RESUMEN

Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a non-covalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives.

Asunto(s)

Antioxidantes/química , Cisteína/genética , Albúmina Sérica Humana/genética , Compuestos de Sulfhidrilo/química , Cromatografía por Intercambio Iónico , Cisteína/química , Expresión Génica/genética , Humanos , Dominios Proteicos/genética , Multimerización de Proteína , Saccharomycetales/genética , Albúmina Sérica Humana/química

RESUMEN

Thiol groups in protein cysteine (Cys) residues can undergo one- and two-electron oxidation reactions leading to the formation of thiyl radicals or sulfenic acids, respectively. In this mini-review we summarize the mechanisms and kinetics of the formation of these species by biologically relevant oxidants. Most of the latter react with the deprotonated form of the thiol. Since the pKa of the thiols in protein cysteines are usually close to physiological pH, the thermodynamics and the kinetics of their oxidation in vivo are affected by the acidity of the thiol. Moreover, the protein microenvironment has pronounced effects on cysteine residue reactivity, which in the case of the oxidation mediated by hydroperoxides, is known to confer specificity to particular protein cysteines. Despite their elusive nature, both thiyl radicals and sulfenic acids are involved in the catalytic mechanism of several enzymes and in the redox regulation of protein function and/or signaling pathways. They are usually short-lived species that undergo further reactions that converge in the formation of different stable products, resulting in several post-translational modifications of the protein. Some of these can be reversed through the action of specific cellular reduction systems. Others damage the proteins irreversibly, and can make them more prone to aggregation or degradation.

Asunto(s)

Cisteína/química , Proteínas/metabolismo , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Proteínas/química

RESUMEN

Inflammation plays a major role in the onset and development of chronic non-communicable diseases like obesity, cardiovascular diseases and cancer. Combined, these diseases represent the most common causes of death worldwide, thus development of novel pharmacological approaches is crucial. Electrophilic nitroalkenes derived from fatty acids are formed endogenously and exert anti-inflammatory actions by the modification of proteins involved in inflammation signaling cascades. We have developed novel nitroalkenes derived from α-tocopherol aiming to increase its salutary actions by adding anti-inflammatory properties to a well-known nutraceutical. We synthesized and characterized an α-tocopherol-nitroalkene (NATOH) and two hydrosoluble analogues derived from Trolox (NATxME and NATx0). We analyzed the kinetics of the Michael addition reaction of these compounds with thiols in micellar systems aiming to understand the effect of hydrophobic partition on the reactivity of nitroalkenes. We studied NATxME in vitro showing it exerts non-conventional anti-inflammatory responses by inducing Nrf2-Keap1-dependent gene expression and inhibiting the secretion of NF-κB dependent pro-inflammatory cytokines. NATxME was also effective in vivo, inhibiting neutrophil recruitment in a zebrafish model of inflammation. This work lays the foundation for the rational design of a new therapeutic strategy for the prevention and treatment of metabolic and inflammation-related diseases.

Asunto(s)

Alquenos/síntesis química , Alquenos/farmacología , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacología , Transducción de Señal , Tocoferoles/síntesis química , Tocoferoles/farmacología , Alquenos/química , Animales , Antiinflamatorios/química , Cromanos/síntesis química , Cromanos/química , Cromanos/farmacología , Cinética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Micelas , Infiltración Neutrófila/efectos de los fármacos , Células RAW 264.7 , Tocoferoles/química , Pez Cebra

RESUMEN

Glutathione transferases (GSTs) comprise a major detoxification system in helminth parasites, displaying both catalytic and non-catalytic activities. The kinetic mechanism of these enzymes is complex and depends on the isoenzyme which is being analyzed. Here, we characterized the kinetic mechanism of rEgGST1, a recombinant form of a cytosolic GST from Echinococcus granulosus (EgGST1), which is related to the Mu-class of mammalian enzymes, using the canonical substrates glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). Initial rate and product inhibition studies were consistent with a steady-state random sequential mechanism, where both substrates are bound to the enzyme before the products are released. Kinetic constants were also determined (pH 6.5 and 30 °C). Moreover, rEgGST1 lowered the pKa of GSH from 8.71 ± 0.07 to 6.77 ± 0.08, and enzyme-bound GSH reacted with CDNB 1 × 105 times faster than free GSH at pH 7.4. Finally, the dissociation of the enzyme-GSH complex was studied by means of intrinsic fluorescence, as well as that of the complex with the anthelminth drug mebendazole. This is the first report on mechanistic issues related to a helminth parasitic GST.

