RESUMO
Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori, the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H. pylori. Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H. pylori. In vitro, SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo, a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H. pylori.
Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Metalochaperonas/metabolismo , Níquel/metabolismo , Peptidilprolil Isomerase/metabolismo , Animais , Infecções por Helicobacter/microbiologia , Camundongos , Urease/metabolismoRESUMO
[NiFe]-hydrogenase enzymes catalyze the reversible oxidation of hydrogen at a bimetallic cluster and are used by bacteria and archaea for anaerobic growth and pathogenesis. Maturation of the [NiFe]-hydrogenase requires several accessory proteins to assemble and insert the components of the active site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase enzyme precursor protein, a process that is accomplished by two nickel metallochaperones, the accessory protein HypA and the GTPase HypB. Recent work demonstrated that nickel is rapidly transferred to HypA from GDP-loaded HypB within the context of a protein complex in a nickel selective and unidirectional process. To investigate the mechanism of metal transfer, we examined the allosteric effects of nucleotide cofactors and partner proteins on the nickel environments of HypA and HypB by using a combination of biochemical, microbiological, computational, and spectroscopic techniques. We observed that loading HypB with either GDP or a nonhydrolyzable GTP analogue resulted in a similar nickel environment. In addition, interaction with a mutant version of HypA with disrupted nickel binding, H2Q-HypA, does not induce substantial changes to the HypB G-domain nickel site. Instead, the results demonstrate that HypB modifies the acceptor site of HypA. Analysis of a peptide maquette derived from the N-terminus of HypA revealed that nickel is predominately coordinated by atoms from the N-terminal Met-His motif. Furthermore, HypA is capable of two nickel-binding modes at the N-terminus, a HypB-induced mode and a binding mode that mirrors the peptide maquette. Collectively, these results reveal that HypB brings about changes in the nickel coordination of HypA, providing a mechanism for the HypB-dependent control of the acquisition and release of nickel by HypA.
Assuntos
Proteínas de Transporte/química , Complexos de Coordenação/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Níquel/química , Proteínas de Transporte/metabolismo , Complexos de Coordenação/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Níquel/metabolismoRESUMO
The biosynthesis of the dinuclear metal cluster at the active sites of the [NiFe]-hydrogenase enzymes is a multi-step process executed by a suite of accessory proteins. Nickel insertion during maturation of Escherichia coli [NiFe]-hydrogenase 3 is achieved by the metallochaperones HypA, SlyD and the GTPase HypB, but how these proteins cooperate to ensure nickel delivery is not known. In this study, the complexes formed between the individual purified proteins were examined by using several methods. Size exclusion chromatography (SEC) indicated that SlyD and HypB interact primarily in a 1:1 complex. The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP. A ternary complex between all three proteins was not detected, and instead SlyD blocked the interaction of HypA with HypB in competitive binding experiments. Furthermore, cross-linking experiments suggest a weak interaction between HypA and SlyD, which is not detectable by SEC. Electrochemical analysis confirmed each of the pairwise interactions and that the relative affinities of these complexes are on the order of HypB-SlyD > HypB-HypA > HypA-SlyD. These results indicate a hierarchy of interactions, as opposed to a single multiprotein complex, and provide insight into the nickel delivery process during hydrogenase enzyme maturation.
Assuntos
Escherichia coli/enzimologia , Hidrogenase/metabolismo , Níquel/metabolismo , Cromatografia em Gel , Hidrogenase/química , Metalochaperonas/química , Metalochaperonas/metabolismo , Níquel/químicaRESUMO
In this study, a bimetallic palladium-copper aerogel was synthesized and used for modification of a graphite paste electrode (Pd-Cu/GPE), allowing the sensitive determination of bisphenol A (BPA). Different techniques, such as SEM, TEM, XPS, and AFM, were used for characterization of the Pd-Cu aerogel. To elucidate the properties of the Pd-Cu/GPE, the electrochemistry methods such as differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy were used. DPV measurements were conducted in phosphate electrolyte and buffer solution (0.2 M PBS, pH 5) at a potential range from 0.4 to 0.9 V vs. Ag/AgCl. The DPVs peaks currents increased linearly with BPA concentrations in the 0.04-85 and 85-305 µM ranges, with a limit of detection of 20 nM. The modified electrode was successfully used in real samples to determine BPA, and the results were compared to the standard HPLC method. The results showed that the Pd-Cu/GPE had good selectivity, stability, and sensitivity for BPA determination.
