[Methods in Enzymology] Thiol Redox Transitions in Cell Signaling, Part B: Cellular Localization and Signaling Volume 474 || Mitochondrial Thioredoxin Reductase

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Mitochondrial Thioredoxin

Reductase: Purification, Inhibitor

Studies, and Role in Cell Signaling

Maria Pia Rigobello* and Alberto Bindoli†

Contents

1. In

in

076

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troduction

Enzymology, Volume 474 # 2010

-6879, DOI: 10.1016/S0076-6879(10)74007-6 All rig

ent of Biological Chemistry, University of Padova, Padova, Italyof Neuroscience (CNR), Section of Padova, c/o Department of Biological Chemis

Else

hts

try,

110

2. P
urification of Thioredoxin Reductase from Isolated Mitochondria,
Cultured Cells, and Whole Organs
111
2
.1. P reparation and purification of mitochondria 111
2
.2. F reeze/thaw cycles and disruption of mitochondria 112
2
.3. H eat treatment 113
2
.4. A mmonium sulfate fractionation 113
2
.5. D EAE-Sephacel chromatography 113
2
.6. 2 0,50-ADP-Sepharose 4B affinity chromatography 114
2
.7. o -Aminohexyl-Sepharose 4B 114
2
.8. R echromatography on 20,50-ADP-Sepharose 4B 114
2
.9. P urification of TrxR2 from whole organs or cultured cells 116
3. E
stimation of Thioredoxin Reductase Activity 117
4. In
hibitor Studies of Thioredoxin Reductase 118
5. R
ole in Cell Signaling 118
Refe
rences 120
Abstract

Mitochondrial thioredoxin reductase (TrxR2) maintains thioredoxin (Trx2) in a

reduced state and plays a critical role in mitochondrial and cellular functions.

TrxR2 has been identified in many different tissues and can be purified to

homogeneity from whole organs and isolated mitochondria.

Here we describe the detailed steps required to purify this enzyme.

A different initial procedure is needed, according to whether purification starts

from whole organs or from isolated and purified mitochondria. In the first case,

acid precipitation is a critical preliminary step to separate mitochondrial

vier Inc.

reserved.

Padova,

109

110 Maria Pia Rigobello and Alberto Bindoli

thioredoxin reductase from the cytosolic isoform. Preparation involves ammo-

nium sulfate fractionation, heating, and freeze/thaw cycles, followed by chro-

matographic passages involving DEAE-Sephacel, 20,50-ADP-Sepharose 4B

affinity, and o-Aminohexyl-Sepharose 4B columns. The 20,50-ADP-Sepharose4B affinity step can be repeated to remove any contaminating glutathione

reductase completely. Although several methods are available to detect the

activity of this enzyme, reduction of DTNB is an easy and inexpensive test that

can be applied not only to the highly purified enzyme but also to lysed

mitochondria, provided non-TrxR2-dependent reaction rates are subtracted.

TrxR2, like TrxR1, can be inhibited by several different and chemically unrelated

substances, usually acting on the C-terminal containing the cysteine–

selenocysteine active site. Many of these inhibitors react preferentially with

the reduced form of the C-terminal tail. This condition can be evaluated by

estimating enzyme activity after removal of the inhibitor by gel filtration of the

enzyme preincubated in oxidizing or reducing conditions. Inhibition of thiore-

doxin reductase has important consequences for cell viability and can lead to

apoptosis. Inhibition of TrxR2 causes large production of hydrogen peroxide,

which diffuses from the mitochondrion to the cytosol and is responsible for

most of the signaling events observed. Methods to measure hydrogen peroxide

in isolated mitochondria or cultured cells are described.

1. Introduction

Thioredoxin reductases (TrxR; EC 1.8.1.9) belong to the pyridinenucleotide disulfide oxidoreductase family, and their major functionis to maintain thioredoxins in a reduced state. Mammalian thioredoxinreductases (high-Mr thioredoxin reductases) are homodimeric enzymescontaining a C-terminal selenocysteine involved in catalytic activity(Arner and Holmgren, 2000; Mustacich and Powis, 2000; Tamura andStadtman, 1996). In mammals, three major isoforms of thioredoxin reduc-tase have been found, cytosolic (TrxR1), mitochondrial (TrxR2, also calledTR3 or TRb), and testis-specific (TGR) (Arner, 2009). However, severalsplice variants of TrxR1 and TrxR2 have been also identified (Arner, 2009).Interestingly, at variance with the predominant isoform, splice variants ofTrxR2 lacking the mitochondrial signaling peptide have been found locatedin the cytosol (Turanov et al., 2006). Other splice forms of TrxR2 are aprotein variant subunit with a shorter interface domain (Miranda-Vizueteand Spyrou, 2002) and another version whose overexpression induces cellapoptosis (Chang et al., 2005). The presence of several different splicevariants for thioredoxin reductase makes analysis of its expressionand functions rather complex. Disruption of TrxR2 gene is associatedwith embryonic death, suggesting a crucial role played by this enzyme,

