Sulfenic acid formation in human serum albumin by hydrogen peroxide and peroxynitrite

Human serum albumin (HSA), the most abundant protein in plasma, has been proposed to have an antioxidant role. The main feature responsible for this property is its only thiol, Cys34, which comprises approximately 80% of the total free thiols in plasma and reacts preferentially with reactive oxygen and nitrogen species. Herein, we show that the thiol in HSA reacted with hydrogen peroxide with a second-order rate constant of 2.26 M(-1) s(-1) at pH 7.4 and 37 degrees C and a 1:1 stoichiometry. The formation of intermolecular disulfide dimers was not observed, suggesting that the thiol was being oxidized beyond the disulfide. With the reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl), we were able to detect the formation of sulfenic acid (HSA-SOH) from the UV-vis spectra of its adduct. The formation of sulfenic acid in Cys34 was confirmed by mass spectrometry using 5,5-dimethyl-1,3-cyclohexanedione (dimedone). Sulfenic acid was also formed from exposure of HSA to peroxynitrite, the product of the reaction between nitric oxide and superoxide radicals, in the absence or in the presence of carbon dioxide. The latter suggests that sulfenic acid can also be formed through free radical pathways since following reaction with carbon dioxide, peroxynitrite yields carbonate radical anion and nitrogen dioxide. Sulfenic acid in HSA was remarkably stable, with approximately 15% decaying after 2 h at 37 degrees C under aerobic conditions. The formation of glutathione disulfide and mixed HSA-glutathione disulfide was determined upon reaction of hydrogen peroxide-treated HSA with glutathione. Thus, HSA-SOH is proposed to serve as an intermediate in the formation of low molecular weight disulfides, which are the predominant plasma form of low molecular weight thiols, and in the formation of mixed HSA disulfides, which are present in approximately 25% of circulating HSA.     

Reactivity of sulfenic acid in human serum albumin

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.     

Sulfenic acids as reactive intermediates in xenobiotic metabolism

Sulfenic acid reactive intermediates are formed during the oxidation of cysteine residues of proteins and play key roles in enzyme catalysis, redox homeostasis and regulation of cell signalling. However few data are presently available on the formation and fate of sulfenic acids as reactive intermediates during the metabolism of xenobiotics. This article is a review of the xenobiotic metabolism situations in which the intermediate formation of a sulfenic acid has been reported. Formation of these intermediates has been either proposed on the basis of the isolation of products possibly deriving from sulfenic acids or shown after trapping of the sulfenic acid by specific nucleophiles. This review indicates the different mechanisms by which a sulphur-containing xenobiotic can be metabolized with the intermediate formation of a sulfenic acid. It also indicates the different possible fates of these sulfenic acids that have been reported in the literature. Finally, it discusses the possible implications of the formation of xenobiotic-derived sulfenic acid reactive metabolites in pharmacology and toxicology.     

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.     

Synthesis of chemical probes to map sulfenic acid modifications on proteins

Cysteine sulfenic acids in proteins can be identified by their ability to form adducts with dimedone, but this reagent imparts no spectral or affinity tag for subsequent analyses of such tagged proteins. Given its similar reactivity toward cysteine sulfenic acids, 1,3-cyclohexadione was synthetically modified to an alcohol derivative and linked to fluorophores based on isatoic acid and 7-methoxycoumarin. The resulting compounds retain full reactivity and specificity toward cysteine sulfenic acids in proteins, allowing for incorporation of the fluorescent label into the protein and "tagging" it based on its sulfenic acid redox state. Control experiments using dimedone further show the specificity of the reaction of 1,3-diones with protein sulfenic acids in aqueous media. These new compounds provide the basis for an improved method for the detection of protein sulfenic acids.     