RESUMO
To investigate the chemotherapeutic and pharmacokinetic aspects of two lanthanide complexes (Tb(III) and La(III) containing 2,2'-bipyridine ligand), in vitro binding studies were carried out with BSA by employing multiple biophysical methods and molecular modeling study. There are different techniques containing fluorescence, absorption spectroscopy and competitive experiments to determine the interaction mode between BSA and these complexes. These complexes efficiently quenched the BSA emission through a static procedure. The results showed that the terbium and lanthanum complexes exhibited a high propensity for BSA interaction via van der Waals force. Further, competitive examination and docking study showed that the interaction site of these complexes on BSA is site III. The results of docking calculations were in good agreement with experimental examinations. Also, the energy transfer from BSA to these complexes has happened with high possibility. Moreover, antimicrobial studies of different bacterial and fungi indicated its promising antibacterial activity. In vitro cytotoxicity of the Tb complex and La complex was carried out in MCF-7 and A-549 cell lines, which revealed significantly good activity.Communicated by Ramaswamy H. Sarma.
Assuntos
Anti-Infecciosos , Elementos da Série dos Lantanídeos , 2,2'-Dipiridil/farmacologia , Anti-Infecciosos/farmacologia , Sítios de Ligação , Simulação de Acoplamento Molecular , Ligação Proteica , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência , Térbio , TermodinâmicaRESUMO
To determine the chemotherapeutic and pharmacokinetic aspects of an ytterbium complex containing 2,9-dimethyl-1,10-phenanthroline (Me2Phen), in vitro binding studies were carried out with FS-DNA/BSA by employing multiple biophysical methods and a molecular modeling study. There are different techniques including absorption spectroscopy, fluorescence spectroscopy, circular dichroism studies, viscosity experiments (only in the case of DNA), and competitive experiments used to determine the interaction mode between DNA/BSA and the ytterbium-complex. The results showed that the Yb-complex exhibited a high propensity for the interaction of BSA and DNA via hydrophobic interactions and van der Waals forces. Further, a competitive examination and docking study showed that the interaction site of the ytterbium complex on BSA is site III. The results of docking calculations for DNA/BSA were in good agreement with experimental findings. The complex displays efficient DNA cleavage in the presence of hydrogen peroxide. Moreover, antimicrobial studies of different bacteria and fungi indicated its promising antibacterial activity. In vitro cytotoxicity studies of the Yb-complex, starch nano-encapsulated, and lipid nano-encapsulated were carried out in MCF-7 and A-549 cell lines, which revealed significantly good activity. The results of anticancer activity studies showed that the cytotoxic activity of the Yb-complex was increased when encapsulated with nanocarriers. Based on biological applications of the Yb-complex, it can be concluded that this complex and its nanocarriers can act as novel anticancer and antimicrobial candidates.
RESUMO
In order to estimate the biological potential of a synthesized complex [Yb(bpy)2Cl3.OH2] where bpy is 2,2'-bipyridine, its binding behavior with fish salmon-DNA (FS-DNA) and bovine serum albumin (BSA) were studied by different kinds of spectroscopy and molecular modeling methods. This complex was selected for its antibacterial and antifungal activities as well as the DNA cleavage activities were examined by agarose gel electrophoresis. The analyses of fluorescence data at four temperatures were done in order to evaluate the binding and thermodynamic parameters of the interaction of Yb(III) complex with DNA and BSA. The experimental results indicated that the major binding modes were based on groove binding with DNA and BSA. In addition, iodide quenching studies, ethidium bromide (EtBr) exclusion assay, ionic strength effect, circular dichroism, and viscosity studies reflected the binding of Yb(III) complex explicitly with the FS-DNA mainly in a groove binding mode. Moreover, molecular docking studies indicated that this complex was bound to the minor groove of DNA and to polar and apolar residues located in the subdomain IB of BSA (site 3). Also, the results of competitive experiments assessed site 3 of BSA as the most probable binding site for this complex. The molecular docking results were in good agreement with our experimental results. From both experimental and docking results, the binding constant values displayed the remarkably high affinity of Yb(III) complex to DNA as well as BSA.Communicated by Ramaswamy H. Sarma.
