RESUMEN
Glutathione (GSH) is indispensable for maintaining redox homeostasis in biological fluids and serves as a key component in cellular defense mechanisms. Accurate assessment of GSH relative to its oxidized counterpart, glutathione disulfide (GSSG), is critical for the early diagnosis and understanding of conditions related to oxidative stress. Despite existing methods for their quantification, the label-free and simultaneous measurement of GSH and GSSG in biological fluid presents significant challenges. Herein, we report the use of an alpha-hederin (Ah) nanopore for the direct measurement of the GSH:GSSG ratio in simulated biological fluid, containing fetal bovine serum (FBS). This system hinges on detecting characteristic relative ion blockades (ΔI/Io) as GSH and GSSG molecules pass through the Ah nanopore under an applied electric field. The distinct current blockage signals derived from the translocation of GSH and GSSG enabled us to determine the molar ratio of GSH and its oxidized form. Notably, the interactions between the hydroxyl groups of the sugar moiety lining the nanopore's inner surface and the sulfhydryl group of GSH significantly influence the translocation dynamics, resulting in a longer translocation time for GSH compared to GSSG. The Ah nanopore technology proposed in this study offers a promising approach for real-time, single molecule-level monitoring of glutathione redox status in biological fluids, eliminating the need for labeling or extensive sample preparation.
Asunto(s)
Técnicas Biosensibles , Disulfuro de Glutatión , Glutatión , Nanoporos , Oxidación-Reducción , Glutatión/química , Glutatión/análisis , Glutatión/sangre , Técnicas Biosensibles/métodos , Disulfuro de Glutatión/análisis , Disulfuro de Glutatión/química , Disulfuro de Glutatión/sangre , Animales , Humanos , Bovinos , Estrés OxidativoRESUMEN
Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30µM (R2=0.9932±0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.
Asunto(s)
Glutatión , Oxidación-Reducción , o-Ftalaldehído , o-Ftalaldehído/química , Glutatión/análisis , Glutatión/química , Glutatión/metabolismo , Humanos , Animales , Disulfuro de Glutatión/análisis , Disulfuro de Glutatión/metabolismo , Disulfuro de Glutatión/química , Fosfinas/químicaRESUMEN
A novel method is introduced for extracting and enriching Cd(II) and Pb(II) from edible oils using glutathione disulfide (GSSG) as both an extractant and a phase-separation agent. The ions in the oils were initially extracted into an aqueous solution containing GSSG. After mixing the solution with acetonitrile at the appropriate volume ratio, a new phase formed, resulting in enrichment of the analytes. The experimental conditions were optimized using response surface methodology with a central composite design. Under optimal conditions, the method offered a combined enrichment factor of >660, with combined extraction efficiencies of 84.31% and 83.35% for Cd(II) and Pb(II), respectively. Finally, the method was conjugated to capillary electrophoresis to determine Cd(II) and Pb(II) in edible oil samples, with detection limits of 0.45 and 1.24 ppb, respectively. In comparison to traditional approaches, the GSSG-based method demonstrates rapidity, efficiency, and recyclability in extracting heavy metal ions from complex matrices.
Asunto(s)
Cadmio , Electroforesis Capilar , Contaminación de Alimentos , Disulfuro de Glutatión , Plomo , Aceites de Plantas , Cadmio/aislamiento & purificación , Cadmio/química , Cadmio/análisis , Electroforesis Capilar/métodos , Plomo/aislamiento & purificación , Plomo/análisis , Plomo/química , Aceites de Plantas/química , Aceites de Plantas/aislamiento & purificación , Contaminación de Alimentos/análisis , Disulfuro de Glutatión/análisis , Disulfuro de Glutatión/química , Disulfuro de Glutatión/aislamiento & purificación , Fraccionamiento Químico/métodosRESUMEN
Glutathione (GSH) is one of the main antioxidant molecules present in cells. It harbors a thiol group responsible for sustaining cellular redox homeostasis. This moiety can react with cellular electrophiles such as formaldehyde yielding the compound S-hydroxymethyl-GSH (HSMGSH). HSMGSH is the substrate of the enzyme alcohol dehydrogenase 5 (ADH5) and thus a key intermediate in formaldehyde metabolism. In this work, we describe a method for the chemical synthesis of HSMGSH and a pipeline to identify this compound in complex cell extracts by means of ultra-high-performance liquid chromatography coupled to high-resolution spectrometry (UHPLC-HRMS). This method also allows determining GSH and oxidized disulfide (GSSG) in the same samples, thus providing broad information about formaldehyde-GSH metabolism.
