Selenodiglutathione is a highly efficient oxidant of reduced thioredoxin and a substrate for mammalian thioredoxin reductase.

Selenium compounds like selenite (SeO3(2-) may form a covalent adduct with glutathione (GSH) in the form of selenodiglutathione (GS-Se-SG), which is assumed to be important in the metabolism of selenium. We have isolated GS-Se-SG and studied its reactions with NADPH and thioredoxin reductase from calf thymus or with thioredoxin reductase and thioredoxin from Escherichia coli. Incubation of 0.1 microM calf thymus thioredoxin reductase or 0.1 microM thioredoxin reductase and 1 microM thioredoxin from E. coli with 5, 10, or 20 microM GS-Se-SG resulted in a fast initial reaction, followed by a large and continued oxidation of NADPH. However, anaerobic incubation of 0.1 microM calf thymus thioredoxin reductase and 20 microM GS-Se-SG resulted only in oxidation of a stoichiometric amount of NADPH; admission of oxygen started continuous NADPH oxidation. Contrary to the mammalian enzyme, GS-Se-SG was not a substrate for thioredoxin reductase from E. coli. The rate of the oxygen-dependent reaction between calf thymus thioredoxin reductase and GS-Se-SG was increased 2-fold in the presence of 4 mM GSH, indicating that HSe- was the reactive intermediate. Glutathione reductase from rat liver reduced GS-Se-SG with a very slow continued oxidation of NADPH, and the presence of the enzyme did not affect the oxygen-dependent nonstoichiometric oxidation of NADPH by GS-Se-SG and thioredoxin reductase. Fluorescence spectroscopy showed GS-Se-SG to be a very efficient oxidant of reduced thioredoxin from E. coli and kinetically superior to insulin disulfides. Thioredoxin-dependent reduction of CDP to dCDP by ribonucleotide reductase was effectively inhibited by GS-Se-SG.     

Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.     

Methaneseleninic acid is a substrate for truncated mammalian thioredoxin reductase: implications for the catalytic mechanism and redox signaling

Mammalian thioredoxin reductase is a homodimeric pyridine nucleotide disulfide oxidoreductase that contains the rare amino acid selenocysteine (Sec) on a C-terminal extension. We previously have shown that a truncated version of mouse mitochondrial thioredoxin reductase missing this C-terminal tail will catalyze the reduction of a number of small molecules. Here we show that the truncated thioredoxin reductase will catalyze the reduction of methaneseleninic acid. This reduction is fast at pH 6.1 and is only 4-fold slower than that of the full-length enzyme containing Sec. This finding suggested to us that if the C-terminal Sec residue in the holoenzyme became oxidized to the seleninic acid form (Sec-SeO(2)(-)) that it would be quickly reduced back to an active state by enzymic thiols and further suggested to us that the enzyme would be very resistant to irreversible inactivation by oxidation. We tested this hypothesis by reducing the enzyme with NADPH and subjecting it to high concentrations of H(2)O(2) (up to 50 mM). The results show that the enzyme strongly resisted inactivation by 50 mM H(2)O(2). To determine the redox state of the C-terminal Sec residue, we attempted to inhibit the enzyme with dimedone. Dimedone alkylates protein sulfenic acid residues and presumably will alkylate selenenic acid (Sec-SeOH) residues as well. The enzyme was not inhibited by dimedone even when a 150-fold excess was added to the reaction mixture containing the enzyme and H(2)O(2). We also tested the ability of the truncated enzyme to resist inactivation by oxidation as well and found that it also was resistant to high concentrations of H(2)O(2). One assumption for the use of Sec in enzymes is that it is catalytically superior to the use of cysteine. We and others have previously suggested that there are reasons for the use of Sec in enzymes that are unrelated to the conversion of substrate to product. The data presented here support this assertion. The results also imply that the redox signaling function of the thioredoxin system can remain active under oxidative stress.     

The mammalian cytosolic selenoenzyme thioredoxin reductase reduces ubiquinone. A novel mechanism for defense against oxidative stress

The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was time- and dose-dependent, with an apparent K(m) of 22 microm and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.     

