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1.
Ecotoxicol Environ Saf ; 205: 111293, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32949840

RESUMO

Wastewater from printing and dyeing processes often contains aniline and high salinity, which are hazardous to aquatic species. Glycophytic plants cannot survive under high-salinity conditions, whereas halophytes grow well in such an environment. In this study, we investigated the influence of NaCl on the antioxidant level in Suaeda salsa affected by aniline stress. The seedlings showed various growth toxicity effects under different concentrations of aniline. The results showed that the effect of the aniline was more severe for the root growth compared to that for the shoot growth. Aniline exposure significantly increased the total free radicals and ·OH radicals in the plants. Suaeda salsa exposure to aniline caused oxidative stress by altering the superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity, which resulted in the overproduction of H2O2 and the inducement of lipid peroxidation. Analysis revealed that the malondialdehyde (MDA) content was enhanced after aniline exposure and that the chlorophyll content was significantly decreased. The results showed that aniline induced the production of free radicals and reactive oxygen species (ROS), and changed the antioxidant defense system. This ultimately resulted in oxidative damage in S. salsa; however, it was found that moderate salinity could mitigate the effects. In conclusion, salinity may alleviate the growth inhibition caused by aniline by regulating the antioxidant capacity of S. salsa.


Assuntos
Compostos de Anilina/toxicidade , Antioxidantes/metabolismo , Chenopodiaceae/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Poluentes Químicos da Água/toxicidade , Catalase/metabolismo , Chenopodiaceae/enzimologia , Chenopodiaceae/crescimento & desenvolvimento , Clorofila/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Salinidade , Plantas Tolerantes a Sal/efeitos dos fármacos , Plantas Tolerantes a Sal/enzimologia , Plantas Tolerantes a Sal/crescimento & desenvolvimento , Plântula/efeitos dos fármacos , Plântula/enzimologia , Plântula/crescimento & desenvolvimento , Superóxido Dismutase/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(34): 20566-20575, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788347

RESUMO

The complexity of the cellular medium can affect proteins' properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer-monomer dissociation constant K D was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.


Assuntos
Multimerização Proteica , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Dimerização , Espectroscopia de Ressonância de Spin Eletrônica , Células HeLa , Humanos , Conformação Proteica
3.
Proc Natl Acad Sci U S A ; 117(32): 19178-19189, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32723819

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x2-y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.


Assuntos
Bacillus licheniformis/enzimologia , Proteínas de Bactérias/química , Quitina/metabolismo , Oxigenases de Função Mista/química , Oxigênio/metabolismo , Bacillus licheniformis/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Quitina/química , Cobre/química , Cobre/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Imagem por Ressonância Magnética , Oxigenases de Função Mista/metabolismo , Oxigênio/química , Especificidade por Substrato
4.
Food Chem ; 331: 127314, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32590264

RESUMO

The formation of short-lived and stable radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy and compared with hydroperoxides and hexanal in complex starch-protein-lipid model systems, as well as in corn extrudates. Stable radicals were detected directly in ground samples. Short-lived lipid radicals were measured ex situ in ethyl acetate extracts of model systems and extrudates by the use of the spin trap PBN. Significant adduct formation was found after 30 min at 50 °C. During storage, lipid radicals (PBN adducts) increased in model systems. Simulation of EPR spectra from bulk oil demonstrated that mainly alkoxyl radical adducts were detected, to which rapidly decomposing peroxyl radical adducts also contributed. Stable radicals in extrudates were attributed to protein radicals based on g-value of 2.00467 compared with 2.00474 found in model system prepared with zein. The signal intensity of the stable radical remained constant during storage, but increased during extrusion.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Lipídeos/química , Proteínas de Plantas/química , Amido/química , Zea mays/química
5.
Chemosphere ; 258: 127120, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32544808