Asunto(s)
Antioxidantes , Glutatión , Humanos , Disulfuro de Glutatión/química , Cromatografía Líquida de Alta Presión/métodos , Glutatión/metabolismo , Antioxidantes/metabolismo , Compuestos de Sulfhidrilo , Oxidación-ReducciónRESUMEN
Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining redox cellular homeostasis. The thiols of two GSH molecules can be oxidized to the disulfide GSSG. The cytosolic GSH/GSSG ratio is very high (>100), and its reduction can lead to apoptosis or necrosis, which are of interest in cancer research. CuII ions are very efficient oxidants of thiols, but with an excess of GSH, CuIn(GS)m clusters are formed, in which CuI is very slowly reoxidized by O2 at pH 7.4 and even more slowly at lower pH. Here, the aerobic oxidation of GSH by CuII was investigated at different pH values in the presence of the anticancer thiosemicarbazone Dp44mT, which accumulates in lysosomes and induces lysosomal membrane permeabilization in a Cu-dependent manner. The results showed that CuII-Dp44mT catalyzes GSH oxidation faster than CuII alone at pH 7.4 and hence accelerates the production of very reactive hydroxyl radicals. Moreover, GSH oxidation and hydroxyl radical production by CuII-Dp44mT were accelerated at the acidic pH found in lysosomes. To decipher this unusually faster thiol oxidation at lower pH, density functional theory (DFT) calculations, electrochemical and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of CuII-Dp44mT on the hydrazinic nitrogen, which favors the rate-limiting reduction step without subsequent dissociation of the CuI intermediate. Furthermore, preliminary biological studies in cell culture using the proton pump inhibitor bafilomycin A1 indicated that the lysosomal pH plays a role in the activity of CuII-Dp44mT.
Asunto(s)
Cobre , Tiosemicarbazonas , Animales , Catálisis , Cobre/química , Glutatión/química , Disulfuro de Glutatión/química , Disulfuro de Glutatión/metabolismo , Concentración de Iones de Hidrógeno , Mamíferos/metabolismo , Oxidación-Reducción , Compuestos de Sulfhidrilo/química , Tiosemicarbazonas/química , Tiosemicarbazonas/farmacologíaRESUMEN
Glutathione disulfide (GSSG) has been monitored in human saliva samples by an optimized and validated method that is based on liquid chromatography coupled to on-line post column derivatization. The analyte was separated from the sample matrix using a 100% aqueous mobile phase through a core-shell reversed phase column. Following optimization of the reaction using Box- Behnken experimental design and validation, GSSG was quantified accurately and selectively in the range of 100-2000 nmol L-1 with a LOD of 20 nmol L-1. GSSG was quantified in 15 out of 20 human saliva samples (75%) with a mean value of 860 nmol L-1 (150-4600 nmol L-1). Blocking of reduced Glutathione with N-ethylmaleimide ensured stability of the samples for at least 72 h at all temperatures examined.
Asunto(s)
Glutatión , o-Ftalaldehído , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Glutatión/química , Disulfuro de Glutatión/química , Humanos , o-Ftalaldehído/químicaRESUMEN
Although peptides were widely used in many fields, their interface behaviors as surfactants have not been explored. The results of the surface tension experiments by an automatic surface tension meter indicate that the stability and emulsifying ability of glutathione disulfide (GSSG) under alkaline conditions were stronger than those under acidic conditions. With encoding the different oxygen and nitrogen atoms in GSSG, as well as the different hydrogen atoms bonded with oxygen and nitrogen atoms. The pH Dependence of the number of hydrogen bonds, affected by the protonation and deprotonation of the functional groups in GSSG, is calculated by LAMMPS software. The results demonstrate that GSSG forms twice as many hydrogen bonds under alkaline conditions as under acidic conditions, resulting in a better surface-interface activity in alkaline conditions. The interface properties of the GSSG surfactant can be regulated by pH. Therefore, GSSG is a potential pH-responsive surfactant.