Prediction of protein homo-oligomer types by pseudo amino acid composition: Approached with an improved feature extraction and Naive Bayes Feature Fusion

Summary. The interaction of non-covalently bound monomeric protein subunits forms oligomers. The oligomeric proteins are superior to the monomers within the scope of functional evolution of biomacromolecules. Such complexes are involved in various biological processes, and play an important role. It is highly desirable to predict oligomer types automatically from their sequence. Here, based on the concept of pseudo amino acid composition, an improved feature extraction method of weighted auto-correlation function of amino acid residue index and Naive Bayes multi-feature fusion algorithm is proposed and applied to predict protein homo-oligomer types. We used the support vector machine (SVM) as base classifiers, in order to obtain better results. For example, the total accuracies of A, B, C, D and E sets based on this improved feature extraction method are 77.63, 77.16, 76.46, 76.70 and 75.06% respectively in the jackknife test, which are 6.39, 5.92, 5.22, 5.46 and 3.82% higher than that of G set based on conventional amino acid composition method with the same SVM. Comparing with Chou’s feature extraction method of incorporating quasi-sequence-order effect, our method can increase the total accuracy at a level of 3.51 to 1.01%. The total accuracy improves from 79.66 to 80.83% by using the Naive Bayes Feature Fusion algorithm. These results show: 1) The improved feature extraction method is effective and feasible, and the feature vectors based on this method may contain more protein quaternary structure information and appear to capture essential information about the composition and hydrophobicity of residues in the surface patches that buried in the interfaces of associated subunits; 2) Naive Bayes Feature Fusion algorithm and SVM can be referred as a powerful computational tool for predicting protein homo-oligomer types.     

Redox properties of serum albumin

Background

Oxidative damage results in protein modification, and is observed in numerous diseases. Human serum albumin (HSA), the most abundant circulating protein in the plasma, exerts important antioxidant activities against oxidative damage.

Scope of review

The present review focuses on the characterization of chemical changes in HSA that are induced by oxidative damage, their relevance to human pathology and the most recent advances in clinical applications.

Major conclusions

The antioxidant properties of HSA are largely dependent on Cys34 and its contribution to the maintenance of intravascular homeostasis, including protecting the vascular endothelium under disease conditions related to oxidative stress. Recent studies also evaluated the susceptibility of other important amino acid residues to free radicals. The findings suggest that a redox change in HSA is related to the oxidation of several amino acid residues by different oxidants. Further, Cys34 adducts, such as S-nitrosylated and S-guanylated forms also play an important role in clinical applications. On the other hand, the ratio of the oxidized form to the normal form of albumin (HMA/HNA), which is a function of the redox states of Cys34, could serve as a useful marker for evaluating systemic redox states, which would be useful for the evaluation of disease progression and therapeutic efficacy.

General significance

This review provides new insights into our current understanding of the mechanism of HSA oxidation, based on in vitro and in vivo studies.This article is part of a Special Issue entitled Serum Albumin.     

Facile synthesis and biological evaluation of a cell-permeable probe to detect redox-regulated proteins

We have developed an improved synthesis for the cell-permeable, sulfenic acid probe DAz-1. Using DAz-1, we detect sulfenic acid modifications in the cell-cycle regulatory phosphatase Cdc25A. In addition, we show that DAz-1 has superior potency in cells compared to a biotinylated derivative. Collectively, these findings set the stage for the development of activity-based inhibitors of Cdc25 cell-cycle phosphatases, which are sensitive to the redox state of the active-site cysteine and demonstrate the advantage of bioorthogonal conjugation methods to detect protein sulfenic acids in cells.     

Mechanisms of homocysteine toxicity in humans

Summary. Homocysteine, a non-protein amino acid, is an important risk factor for ischemic heart disease and stroke in humans. This review provides an overview of homocysteine influence on endothelium function as well as on protein metabolism with a special respect to posttranslational modification of protein with homocysteine thiolactone. Homocysteine is a pro-thrombotic factor, vasodilation impairing agent, pro-inflammatory factor and endoplasmatic reticulum-stress inducer. Incorporation of Hcy into protein via disulfide or amide linkages (S-homocysteinylation or N-homocysteinylation) affects protein structure and function. Protein N-homocysteinylation causes cellular toxicity and elicits autoimmune response, which may contribute to atherogenesis. Present address: Department of Biochemistry and Biotechnology, Agricultural University, 60637 Poznań, Poland     

Cytoplasmic/nuclear localization of tuberin in different cell lines

Summary. Tuberous sclerosis (TSC) is an autosomal dominantly inherited disease affecting 1 in 6000 individuals. The TSC gene products, hamartin and tuberin, form a complex, of which tuberin is assumed to be the functional component being involved in a wide variety of different cellular processes. Tuberin has been demonstrated to be localized to both, the cytoplasm and the nucleus. The cytoplasmic/nuclear localization of tuberin is known to be regulated by the serine/threonine protein kinase Akt. Akt also regulates the cytoplasmic/nuclear localization of the cyclin-dependent kinase inhibitor p27. In this study the localization of these two Akt-regulated proteins was analysed in different cell lines.     