Mitochondrial Thioredoxin Reductase 111

particularly in hematopoiesis and heart function (Conrad et al., 2004).The three-dimensional structure of mouse TrxR2 has been solved andfound comparable to that of TrxR1 and glutathione reductase (Biterovaet al., 2005). In addition to thioredoxin, and like its cytosolic counterpart,TrxR2 is able to reduce several different unrelated substrates such as DTNB(5,50-dithiobis(2-nitrobenzoic acid)), selenite, and alloxan (Rigobello et al.,1998). It has also been identified in many different tissues (Kawai et al.,2000; Kim et al., 1999; Lescure et al., 1999; Miranda-Vizuete et al., 1999a,b)and isolated and purified to homogeneity from rat liver (Lee et al., 1999), ratliver mitochondria (Rigobello et al., 1998), and mitochondria from bovineadrenal cortex (Watabe et al., 1999).

2. Purification of Thioredoxin Reductase from

Isolated Mitochondria, Cultured Cells,

and Whole Organs

Mitochondrial thioredoxin reductase can be purified after preliminarypreparation of isolated mitochondria or starting directly from whole organsor cultured cells (Fig. 7.1).

2.1. Preparation and purification of mitochondria

Mitochondria can be isolated from tissue homogenates or disrupted cellswith conventional procedures involving differential centrifugation (seeprevious volumes of this series, e.g., vol. 10, describing detailed preparationsof mitochondria). Briefly, after centrifugation of nuclei and unbroken cellsat 800�g for 5 min, mitochondria can be obtained from the supernatantafter centrifugation at 8000�g for 10 min. Pellets can be resuspended andrecentrifuged at 10,000�g to wash the mitochondria, which are finallyresuspended in a small volume of medium.

Crude rat liver mitochondrial suspensions can be further purified by thesilica colloid Percoll which, by centrifugation, results in a density gradient. Themethod of Hovius et al. (1990) with a few modifications (Rigobello et al.,2001) is described. Mitochondria (5 ml of about 60 mg ml�1 suspension) arelayered on top of centrifuge tubes containing 45 ml of 30% (v/v) Percoll in0.225Mmannitol and 1 mM EGTA buffered with 25 mMHEPES (pH 7.4).Samples are centrifuged at 95,000�g for 30 min. Mitochondria can be col-lected in the lower fraction at the relative density of 1.070/1.100 g ml�1 andwashed twice with the desired medium, as previously described.

Isolated organsliver, heart, kidney, brain

Tissuehomogenate

Cellcultures

Cell lysate

Acidprecipitation

Acidprecipitation

DEAE-sephacellinear gradient

2�,5�-ADP-sepharosestep gradient

2�,5�-ADP-sepharosestep gradient

2�,5�-ADP-sepharoselinear gradient TrxR2

w -aminohexyl-sepharoselinear gradient

Isolated mitochondria

Percoll purification

Detergent treatment

Heat treatment

Ammonium sulfatefractionation

Figure 7.1 Diagram showing major steps for purification of TrxR2 from wholeorgans, isolated mitochondria or cell cultures.


2.2. Freeze/thaw cycles and disruption of mitochondria

To obtain a sufficient amount of TrxR2 from isolated mitochondria, it isnecessary to start from a concentration of 5–6 g of mitochondrial proteins,based on the biuret procedure. We usually start from about 100 ml ofa suspension of 50–60 mg protein ml�1. Preparations can be collectedover time and stored at �20 �C, even for months. Further processing ofmitochondria before the chromatographic steps can be based on sonicirradiation followed by ammonium sulfate and heat treatment, as describedin a previous chapter in this series (Bindoli and Rigobello, 2002). Here wedescribe an alternative procedure based on mitochondria disruption withTriton X-100 (Watabe et al., 1999) which provides higher yields. Frozenstored mitochondria are thawed, pooled, and diluted with distilled water(1:1) containing an antiprotease cocktail (‘‘Complete’’ Roche, Mannheim,Germany) and 0.2% Triton X-100. They are then subjected to three freeze/thaw cycles (at �70 �C), followed by homogenization in Ultra Turrax( Janke & Kunkel, Staufen, Germany), twice for 30 s each. The resulting


suspension is centrifuged at 12,000�g for 60 min at 5 �C. Pellets arediscarded, and the supernatant used for further purification of the enzyme.