Asunto(s)
Tensoactivos , Agua , Glutatión , Disulfuro de Glutatión/química , Concentración de Iones de Hidrógeno , Nitrógeno , Oxígeno , Tensoactivos/química , Agua/químicaRESUMEN
Nickel serves critical roles in the metabolism of E. coli and many prokaryotes. Many details of nickel trafficking are unestablished, but a nonproteinaceous low-molecular-mass (LMM) labile nickel pool (LNiP) is thought to be involved. The portion of the cell lysate that flowed through a 3 kDa cutoff membrane, which ought to contain this pool, was analyzed by size-exclusion and hydrophilic interaction chromatographies (SEC and HILIC) with detection by inductively coupled plasma (ICP) and electrospray ionization (ESI) mass spectrometries. Flow-through-solutions (FTSs) contained 11-15 µM Ni, which represented most Ni in the cell. Chromatograms exhibited 4 major Ni-detected peaks. MS analysis of FTS and prepared nickel complex standards established that these peaks arose from Ni(II) coordinated to oxidized glutathione, histidine, aspartate, and ATP. Surprisingly, Ni complexes with reduced glutathione or citrate were not members of the LNiP under the conditions examined. Aqueous Ni(II) ions were absent in the FTS. Detected complexes were stable in chelator-free buffer but were disrupted by treatment with 1,10-phenanthroline or citrate. Titrating FTS with additional NiSO4 suggested that the total nickel-binding capacity of cytosol is approximately 20-45 µM. Members of the LNiP are probably in rapid equilibrium. Previously reported binding constants to various metalloregulators may have overestimated the relevant binding strength in the cell because aqueous metal salts were used in those determinations. The LNiP may serve as both a Ni reservoir and buffer, allowing cells to accommodate a range of Ni concentrations. The composition of the LNiP may change with cellular metabolism and nutrient status.
Asunto(s)
Escherichia coli/química , Escherichia coli/metabolismo , Níquel/química , Níquel/metabolismo , Glutatión/química , Disulfuro de Glutatión/química , Disulfuro de Glutatión/metabolismo , Modelos Moleculares , Estructura Molecular , Oligoelementos/química , Oligoelementos/metabolismoRESUMEN
Silk fibroin (SF), derived from Bombyx mori, is a category of fibrous protein with outstanding potential for applications in the biomedical and biotechnological fields. In spite of its many advantageous properties, the exploration of SF as a versatile nanodrug precursor for tumor therapy has still been restricted in recent years. Herein, a multifunctional SF-derived nanoplatform was facilely developed via encapsulating the photosensitizer chlorin e6 (Ce6) into MnO2-capped SF nanoparticles (NPs). SF@MnO2 nanocarriers were synthesized through a surface crystallization technique, using SF as a reductant and sacrificial template. Afterwards, Ce6 was covalently incorporated into the loose structure of the SF@MnO2 nanocarrier on the basis of adsorption to abundant peptide-binding sites. To modulate the tumor microenvironment (TME), SF@MnO2/Ce6 (SMC) NPs were capable of catalyzing the decomposition of H2O2 into O2, which can be converted into cytotoxic reactive oxygen species (ROS) during photodynamic therapy (PDT). Moreover, the MnO2 component was able to oxidize intracellular glutathione (GSH) into non-reducing glutathione disulfide (GSSG), and the consumption of GSH could significantly protect the local ROS from being reduced, which further augmented the therapeutic outcome of PDT. Via another angle, SMC NPs can produce strong hyperthermia under near-infrared (NIR) light activation, which was highly desirable for efficient photothermal therapy (PTT). Both in vitro and in vivo studies demonstrated the intense tumor inhibitory effects as a result of augmented PTT/PDT mediated by SMC NPs. We believe that this study may provide useful insights for employing SF-based nanocomposites for more medical applications in the near future.