Semisynthesis and characterization of mammalian thioredoxin reductase

Thioredoxin reductase and thioredoxin constitute the cellular thioredoxin system, which provides reducing equivalents to numerous intracellular target disulfides. Mammalian thioredoxin reductase contains the rare amino acid selenocysteine. Known as the "21st" amino acid, selenocysteine is inserted into proteins by recoding UGA stop codons. Some model eukaryotic organisms lack the ability to insert selenocysteine, and prokaryotes have a recoding apparatus different from that of eukaryotes, thus making heterologous expression of mammalian selenoproteins difficult. Here, we present a semisynthetic method for preparing mammalian thioredoxin reductase. This method produces the first 487 amino acids of mouse thioredoxin reductase-3 as an intein fusion protein in Escherichia coli cells. The missing C-terminal tripeptide containing selenocysteine is then ligated to the thioester-tagged protein by expressed protein ligation. The semisynthetic version of thioredoxin reductase that we produce in this manner has k(cat) values ranging from 1500 to 2220 min(-)(1) toward thioredoxin and has strong peroxidase activity, indicating a functional form of the enzyme. We produced the semisynthetic thioredoxin reductase with a total yield of 24 mg from 6 L of E. coli culture (4 mg/L). This method allows production of a fully functional, semisynthetic selenoenzyme that is amenable to structure-function studies. A second semisynthetic system is also reported that makes use of peptide complementation to produce a partially active enzyme. The results of our peptide complementation studies reveal that a tetrapeptide that cannot ligate to the enzyme (Ac-Gly-Cys-Sec-Gly) can form a noncovalent complex with the truncated enzyme to form a weak complex. This noncovalent peptide-enzyme complex has 350-500-fold lower activity than the semisynthetic enzyme produced by peptide ligation.     

Ethaselen: a potent mammalian thioredoxin reductase 1 inhibitor and novel organoselenium anticancer agent

Mammalian thioredoxin reductase 1 (TrxR1) is considered to be an important anticancer drug target and to be involved in both carcinogenesis and cancer progression. Here, we report that ethaselen, a novel organoselenium compound with anticancer activity, specifically binds to the unique selenocysteine-cysteine redox pair in the C-terminal active site of mammalian TrxR1. Ethaselen was found to be a potent inhibitor rather than an efficient substrate of mammalian TrxR1. It effectively inhibits wild-type mammalian TrxR1 at submicromolar concentrations with an initial mixed-type inhibition pattern. By using recombinant human TrxR1 variants and human glutathione reductase, we prove that ethaselen specifically targets the C-terminal but not the N-terminal active site of mammalian TrxR1. In A549 human lung cancer cells, ethaselen significantly suppresses cell viability in parallel with direct inhibition of TrxR1 activity. It does not, however, alter either the disulfide-reduction capability of thioredoxin or the activity of glutathione reductase. As a downstream effect of TrxR1 inactivation, ethaselen causes a dose-dependent thioredoxin oxidation and enhances the levels of cellular reactive oxygen species in A549 cells. Thus, we propose ethaselen as the first selenium-containing inhibitor of mammalian TrxR1 and provide evidence that selenium compounds can act as anticancer agents based on mammalian TrxR1 inhibition.     

Small molecule inhibitors of mammalian thioredoxin reductase

Mammalian thioredoxin reductases (TrxRs) are a family of NADPH-dependent flavoproteins with a penultimate selenocysteine residue at the carboxy-terminus. Besides their native substrate thioredoxins (Trx), the enzymes show a broad substrate specificity, at least partially, because of the C-terminal redox-active site that is easily accessible in the reduced form. TrxRs are ubiquitous in all kinds of cells and have a critical role in regulating intracellular redox signaling. In recent years, a wealth of evidence has revealed that overactivation/dysfunction of TrxRs is closely related to various diseases, especially in tumor development, and thus the past decades have witnessed an expanding interest in finding TrxRs inhibitors, which might be promising agents for cancer chemotherapy. Herein we reviewed the small molecule inhibitors of mammalian TrxRs, with an emphasis on those that have potential anticancer activity. This review includes the nonpatent references up to 2010 that deal with mammalian TrxR inhibitors.     

Interactions of quinones with thioredoxin reductase: a challenge to the antioxidant role of the mammalian selenoprotein

Mammalian thioredoxin reductases (TrxR) are important selenium-dependent antioxidant enzymes. Quinones, a wide group of natural substances, human drugs, and environmental pollutants may act either as TrxR substrates or inhibitors. Here we systematically analyzed the interactions of TrxR with different classes of quinone compounds. We found that TrxR catalyzed mixed single- and two-electron reduction of quinones, involving both the selenium-containing motif and a second redox center, presumably FAD. Compared with other related pyridine nucleotide-disulfide oxidoreductases such as glutathione reductase or trypanothione reductase, the k(ca)(t)/K(m) value for quinone reduction by TrxR was about 1 order of magnitude higher, and it was not directly related to the one-electron reduction potential of the quinones. A number of quinones were reduced about as efficiently as the natural substrate thioredoxin. We show that TrxR mainly cycles between the four-electron reduced (EH(4)) and two-electron reduced (EH(2)) states in quinone reduction. The redox potential of the EH(2)/EH(4) couple of TrxR calculated according to the Haldane relationship with NADPH/NADP(+) was -0.294 V at pH 7.0. Antitumor aziridinylbenzoquinones and daunorubicin were poor substrates and almost inactive as reversible TrxR inhibitors. However, phenanthrene quinone was a potent inhibitor (approximate K(i) = 6.3 +/- 1 microm). As with other flavoenzymes, quinones could confer superoxide-producing NADPH oxidase activity to mammalian TrxR. A unique feature of this enzyme was, however, the fact that upon selenocysteine-targeted covalent modification, which inactivates its normal activity, reduction of some quinones was not affected, whereas that of others was severely impaired. We conclude that interactions with TrxR may play a considerable role in the complex mechanisms underlying the diverse biological effects of quinones.     

Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase

Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.     

Annexin A2-S100A10 heterotetramer, a novel substrate of thioredoxin

The binding of plasminogen activators and plasminogen to the cell surface results in the rapid generation of the serine protease plasmin. Plasmin is further degraded by an autoproteolytic reaction, resulting in the release of an angiostatin, A61 (Lys78-Lys468). Previously, we demonstrated that the annexin A2-S100A10 heterotetramer (AIIt) stimulates the release of A61 from plasmin by promoting the autoproteolytic cleavage of the Lys468-Gly469 bond and reduction of the plasmin Cys462-Cys541 disulfide (Kwon, M., Caplan, J. F., Filipenko, N. R., Choi, K. S., Fitzpatrick, S. L., Zhang, L., and Waisman, D. M. (2002) J. Biol. Chem. 277, 10903-10911). Mechanistically, it was unclear if AIIt promoted a conformational change in plasmin, resulting in contortion of the plasmin disulfide, or directly reduced the plasmin disulfide. In the present study, we show that AIIt thiols are oxidized during the reduction of plasmin disulfides, establishing that AIIt directly participates in the reduction reaction. Incubation of HT1080 cells with plasminogen resulted in the rapid loss of thiol-specific labeling of AIIt by 3-(N-maleimidopropionyl)biocytin. The plasminogen-dependent oxidation of AIIt could be attenuated by thioredoxin. Thioredoxin reductase catalyzed the transfer of electrons from NADPH to the oxidized thioredoxin, thus completing the flow of electrons from NADPH to AIIt. Therefore, we identify AIIt as a substrate of the thioredoxin system and propose a new model for the role of AIIt in the redox-dependent processing of plasminogen and generation of an angiostatin at the cell surface.     

A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.     

Roles of N-terminal active cysteines and C-terminal cysteine-selenocysteine in the catalytic mechanism of mammalian thioredoxin reductase

Mammalian thioredoxin reductase [EC 1.6.4.5], a homodimeric flavoprotein, has a marked similarity to glutathione reductase. The two cysteines in the N-terminal FAD domain (-Cys59-x-x-x-x-Cys64-) and histidine (His472) are conserved between them at corresponding positions, but the mammalian thioredoxin reductase contains a C-terminal extension of selenocysteine (Sec or U) at the penultimate position and a preceding cysteine (-Gly-Cys497-Sec498-Gly). Introduction of mutations into the cloned rat thioredoxin reductase gene revealed that residues Cys59, Cys64, His472, Cys497, and Sec498, as well as the sequence of Cys497 and Sec498 were essential for thioredoxin-reducing activity. To analyze the catalytic mechanism of the mammalian thioredoxin reductase, the wild-type, U498C, U498S, C59S, and C64S were overproduced in a baculovirus/insect cell system and purified. The wild-type thioredoxin reductase produced in this system, designated as WT, was found to lack the Sec residue and to terminate at Cys497. A Sec-containing thioredoxin reductase, which was purified from COS-1 cells transfected with the wild-type cDNA, was designated as SecWT and was used as an authentic enzyme. Among mutant enzymes, only U498C retained a slight thioredoxin-reducing activity at about three orders magnitude lower than SecWT. WT, U498C, and U498S showed some 5,5'-dithiobis(2-nitrobenzoic acid)-reducing activity and transhydrogenase activity, and C59S and C64S had substantially no such activities. These data and spectral analyses of these enzymes suggest that Cys59 and Cys64 at the N-terminus, in conjunction with His472, function as primary acceptors for electrons from NADPH via FAD, and that the electrons are then transferred to Cys497-Sec498 at the C-terminus for the reduction of oxidized thioredoxin in the mammalian thioredoxin reductase.     

Synthesis of peptide substrates for mammalian thioredoxin reductase.