2.3. Heat treatment

The obtained preparation is heated at 60 �C for 3 min and rapidly cooledin an ice bath at 4 �C. Precipitated proteins are then removed by centrifu-gation at 105,000�g for 1 h at 5 �C.

2.4. Ammonium sulfate fractionation

The following purification steps are based on modifications of the methodsoriginally described by Luthman and Holmgren (1982) for thioredoxinreductase from rat liver cytosol and Williams et al. (1967) for Escherichia coli.The clear supernatant obtained after heating is fractionated with ammoniumsulfate in two saturation steps. The first stage is 0–50% (w/v) ammoniumsulfate fractionation. Precipitated proteins are centrifuged at 37,000�g for30 min at 5 �C. Pellets are dissolved in 10 mM Tris–HCl (pH 7.5) containing1 mM EDTA and extensively dialyzed overnight against the same buffer. Thisenzyme preparation is collected and used for the further chromatographicpurification steps. The supernatant obtained after the previous centrifugationcan be subjected to a further fractionation step with ammonium sulfate,achieving 85% saturation. Thioredoxin reductase activity is still present inthe 50–85% fraction, but is heavily contaminated with glutathione reductase.It is therefore preferable to avoid using it for further purification.

2.5. DEAE-Sephacel chromatography

The dialyzed enzyme preparation is concentrated by ultrafiltration underargon through an Amicon YM/10 membrane and transferred to an anionexchange DEAE-Sephacel column (4 � 16 cm) preequilibrated with10 mM Tris–HCl (pH 7.5) containing 1 mM EDTA. The column is elutedwith 500 ml linear gradient (from 0.0 to 0.3M) of NaCl in the same buffer.Fractions of 10 ml are collected. Aliquots of these fractions are usedto estimate thioredoxin reductase by DTNB method (see below) andprotein content by measuring absorbance at 280 nm. Fractions eluted inthe 0.13–0.15 M NaCl interval reveal the highest activity of thioredoxinreductase and are pooled and concentrated by ultrafiltration under argonthrough an Amicon YM/10 membrane. Concentrated samples are dialyzedovernight against 50 mM Tris–HCl (pH 7.5) buffer containing 1 mMEDTA.


2.6. 20,50-ADP-Sepharose 4B affinity chromatography

The resulting dialyzed enzyme solution is applied to a 20,50-ADP-Sepharose4B affinity chromatography column (0.8 � 10 cm), preequilibrated with50 mM Tris–HCl buffer (pH 7.5) containing 1 mM EDTA. The enzymeis then eluted with three discontinuous gradient steps of Na,K-phosphate(0.3 and 0.5 M) and NaCl (0.8 M), in 50 mM Tris–HCl buffer (pH 7.5)containing 1 mM EDTA (Fig. 7.2A). Fractions of 2.5 ml are collectedand aliquots used to estimate TrxR2 by DTNBmethod and protein contentat 280 nm. The Na,K-phosphate steps allow the residual TrxR1 tobe eluted. Mitochondrial thioredoxin reductase fractions are eluted at0.8MNaCl (Fig. 7.2A), pooled, and concentrated, as described previously,in the presence of 0.2% (w/v) octylglucoside (n-octyl-b-D-glucopyrano-side) to prevent loss of enzyme activity. Subsequently, the concentratedfraction is dialyzed with 50 mM Tris–HCl buffer (pH 7.5) containing 1 mMEDTA. At this stage, the degree of purity can be assessed by SDS–PAGE.

2.7. o-Aminohexyl-Sepharose 4B

Enzyme preparation can be further purified in ano-Aminohexyl-Sepharose4B column (0.8 � 5 cm) equilibrated with 50 mM Tris–HCl buffer(pH 7.5) and 1 mM EDTA. The enzymatic fraction is eluted with a lineargradient of NaCl (0.0–0.8 M). The flow rate is 0.8 ml min�1 and fractionsof 2 ml are collected. Pooled fractions are concentrated and dialyzed asdescribed above. Another way of purification of the dialyzed preparationobtained after 20,50-ADP-Sepharose 4B affinity is based on fast proteinliquid chromatography (FPLC, Pharmacia, Piscataway, NJ). Samplesare applied to a Superdex-200 column equilibrated with 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 10 mM mercaptoethanol,and 50 mM PMSF (phenylmethylsulfonyl fluoride). Fractions are collectedat a flow rate of 0.4 ml min�1.

