. NTA could adsorb on fluorite in three ways, while the dominant two ways were the complex between double O of NTA and Ca of fluorite in a vertical model and the complex between double O of NTA and Ca of fluorite in a horizontal model.

The role of ligand in the activation of peroxymonosulfate by Fe3O4 for the degradation of organic pollutants

Advanced oxidation processes based on peroxymonosulfate (PMS) hold remarkable promise for addressing emerging organic pollutants (EOPs) and are worthy of systematic investigation. However, the existing technology has not been widely applied due to bottlenecks such as reaction efficiency. Herein, we proposed a novel system incorporating nitrilotriacetic acid (NTA) as a functional ligand to enhance the activation of PMS by Fe3O4, thereby facilitating a better efficiency for the degradation of EOPs. Results indicated that the system can achieve an optimal removal rate of 99.6% for atrazine (ATZ) in the presence of NTA. Quenching experiments and electron paramagnetic resonance techniques were employed for mechanism exploration. The results revealed that the enhanced Fe(II)/Fe(III) cycling on the surface of Fe3O4, triggered by the addition of NTA, accelerates the reduction of trivalent iron and continuously activates PMS to generate radicals, particularly SO4−. The mechanistic analysis showed that in the first stage, the lower degradation efficiency could due to the self-quenching of the free radicals, with the uncomplexed Fe(II) and Fe(III) on the surface, the free NTA in the solution, or with the PMS, in addition to reacting with the pollutants. In the second stage, the ATZ achieved rapid degradation as the concentration of substances competing for free radicals decreased. Our work elucidates the key role of NTA functional ligand in the Fe3O4/PMS advanced oxidation processes, which may provide new approach for the efficient degradation of EOPs.

Carrier-based immobilization of Aerococcus viridansl-lactate oxidase

l-Lactate oxidase has important applications in biosensing and finds increased use in biocatalysis. The enzyme has been characterized well, yet its immobilization has not been explored in depth. Here, we studied immobilization of Aerococcus viridansl-lactate oxidase on porous carriers of variable matrix material (polymethacrylate, polyurethane, agarose) and surface functional group (amine, Ni2+-loaded nitrilotriacetic acid (NiNTA), epoxide). Carrier activity (Ac) and immobilized enzyme effectiveness (ɳ) were evaluated in dependence of protein loading. Results show that efficient immobilization (Ac: up to 1450 U/g carrier; ɳ: up to 65%) requires a hydrophilic carrier (agarose) equipped with amine groups. The value of ɳ declines sharply as Ac increases, probably due to transition into diffusional regime. Untagged l-lactate oxidase binds to NiNTA carrier similarly as N-terminally His-tagged enzyme. Lixiviation studies reveal quasi-irreversible enzyme adsorption on NiNTA carrier while partial release of activity (≤ 25%) is shown from amine carrier. The desorbed enzyme exhibits the same specific activity as the original l-lactate oxidase. Collectively, our study identifies basic requirements of l-lactate oxidase immobilization on solid carrier and highlights the role of ionic interactions in enzyme-surface adsorption.

Decorated Magnetic Nanocatalyst with Trinitriloacetic Acid as a Heterogeneous Catalyst used in the Synthesis of Pyrimido[4,5]quinoline‐2,4‐diones

AbstractTo answer the challenges of using nitrilotriacetic acid (NTAA) as an efficient, recyclable, and durable acidic nanocatalyst, a novel, versatile, engineerable, affordable, eco‐friendly acidic heterogeneous magnetic nanocatalyst has been synthesized with high thermal stability and excellent catalytic efficiency through immobilization of NTTA on the surface of the functionalized magnetite core‐shell. Fe3O4@SiO2‐NH‐NTAA, has been synthesized and presented here. This nanomagnetic catalyst, Fe3O4@SiO2‐NH‐NTAA, was fully identified and characterized through analytical techniques such as FTIR, FESEM, TEM, EDX, elemental mapping, TGA‐DSC, DLS, VSM, and XRD, in addition to theoretical calculations based on DFT/B3LYP/6‐311++G(d, p). Its catalytic capability as well as easy separation, recoverability, and reuseability were examined in the one‐pot preparation of pyrimido[4,5]quinolone‐2,4‐diones under mild conditions, where the high performance of the Fe3O4@SiO2‐NH‐NTAA magnetic nano‐catalyst was proved by excellent reaction yield (&gt;98 %) and the catalyst's recovery efficiency (&gt;98 %). Due to the presence of NTAA's zwitterionic forms in the outermost layer of Fe3O4@SiO2‐NH‐NTAA, this acidic catalyst causes the progress and control of the three‐component reaction in this article well and in the desired direction.

Transition Metal Ion FRET-Based Probe to Study Cu(II)-Mediated Amyloid-β Ligand Binding.