Asunto(s)

Echinococcus granulosus/química , Glutatión Transferasa/metabolismo , Glutatión/metabolismo , Proteínas del Helminto/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Antihelmínticos/farmacología , Clonación Molecular , Dinitroclorobenceno/metabolismo , Echinococcus granulosus/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutatión Transferasa/antagonistas & inhibidores , Glutatión Transferasa/genética , Proteínas del Helminto/antagonistas & inhibidores , Proteínas del Helminto/genética , Concentración de Iones de Hidrógeno , Inactivación Metabólica/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mebendazol/farmacología , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Especificidad por Sustrato

RESUMEN

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Albúmina Sérica/química , Albúmina Sérica/aislamiento & purificación , Anciano , Humanos , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/análisis , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Albúmina Sérica/análisis

RESUMEN

The plasma compartment has particular features regarding the nature and concentration of low and high molecular weight thiols and oxidized derivatives. Plasma is relatively poor in thiol-based antioxidants; thiols are in lower concentrations than in cells and mostly oxidized. The different thiol-disulfide pairs are not in equilibrium and the steady-state concentrations of total thiols as well as reduced versus oxidized ratios are maintained by kinetic barriers, including the rates of reactions and transport processes. The single thiol of human serum albumin (HSA-SH) is the most abundant plasma thiol. It is an important target for oxidants and electrophiles due to its reactivity with a wide variety of species and its relatively high concentration. A relatively stable sulfenic (HSA-SO3H) acid can be formed in albumin exposed to oxidants. Plasma increases in mixed disulfides (HSA-SSR) or in sulfinic (HSA-SO2H) and sulfonic (HSA-SO3H) acids are associated with different pathologies and may constitute biomarkers of the antioxidant role of the albumin thiol. In this work we provide a critical review of the plasma thiol pool with a focus on human serum albumin.

Asunto(s)

Plasma/química , Albúmina Sérica/metabolismo , Humanos , Oxidación-Reducción , Compuestos de Sulfhidrilo/metabolismo

RESUMEN

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ∼threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.

Asunto(s)

Ácidos Grasos/farmacología , Albúmina Sérica/química , Cristalografía por Rayos X , Ácido Ditionitrobenzoico/metabolismo , Ácido Ditionitrobenzoico/farmacología , Ácidos Grasos/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Modelos Moleculares , Oxidación-Reducción , Unión Proteica , Estabilidad Proteica , Albúmina Sérica/efectos de los fármacos , Albúmina Sérica/metabolismo , Ácidos Sulfénicos/química , Ácidos Sulfénicos/metabolismo , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/metabolismo , Reactivos de Sulfhidrilo/metabolismo , Reactivos de Sulfhidrilo/farmacología

RESUMEN

Protein sulfenic acids (R-SOH) are receiving increased interest as intermediates in redox processes. Human serum albumin, the most abundant protein in plasma, possesses a single free thiol. We describe herein the different methodologies that we have employed to study the formation of sulfenic acid in this protein and characterize some of its properties, including reactions that lead to the formation of mixed disulfides and the sulfinic acid derivative. The thiol of albumin is oxidized by hydrogen peroxide and peroxynitrite to a relatively stable sulfenic acid, which can be detected through different strategies including reduction with sodium arsenite and reaction with glutathione. Dimedone trapping followed by mass spectrometry analysis confirmed the modification. The challenge of obtaining quantitative data regarding albumin sulfenic acid has been approached using the yellow thiol thionitrobenzoate. A careful analysis has led to the determination of the rate constants of the reactions of sulfenic acid with analytical probes and with possible biological targets such as plasma thiols, which lead to mixed disulfides, and hydrogen peroxide, which overoxidizes the sulfenic to sulfinic acid. Our results support the concept that sulfenic acid is a central intermediate in the formation of oxidized albumin species that are present in circulating albumin and increase under pathological conditions.