2.8. Rechromatography on 20,50-ADP-Sepharose 4B

This step is designed to remove contaminating glutathione reductase. If atthe end of the o-Aminohexyl-Sepharose 4B step, the sample still retainsglutathione reductase activity, further chromatography on 20,50-ADP-Sepharose 4B is required. The dialyzed enzyme is eluted with 50 mM Trisbuffer (pH 7.5) containing 1 mM EDTA at a linear gradient from 0.6 to1.0MNaCl in the same buffer (Fig. 7.2B). Fractions of 1.2 ml are collectedat a flow rate of 0.5 ml min�1.

Fraction number

1.8

1.6

1.4

1.2

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0.4

0.2

0.00 10 20 30 40 50 60 70 80 90

3.0A

2.5

2.0

1.5

1.0

0.5

0.0

Na,K-Pi 0.3M Na,K-Pi 0.5M NaCl 0.8M

Fraction number

Mitochondrial thioredoxin reductase (mmol � min–1� ml–1)Glutathione reductase (mmol � min–1� ml–1)

Absorbance at 280nm

Mitochondrial thioredoxin reductase (mmol � min–1� ml–1)Glutathione reductase (mmol � min–1� ml–1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 20 40 60 80 100 120 140

B 1.2

1.0

0.8

0.6

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1.0M

0.6M

NaC

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Figure 7.2 (A) Chromatographic purification of mitochondrial thioredoxin reductaseby 20,50-ADP-Sepharose 4B affinity chromatography. (B) The o-hexyl column eluate isapplied to a 20,50-ADP-Sepharose column with a continuous gradient of NaCl. Asshown in (B), thioredoxin reductase can be completely separated by glutathionereductase (GR). TrxR2 is estimated by DTNB method, and GR is measured in 0.2 MTris–HCl buffer (pH 8.1), 1 mM EDTA, 1 mM GSSG and 0.25 mM NADPH. Absor-bance is estimated at 340 nm (eM ¼ 6220 M�1 cm�1).



2.9. Purification of TrxR2 from whole organs or cultured cells

Lee et al. (1999) describe a procedure involving acidification to pH 5 of ratliver homogenate, which allows separation and purification of TrxR2directly from the liver without any preliminary purification of mitochon-dria. Rat liver is extensively homogenized with Ultra Turrax in 20 mMTris–HCl buffer (pH 7.8) in the presence of 1 mM EDTA and proteaseinhibitors (‘‘Complete’’ Roche), followed by further homogenization in aglass-teflon tissue grinder. After centrifugation at 70,000�g for 30 min, theresulting supernatant is brought to pH 5.0 by adding 1 M acetic aciddropwise. The resulting cloudy suspension is again centrifuged at70,000�g for 30 min and the pellet, resuspended and neutralized, is usedas a source of TrxR2. The supernatant contains most of TrxR1. Thismethod is convenient when organs are available in small quantity as mayoccur with brain, kidney, and heart, and it is consequently difficult to obtaina sufficient amount of mitochondria. The resulting enzyme is slightly lesspure, but the yield is satisfactory. Figure 7.3 shows SDS–PAGE and West-ern blotting separation of TrxR2 obtained from various tissues; by compar-ison, a sample obtained after preliminary preparation of rat livermitochondria is also shown (Liver*). This procedure can also be used forcultured cells. Both tissues and cultured cells can be frozen. Cells (at 108

density) are lysed with RIPA buffer modified as follows: 150 mM NaCl,50 mM Tris–HCl (pH 7.4), 1% Triton X-100, 0.1% SDS, 0.5% DOC,1 mM NaF, 1 mM EDTA, and immediately before use, an antiproteasecocktail (‘‘Complete’’ Roche) containing PMSF is added. Samples aresubjected to acid precipitation as described above for rat liver.

55kDa

A

B

MW Liver*Liver

Kidney

HeartBrain

Figure 7.3 SDS–PAGE (A) and Western blotting (B) of mitochondrial thioredoxinreductase prepared from various organs. Purified enzyme obtained from 20,50-ADPSepharose column is separated by polyacrylamide gel electrophoresis, followed byCoomassie brilliant blue staining (A). Polypeptide bands showing molecular weightof about 54 kDa are detected. In (B) proteins are transferred to nitrocellulose mem-brane and revealed with anti-TrxR2 antibody. Asterisk (*): TrxR2 obtained frompreviously isolated rat liver mitochondria.


