Lucía Turell

The thiol pool in human plasma: the central contribution of albumin to redox processes.

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.

Sulfenic acid in human serum albumin

Summary.Sulfenic acid (RSOH) is a central intermediate in both the reversible and irreversible redox modulation by reactive species of an increasing number of proteins involved in signal transduction and enzymatic pathways. In this paper we focus on human serum albumin (HSA), the most abundant plasma protein, proposed to serve antioxidant functions in the vascular compartment. Sulfenic acid in HSA has been previously detected using different methods after oxidation of its single free thiol Cys34 through one- or two-electron mechanisms. Since recent evidence suggests that sulfenic acid in HSA is stabilized within the protein environment, this derivative represents an appropriate model to examine protein sulfenic acid biochemistry, structure and reactivity. Sulfenic acid in HSA could be involved in mixed disufide formation, supporting a role of HSA-Cys34 as an important redox regulator in extracellular compartments.

Thiol and Sulfenic Acid Oxidation of AhpE, the One-Cysteine Peroxiredoxin from Mycobacterium tuberculosis: Kinetics, Acidity Constants, and Conformational Dynamics

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogens 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.

Oxidation of the albumin thiol to sulfenic acid and its implications in the intravascular compartment

Human serum albumin (HSA) is the most abundant protein in the intravascular compartment. It possesses a single thiol, Cys34, which constitutes ~80% of the total thiols in plasma. This thiol is able to scavenge plasma oxidants. A central intermediate in this potential antioxidant activity of human serum albumin is sulfenic acid (HSA-SOH). Work from our laboratories has demonstrated the formation of a relatively stable sulfenic acid in albumin through complementary spectrophotometric and mass spectrometric approaches. Recently, we have been able to obtain quantitative data that allowed us to measure the rate constants of sulfenic acid reactions with molecules of analytical and biological interest. Kinetic considerations led us to conclude that the most likely fate for sulfenic acid formed in the plasma environment is the reaction with low molecular weight thiols to form mixed disulfides, a reversible modification that is actually observed in ~25% of circulating albumin. Another possible fate for sulfenic acid is further oxidation to sulfinic and sulfonic acids. These irreversible modifications are also detected in the circulation. Oxidized forms of albumin are increased in different pathophysiological conditions and sulfenic acid lies in a mechanistic junction, relating oxidizing species to final thiol oxidation products.

Sulfenic acid—A key intermediate in albumin thiol oxidation

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.

Formation and Reactions of Sulfenic Acid in Human Serum Albumin

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.

HPLC separation of human serum albumin isoforms based on their isoelectric points

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.

The Chemical Basis of Thiol Addition to Nitro-conjugated Linoleic Acid, a Protective Cell-signaling Lipid

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 β-mercaptoethanol) and human serum albumin. Reactions followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2-CLA located on the β- 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 β-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.

The thiol of human serum albumin: Acidity, microenvironment and mechanistic insights on its oxidation to sulfenic acid

Abstract 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 0.1 M 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 &Dgr;‡G and &Dgr;‡H barriers at 298 K of respectively 59 and 54 kJ mol−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. Graphical abstract Figure. No Caption available. HighlightsThe pKa for delipidated HSA thiol is 8.1 at 37 °C and 0.1 M ionic strength.HSA thiolate is stabilized by H‐bonding with Tyr84 and waters bridging to Asp38.Calculated &Dgr;H‡ and &Dgr;G‡ to oxidize Cys34 by H2O2 respectively are 54 and 59 kJ mol−1.A post‐transition state proton transfer leads to HSA sulfenate and water.Theoretical data is in excellent agreement with temperature dependency of kinetics.

The chemical foundations of nitroalkene fatty acid signaling through addition reactions with thiols

Nitroalkene fatty acids can be formed in vivo and administered exogenously. They exert pleiotropic signaling actions with cytoprotective and antiinflammatory effects. The presence of the potent electron withdrawing nitro group confers electrophilicity to the adjacent β-carbon. Thiols (precisely, thiolates) are strong nucleophiles and can react with nitroalkene fatty acids through reversible Michael addition reactions. In addition, nitroalkene fatty acids can undergo several other processes including metabolic oxidation, reduction, esterification, nitric oxide release and partition into hydrophobic compartments. The signaling actions of nitroalkenes are mainly mediated by reactions with critical thiols in regulatory proteins. Thus, the thio-Michael addition reaction provides a framework for understanding the molecular basis of the biological effects of nitroalkene fatty acids at the crossroads of thiol signaling and electrophilic lipid signaling. In this review, we describe the reactions of nitroalkene fatty acids in biological contexts. We focus on the Michael addition-elimination reaction with thiols and its mechanism, and extrapolate kinetic and thermodynamic considerations to in vivo settings.