Asunto(s)
Antineoplásicos/química , Fibroínas/química , Compuestos de Manganeso/química , Nanopartículas/química , Óxidos/química , Fármacos Fotosensibilizantes/química , Porfirinas/química , Microambiente Tumoral/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Materiales Biocompatibles/química , Línea Celular Tumoral , Clorofilidas , Cristalización , Femenino , Glutatión/química , Disulfuro de Glutatión/química , Humanos , Peróxido de Hidrógeno/química , Rayos Infrarrojos , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales , Oxidación-Reducción , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Porfirinas/farmacología , Especies Reactivas de Oxígeno/química , Propiedades de SuperficieRESUMEN
Glutathionyl hemoglobin is a minor form of hemoglobin with intriguing properties. The measurement of the redox potential of its reactive ß-93-Cysteine is useful to improve understanding of the response of erythrocytes to transient and chronic conditions of oxidative stress, where the level of glutathionyl hemoglobin is increased. An independent literature experiment describes the recovery of human erythrocytes exposed to an oxidant burst by measuring glutathione, glutathione disulfide and glutathionyl hemoglobin in a two-hour period. This article calculates a value for the redox potential E0 of the ß-93-Cysteine, considering the erythrocyte as a closed system at equilibrium described by the Nernst equation and using the measurements of the literature experiment. The obtained value of E0 of -121 mV at pH 7.4 places hemoglobin as the most oxidizing thiol of the erythrocyte. By using as synthetic indicators of the concentrations the electrochemical potentials of the two main redox pairs in the erythrocytes, those of glutathione-glutathione disulfide and of glutathionyl-hemoglobin, the mechanism of the recovery phase can be hypothesized. Hemoglobin acts as the redox buffer that scavenges oxidized glutathione in the oxidative phase and releases it in the recovery phase, by acting as the substrate of the NAD(P)H-cofactored enzymes.
Asunto(s)
Cisteína/química , Hemoglobinas/química , Oxidantes/química , Oxidación-Reducción , Compuestos de Sulfhidrilo/química , Fenómenos Químicos , Eritrocitos/química , Eritrocitos/metabolismo , Glutatión/química , Glutatión/metabolismo , Disulfuro de Glutatión/química , Hemoglobinas/metabolismo , Humanos , Peróxido de Hidrógeno/química , Cinética , Modelos Biológicos , Oxidantes/farmacología , Oxidación-Reducción/efectos de los fármacos , Estrés OxidativoRESUMEN
BACKGROUND: We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H2O2 and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated. METHODS: Human recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH. RESULTS: S-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S. CONCLUSIONS: S-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1. GENERAL SIGNIFICANCE: The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.
Asunto(s)
Catálisis , Disulfuros/química , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/química , Estrés Oxidativo/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Glutatión/química , Disulfuro de Glutatión/química , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Humanos , Peróxido de Hidrógeno/química , Oxidación-Reducción/efectos de los fármacosRESUMEN
Protein aggregation is a complex physiological process, primarily determined by stress-related factors revealing the hidden aggregation propensity of proteins that otherwise are fully soluble. Here we report a mechanism by which glycolytic glyceraldehyde-3-phosphate dehydrogenase of Arabidopsis thaliana (AtGAPC1) is primed to form insoluble aggregates by the glutathionylation of its catalytic cysteine (Cys149). Following a lag phase, glutathionylated AtGAPC1 initiates a self-aggregation process resulting in the formation of branched chains of globular particles made of partially misfolded and totally inactive proteins. GSH molecules within AtGAPC1 active sites are suggested to provide the initial destabilizing signal. The following removal of glutathione by the formation of an intramolecular disulfide bond between Cys149 and Cys153 reinforces the aggregation process. Physiological reductases, thioredoxins and glutaredoxins, could not dissolve AtGAPC1 aggregates but could efficiently contrast their growth. Besides acting as a protective mechanism against overoxidation, S-glutathionylation of AtGAPC1 triggers an unexpected aggregation pathway with completely different and still unexplored physiological implications.