3. Estimation of Thioredoxin Reductase Activity

In mitochondria prepared from tissues or lysed cells, TrxR2 activitycan easily be estimated by DTNB reduction. It has long been knownthat mitochondria are able to reduce low molecular weight disulfides. Inparticular, the NADPH-dependent DTNB reductase activity of crude mito-chondrial fractions or purified mitochondrial matrix is essentially attributed tothioredoxin reductase activity (Lenartowicz and Wudarczyk, 1995). Eitherfreshly isolated or previously frozen mitochondria can be used for TrxR2estimation. Mitochondria (5 mg in 200 ml of 0.2M phosphate buffer (pH 7.4)containing 5 mM EDTA) are treated for 1 h at 0 �C with 75 mM CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and sub-jected to occasional vortexing. Then, 1 mg protein is transferred to bothsample and reference cuvettes containing the same medium and added with1 mM DTNB. The reaction is started by adding 0.25 mM NADPH to thesample cuvette and absorbance is determined for a few minutes at 412 nm(eM 13,600 M�1 cm�1). Enzyme activity is expressed as nmol min�1 mg�1

protein and is calculated by taking into account the fact that 1 mol of NADPHyields 2 mol of TNB anion (reduced DTNB). DTNB stock solution can beprepared by bringing the acidic suspension to pH 7.0 by careful addition of1 M Tris-base, avoiding any local rise above pH 9 to prevent cleavage of thedisulfide. Freeze/thaw cycles in the presence of detergent can improve theresult of the assay. Mitochondria can also be disrupted by sonic irradiationinstead of detergent. Besides, TrxR2 can be measured in the mitochondrialmatrix fraction obtained after sonic irradiation followed by centrifugationat 100,000�g for 60 min, to remove mitochondrial membranes. Lowconcentrations of gold(I) complexes such as auranofin or arsenite are stronginhibitors of thioredoxin reductases (Gromer et al., 1998; Hill et al., 1997;Luthman and Holmgren, 1982; Tamura and Stadtman, 1996) and this prop-erty can therefore be exploited to estimate NADPH-dependent DTNBreductase activity other than that of thioredoxin reductase (Hill et al., 1997).Therefore, to a second sample containing all the above reagents, 1–2 mMauranofin (or aurothioglucose, or any other gold(I)complex) is added. Theabsorbance after addition of NADPH can be subtracted from that of thesample run in the absence of inhibitor. The resulting differential absorbanceallows calculation of enzyme activity due solely to TrxR2.

To estimate the activity of the purified enzyme, the same methodsemployed for cytosolic thioredoxin reductase can be used. They have beenthoroughly described in previous issues of this series (Arner et al., 1999;Gromer et al., 2002; Holmgren and Bjornstedt, 1995). The simplest andleast expensive method to assess purified TrxR2 is reduction of DTNB,which can be performed in the same conditions as those described above for


lysed mitochondria. Other methods rely upon NADPH oxidation or insulinreduction mediated by thioredoxin (Arner et al., 1999). Spectrophotometricmethods can be properly modified to be adapted to microplate readers. Thisprocedure is used for the samples eluted from chromatographic columns.

4. Inhibitor Studies of Thioredoxin Reductase

Due to the highly accessible and reactive C-terminal residue contain-ing a cysteine–selenocysteine group, thioredoxin reductase can easily beinhibited by several chemically unrelated substances. Inhibitors includeheavy and transition metals and metal complexes, alkylating agents, dini-trohalobenzenes, quinones, flavonoids, and other polyphenols (Arner,2009). In particular, gold complexes are potent inhibitors acting in thenanomolar range of concentration (Gromer et al., 1998). Many of theseinhibitors are already established or potential antitumor agents, making thisenzyme an interesting molecular target for cancer chemotherapy (Arner,2009; Bindoli et al., 2009; Urig and Becker, 2006). The preventive reduc-tion of TrxR2 by NADPH is a critical condition which makes mostinhibitors effective. In contrast, oxidized enzyme prevents inhibition. Onthis basis, it is possible to assess the potential interaction of several differentinhibitors with the C-terminal active site of TrxR.