Characterization of the yeast peroxiredoxin Ahp1 in its reduced active and overoxidized inactive forms using NMR

Peroxiredoxins (Prx's) are a superfamily of thiol-specific antioxidant proteins present in all organisms and involved in the hydroperoxide detoxification of the cell. The catalytic cysteine of Prx's reduces hydroperoxides and is transformed into a transient sulfenic acid (Cys-SOH). At high hydroperoxide concentration, the sulfenic acid can be overoxidized into a sulfinate, or even a sulfonate. We present here the first peroxiredoxin characterization by solution NMR of the Saccharomyces cerevisiae alkylhydroperoxide reductase (Ahp1) in its reduced and in vitro overoxidized forms. NMR (15)N relaxation data and ultracentrifugation experiments indicate that the protein behaves principally as a homodimer (2 x 19 kDa) in solution, regardless of the redox state. In vitro treatment of Ahp1 by a large excess of tBuOOH leads to an inactive form, with the catalytic cysteine overoxidized into sulfonate, as demonstrated by (13)C NMR. Depending on the amino acid sequence of their active site, Prx's are classified into five different families. In this classification, Ahp1 is a member of the scarcely studied D-type Prx's. Ahp1 is unique among the D-type Prx's in its ability to form an intermolecular disulfide. The peptidic sequence of Ahp1 was analyzed and compared to other D-type Prx sequences.     

The effects of thiopentone on free intracellular amino acids in polymorphonuclear leucocytes

Summary. Previous studies have shown the inhibitory effects of thiopentone on polymorphonuclear leucocyte (PML) function. However, major biochemical mechanisms which have been involved are still unknown. The aim of this study was therefore to investigate thiopentone's effects on intracellular amino acid metabolism in PML using both advanced PML separation – and HPLC techniques, especially developed for this purpose and precisely validated in our institute. Overall, our study indicates important dose-dependent alterations of free intracellular amino acid metabolism following thiopentone treatment and draw attention to the biochemical mechanisms which may be involved in both thiopentone-induced modulation in PML function and cellular immunocompetence. Received April 4, 1999     

Using cellular automata images and pseudo amino acid composition to predict protein subcellular location

Summary. The avalanche of newly found protein sequences in the post-genomic era has motivated and challenged us to develop an automated method that can rapidly and accurately predict the localization of an uncharacterized protein in cells because the knowledge thus obtained can greatly speed up the process in finding its biological functions. However, it is very difficult to establish such a desired predictor by acquiring the key statistical information buried in a pile of extremely complicated and highly variable sequences. In this paper, based on the concept of the pseudo amino acid composition (Chou, K. C. PROTEINS: Structure, Function, and Genetics, 2001, 43: 246–255), the approach of cellular automata image is introduced to cope with this problem. Many important features, which are originally hidden in the long amino acid sequences, can be clearly displayed through their cellular automata images. One of the remarkable merits by doing so is that many image recognition tools can be straightforwardly applied to the target aimed here. High success rates were observed through the self-consistency, jackknife, and independent dataset tests, respectively.     