Asunto(s)
Arabidopsis/metabolismo , Glutatión/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Anotación de Secuencia Molecular , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dominio Catalítico , Glutarredoxinas/metabolismo , Glutatión/química , Disulfuro de Glutatión/química , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/química , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Cinética , Simulación de Dinámica Molecular , Oxidación-Reducción , Pliegue de Proteína , Solubilidad , Tiorredoxinas/metabolismoRESUMEN
The superoxide-generating reaction of adrenaline autoxidation in an alkaline medium, used in vitro to identify the antioxidant properties of various compounds, simulates the complex multistep process of quinoid oxidation of catecholamines (CA) in the body. Sulfur-containing cysteine (Cys) and reduced glutathione (GSH), as well as oxidized glutathione (GSSG), have been shown to inhibit this process. The studied substances were considered as inhibitors of quinoid oxidation and are evaluated as antioxidants. The IC50 values for Cys and GSH were close to 7.5 mM. Inhibition by GSSG was weaker; represented approximately 50-70% of Cys and GSH. Other sulfur-containing compounds that differ in chemical structure, the amino acids taurine and methionine were ineffective. The interest in this model and the search for effective compounds acting on this reaction is associated with one of the mechanisms of the etiopathogenesis of Parkinson's disease (PD) discussed in the literature, which occurs when the biochemical transformations of dopamine CA and its quinoid oxidation process are violated. Cys, GSH and GSSG in the model system inhibit quinoid oxidation of adrenaline, as a result of which the formation of superoxide (O2 ·-) is also inhibited. Experiments with the superoxide-generating enzymatic reaction xanthine xanthioxidase, the chemistry of which is different and not related to formation of quinoid metabolites, showed that the studied substances did not inhibit O2 ·- formation in this model. Thus, it was established that the biologically active sulfur-containing compounds Cys, GSH and GSSG are specific inhibitors of quinoid oxidation of CA, and are likely to be able to play the role of a neuroprotector. It is proposed to use these compounds in the treatment and prevention of PD by activating their biosynthesis in the body.
Asunto(s)
Epinefrina/metabolismo , Compuestos de Azufre/química , Antioxidantes/química , Cisteína/química , Disulfuro de Glutatión/química , Humanos , Fármacos Neuroprotectores/química , Oxidación-ReducciónRESUMEN
Protein-glutathione mixed disulphides (PSSG) are an important redox-sensitive posttranslational modification. Quantitation of protein-glutathione mixed disulphides (PSSG) is achieved by the reduction of the disulphide bond to liberate glutathione (GSH); however, this method leaves the assay susceptible to contamination by cytosolic GSH and glutathione disulphide (GSSG) captured during protein precipitation. The method herein describes a workflow in which protein from mouse liver is precipitated and adventitious GSH contamination is removed by reaction with N-ethylmaleimide. The sample is divided into two equal aliquots, a control aliquot that allows for direct quantitation of adventitious GSSG and a chemically reduced aliquot that contains GSH from both the GSSG and PSSG disulphides. Determining the concentration of adventitious GSSG allows for correction of the latter value to provide an accurate assay of PSSG. This assay also provides quantitation of cytosolic GSH and GSSG.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Disulfuro de Glutatión/química , Glutatión/química , Espectrometría de Masas en Tándem/métodos , Animales , Disulfuros/química , Glutatión/genética , Disulfuro de Glutatión/genética , Ratones , Estrés Oxidativo/genética , Procesamiento Proteico-Postraduccional/genética , Proteínas/química , Proteínas/genéticaRESUMEN
The hexapeptide glutathione disulfide (GSSG) has six ionizable groups with six associated dissociation constants. The experimentally measured pH-titration curve, however, does not exhibit the six corresponding equivalence points and bears little resemblance to standard textbook examples of acid-base pH-titration curves. The curve highlights the difficulties in determining multiple pKa values of polyprotic acids - typically proteins and peptides - from experiment. The six pKa values of GSSG can, however, be estimated using Car-Parrinello molecular dynamics (CPMD) simulations in conjunction with metadynamics sampling of the underlying free energy landscape of the dissociation reactions. Ab initio MD simulations were performed on a GSSG molecule solvated by 200 water molecules. Using the estimated pKa values the theoretical titration curve was calculated and found to be in good agreement with experiment. The results clearly highlight how dissociation constants estimated from ab initio MD simulations can facilitate the interpretation of the pH-titration curves of complex chemical and biological systems.