RESUMO

Diclofenac (DCF) control measures have become an area of increased interest for environmental researchers due to the high environmental concentration and risk of DCF. Adsorption seems to be promising for DCF removal from the aqueous phase because of its specific superiority in comparison with biodegradation, membrane separation, and advanced oxidation or reduction. In this study, OMS-2 and metal-doped OMS-2 ((Me-OMS-2, with Me = Co, Cu or Ce) were prepared and tested as adsorbents for the removal of DCF. It was evident that the maximum adsorption capacity and rate of Ce-OMS-2 were much higher than those of the other adsorbents, which could be attributed to its large specific surface area and stereoscopic aperture structure. The experimental data are fitted the pseudo-second-order model, the Elovich equation and the Langmuir model well; moreover, the process is an endothermic and spontaneous thermodynamic process, during which the entropy increased, based on the experimental results, indicating that chemisorption was dominant during the DCF adsorption process onto Ce-OMS-2. By the integral of the peak deconvoluted from the XPS spectrum, the ratio of Mn3+/Mn4+ increased from 0.393 to 0.407, revealing that Mn(IV) is rarely reduced into Mn(III) during the DCF adsorption process.


Assuntos
Diclofenaco/química , Diclofenaco/isolamento & purificação , Manganês/química , Poluentes Químicos da Água/isolamento & purificação , Adsorção , Espectroscopia de Ressonância de Spin Eletrônica , Substâncias Húmicas , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Termodinâmica , Água , Poluentes Químicos da Água/química
6.
PLoS One ; 15(5): e0232555, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392255

RESUMO

We report an easy, efficient and reproducible way to prepare Rapid-Freeze-Quench samples in sub-millimeter capillaries and load these into the probe head of a 275 GHz Electron Paramagnetic Resonance spectrometer. Kinetic data obtained for the binding reaction of azide to myoglobin demonstrate the feasibility of the method for high-frequency EPR. Experiments on the same samples at 9.5 GHz show that only a single series of Rapid-Freeze-Quench samples is required for studies at multiple microwave frequencies.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Animais , Espectroscopia de Ressonância de Spin Eletrônica/instrumentação , Congelamento , Cavalos , Cinética , Micro-Ondas , Mioglobina/química
7.
Bioresour Technol ; 312: 123555, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32447123

RESUMO

In-situ detection on primary volatiles and stable radicals is of great importance for better understanding of lignin pyrolysis mechanisms and utilization. In this study, a novel in-situ pyrolysis time-of-flight mass spectrometry with double ionization sources was taken to in-situ detect primary volatiles and gas products, and the evolution of stable radicals in lignin pyrolysis residues was explored by EPR spectroscopy. The results show that the cleavage of ß-O-4 linkage is mainly responsible for lignin depolymerization at 100-300 °C, releasing the G-type compounds. And these G-type compounds can further undergo O-CH3, Car-OCH3 and Car-OH bonds cleavage to form biphenolic hydroxyl compounds, phenols and aromatic hydrocarbons. According to the EPR analysis, the radical concentration increased from 1017 to 1019 spins/g with the temperature, and stable free-radical species are mainly composed of the o-methoxy and hydroxyl substituted phenoxy radicals and carbon-centered aromatic radicals, which can well interpret the demethylation, demethoxylation and dehydroxylation mechanisms.


Assuntos
Lignina , Pirólise , Espectroscopia de Ressonância de Spin Eletrônica , Temperatura Alta , Espectrometria de Massas
8.
Nat Commun ; 11(1): 2315, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385229

RESUMO

As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.


Assuntos
Colágeno/química , Tendões/química , Animais , Materiais Biocompatíveis/química , Biopolímeros/química , Di-Hidroxifenilalanina/química , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Oxirredução , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio/química
9.
Proc Natl Acad Sci U S A ; 117(22): 11916-11922, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414932

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with both [Formula: see text] and [Formula: see text] as cosubstrates. In this study, the [Formula: see text] reaction with reduced Hypocrea jecorina LPMO9A (CuI-HjLPMO9A) is demonstrated to be 1,000-fold faster than the [Formula: see text] reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze-quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce CuII-HjLPMO9A and indicates homolytic O-O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the CuII sites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.