Assuntos
Anti-Infecciosos , Clivagem do DNA , 2,2'-Dipiridil , Animais , Sítios de Ligação , DNA/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência , Termodinâmica , ItérbioRESUMO
BACKGROUND: There is a crucial need for finding and developing new compounds as the anticancer and antimicrobial agents with better activity, specific target, and less toxic side effects. OBJECTIVES: Base on the potential anticancer properties of lanthanide complexes, in the paper, the biological applications of terbium (Tb) complex, containing 2,9-dimethyl- 1,10-phenanthroline (Me2Phen) such as anticancer, antimicrobial, DNA cleavage ability, the interaction with FS-DNA (Fish-Salmon DNA) and BSA (Bovine Serum Albumin) was examined. METHODS: The interaction of Tb-complex with BSA and DNA was studied by emission spectroscopy, absorption titration, viscosity measurement, CD spectroscopy, competitive experiments, and docking calculation. Also, the ability of this complex to cleave DNA was reported by gel electrophoresis. Tb-complex was concurrently screened for its antibacterial activities by different methods. Besides, the nanocarriers of Tb-complex (lipid nanoencapsulation (LNEP) and the starch nanoencapsulation (SNEP)), as active anticancer candidates, were prepared. MTT technique was applied to measure the antitumor properties of these compounds on human cancer cell lines. RESULTS: The experimental and docking results suggest significant binding between DNA as well as BSA with terbium-complex. Besides, groove binding plays the main role in the binding of this compound with DNA and BSA. The competitive experiment with hemin demonstrated that the terbium complex was bound at site III of BSA, which was confirmed by the docking study. Also, Tb-complex was concurrently screened for its DNA cleavage, antimicrobial, and anticancer activities. The anticancer properties of LNEP and SNEP are more than the terbium compound. CONCLUSIONS: Tb-complex can bond to DNA/BSA with high binding affinity. Base on biological applications of Tb-complex, it can be concluded that this complex and its nanocarriers can suggest as novel anticancer, antimicrobial candidates.
RESUMO
In this study, the interactions of a novel metal complex [Dy(bpy)2Cl3.OH2] (bpy is 2,2'-bipyridine) with fish salmon DNA (FS-DNA) and bovine serum albumin (BSA) were investigated by experimental and theoretical methods. All results suggested significant binding between the Dy(III) complex with FS-DNA and BSA. The binding constants (Kb), Stern-Volmer quenching constants (KSV) of Dy(III)-complex with FS-DNA and BSA at various temperatures as well as thermodynamic parameters using Van't Hoff equation were obtained. The experimental results from absorption, ionic strength, iodide ion quenching, ethidium bromide (EtBr) quenching studies and positive ΔHË and ΔSË suggested that hydrophobic groove-binding mode played a predominant role in the binding of Dy(III)-complex with FS-DNA. Indeed, the molecular docking results for DNA-binding were in agreement with experimental data. Besides, the results found from experimental and molecular modeling indicated that the Dy(III)-complex bound to BSA via Van der Waals interactions. Moreover, the results of competitive tests by phenylbutazone, ibuprofen, and hemin (as a site-I, site-II and site-III markers, respectively) considered that the site-III of BSA is the most possible binding site for Dy(III)-complex. In addition, Dy(III) complex was concurrently screened for its antimicrobial activities. The presented data provide a promising platform for the development of novel metal complexes that target nucleic acids and proteins with antimicrobial activity.Communicated by Ramaswamy H. Sarma.