Asunto(s)
Técnicas de Química Analítica , Simulación por Computador , Disulfuro de Glutatión/química , Concentración de Iones de HidrógenoRESUMEN
PURPOSE: Investigate the possibility of measuring changes in glutathione (GSH) concentration using the MRS PRESS and MEGA-PRESS sequences by tracking the natural oxidation of GSH, and to examine the accuracy of the two methods. METHODS: 122 GSH edited MEGA-PRESS and PRESS acquisitions were acquired on a "braino" based phantom +3.0â¯mM GSH during a period of 11â¯days. All spectra were analyzed in LCModel. (The MEGA-PRESS data were first preprocessed in Matlab). Degradation curves were modeled. A one year follow-up on the same phantom and measurements from a similar phantom without GSH and one pure GSH phantom were also included. RESULTS: Both MEGA-PRESS and PRESS showed degradation of the measured GSH signal. Modeling the exponential decay of the GSH signal in MEGA-PRESS and PRESS gave for tâ¯=â¯0; 2.9 i.u. for MEGA-PRESS and 2.3 i.u. for PRESS. As t increased, the GSH concentration converged to zero for MEGA-PRESS but not for PRESS (0.7 i.u.). GSH for the one year follow up were 0.0 i.u. for MEGA-PRESS and 0.6 i.u. for PRESS. Similar phantom without GSH yielded 0.0 i.u. for both MEGA-PRESS and PRESS. CONCLUSION: It is possible to measure changes in GSH concentration in a phantom using both PRESS and MEGA-PRESS techniques, however the PRESS spectrum appears to include oxidized GSH (GSSG). In addition, GSH edited MEGA-PRESS measurement gives more precise values at lower GSH concentrations.
Asunto(s)
Glutatión/química , Espectroscopía de Resonancia Magnética , Oxígeno/química , Antioxidantes/química , Encéfalo/diagnóstico por imagen , Radicales Libres , Disulfuro de Glutatión/química , Humanos , NADP/química , Fantasmas de Imagen , Sustancias Reductoras/química , Reproducibilidad de los Resultados , Relación Señal-RuidoRESUMEN
Angiotensin converting enzyme (ACE, peptidyldipeptidase A, EC 3.4.15.1) plays an important role in the regulation of blood pressure. In this study, ACE was purified from human plasma by affinity chromatography in single step. The enzyme purified in 5367-fold from human plasma and specific activity was found to be 1208 EU/mg protein. The purity and molecular weight of ACE were determined by SDS-PAGE, which indicated two bands at around 60â¯kDa and 70â¯kDa on the gel. Effect of oxidized glutathione (GSSG) peptide and reduced glutathione (GSH) peptide on purified ACE activity were also investigated in which lisinopril was used as reference inhibitor. GSSG showed activation effect on ACE activity whereas GSH provided inhibition effect. In the lights of activity (%) versus activator graph for GSSG and activity (%) versus inhibitor graphs for GSH and lisinopril; IC50 values for GSH and lisinopril were determined to be 16.2⯵M and 0.781â¯nM, respectively. Type of inhibition for GSH and lisinopril from graph Lineweaver-Burk was found to be reversible non-competitive inhibition and Ki constants for GSH and lisinopril were calculated as 11.7⯵M and 0.662â¯nM, respectively.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Disulfuro de Glutatión/metabolismo , Glutatión/metabolismo , Fragmentos de Péptidos/metabolismo , Peptidil-Dipeptidasa A , Inhibidores de la Enzima Convertidora de Angiotensina/química , Cromatografía de Afinidad , Glutatión/química , Disulfuro de Glutatión/química , Humanos , Modelos Lineales , Fragmentos de Péptidos/química , Peptidil-Dipeptidasa A/sangre , Peptidil-Dipeptidasa A/aislamiento & purificación , Peptidil-Dipeptidasa A/metabolismoRESUMEN