Assuntos
Aminoácidos/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxigenases de Função Mista/química , Polissacarídeos/metabolismo , Sítios de Ligação , Biocombustíveis , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Hypocrea/metabolismo , Cinética , Espectroscopia de Ressonância Magnética/métodos , Oxigenases de Função Mista/metabolismo , Oxirredução , Peróxidos/metabolismo , Triptofano/metabolismo , Tirosina/metabolismo
10.
Igaku Butsuri ; 40(1): 13-18, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32238677

RESUMO

Hypoxia has been known to be a feature associated with tumor radioresistance. So far, clinical strategies to overcome chronic hypoxia due to the limitation of the oxygen diffusion have been designed. However, intermittent or acute/cycling hypoxia, whose frequency can range between a few cycles per minutes to hours, is receiving increased attention, because this type of hypoxia has been reported to have an influence on tumor malignancy as well as treatment resistance via increased expression of pro-survival pathways. Therefore, a priori information on fluctuating hypoxia can be important in clinical treatment planning, but complicated dynamics makes it difficult to elucidate biological significance of intermittent hypoxia.Here, we illustrate the use of pulsed electron spin resonance imaging (ESRI) as a novel imaging method to directly monitor fluctuating oxygenation i.e. cycling hypoxia in transplanted tumors. A common resonator platform for both ESRI and magnetic resonance imaging (MRI) provided pO2 maps with anatomical guidance without positional movement. Oxygen images every 3 min in pO2 could visualize the rapid oxygen fluctuation and distinguish the cycling hypoxia and chronic hypoxia. Furthermore, we have examined the vascular renormalization process by longitudinally pO2 mapping during treatments with a multi-tyrosine kinase inhibitor sunitinib. Transient improvement in tumor oxygenation and the decrease of cycling tumor hypoxia were visualized by ESRI 2 to 4 days following antiangiogenic treatments. Radiation treatment during this time period of improved oxygenation by antiangiogenic therapy resulted in a synergistic delay in tumor growth.In conclusion, this ESRI technique combined with MRI, may offer a powerful clinical tool to noninvasively detect variable hypoxic status in tumors and to identify a window of vascular renormalization to maximize the effects of combination therapy with antiangiogenic drugs.


Assuntos
Neoplasias , Radiobiologia , Hipóxia Tumoral , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Hipóxia , Neoplasias/diagnóstico por imagem , Neoplasias/fisiopatologia , Oxigênio
11.
Nat Commun ; 11(1): 1757, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273505

RESUMO

NifB is a radical S-adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this MaNifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical.


Assuntos
Proteínas de Bactérias/metabolismo , Methanosarcina/metabolismo , Nitrogênio/metabolismo , Nitrogenase/biossíntese , Alanina/genética , Alanina/metabolismo , Proteínas de Bactérias/genética , Espectroscopia de Ressonância de Spin Eletrônica , Histidina/genética , Histidina/metabolismo , Ligantes , Methanosarcina/genética , Mutagênese , Nitrogenase/genética , Espectroscopia por Absorção de Raios X
12.
Chemosphere ; 253: 126662, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32268253

RESUMO

In this study, pyrite (FeS2) was used as a novel activator of calcium peroxide (CaO2) for the degradation of diethyl phthalate (DEP) in both aqueous solution and soil. DEP (10 mg/L) in aqueous solution was completely degraded within 5.0 min by the FeS2 (0.30 g/L)/CaO2 (1.0 mM) system at pH 3.5. X-ray diffraction (XRD), scanning electron microscopy (SEM), electron paramagnetic resonance (EPR), free radical quenching, and X-ray photoelectron spectroscopy (XPS) were used to elucidate the mechanism of the catalytic decomposition of CaO2, radical formation and DEP degradation in the presence of by pyrite. The results show that hydroxyl radicals (OH) are the dominant active species responsible for DEP degradation. Surface or lattice Fe(II) of FeS2 readily activates H2O2 generated by CaO2 decomposition to produce OH, while the reducing sulfur species of FeS2 promotes the regeneration of surface of Fe(II) that catalyzes the production of additional OH, leading to the efficiently oxidative degradation of DEP. Although high concentration of common anions, such as Cl-, NO3-, SO42-, and HCO3-, exert inhibitory effects on DEP degradation by pyrite/CaO2, the reaction system can still efficiently degrade DEP in realistic soil. It was observed that 78% of DEP (25 mg kg-1) was degraded by 2.5% CaO2 (w/w) and 0.5% FeS2 (w/w) within 24 h. These results provide new insight into the mechanistic processes of CaO2 activation and OH formation by the novel FeS2 catalyst, demonstrating a promising alternative to the traditional H2O2-base Fenton process for contaminated soil remediation.