Assuntos
Anti-Infecciosos , Soroalbumina Bovina , 2,2'-Dipiridil , Animais , Sítios de Ligação , DNA/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência , TermodinâmicaRESUMO
In this study, fluorescence emission spectra, UV-vis absorption spectra, ethidium bromide (EB)-competition experiment, and iodide quenching experiment were used for the interaction study of the Fish salmon DNA (FS-DNA) with [Pr(dmp)2Cl3(OH2)] where dmp is 2,9-dimethyl 1,10-phenanthroline. The binding constant and the number of binding sites of the complex with FS-DNA were 6.09 ± 0.04 M-1 and 1.18, respectively. The free energy, enthalpy, and entropy changes (ΔG°, ΔH°, and ΔS°) in the binding process of the Pr(III) complex with FS-DNA were -8.02 kcal mol-1, +39.44 kcal mol-1, and +159.56 cal mol-1 K-1, respectively. Based on these results, the interaction process between FS-DNA with [Pr(dmp)2Cl3(OH2)] was spontaneous and the main binding interaction force was groove binding mode. Also, Fluorescence and electronic absorption spectroscopy were used in order to evaluate the binding characteristics, stoichiometry, and interaction mode of praseodymium(III) (Pr(III)) complex with bovine serum albumin (BSA). Title complex showed good binding propensity to BSA presenting moderately high Kb values. The fluorescence quenching of BSA by Pr(III) complex has been observed to be the static process. The positive ΔH° and ΔS° values showed that the hydrophobic interaction is the main force in the binding of Pr(III) complex and BSA. Eventually, the average aggregation number,
Assuntos
Antibacterianos/química , DNA/química , Fenantrolinas/química , Praseodímio/química , Soroalbumina Bovina/química , Espectrometria de Fluorescência/métodos , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Etídio/química , Fenantrolinas/metabolismo , Praseodímio/metabolismo , Ligação Proteica , Soroalbumina Bovina/metabolismo , Especificidade da Espécie , Staphylococcus aureus/efeitos dos fármacosRESUMO
In this study, the interaction of Holmium (Ho) complex including 2, 9-dimethyl-1,10-phenanthroline, also called Neocuproine (Neo), [Ho(Neo)2Cl3.H2O], as fluorescence probe with fish-salmon DNA (FS-DNA) is studied during experimental investigations. Multi-spectroscopic methods are utilized to determine the affinity binding constants (Kb) of complex-FS-DNA. It is found that fluorescence of Ho complex is strongly quenched by the FS-DNA through a static quenching procedure. Under optimal conditions in Tris(trishydroxymethyl-aminomethane)-HCl buffer at 25 °C with pH ≈ 7.2, intrinsic binding constant Kb of Ho complex is 6.12 ± 0.04 × 105 M-1. Also, the binding site number and Stern-Volmer quenching constant are calculated. There are different approaches, including iodide quenching assay, salt effect and thermodynamical assessment to determine the features of the binding mode between Ho complex and FS-DNA. Also, the parent and starch and lipid nanoencapsulated Ho complex, as potent antitumor candidates, were synthesized. The main structure of Ho complex is maintained after encapsulation using starch and lipid nanoparticles. 3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method was used to assess the anticancer properties of Ho complex and its encapsulated forms on human cancer cell lines of human lung carcinoma cell line and breast cancer cell line. In conclusion, these compounds could be considered as new antitumor candidates. Communicated by Ramaswamy H. Sarma.
Assuntos
Antibacterianos/farmacologia , DNA/metabolismo , Nanopartículas/química , Fenantrolinas/toxicidade , Absorção Fisico-Química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Etídio/metabolismo , Concentração Inibidora 50 , Iodetos/química , Cinética , Lipídeos/química , Testes de Sensibilidade Microbiana , Nanopartículas/ultraestrutura , Fenantrolinas/química , Salmão , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Amido/química , Temperatura , Termogravimetria , ViscosidadeRESUMO
In this work, the terbium(III), dysprosium(III), and ytterbium(III) complexes containing 2, 2'-bipyridine (bpy) ligand have been synthesized and characterized using CHN elemental analysis, FT-IR, UV-Vis and 1H-NMR techniques and their binding behavior with human serum albumin (HSA) was studied by UV-Vis, fluorescence and molecular docking examinations. The experimental data indicated that all three lanthanide complexes have high binding affinity to HSA with effective quenching of HSA fluorescence via static mechanism. The binding parameters, the type of interaction, the value of resonance energy transfer, and the binding distance between complexes and HSA were estimated from the analysis of fluorescence measurements and Förster theory. The thermodynamic parameters suggested that van der Waals interactions and hydrogen bonds play an important role in the binding mechanism. While, the energy transfer from HSA molecules to all these complexes occurs with high probability, the order of binding constants (BpyTb > BpyDy > BpyYb) represents the importance of radius of Ln3+ ion in the complex-HSA interaction. The results of molecular docking calculation and competitive experiments assessed site 3 of HSA, located in subdomain IB, as the most probable binding site for these ligands and also indicated the microenvironment residues around the bound mentioned complexes. The computational results kept in good agreement with experimental data.
