Review ArticleThe thiol pool in human plasma: The central contribution of albumin to redox processes
Graphical abstract
Introduction
In biological systems, thiols are found in cysteine and derived molecules of low and high molecular weight. Thiols are good reductants and good nucleophiles; they can react by one- and two-electron mechanisms and they are susceptible to reversible and irreversible modifications. In most proteins, cysteine accounts for less than 3% of the amino acid composition. However, its chemical versatility allows this residue to participate in several processes such as catalysis, signaling, antioxidant defense, metal complexing, and structural stabilization.
The plasma compartment is characterized by having relatively low concentrations of thiols and by the presence of human serum albumin (HSA) as the most abundant one. In the first part of this review we will discuss the thiol pool of plasma, and in the second part we will focus on HSA and its thiol (HSA-SH).
Section snippets
Redox versatility of thiols
Thiols (RSH) can undergo oxidation processes to yield a wide range of products (Fig. 1). They can react with disulfides (RSSR) through reversible thiol-disulfide exchange reactions (pathway a in Fig. 1). These reactions can occur alone at appreciable rates or they can be catalyzed by enzymes of the thioredoxin family. Thiols can react with two-electron oxidants such as hydroperoxides (POOH) or hypohalous acids (i.e., HOCl) yielding sulfenic acids (RSOH) (pathway b in Fig. 1). Sulfenic acids are
The reacting species is the thiolate
For most chemical and enzymatic reactions of thiols, reactivity involves the nucleophilic attack of the ionized thiolate (RS−) on an electrophile. Thus, the observed rate constant for a certain reaction increases as the pH alkalinizes because more thiolate is available. In turn, if we compare different compounds, we can see that the availability of thiolate at a fixed pH depends on the pKa of the corresponding thiol; as the pKa of the thiol decreases, more thiol is ionized to thiolate.
The fact
In plasma, total thiols are at a lower concentration than in cells and the predominant thiol is HSA
In cells, thiols are present at millimolar concentrations and highly reduced. The total glutathione concentration is 2–17 mM [6] and the percentage of reduced glutathione is 91% for whole cells [7]. Specifically in the cytosolic compartment, glutathione is even more reduced, with recent evaluations yielding values of~99.97% [8], [9]. Protein thiols are more abundant than glutathione (10–50 mM) [6]. They are~90% reduced and they represent ~70% of the total pool of reduced thiols [7]. Two important
The different thiol-disulfide pairs are not in equilibrium with each other in plasma
If the different thiols and disulfides were in equilibrium in the circulation with regard to the thiol disulfide exchange reactions, the concentration quotients would equal the value of the equilibrium constants. For example, the exchange between cysteine and glutathione is represented by (actually, the reacting species are the thiolates)CysSH + GSSG ⇌ CysSSG + GSHCysSH + CysSSG ⇌ CysSSCys + GSH.
The overall reaction is described by2CysSH + GSSG ⇌ CysSSCys + 2GSH.
The corresponding equilibrium
Possible kinetic barriers that keep plasma thiol and disulfide concentrations away from equilibrium
Since thiols and disulfides are not in equilibrium in plasma, it is clear that kinetic barriers exist. The steady-state concentrations are the result of several processes occurring simultaneously that affect not only the total concentrations but also the ratios of oxidized versus reduced thiols. Although qualitative information about these processes exists, precise quantitative information is mostly lacking. The plasma concentrations of thiols and oxidized derivatives are influenced by: (i) the
The extracellular low molecular weight thiol pool is not an inert bystander but actively affects cell processes
The extracellular concentrations of different thiols and oxidized derivatives are tightly regulated, and its alterations are associated with cellular responses. Cells in culture respond to increased ratios of cysteine disulfide to cysteine in the extracellular medium through downstream events that impact on proliferation, apoptosis, and proinflammatory signaling (for review see [29]). In line with this concept, the in vivo concentrations of plasma low molecular weight thiols and disulfides have
Antioxidants are scarce in plasma and HSA is in the spotlight as the predominant thiol
Plasma low molecular weight antioxidants include reduced thiols, ascorbate, urate, among others (Table 1, Table 2). Reduced low molecular weight thiols are in very low concentrations, adding up to a total of 12–20 µM.
Despite the plasma proteome comprising more than 3000 components, proteins with potential antioxidant function are also scarce in this compartment. Among the 150 most abundant polypeptides, only two possess antioxidant capacity: human serum albumin and glutathione peroxidase 3 [33] (
HSA is a monomeric, three-domain, allosteric protein with one free cysteine
HSA is a monomeric, nonglycosylated, three-domain protein. It is present in plasma at a concentration of ~43 g/L (~0.6 mM), constituting about 60% of total plasma proteins. It has 585 amino acids (66438 Da) and 17 disulfide bridges leaving only one free cysteine, Cys34. At pH 7.4, HSA possesses 215 ions with a net charge of -19 conferring HSA a high solubility [34].