Asunto(s)

Albúmina Sérica/metabolismo , Ácidos Sulfénicos/metabolismo , Arsenitos/química , Arsenitos/farmacología , Catálisis , Ciclohexanonas/química , Ciclohexanonas/farmacología , Glutatión/metabolismo , Glutatión/farmacología , Humanos , Espectrometría de Masas , Modelos Biológicos , Oxidación-Reducción , Albúmina Sérica/análisis , Albúmina Sérica/química , Compuestos de Sodio/química , Compuestos de Sodio/farmacología , Ácidos Sulfénicos/química , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/metabolismo

RESUMEN

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen's antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhpE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Asunto(s)

Cisteína/química , Mycobacterium tuberculosis/enzimología , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Ácidos Sulfénicos/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Mycobacterium tuberculosis/patogenicidad , Oxidación-Reducción , Peróxidos/antagonistas & inhibidores , Peróxidos/metabolismo , Peróxidos/toxicidad , Ácido Peroxinitroso/metabolismo , Conformación Proteica , Especificidad por Sustrato , Ácidos Sulfénicos/química , Compuestos de Sulfhidrilo/química , Termodinámica

RESUMEN

The single thiol of human serum albumin (HSA-SH) is the predominant plasma thiol. Both circulating albumin and pharmaceutical preparations are heterogeneous regarding the thiol redox status, as revealed by anion-exchange-hydrophobic interaction chromatography. Sulfenic acid (HSA-SOH) is an intermediate in HSA-SH oxidation processes that was detected through different techniques including mass spectrometry. Recently, quantitative data led to the determination of rate constants. The preferred fate of HSA-SOH is the formation of mixed disulfides. Alternatively, HSA-SOH can be further oxidized to sulfinic and sulfonic acids. Oxidized forms increase under disease conditions, underscoring the importance of HSA-SH as a plasma scavenger of intravascular oxidants. We here provide a critical review of the oxidation of HSA-SH in the context of the intravascular compartment, with emphasis in the methodological approaches of mass spectrometry and chromatography for the analysis of albumin thiol redox states.

Asunto(s)

Albúmina Sérica/química , Ácidos Sulfénicos/química , Compuestos de Sulfhidrilo/química , Cromatografía , Humanos , Espectrometría de Masas , Oxidación-Reducción , Albúmina Sérica/metabolismo

RESUMEN

Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was approximately 20-fold faster than the second phase and first order in HSA-SOH and TNB (105 +/- 11 M-1 s-1, 25 degrees C, pH 7.4), allowing quantitative data on HSA-SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 degrees C, 4 min) yielded 0.18 +/- 0.02 mol of HSA-SOH per mol of HSA. HSA-SH reacted with hydrogen peroxide at 2.7 +/- 0.7 M-1 s-1 (37 degrees C, pH 7.4), while HSA-SOH reacted at 0.4 +/- 0.2 M-1 s-1, yielding sulfinic acid (HSA-SO2H), as detected by mass spectrometry. The rate constants of HSA-SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA-SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA-SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 +/- 0.2, 2.9 +/- 0.5, 9.3 +/- 0.9, and 55 +/- 3 M-1 s-1 (25 degrees C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.

Asunto(s)

Albúmina Sérica/química , Ácidos Sulfénicos/química , Arsenitos/química , Ciclohexanonas/química , Humanos , Peróxido de Hidrógeno/química , Estructura Molecular , Nitrobenzoatos/química , Oxidación-Reducción , Compuestos de Sodio/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Compuestos de Sulfhidrilo/química