Recent therapeutic strategies suggest that small peptides can act as aggregation inhibitors of monomeric amyloid-β (Αβ) by inducing structural rearrangements upon complexation. However, characterizing the binding events in such dynamic and transient noncovalent complexes, especially in the presence of natively occurring metal ions, remains a challenge. Here, we deploy a combined transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) approach to characterize the structure of mass- and charge-selected Aβ complexes with Cu(II) ions (a quencher) and a potential aggregation inhibitor, a small neuropeptide named leucine enkephalin (LE). We show conformational changes of monomeric Αβ species upon Cu(II)-binding, indicating an uncoiled N-terminus and a close interaction between the C-terminus and the central hydrophobic region. Furthermore, we introduce LE labeled at the N-terminus with a metal-chelating agent, nitrilotriacetic acid (NTA). This allows us to employ tmFRET to probe the binding even in low-abundance and transient Aβ-inhibitor-metal ion complexes. Complementary intramolecular distance and global shape information from tmFRET and native IM-MS, respectively, confirmed Cu(II) displacement toward the N-terminus of Αβ, which discloses the binding region and the inhibitor's orientation.

Anaerobic ammonium oxidation coupled to iron(III) reduction catalyzed by a lithoautotrophic nitrate-reducing iron(II) oxidizing enrichment culture.

The last two decades have seen nitrogen/iron-transforming bacteria at the forefront of new biogeochemical discoveries, such as anaerobic ammonium oxidation coupled to ferric iron reduction (feammox) and lithoautotrophic nitrate-reducing ferrous iron-oxidation (NRFeOx). These emerging findings continue to expand our knowledge of the nitrogen/iron cycle in nature and also highlight the need to re-understand the functional traits of the microorganisms involved. Here, as a proof-of-principle, we report compelling evidence for the capability of an NRFeOx enrichment culture to catalyze the feammox process. Our results demonstrate that the NRFeOx culture predominantly oxidizes NH4+ to nitrogen gas, by reducing both chelated nitrilotriacetic acid (NTA)-Fe(III) and poorly soluble Fe(III)-bearing minerals (γ-FeOOH) at pH 4.0 and 8.0, respectively. In the NRFeOx culture, Fe(II)-oxidizing bacteria of Rhodanobacter and Fe(III)-reducing bacteria of unclassified_Acidobacteriota coexisted. Their relative abundances were dynamically regulated by the supplemented iron sources. Metagenomic analysis revealed that the NRFeOx culture contained a complete set of denitrifying genes along with hao genes for ammonium oxidation. Additionally, numerous genes encoding extracellular electron transport-associated proteins or their homologs were identified, which facilitated the reduction of extracellular iron by this culture. More broadly, this work lightens the unexplored potential of specific microbial groups in driving nitrogen transformation through multiple pathways and highlights the essential role of microbial iron metabolism in the integral biogeochemical nitrogen cycle.

Multiresponsive Core-Shell Microgels Functionalized by Nitrilotriacetic Acid.

Stimuli-responsive microgels with ionizable functional groups offer versatile applications, e.g., by the uptake of oppositely charged metal ions or guest molecules such as drugs, dyes, or proteins. Furthermore, the incorporation of carboxylic groups enhances mucoadhesive properties, crucial for various drug delivery applications. In this work, we successfully synthesized poly{N-vinylcaprolactam-2,2'-[(5-acrylamido-1-carboxypentyl)azanediyl]diacetic acid} [p(VCL/NTAaa)] microgels containing varying amounts of nitrilotriacetic acid (NTA) using precipitation polymerization. We performed fundamental characterization by infrared (IR) spectroscopy and dynamic and electrophoretic light scattering. Despite their potential multiresponsiveness, prior studies on NTA-functionalized microgels lack in-depth analysis of their stimuli-responsive behavior. This work addresses this gap by assessing the microgel responsiveness to temperature, ionic strength, and pH. Morphological investigations were performed via NMR relaxometry, nanoscale imaging (AFM and SEM), and reaction calorimetry. Finally, we explored the potential application of the microgels by conducting cytocompatibility experiments and demonstrating the immobilization of the model protein cytochromec in the microgels.

Enzymatically-induced dynamic assemblies from surface functional stomatocyte nanoreactors.

Collective behavior has become a recent topic of investigation in systems chemistry. In pursuing this phenomenon, we present polymersome stomatocytes loaded with the enzyme urease, which show basic stigmergy-based communication and are capable of signal production, reception, and response by clustering with surface complementary binding partners. The collective behavior is transient and based on the widely known pH-sensitive non-covalent interactions between nitrilotriacetic acid (NTA) and histidine (His) moieties attached to the surface of urease-loaded and empty stomacytes, respectively. Upon the addition of the substrate urea, the urease stomatocytes are able to increase the environmental pH, allowing the NTA units to interact with the surface histidines on the complementary species, triggering the formation of transient clusters. The stomatocytes display a maximum clustering interaction at a pH between 6.3 and 7.3, and interparticle repulsive behavior outside this range. This leads to oscillating behavior, as the aggregates disassemble when the pH increases due to high local urease activity. After bulk pH conditions are restored, clustering can take place again. Within the detectable region of dynamic light scattering, individual stomatocytes can aggregate to agglomerates with 10 times their volume. Understanding and designing population behavior of active colloids can facilitate the execution of cooperative tasks, which are not feasible for individual colloids.