The crystal structure of HSA has already been solved and it shows that it is a heart-shaped protein with 67% α-helixes and no β-sheets
HSA is synthesized in the liver and present in intravascular and extravascular compartments
HSA synthesis takes place in the hepatocytes accounting for 10% of total protein synthesis in the liver [39]. HSA follows the rule “one gene–one protein” and as a monomeric protein no chain assembly is needed. When isolated from plasma, HSA contains 585 amino acids; however, when first translated from RNA it contains a prepropeptide at the N-terminus which is removed in the hepatocyte before secretion. Disulfide formation starts before the chain is completed and proceeds in the N-terminal to
HSA has several functions including the binding of exogenous and endogenous compounds
HSA plays several roles in the circulation. HSA is responsible for 80% of the oncotic pressure in plasma. Due to the 19 negative charge of HSA at pH 7.4, it constitutes the principal macromolecular anion in plasma [34].
The multidomain structure allows HSA to bind and transport a wide variety of endogenous and exogenous ligands. Long and medium chain fatty acids constitute some of the most important endogenous ligands. These relatively insoluble and metabolically active molecules are transported
HSA is heterogeneous regarding the oxidation state of Cys34 in vitro and in vivo
The presence of a free thiol in HSA, Cys34, gives rise to heterogeneity. In plasma, HSA is found as a mixture of mercaptalbumin with the thiol in the reduced form (HSA-SH) and nonmercaptalbumin with the thiol modified (Table 1). In healthy adults, about 75% of HSA is found as mercaptalbumin. The nonmercaptalbumin fraction consists mainly of mixed disulfides of HSA with low molecular weight thiols [67], [68]. About 1–2% of total HSA is found with the thiol oxidized to higher oxidation states
The HSA thiol reacts with a wide variety of oxidant species
Honoring its thiol nature, HSA-SH can react in the test tube with most typical radical and nonradical oxidants. The occurrence of these reactions in vivo is evidenced by the detection of thiol oxidized isoforms of albumin in the circulation, that increase in conditions related to oxidative stress. The exact nature of the oxidant species involved cannot be elucidated from product characterization studies, since the final products of the reactions are expected to overlap. So what oxidants could
Cys34 pKa: A long-standing controversy
The HSA-SH acidity constant (pKa) has been reported as <7 [36] or even <5 [88]. Other reports have shown values of about 8 [85], [80], [89]. Three aspects may contribute to the complexities regarding the determination of this pKa and the diversity of results obtained. First, according to NMR experiments, the thiol exists in two conformations and the reduced thiol predominates in a buried conformation that shifts to an exposed one upon chemical modification [90], [91]. Second, HSA suffers
The reactivity of HSA-SH is restricted by the protein environment
When we compare HSA-SH against low molecular weight thiols, its diminished reactivity becomes evident. With hydrogen peroxide, a small oxidant, the pH-independent rate constant (when all the thiol is in the thiolate form) for HSA is fourfold lower (5.3±0.2 M−1 s−1) [80] than those for low molecular weight thiolates of comparable pKa (18–26 M−1 s−1) [94]. With peroxynitrous acid, the HSA thiolate reacts at 7.9×104 M−1 s−1 [85] while low molecular weight thiolates react at 1.3×105–2×105 M−1 s−1 [95].
Ligand binding affects thiol reactivity
As noted before HSA plays a key role in fatty acid transport between tissues. Despite the fact that Cys34 is not directly located in a fatty acid binding site, its oxidizability is affected [55], [98], [99]. When we studied the effect of fatty acid binding on Cys34 reactivity we found that the stearic acid-HSA (5/1) complex reacted sixfold faster than fatty acid-free HSA at pH 7.4 with DTNB and twofold faster with hydrogen peroxide and peroxynitrite. We also observed a decrease in pKa by 0.5 pH
A relatively stable sulfenic acid is formed in HSA
Sulfenic acid formation in HSA was observed more than 60 years ago [100] and confirmed with different analytical techniques [79], [85], [101], [102], [103], [104]. However, for a comprehensive characterization of its reactivity and stability it was necessary to obtain quantitative data on its formation. We have developed a strategy for quantifying sulfenic acid (HSA-SOH) formed after exposure of HSA-SH to oxidants [81]. Thionitrobenzoate (TNB) reacts with HSA-SOH, yielding the mixed disulfide
Oxidized forms of HSA are increased in several pathophysiological conditions
Oxidative stress can be defined as an imbalance between prooxidants and antioxidants in favor of the former [105] or, from a mechanistic standpoint, as a disruption of redox signaling and control [106]. This condition is linked to a growing number of human diseases and pathophysiological processes. HSA-SH, as the most abundant thiol in plasma, is expected to be oxidatively modified in these situations.
The HSA redox state has been assessed in diverse conditions using mass spectrometry,
Acknowledgments
This work was supported by grants from ANII, Agencia Nacional de Investigación e Innovación, Uruguay, FCE_2009_1988 (to L.T.), CSIC, Universidad de la República, Uruguay (to R.R. and B.A.), and the National Institutes of Health (R01AI095173, to R.R.). L.T. was partially supported by a fellowship from ANII. We thank María José Torres, Horacio Botti, and Gerardo Ferrer-Sueta for helpful discussions.
References (135)
- et al.