Variability in the levels of GSH and GSSG in plasma is suggested to derive from inadequate pre-processing methods. The aim of this study was to develop a protocol for comparable and reliable measurements of GSH/GSSG. Venous blood from 8 healthy individuals were collected and divided into 7 different pre-processing procedures. For three of the samples an extraction mixture was added after 0 (baseline), 4 and 8â¯min and for three of the samples the extraction mixture was added at different times during defrost. A worst case scenario where a sample was left in a cool box during 6â¯h was also included. The samples were analyzed with UHPLC-ESI-MSMS. A large difference in the levels of GSH and GSSG were identified and it was clearly associated with the sample handling procedures. A sample left untreated for 4â¯min will have significantly reduced amount of GSH. Stability tests showed that the level of GSH was reduced after 3â¯months in -80⯰C.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Disulfuro de Glutatión/sangre , Disulfuro de Glutatión/química , Glutatión/sangre , Glutatión/química , Estabilidad de Medicamentos , Humanos , Modelos Lineales , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem/métodosRESUMEN
Many proteins provided with disulfide bridges in the native state undergo amorphous irreversible aggregation when these bonds are not formed. Here we show that egg lysozyme displays a clever strategy to prevent this deleterious aggregation during the nascent phase when disulfides are still absent. In fact, when the reduced protein assembles into a molten globule state, its cysteines acquire strong hyper-reactivity towards natural disulfides. The most reactive residue, Cys94, reacts with oxidized glutathione (GSSG) 3000 times faster than an unperturbed protein cysteine. A low pKa of its sulfhydryl group (6.6/7.1) and a productive complex with GSSG (KD = 0.3 mM), causes a fast glutathionylation of this residue (t1/2 = 3 s) and a complete inhibition of the protein aggregation. Other six cysteines display 70 times higher reactivity toward GSSG. The discovery of extreme hyper-reactivity in cysteines only devoted to structural roles opens new research fields for Alzheimer's and Parkinson diseases.
Asunto(s)
Cisteína/química , Disulfuros/química , Muramidasa/química , Agregado de Proteínas , Disulfuro de Glutatión/química , Isoenzimas/química , Isoenzimas/metabolismo , Muramidasa/metabolismo , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
In plant cells, many stresses, including low oxygen availability, result in a higher production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules can lead to redox-dependent post-translational modification of proteins Cys residues. Here, we studied the effect of different redox modifications on alcohol dehydrogenase (ADH) from Arabidopsis thaliana. ADH catalyzes the last step of the ethanol fermentation pathway used by plants to cope with energy deficiency during hypoxic stress. Arabidopsis suspension cell cultures showed decreased ADH activity upon exposure to H2O2, but not to the thiol oxidizing agent diamide. We purified recombinant ADH and observed a significant decrease in the enzyme activity by treatments with H2O2 and diethylamine NONOate (DEA/NO). Treatments leading to the formation of a disulfide bond between ADH and glutathione (protein S-glutathionylation) had no negative effect on the enzyme activity. LC-MS/MS analysis showed that Cys47 and Cys243 could make a stable disulfide bond with glutathione, suggesting redox sensitivity of these residues. Mutation of ADH Cys47 to Ser caused an almost complete loss of the enzyme activity while the Cys243 to Ser mutant had increased specific activity. Incubation of ADH with NAD+ or NADH prevented inhibition of the enzyme by H2O2 or DEA/NO. These results suggest that binding of ADH with its cofactors may limit availability of Cys residues to redox modifications. Our study demonstrates that ADH from A. thaliana is subject to different redox modifications. Implications of ADH sensitivity to ROS and RNS during hypoxic stress conditions are discussed.