Assuntos
2,2'-Dipiridil/química , Fenômenos Químicos , Técnicas de Química Sintética , Elementos da Série dos Lantanídeos/química , Modelos Moleculares , Compostos Organometálicos/química , Albumina Sérica Humana/química , Sítios de Ligação , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/metabolismo , Ligação Proteica , Albumina Sérica Humana/metabolismo , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , TermodinâmicaRESUMO
In this study, a rapid flow injection-flame atomic absorption spectrometry for cyanide detection was developed. Different AgX (where X is Cl(-), Br(-), I(-) and N(3)(-)) solid-phase reagents (SPR) were tested for indirect determination of cyanide. In a single-line FIA system, the cyanide was allowed to react with AgX SPR, which in turn changed Ag ions in AgX to silver cyanide complexes in a sodium hydroxide carrier stream. The eluent containing the analyte as silver cyanide complexes was measured by FAAS. The calibration curve was linear up to 30 mg l(-1) with a detection limit of 0.05 mg l(-1) for cyanides. The sampling rate and the relative standard deviation were <1.09% and >200 h(-1), respectively. The method was applied to the determination of cyanide in electroplating wastewater.
Assuntos
Cianetos/análise , Análise de Injeção de Fluxo/métodos , Halogênios/química , Compostos de Nitrogênio/análise , Compostos de Prata/análise , Extração em Fase Sólida/métodos , Poluentes Químicos da Água/análise , Cianetos/química , Galvanoplastia , Análise de Injeção de Fluxo/instrumentação , Compostos de Nitrogênio/química , Compostos de Prata/química , Extração em Fase Sólida/instrumentação , Poluentes Químicos da Água/químicaRESUMO
In order to evaluate biological potential of a novel synthesized complex [Nd(dmp)2Cl3.OH2] where dmp is 29-dimethyl 110-phenanthroline, the DNA-binding, cleavage, BSA binding, and antimicrobial activity properties of the complex are investigated by multispectroscopic techniques study in physiological buffer (pH 7.2).The intrinsic binding constant (Kb) for interaction of Nd(III) complex and FS-DNA is calculated by UV-Vis (Kb = 2.7 ± 0.07 × 105) and fluorescence spectroscopy (Kb = 1.13 ± 0.03 × 105). The Stern-Volmer constant (KSV), thermodynamic parameters including free energy change (ΔG°), enthalpy change (∆H°), and entropy change (∆S°), are calculated by fluorescent data and Vant' Hoff equation. The experimental results show that the complex can bind to FS-DNA and the major binding mode is groove binding. Meanwhile, the interaction of Nd(III) complex with protein, bovine serum albumin (BSA), has also been studied by using absorption and emission spectroscopic tools. The experimental results show that the complex exhibits good binding propensity to BSA. The positive ΔH° and ∆S° values indicate that the hydrophobic interaction is main force in the binding of the Nd(III) complex to BSA, and the complex can quench the intrinsic fluorescence of BSA remarkably through a static quenching process. Also, DNA cleavage was investigated by agarose gel electrophoresis that according to the results cleavage of DNA increased with increasing of concentration of the complex. Antimicrobial screening test gives good results in the presence of Nd(III) complex system.