Carbon dioxide stimulates the production of thiyl, sulfinyl, and disulfide radical anion from thiol oxidation by peroxynitrite
J. Biol. Chem.
(2001) Superoxide as an intracellular radical sink
Free Radic. Biol. Med.
(1993)- et al.
Homocysteine metabolism in pregnancies complicated by neural-tube defects
Lancet
(1995) - et al.
Chemical biology of homocysteine thiolactone and related metabolites
Adv. Clin. Chem.
(2011) - et al.
Redox biochemistry of hydrogen sulfide
J. Biol. Chem.
(2010) - et al.
Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest
Free Radic. Biol. Med.
(2011) The effect of cyst(e)ine on the auto-oxidation of homocysteine
Free Radic. Biol. Med
(1999)- et al.
Relative roles of albumin and ceruloplasmin in the formation of homocystine, homocysteine-cysteine-mixed disulfide, and cystine in circulation
J. Biol. Chem.
(2001) - et al.
Albumin thiolate anion is an intermediate in the formation of albumin-S-S-homocysteine
J. Biol. Chem.
(2001) - et al.
N-Acetylcysteine—a safe antidote for cysteine/glutathione deficiency
Curr. Opin. Pharmacol.
(2007)
N -Acetyl-cysteine reduces homocysteine plasma levels after single intravenous administration by increasing thiols urinary excretion
Pharmacol. Res.
Redox clamp model for study of extracellular thiols and disulfides in redox signaling
Methods Enzymol
Cysteine/cystine redox signaling in cardiovascular disease
Free Radic. Biol. Med.
The undertow of sulfur metabolism on glutamatergic neurotransmission
Trends Biochem. Sci.
Cerebral cystine uptake: a tale of two transporters
Trends Pharmacol. Sci.
Allostery in a monomeric protein: the case of human serum albumin
Biophys. Chem.
Fatty acid binding to human serum albumin: new insights from crystallographic studies
Biochim. Biophys. Acta
Albondin-mediated capillary permeability to albumin. Differential role of receptors in endothelial transcytosis and endocytosis of native and modified albumins
J. Biol. Chem
Albumin turnover: experimental approach and its application in health and renal diseases
Clin. Chim. Acta
Preferential interaction of albumin-binding proteins, gp30 and gp18, with conformationally modified albumins. Presence in many cells and tissues with a possible role in catabolism
J. Biol. Chem.
Oxidation of Arg-410 promotes the elimination of human serum albumin
Biochim. Biophys. Acta
Structure and lipid binding properties of serum albumin
Methods Enzymol.
Crystallographic analysis reveals common modes of binding of medium and long-chain fatty acids to human serum albumin
J. Mol. Biol
Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis
J. Mol. Biol.
Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants
Arch. Biochem. Biophys.
Hydrogen peroxide-mediated degradation of protein: different oxidation modes of copper- and iron-dependent hydroxyl radicals on the degradation of albumin
Biochim. Biophys. Acta
Metal-catalyzed oxidation of human serum albumin: conformational and functional changes. Implications in protein aging
J. Biol. Chem.
Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles
J Controlled Release
Pharmaceutical aspects of the recombinant human serum albumin dimer: structural characteristics, biological properties, and medical applications
J. Pharm. Sci.
Summary of recombinant human serum albumin development
Biological
Determination of the in vivo redox status of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma
Anal. Biochem.
Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry
Anal. Biochem.
Characterization of oxidation end product of plasma albumin ‘in vivo’
Biochem. Biophys. Res. Commun.
Redox state of human serum albumin in terms of cysteine-34 in health and disease
Methods Enzymol
Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues
Free Radic. Biol. Med.
Nitro-fatty acid metabolome: saturation, desaturation, beta-oxidation, and protein adduction
J. Biol. Chem.
Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids
Arch. Biochem. Biophys.
The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2
Arch. Biochem. Biophys.
Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations
Arch. Biochem. Biophys.
Kinetics of peroxynitrite reaction with amino acids and human serum albumin
J. Biol. Chem.
Nitrogen dioxide and carbonate radical anion: two emerging radicals in biology
Free Radic. Biol. Med.
Sulfenic acid-a key intermediate in albumin thiol oxidation
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
1H NMR of albumin in human blood plasma: drug binding and redox reactions at Cys34
FEBS Lett.
Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide
Free Radic. Biol. Med.
Peroxynitrite reaction with the reduced and the oxidized forms of lipoic acid: new insights into the reaction of peroxynitrite with thiols
Arch. Biochem. Biophys
S-Nitrosylated human serum albumin-mediated cytoprotective activity is enhanced by fatty acid binding
J. Biol. Chem.
Pathways of peroxynitrite oxidation of thiol groups
Biochem. J.
Evaluation of the "radical sink" hypothesis from a chemical-kinetic viewpoint
J. Radianal. Nucl. Chem
Factors affecting protein thiol reactivity and specificity in peroxide reduction
Chem. Res. Toxicol.
Cysteine residues exposed on protein surfaces are the dominant intramitochondrial thiol and may protect against oxidative damage
FEBS J.
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