Preparation of a water-soluble molybdenum(VI) complex with nitrilotriacetic acid and monoethano-lamine. Molecular structure of triammonium [trioxo(nitrilotriacetato)molybdate] monohydrate (NH4)3[MoO3L]·H2O. Application of its solution to increase the productivity of red clover.

Preparation of a water-soluble molybdenum(VI) complex with nitrilotriacetic acid and monoethano-lamine. Molecular structure of triammonium [trioxo(nitrilotriacetato)molybdate] monohydrate (NH4)3[MoO3L]·H2O. Application of its solution to increase the productivity of red clover.

Isolation of yeast mitochondria by affinity purification using magnetic beads.

Isolated mitochondria have been widely utilized in various model organisms to investigate the diverse functions of the organelle. Techniques such as differential centrifugation, density gradient ultracentrifugation and antibody-coated magnetic beads are employed for isolation of the organelle from whole cells. However, mitochondria isolated using differential centrifugation are often contaminated with other organelles; isolation using density gradient ultracentrifugation can reduce contamination but is time-intensive and requires large amounts of starting materials; and mitochondria isolated using antibody-coated magnetic beads are irreversibly bound to the beads. Here, we provide a step-by-step protocol for the isolation of highly pure mitochondria from Saccharomyces cerevisiae using a magnetic bead affinity purification method that overcomes these limitations. This protocol describes how to isolate mitochondria, tagged by insertion of 6 histidines (6xHis) into the chromosomal copy of the TOM70 (Translocase of outer membrane 70) gene using Ni-NTA (nickel(II) nitrilotriacetic acid) paramagnetic beads, and the subsequent release of mitochondria from the beads using a buffer containing imidazole. We provide examples of expected results, highlighting the purity, integrity and import activity of isolated mitochondria. These affinity-purified mitochondria are intact and functional, containing less contamination with cytosol and other organelles compared to mitochondria isolated by other methods. Our method is adaptable and can be applied to other model organisms that can be genetically manipulated using CRISPR or other methods.

Functional nanochaperones for PEGylated insulin delivery in long-term glycemic control.

PEGylation is a promising strategy for modulating the physicochemical properties and improving the therapeutic efficacy of protein drugs. However, the application of multi-PEGylation frequently results in diminished protein activity. A single low molecular weight PEG (5 kDa) modified at the amino terminus of the B chain preserves the biological activity of insulin and moderately improves its pharmacokinetics. Nonetheless, this modification offers limited protein stabilization. Furthermore, overdoses still carry the risk of hypoglycemia, posing challenges for the clinical application of PEGylated insulin. Here, we constructed multifunctional nanochaperones featuring phenylboronic acid (PBA) modified hydrophobic microdomains and nitrilotriacetic acid (NTA)-based coordination domains (PN-nChaps) for PEGylated insulin delivery. This delivery strategy effectively overcomes the limitations associated with PEGylation by enhancing the stability and reducing the immunogenicity of PEGylated insulin, while enabling glucose-responsive controlled release. PEGylated insulin with nanochaperone carrier demonstrates a prolonged half-life (t1/2 = 18.66 h), facilitates on-demand release, and minimizes the risk of hypoglycemia. This approach provides a safe and effective strategy for long-term glycemic management in diabetic patients.

Spin labels for 19F ENDOR distance determination: resolution, sensitivity and distance predictability.

19F electron-nuclear double resonance (ENDOR) has emerged as an attractive method for determining distance distributions in biomolecules in the range of 0.7-2 nm, which is not easily accessible by pulsed electron dipolar spectroscopy. The 19F ENDOR approach relies on spin labeling, and in this work, we compare various labels' performance. Four protein variants of GB1 and ubiquitin bearing fluorinated residues were labeled at the same site with nitroxide and trityl radicals and a Gd(III) chelate. Additionally, a double-histidine variant of GB1 was labeled with a Cu(II) nitrilotriacetic acid chelate. ENDOR measurements were carried out at W-band (95 GHz) where 19F signals are well separated from 1H signals. Differences in sensitivity were observed, with Gd(III) chelates providing the highest signal-to-noise ratio. The new trityl label, OXMA, devoid of methyl groups, exhibited a sufficiently long phase memory time to provide an acceptable sensitivity. However, the longer tether of this label effectively reduces the maximum accessible distance between the 19F and the Cα of the spin-labeling site. The nitroxide and Cu(II) labels provide valuable additional geometric insights via orientation selection. Prediction of electron-nuclear distances based on the known structures of the proteins were the closest to the experimental values for Gd(III) labels, and distances obtained for Cu(II) labeled GB1 are in good agreement with previously published NMR results. Overall, our results offer valuable guidance for selecting optimal spin labels for 19F ENDOR distance measurement in proteins.