RESUMO
Human serum albumin (HSA) contains 17 disulfides and only one reduced cysteine, Cys34, which can be oxidized to a relatively stable sulfenic acid (HSA-SOH). This derivative has been previously detected and quantified. However, its properties are poorly understood. Herein, HSA-SOH formation from the exposure of HSA to hydrogen peroxide was confirmed using the sulfenic acid probe bicyclo [6.1.0]nonyne-biotin (BCN-Bio1), and by direct detection by whole protein mass spectrometry. The decay pathways of HSA-SOH were studied. HSA-SOH reacted with a thiol leading to the formation of a mixed disulfide. The reaction occurred through a concerted or direct displacement mechanism (SN2) with the thiolate (RS-) as nucleophile towards HSA-SOH. The net charge of the thiolate affected the value of the rate constant. In the presence of hydrogen peroxide, HSA-SOH was further oxidized to sulfinic acid (HSA-SO2H) and sulfonic acid (HSA-SO3H). The rate constants of these reactions were estimated. Lastly, HSA-SOH spontaneously decayed in solution. Mass spectrometry experiments suggested that the decay product is a sulfenylamide (HSA-SN(R')Râ³). Chromatofocusing analysis showed that the overoxidation with hydrogen peroxide predominates at alkaline pH whereas the spontaneous decay predominates at acidic pH. The present findings provide insights into the reactivity and fate of the sulfenic acid in albumin, which are also of relevance to numerous sulfenic acid-mediated processes in redox biology and catalysis.
Assuntos
Ácidos Sulfênicos , Compostos de Sulfidrila , Cisteína , Humanos , Oxirredução , Albumina Sérica/metabolismo , Albumina Sérica HumanaRESUMO
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.
Assuntos
Antioxidantes/química , Cisteína/genética , Albumina Sérica Humana/genética , Compostos de Sulfidrila/química , Cromatografia por Troca Iônica , Cisteína/química , Expressão Gênica/genética , Humanos , Domínios Proteicos/genética , Multimerização Proteica , Saccharomycetales/genética , Albumina Sérica Humana/químicaRESUMO
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.
Assuntos
Cisteína/química , Proteínas/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional , Proteínas/químicaRESUMO
Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45 ± 0.04)â¯×â¯103 M-1 s-1, koff of (4.4 ± 0.4)â¯×â¯10-4 s-1, and Keq of (1.3 ± 0.1)â¯×â¯10-7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.
Assuntos
Glutationa Redutase/química , Glutationa Redutase/metabolismo , Glutationa/biossíntese , Algoritmos , Alquilação , Sequência de Aminoácidos , Animais , Catálise , Domínio Catalítico , Dissulfeto de Glutationa/metabolismo , Espaço Intracelular , Cinética , Camundongos , Modelos Teóricos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Estresse Oxidativo , Células RAW 264.7 , Espécies Reativas de Oxigênio , Compostos de SulfidrilaRESUMO
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.
Assuntos
Alcenos/síntese química , Alcenos/farmacologia , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Transdução de Sinais , Tocoferóis/síntese química , Tocoferóis/farmacologia , Alcenos/química , Animais , Anti-Inflamatórios/química , Cromanos/síntese química , Cromanos/química , Cromanos/farmacologia , Cinética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Micelas , Infiltração de Neutrófilos/efeitos dos fármacos , Células RAW 264.7 , Tocoferóis/química , Peixe-ZebraRESUMO
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.
Assuntos
Echinococcus granulosus/química , Glutationa Transferase/metabolismo , Glutationa/metabolismo , Proteínas de Helminto/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anti-Helmínticos/farmacologia , Clonagem Molecular , Dinitroclorobenzeno/metabolismo , Echinococcus granulosus/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glutationa Transferase/antagonistas & inibidores , Glutationa Transferase/genética , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/genética , Concentração de Íons de Hidrogênio , Inativação Metabólica/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Mebendazol/farmacologia , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Especificidade por SubstratoRESUMO
Human serum albumin (HSA) has a single reduced cysteine residue, Cys34, whose acidity has been controversial. Three experimental approaches (pH-dependence of reactivity towards hydrogen peroxide, ultraviolet titration and infrared spectroscopy) are used to determine that the pKa value in delipidated HSA is 8.1±0.2 at 37°C and 0.1M ionic strength. Molecular dynamics simulations of HSA in the sub-microsecond timescale show that while sulfur exposure to solvent is limited and fluctuating in the thiol form, it increases in the thiolate, stabilized by a persistent hydrogen-bond (HB) network involving Tyr84 and bridging waters to Asp38 and Gln33 backbone. Insight into the mechanism of Cys34 oxidation by H2O2 is provided by ONIOM(QM:MM) modeling including quantum water molecules. The reaction proceeds through a slightly asynchronous SN2 transition state (TS) with calculated ΔG and ΔH barriers at 298K of respectively 59 and 54kJmol-1 (the latter within chemical accuracy from the experimental value). A post-TS proton transfer leads to HSA-SO- and water as products. The structured reaction site cages H2O2, which donates a strong HB to the thiolate. Loss of this HB before reaching the TS modulates Cys34 nucleophilicity and contributes to destabilize H2O2. The lack of reaction-site features required for differential stabilization of the TS (positive charges, H2O2 HB strengthening) explains the striking difference in kinetic efficiency for the same reaction in other proteins (e.g. peroxiredoxins). The structured HB network surrounding HSA-SH with sequestered waters carries an entropic penalty on the barrier height. These studies contribute to deepen the understanding of the reactivity of HSA-SH, the most abundant thiol in human plasma, and in a wider perspective, provide clues on the key aspects that modulate thiol reactivity against H2O2.