Assuntos
Ferro/química , Peróxidos/química , Ácidos Ftálicos/química , Sulfetos/química , Catálise , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Peróxido de Hidrogênio/química , Radical Hidroxila/química , Oxirredução , Espectroscopia Fotoeletrônica , Enxofre
13.
Arch Biochem Biophys ; 684: 108323, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32126206

RESUMO

Electron Paramagnetic Resonance is a spectroscopic technique which, in combination with site-directed spin-labeling, provides structural and dynamic information about proteins in conditions similar to those of their physiological environment. The information is sequence-resolved, as it is based on probing the local dynamics of a paramagnetic label incorporated as a side chain of a selected amino acid. EPR does not impose a limit on the size of the protein or protein complex, as long as it is amenable to site-directed mutagenesis, and is able to obtain reliable distance distributions between two or more labels (identical or different).. The mean value, width and shape of distance distributions, as well as their dependence upon the state of the protein or interactions with physiological partners, provide insight into order-disorder transitions and the roles of protein flexibility. The main potentialities and limitations of the technique are revised and illustrated with examples of proteins for which order-disorder play an important role.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Proteínas Intrinsicamente Desordenadas/química , Marcadores de Spin , Óxidos N-Cíclicos/química , Cisteína/química , Maleabilidade , Conformação Proteica
14.
Biochim Biophys Acta Proteins Proteom ; 1868(6): 140413, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32179182

RESUMO

Only recently it was discovered that haemoglobin (Hb) belongs to the standard gene repertoire of insects, although their tracheal system is used for respiration. A classical oxygen-carrying function of Hb is only obvious for hexapods living in hypoxic environments. In other insect species, including the common fruit fly Drosophila melanogaster, the physiological role of Hb is yet unclear. Here, we study recombinant haemoglobin from the European honeybee Apis mellifera (Ame) and the malaria mosquito Anopheles gambiae (Aga). Spectroscopic evidence shows that both proteins can be classified as hexacoordinate Hbs with a strong affinity for the distal histidine. AgaHb1 is proposed to play a role in oxygen transport or sensing based on its multimeric state, slow autoxidation, and small but significant amount of five-coordinated haem in the deoxy ferrous form. AmeHb appears to behave more like vertebrate neuroglobin with a complex function given its diversified distribution in the genome.


Assuntos
Anopheles/metabolismo , Abelhas/metabolismo , Hemoglobinas/análise , Sistema Respiratório/metabolismo , Análise Espectral/métodos , Animais , Anopheles/genética , Abelhas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Evolução Molecular , Compostos Férricos/química , Compostos Ferrosos/química , Genoma , Heme/metabolismo , Hemoglobinas/genética , Insetos/genética , Insetos/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Oxigênio
15.
Nucleic Acids Res ; 48(9): e49, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32095832

RESUMO

Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2'dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.


Assuntos
Cobre/química , DNA/química , Espectroscopia de Ressonância de Spin Eletrônica , Aminas/química , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Ácidos Picolínicos/química
16.
Chemistry ; 26(43): 9495-9505, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32059063

RESUMO

The directionality of the hole-transfer processes between DNA backbone and base was investigated by using phosphorodithioate [P(S- )=S] components. ESR spectroscopy in homogeneous frozen aqueous solutions and pulse radiolysis in aqueous solution at ambient temperature confirmed initial formation of G.+ -P(S- )=S. The ionization potential of G-P(S- )=S was calculated to be slightly lower than that of guanine in 5'-dGMP. Subsequent thermally activated hole transfer from G.+ to P(S- )=S led to dithiyl radical (P-2S. ) formation on the µs timescale. In parallel, ESR spectroscopy, pulse radiolysis, and density functional theory (DFT) calculations confirmed P-2S. formation in an abasic phosphorodithioate model compound. ESR investigations at low temperatures and higher G-P(S- )=S concentrations showed a bimolecular conversion of P-2S. to the σ2 -σ*1 -bonded dimer anion radical [-P-2S - . 2S-P-]- [ΔG (150 K, DFT)=-7.2 kcal mol-1 ]. However, [-P-2S - . 2S-P-]- formation was not observed by pulse radiolysis [ΔG° (298 K, DFT)=-1.4 kcal mol-1 ]. Neither P-2S. nor [-P-2S - . 2S-P-]- oxidized guanine base; only base-to-backbone hole transfer occurs in phosphorodithioate.