Mammalian thioredoxin reductase (TR) catalyzes the reduction of the redox-active disulfide bond of thioredoxin (Trx) and is similar in structure and mechanism to glutathione reductase except for a C-terminal 16-amino acid extension containing a rare vicinal selenylsulfide bond. This vicinal selenylsulfide bond is essentially a substrate for the enzyme's N-terminal redox center. Here we report the synthesis of peptide substrates for the truncated enzyme missing the C-terminal redox center. We developed a procedure for the synthesis of peptides containing cyclic vicinal disulfide/selenylsulfide bonds as well as their corresponding acyclic heterodimers. Vicinal disulfide bonds form eight-membered ring structures and are difficult to synthesize owing to their propensity to dimerize during oxidation. Our procedure makes use of two key improvements for on-resin disulfide bond formation presented previously by Galande and coworkers (Galande AK, Weissleder R, Tung C-H. An effective method of on-resin disulfide bond formation in peptides. J. Comb. Chem. 2005; 7: 174-177). First, the addition of an amine base to the deprotection solution allows the complete removal of the StBu group, allowing it to be replaced with a 5-Npys group. The second enhancement is the direct use of a Cys(Mob) or Sec(Mob) derivative as the nucleophilic partner instead of utilizing a naked sulfhydryl or selenol. These improvements result in the formation of a vicinal disulfide (or selenylsulfide) bond in high purity and yield. A direct comparison with the Galande procedure is presented. We also present a novel strategy for the formation of an acyclic, interchain selenylsulfide-linked peptide (linking H-PTVTGC-OH and H-UG-OH). Cysteine analogs of the cyclic and acyclic peptides were also synthesized. The results show that the ring structure contributes a factor of 52 to the rate, but the presence of selenium in the peptide is more important to catalysis than the presence of the ring.     

Protein disulfide-isomerase is a substrate for thioredoxin reductase and has thioredoxin-like activity

We have demonstrated that calf liver protein disulfide-isomerase (Mr 57,000) is a substrate for calf thymus thioredoxin reductase and catalyzes NADPH-dependent insulin disulfide reduction. This reaction can be used as a simple assay for protein disulfide-isomerase during purification in place of the classical method of reactivation of incorrectly oxidized ribonuclease A. Protein disulfide-isomerase contains two redox-active disulfides/molecule which were reduced by NADPH and calf thioredoxin reductase (Km approximately 35 microM). The isomerase was a poor substrate for NADPH and Escherichia coli thioredoxin reductase, but the addition of E. coli thioredoxin resulted in rapid reduction of two disulfides/molecule. Tryptophan fluorescence spectra were shown to monitor the redox state of protein disulfide-isomerase. Fluorescence measurements demonstrated that thioredoxin--(SH)2 reduced the disulfides of the isomerase and allowed the kinetics of the reaction to be followed; the reaction was also catalyzed by calf thioredoxin reductase. Equilibrium measurements showed that the apparent redox potential of the active site disulfide/dithiols of the thioredoxin domains of protein disulfide-isomerase was about 30 mV higher than the disulfide/dithiol of E. coli thioredoxin. Consistent with this, experiments using dithiothreitol or NADPH and thioredoxin reductase-dependent reduction and precipitation of insulin demonstrated differences between protein disulfide-isomerase and thioredoxin, thioredoxin being a better disulfide reductase but less efficient isomerase. Protein disulfide-isomerase is thus a high molecular weight member of the thioredoxin system, able to interact with both mammalian NADPH-thioredoxin reductase and reduced thioredoxin. This may be important for nascent protein disulfide formation and other thiol-dependent redox reactions in cells.     

Efficient selenocysteine-dependent reduction of toxoflavin by mammalian thioredoxin reductase

Background

Toxoflavin (1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7-dione; xanthothricin) is a well-known natural toxin of the pyrimidinetriazinedione type that redox cycles with oxygen under reducing conditions. In mammalian systems, toxoflavin is an inhibitor of Wnt signaling as well as of SIRT1 and SIRT2 activities, but other molecular targets in mammalian cells have been scarcely studied. Interestingly, in a library of nearly 400,000 compounds (PubChem assay ID 588456), toxoflavin was identified as one out of only 56 potential substrates of the mammalian selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1). This activity was here examined in further detail.

Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography.

A scheme is described for the large scale purification of thioredoxin, thioredoxin reductase, and glutathione reductase. The scheme is based on an initial separation of thioredoxin from the two reductases by affinity chromatography on agarose-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (agarose-2',5'-ADP). The two reductases were then separated by hydrophobic chromatography and purified separately to homogeneity. Thioredoxin was purified to homogeneity by immunoadsorption to agarose containing immobilized goat anti-thioredoxin. Overall yields for thioredoxin, thioredoxin reductase, and glutathione reductase exceeded 80% in each case. Both reductases exhibit an absorption band at approximately 320 nm which appears due to a residual amount of tightly bound NADP. Presence of this absorption band has no apparent effect on the specific activity of either enzyme.     

Activity assays of mammalian thioredoxin and thioredoxin reductase: Fluorescent disulfide substrates,mechanisms, and use with tissue samples

Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin–glutathione disulfide (Di-E–GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC–insulin), which displayed higher fluorescence on disulfide reduction. Di-E–GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC–insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.