Mitochondrial thioredoxin reductase is incubated in 0.2 M Na,K-phos-phate buffer (pH 7.4), 5 mM EDTA in the presence or absence of 0.025 mMNADPH. Preincubation is carried out for 1.5 min and then inhibitors areadded at the desired concentrations to a final volume of 60 ml which is appliedto a desalting column (Micro Bio-Spin, Bio-Rad Laboratories) and centri-fuged at 1000�g for 5 min. The filtering procedure can be repeated, in orderto ensure complete removal of the inhibitor. The eluate is directly used toestimate TrxR2 by DTNB method. As shown in Fig. 7.4, only samplespreincubated in reducing conditions show strong inhibition of TrxR2, indi-cating that preliminary reduction of the catalytic site is a critical condition forinhibitor effectiveness. However, a few inhibitors do not require reducingconditions and presumably interactwith a site other than theC-terminal activesite. The tightness of the binding of the inhibitor to the enzyme is also animportant feature to be considered when this assay is performed.

5. Role in Cell Signaling

Inhibition of mitochondrial thioredoxin reductase leads to oxidationof downstream enzymes such as thioredoxin and peroxiredoxin. This oxi-dation is essentially due to a concentration increase in hydrogen peroxide

Gel filtration

−NADPH

S Se SH SeH

TrxR2

+NADPH

TrxR2

10080604020

% T

rxR

2 ac

tivi

ty

0 Control

Inhibitor

10080604020

% T

rxR

2 ac

tivi

ty

0 Control

Inhibitor

Preincubation

Incubation ± Inhibitor± Inhibitor

Figure 7.4 Reducing conditions of TrxR2 are critical for occurrence of inhibition.TrxR2 is incubated with an inhibitor (e.g., 1 mM auranofin), in the absence or presenceof NADPH. Inhibitor is removed by gel filtration. Thioredoxin reductase is estimatedby DTNB procedure. As shown, only prereduction of cysteine–selenocysteine activesite causes a strong inhibition by auranofin.


produced by the respiratory complexes and no longer removed by theinhibited thioredoxin system. In addition, both mitochondrial thioredoxin(Trx2) and peroxiredoxin (Prx3) have been shown to be more sensitive tooxidation than the corresponding cytosolic isoforms (Chen et al., 2006;Cox et al., 2008). The increased concentration of mitochondrial hydrogenperoxide, which in turn can be released to the cytosol, has importantconsequences for cell signaling and apoptosis (Bindoli et al., 2009). Hydro-gen peroxide, forming after inhibition of TrxR2, can be estimated inisolated mitochondria with the highly specific fluorescent probe AmplexRed (10-acetyl-3,7-dihydroxyphenoxazine). The assay is based on oxida-tion of the probe by horseradish peroxidase (HRP), activated by hydrogenperoxide (Mohanty et al., 1997).

Mitochondria (1 mg ml�1), incubated in the presence of inhibitorsof TrxR2 in proper medium (e.g., 0.1M sucrose, 50 mM KCl, 0.5 mM Na,K-phosphate, 20mMHEPES/Tris buffer (pH 7.4) and respiratory substrates),are supplemented with 10 mM Amplex Red and 0.1 units ml�1 of HRP.


The increase in fluorescence is followed spectrofluorometrically at 544 nm(lEx) and 620 nm (lEm). Either a fluorometer or a microplate reader can beused. AH2O2 concentration standard curve can be obtained by adding knownamounts of hydrogen peroxide to the medium, supplemented with AmplexRed and HRP.

In cultured cells, formation of hydrogen peroxide is assessed withthe fluorogenic probes CM-DCFH2-DA (chloromethyl-20,70-dihydrodi-chlorofluorescein) or DHR-123 (dihydrorhodamine 123) (Molecular Probes,Eugene, OR, USA), according to Royall and Ischiropoulos (1993). Cells(at 2 � 104 density) arewashed in phosphate-buffered saline (PBS) containing10 mM glucose and loaded with 10 mM CM-DCFH2-DA or 15 mM DHR-123 for 20 min in the dark. After washing in the same medium, cells areincubated in the desired conditions with the various inhibitors. Fluorescenceincrease is estimated in a multiwell fluorescence plate reader at 485 nm (lEx)and 527 nm (lEm). Interestingly, DHR-123 is considered a probe mainlymonitoring hydrogen peroxide of mitochondrial origin.

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[Methods in Enzymology] Thiol Redox Transitions in Cell Signaling, Part B: Cellular Localization and Signaling Volume 474 || Mitochondrial Thioredoxin Reductase