Assuntos
Ânions/química , DNA/química , Guanina/química , Nucleosídeos/química , Fosfatos/química , Espectroscopia de Ressonância de Spin Eletrônica , Oxirredução , Radiólise de Impulso , Água/química
17.
Chem Pharm Bull (Tokyo) ; 68(2): 150-154, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32009082

RESUMO

Singlet oxygen (1O2) is highly oxidative and exerts strong cytotoxic effects. We tried to establish the best combination of a singlet oxygen generation system and a detection method with ESR, for measurement of the quenching activities of various substances. The photosensitizing reaction of rose bengal or thermal decomposition of 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide, EP) was used for the generation of 1O2, and a sterically hindered secondary amine, 2,2,6,6-tetramethyl-4-piperidone (TEMPD) or 2,2,6,6-tetramethyl-4-piperidinol (TEMP-OH), was used as the 1O2 detection probe. These secondary amines were oxidized by 1O2 to form stable nitroxide radicals, which were detectable by ESR. TEMPD was found to be readily oxidized by air, causing large background signals in comparison with TEMP-OH. The ESR signal obtained by the irradiation of rose bengal with visible light in the presence of TEMP-OH consisted of two kinds of nitroxide radical overlapping. In contrast, only a single nitroxide signal was observed when TEMP-OH was reacted with 1O2 generated from EP. Therefore, the best combination should be EP as the 1O2 generator and TEMP-OH as the detection probe. When using this combination, we found that the concentrations of some organic solvents such as dimethyl sulfoxide and acetonitrile should be kept constant for reliable quantification, because the concentrations of organic solvents affect the ESR signal intensity.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Oxigênio Singlete/análise , Oxirredução , Fármacos Fotossensibilizantes/química , Piperidonas/química , Propionatos/química , Rosa Bengala/química
18.
Environ Sci Technol ; 54(6): 3169-3180, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32083476

RESUMO

Gypsum is the most common sulfate mineral on Earth's surface and is the dominant solid byproduct in a wide variety of mining and industrial processes, thus representing a major source for heavy metal(loid) contamination, including selenium. Gypsum crystals grown from the gel diffusion technique in 0.02 M Na2SeO4 solution at pH 7.5 and 0.02 M Na2SeO3 solutions at pH 7.5 and 9.0 contain 828, 5198, and 5955 ppm Se, respectively. Synchrotron Se K-edge X-ray absorption spectroscopic analyses show that selenite and selenate are the dominant species in Se4+- and Se6+-doped gypsum, respectively. The single-crystal EPR spectra of Se4+- and Se6+-doped gypsum after gamma-ray irradiation reveal five selenium-centered oxyradicals: SeO2-(I), SeO2-(II), SeO2-(III), SeO3-, and HSeO42-. The former three radicals provide unequivocal evidence for the substitution of their paramagnetic precursor SeO32- for SO42- in the gypsum structure, while the latter two confirm the replacement of SeO42- for SO42-. These results demonstrate that gypsum has a significant capacity for sequestrating both selenite and selenate in the structure but has a marked preference for the former, thus confirming important controls on the mobility and bioavailability of selenium oxyanions and pointing to optimal applications of gypsum for remediating selenium contamination under neutral to alkaline conditions.


Assuntos
Compostos de Selênio , Selênio , Sulfato de Cálcio , Espectroscopia de Ressonância de Spin Eletrônica , Ácido Selênico , Ácido Selenioso , Selenito de Sódio , Síncrotrons , Espectroscopia por Absorção de Raios X
19.
Phys Chem Chem Phys ; 22(9): 4875-4879, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32072999

RESUMO

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E. coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.


Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Alquinos/química , Azidas/química , Catálise , Cobre/química , Reação de Cicloadição , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Óxidos de Nitrogênio/química , Marcadores de Spin
20.
Nucleic Acids Res ; 48(6): 2830-2840, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32052020

RESUMO

The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2-10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2' position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2'-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.


Assuntos
DNA/química , Marcadores de Spin , Sequência de Bases , Cristalografia por Raios X , DNA/síntese química , Espectroscopia de Ressonância de Spin Eletrônica , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular
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