Assuntos
Bactérias/efeitos dos fármacos , Proteínas de Ligação a DNA/química , Neodímio/química , Fenantrolinas/química , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bactérias/química , Sítios de Ligação , Proteínas de Ligação a DNA/síntese química , Proteínas de Ligação a DNA/farmacologia , Humanos , Complexos Multiproteicos/química , Neodímio/farmacologia , Fenantrolinas/síntese química , Fenantrolinas/farmacologia , Ligação Proteica , Soroalbumina Bovina/química , Espectrometria de Fluorescência , TermodinâmicaRESUMO
The binding of [Dy(dmp)2Cl3(OH2)], where dmp is 2,9-dimethyl 1,10-phenanthroline, with Fish salmon DNA (FS-DNA) is investigated by absorption and emission spectroscopy, quenching studies, salt dependent, and gel electrophoresis. The binding constant (Kb) of the interaction is calculated as (1.27 ± .05) × 105 M-1 from absorption spectral titration data. The Stern-Volmer constant (KSV), thermodynamic parameters involves ΔG°, ∆H°, and ∆S° are calculated by fluorescent data and Van't Hoff equation. The thermodynamic studies show that the reaction for the binding of the complex with FS-DNA is endothermic and entropically driven (ΔS° > 0, ΔH° > 0). The effect of the complex concentration on FS-DNA cleavage reactions is also investigated by gel electrophoresis. Furthermore, the Dy(III) complex has been screened for its antibacterial activity. The experimental results suggest that the Dy(III) complex binds significantly to FS-DNA by hydrophobic groove binding mode and the complex has more efficient antibacterial activity compared to its metal salt.
Assuntos
Disprósio/química , Disprósio/farmacologia , Fenantrolinas/química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Sítios de Ligação , DNA/química , Clivagem do DNA , Testes de Sensibilidade MicrobianaRESUMO
[NiFe]-hydrogenase, which catalyzes the reversible conversion between hydrogen gas and protons, is a vital component of the metabolism of many pathogens. Maturation of [NiFe]-hydrogenase requires selective nickel insertion that is completed, in part, by the metallochaperones SlyD and HypB. Escherichia coli HypB binds nickel with sub-picomolar affinity, and the formation of the HypB-SlyD complex activates nickel release from the high-affinity site (HAS) of HypB. In this study, the metal selectivity of this process was investigated. Biochemical experiments revealed that the HAS of full length HypB can bind stoichiometric zinc. Moreover, in contrast to the acceleration of metal release observed with nickel-loaded HypB, SlyD blocks the release of zinc from the HypB HAS. X-ray absorption spectroscopy (XAS) demonstrated that SlyD does not impact the primary coordination sphere of nickel or zinc bound to the HAS of HypB. Instead, computational modeling and XAS of HypB loaded with nickel or zinc indicated that zinc binds to HypB with a different coordination sphere than nickel. The data suggested that Glu9, which is not a nickel ligand, directly coordinates zinc. These results were confirmed through the characterization of E9A-HypB, which afforded weakened zinc affinity compared to wild-type HypB but similar nickel affinity. This mutant HypB fully supports the production of [NiFe]-hydrogenase in E. coli. Altogether, these results are consistent with the model that the HAS of HypB functions as a nickel site during [NiFe]-hydrogenase enzyme maturation and that the metal selectivity is controlled by activation of metal release by SlyD.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Hidrogenase/metabolismo , Peptidilprolil Isomerase/metabolismo , Modelos Moleculares , Níquel/metabolismo , Ligação Proteica , Zinco/metabolismoRESUMO
A pneumatic flow injection-tandem spectrometer system, without a delivery pump was used for the speciation of iron. In this system, the suction force of a pneumatic nebulizer of a flame absorption spectrometer was used for solution delivery through the manifold. The Fe(III) and total Fe concentrations were determined using thiocyanate ion in a UV-Vis spectrometer and a FAAS, respectively. The Fe(II) was determined by the difference. The calibration curves were linear up to 18 microg mL(-1) and 25 microg mL(-1) with detection limits of 0.09 microg mL(-1) and 0.07 microg mL(-1) for Fe(III) and Fe(II), respectively. The mid-range precision and accuracy were <2.5% and +/-3% for the two species, respectively, at a sampling rate of 120 h(-1). This system was applied for the determination of Fe(III) and Fe(II) in industrial water, natural water and spiked samples.