Wood formation in poplar: identification, characterization, and seasonal variation of xylem proteins

Proteins that are preferentially produced in developing xylem may play a substantial role in xylogenesis. To reveal the identity of these proteins, comparative two-dimensional polyacrylamide gel electrophoresis was performed on young differentiating xylem, mature xylem, and bark of poplar (Populus trichocarpa Hook. cv. `Trichobel') harvested at different times of the year. The most-abundant xylem proteins were identified by microsequence analysis. For 17 of these proteins a putative function could be assigned based on similarity with previously characterized proteins, and for 15 out of these corresponding expressed sequence tags (ESTs) were found in the poplar EST database. The identified xylem–preferential proteins, defined by comparing the protein patterns from xylem and bark, were all involved in the phenylpropanoid pathway: two caffeoyl-coenzyme A O-methyltransferases (CCoAOMT), one phenylcoumaran benzylic ether reductase (PCBER), one bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT), five S-adenosyl-L-methionine synthetases, and one homologue of glycine hydroxymethyltransferase (GHMT). Remarkably, the biological function of the two most-abundant xylem-preferential proteins (PCBER and a GHMT homologue) remains unclear. In addition, several housekeeping enzymes were identified: two enolases, two glutamine synthetases, one 70-kDa heat-shock cognate, one calreticulin, and one α-tubulin. In comparison to the xylem-preferential proteins, the housekeeping proteins were expressed at significant levels in the bark as well. Also, several additional protein spots were detected for CCoAOMT, PCBER, and COMT by immunoblot. Our data show that for the study of xylogenesis, two-dimensional protein gel comparisons combined with systematic protein sequencing may yield information complementary to that from EST sequencing strategies. Received: 28 June 1999 / Accepted: 3 September 1999     

A DING phosphatase in Thermus thermophilus

Summary. Phosphate transport in bacteria occurs via a phosphate specific transporter system (PSTS) that belongs to the ABC family of transporters, a multisubunit system, containing an alkaline phosphatase. DING proteins were characterized due to the N-terminal amino acid sequence DINGG GATL, which is highly conserved in animal and plant isolates, but more variable in microbes. Most prokaryotic homologues of the DING proteins often have some structural homology to phosphatases or periplasmic phosphate-binding proteins. In E. coli, the product of the inducible gene DinG, possesses ATP hydrolyzing helicase enzymic activity. An alkaline phosphorolytic enzyme of the PSTS system was purified to homogeneity from the thermophilic bacterium Thermus thermophilus. N-terminal sequence analysis of this protein revealed the same high degree of similarity to DING proteins especially to the human synovial stimulatory protein P205, the steroidogenesis-inducing protein and to the phosphate ABC transporter, periplasmic phosphate-binding protein, putative (P. fluorescens Pf-5). The enzyme had a molecular mass of 40 kDa on SDS/PAGE, exhibiting optimal phosphatase activity at pH 12.3 and 70 °C. The enzyme possessed characteristics of a DING protein, such as ATPase, ds endonuclease and 3′ phosphodiesterase (3′-exonuclease) activities and binding to linear dsDNA, displaying helicase activity on supercoiled DNA. Purification and biochemical characterization of a T. thermophilus DING protein was achieved. The biochemical properties, N-terminal sequence similarities of this protein implied that the enzyme belongs to the PSTS family and might be involved in the DNA repair mechanism of this microorganism. Authors’ address: Assist. Prof. A. A. Pantazaki, Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece     

Free glutamine as a major precursor of brown products and fluorophores in Maillard reaction systems

Summary. Glutamine is one of the most abundant free amino acid found in raw food. In this study, the contribution of free glutamine to nonenzymatic browning and fluorescence was investigated using an aqueous model system with methylglyoxal. The results indicated that glutamine contributed to the Maillard reaction via two pathways. First, the hydrolysis of the amide bond of glutamine led to the release of ammonia which was implicated in the formation of brown color and fluorescence. Among other nitrogen donors tested (asparagine, glutamic acid and urea) our results demonstrated that free glutamine was a major source of ammonia during heating. When heated at 120 and 180 °C, 100% of ammonia was released from glutamine after 60 and 10 min, respectively. The second pathway involved a direct Maillard reaction with the α-amino group of glutamine. Both pathways led to a rapid and complete destruction of glutamine when heated in the model systems. With reference to the Maillard browning (absorbance at 420 nm) glutamine turned out to be the most reactive amine, followed by asparagine, glutamate, ammonia and urea. Maximum fluorescence (excitation and emission wavelengths at 330 and 450 nm, respectively) was also observed with glutamine followed by urea and ammonia. Overall this study suggested that free glutamine predominantly contributes to the color and fluorescence formations of foodstuffs.     

Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis

Summary. The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT–PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.