Assuntos
Peróxido de Hidrogênio/metabolismo , Albumina Sérica/metabolismo , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/química , Cisteína/química , Engenharia Genética , Humanos , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Oxirredução , Estresse Oxidativo , Ligação Proteica , Conformação Proteica , Albumina Sérica/química , Ácidos Sulfênicos/químicaRESUMO
Nitroalkene fatty acids are formed in vivo and exert protective and anti-inflammatory effects via reversible Michael addition to thiol-containing proteins in key signaling pathways. Nitro-conjugated linoleic acid (NO2-CLA) is preferentially formed, constitutes the most abundant nitrated fatty acid in humans, and contains two carbons that could potentially react with thiols, modulating signaling actions and levels. In this work, we examined the reactions of NO2-CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine, and ß-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the ß- and δ-carbons with respect to the nitro group. The differential reactivity was confirmed by computational modeling of the electronic structure. The rates (kon and koff) and equilibrium constants for both reactions were determined for different thiols. LC-UV-Visible and LC-MS analyses showed that the fast reaction corresponds to ß-adduct formation (the kinetic product), while the slow reaction corresponds to the formation of the δ-adduct (the thermodynamic product). The pH dependence of the rate constants, the correlation between intrinsic reactivity and thiol pKa, and the absence of deuterium solvent kinetic isotope effects suggested stepwise mechanisms with thiolate attack on NO2-CLA as rate-controlling step. Computational modeling supported the mechanism and revealed additional features of the transition states, anionic intermediates, and final neutral products. Importantly, the detection of cysteine-δ-adducts in human urine provided evidence for the biological relevance of this reaction. Finally, human serum albumin was found to bind NO2-CLA both non-covalently and to form covalent adducts at Cys-34, suggesting potential modes for systemic distribution. These results provide new insights into the chemical basis of NO2-CLA signaling actions.
Assuntos
Ácido Linoleico/química , Nitrocompostos/química , Albumina Sérica/química , Transdução de Sinais , Compostos de Sulfidrila/química , HumanosRESUMO
Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (-SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H2 O2 reduction, a sulfenic acid (-SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S-S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx-MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H2 O2 stress, and its gene expression is clearly induced upon H2 O2 challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Cisteína/metabolismo , Glicopeptídeos/metabolismo , Inositol/metabolismo , Peroxidases/metabolismo , Compostos de Sulfidrila/metabolismo , Corynebacterium glutamicum/genética , Dissulfetos/metabolismo , Peróxido de Hidrogênio/farmacologia , Cinética , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de Proteína , Tiorredoxinas/metabolismoRESUMO
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.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Albumina Sérica/química , Albumina Sérica/isolamento & purificação , Idoso , Humanos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Albumina Sérica/análiseRESUMO
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 â¼threefold 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.
Assuntos
Ácidos Graxos/farmacologia , Albumina Sérica/química , Cristalografia por Raios X , Ácido Ditionitrobenzoico/metabolismo , Ácido Ditionitrobenzoico/farmacologia , Ácidos Graxos/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Modelos Moleculares , Oxirredução , Ligação Proteica , Estabilidade Proteica , Albumina Sérica/efeitos dos fármacos , Albumina Sérica/metabolismo , Ácidos Sulfênicos/química , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/metabolismo , Reagentes de Sulfidrila/farmacologiaRESUMO
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.
Assuntos
Albumina Sérica/metabolismo , Ácidos Sulfênicos/metabolismo , Arsenitos/química , Arsenitos/farmacologia , Catálise , Cicloexanonas/química , Cicloexanonas/farmacologia , Glutationa/metabolismo , Glutationa/farmacologia , Humanos , Espectrometria de Massas , Modelos Biológicos , Oxirredução , Albumina Sérica/análise , Albumina Sérica/química , Compostos de Sódio/química , Compostos de Sódio/farmacologia , Ácidos Sulfênicos/química , Compostos de Sulfidrila/análise , Compostos de Sulfidrila/metabolismoRESUMO
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.
Assuntos
Cisteína/química , Mycobacterium tuberculosis/enzimologia , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Ácidos Sulfênicos/metabolismo , Compostos de Sulfidrila/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Mycobacterium tuberculosis/patogenicidade , Oxirredução , Peróxidos/antagonistas & inibidores , Peróxidos/metabolismo , Peróxidos/toxicidade , Ácido Peroxinitroso/metabolismo , Conformação Proteica , Especificidade por Substrato , Ácidos Sulfênicos/química , Compostos de Sulfidrila/química , TermodinâmicaRESUMO
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.