Assuntos
Compostos Férricos/análise , Compostos Ferrosos/análise , Calibragem , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodos , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta/instrumentação , Espectrofotometria Ultravioleta/métodos , Tiocianatos/análiseRESUMO
The interaction of native fish salmon DNA (FS-DNA) with [Eu(bpy)3Cl2(H2O)]Cl, where bpy is 2,2'-bipyridine, is studied at physiological pH in Tris-HCl buffer by spectroscopic methods, viscometric techniques as well as circular dichroism (CD). These experiments reveal that Eu(III) complex has interaction with FS-DNA. Moreover, binding constant and binding site size have been determined. The value of Kb has been defined 2.46 ± .02 × 10(5) M(-1). The thermodynamic parameters are calculated by Van't Hoff equation, the results show that the interaction of the complex with FS-DNA is an entropically driven phenomenon. CD spectroscopy followed by viscosity as well as fluorescence and UV--Vis measurements indicate that the complex interacts with FS-DNA via groove binding mode. Also, the synthesized Eu(III) complex has been screened for antimicrobial activities.
Assuntos
2,2'-Dipiridil/química , Anti-Infecciosos/química , DNA/química , Európio/química , Animais , Dicroísmo Circular , Testes de Sensibilidade Microbiana , Solventes/química , Espectroscopia de Infravermelho com Transformada de Fourier , Termodinâmica , ViscosidadeRESUMO
Agarose gel electrophoresis, absorption, fluorescence, viscosity, and circular dichroism (CD) have been used in exploring the interaction of terbium(III) complex, [Tb(bpy)2Cl3(OH2)] where bipy is 2,2'-bipyridine, with Fish salmon DNA. Agarose gel electrophoresis assay, along with absorption and fluorescence studies, reveal interaction between the corresponding complex and FS-DNA. Also, the binding constants (Kb) and the Stern-Volmer quenching constants (Ksv) of Tb(III) complex with FS-DNA were determined. The calculated thermodynamic parameters suggested that the binding of mentioned complex to FS-DNA was driven mainly by hydrophobic interactions. A comparative study of this complex with respect to the effect of iodide-induced quenching, ionic strength effect, and ethidium bromide exclusion assay reflects binding of explicit to the FS-DNA primarily in a groove fashion. CD and viscosity data also support the groove binding mode. Furthermore, Tb(III) complex have been simultaneously screened for their antibacterial and antifungal activities.
Assuntos
2,2'-Dipiridil/farmacologia , DNA/metabolismo , Térbio/farmacologia , Absorção de Radiação , Animais , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Sítios de Ligação , Dicroísmo Circular , Clivagem do DNA , Eletroforese em Gel de Ágar , Etídio/química , Etídio/metabolismo , Fungos/efeitos dos fármacos , Cinética , Ligantes , Luminescência , Testes de Sensibilidade Microbiana , Concentração Osmolar , Salmão , Espectrometria de Fluorescência , Temperatura , ViscosidadeRESUMO
A new and simple flow injection method followed by atomic absorption spectrometry has been developed for the indirect determination of ascorbic acid. The proposed method is based on oxidation of ascorbic acid to dehydroascorbic acid using a solid-phase manganese dioxide (30% m/m suspended on silica gel beads) reactor. The flow of the sample through the column reduces the MnO2 to Mn(II) in an acidic carrier stream of 6.3 mM HNO3 (pH 2.2) with flow rate of 4.0 ml/min at room temperature; Mn(II) is measured by atomic absorption spectrometry. The absorbance of Mn(II) is proportional to the concentration of ascorbic acid in the sample. The calibration curve was linear up to 30 mg/L, with a detection limit of 0.2 mg/L for a 220 microL injected sample volume. The developed procedure was found to be suitable for the determination of AsA in pharmaceuticals and foods with a relative standard deviation better than 1.09% and a sampling rate of about 95 h(-1). The results exhibit no interference from the presence of large amounts of organic compounds. The reliability of the method was established by parallel determination against the 2,6-dichlorophenol-indophenol methods.