Review

An introduction to methods for analyzing treagents, and practical considerations

Rosa E. Hansen 1, Jakob R. Winther *

ogy is quenching samples to trap the cellular thioldisulde status.This step not only is crucial for preventing articial oxidation dur-ing cell lysis or sample preparation but also must be so rapid thatperturbation of the thioldisulde equilibria is avoided. This is par-ticularly important in cell extracts that contain redox-active en-zymes that, if not rapidly denatured, can very efciently transferdisuldes between different cellular redox pools. Two different ap-proaches are typically used to quench thioldisulde reactions.One is to block free thiols with a cell-permeable alkylating agent,

ethylenediaminetetraacetic acid; SDS, sodium dodecyl sulfate; IAM, iodoacetamide;PAGE, polyacrylamide gel electrophoresis; NTB, 2-nitro-5-thiobenzoic acid; IAA,iodoacetic acid; VP, 2-vinylpyridine; MMTS, S-methyl methanethiosulfonate; HPLC,high-performance liquid chromatography; ME, b-mercaptoethanol; TCEP, tris(2-carboxyethyl)phosphine; THP, tris(hydroxypropyl)phosphine; BH, sodium borohy-dride; PSSG, proteinGSH mixed disulde; DTNB (or Ellmans reagent), 5,50-dithiobis-(2-nitrobenzoic acid); 4-DPS, 4,40-dithiodipyridine; 4-TP, 4-thiopyridone; mBBr,monobromobimane; SBD-F, ammonium 7-uoro-2,1,3-benzoxadiazole-4-sulfonate;ABD-F, 4-aminosulfonyl-7-uoro-2,1,3-benzoxadiazole; NEM-GS, NEM-glutathioneadduct; AMS, 4-acetamido-40-maleimidylstilbene-2,20-disulfonate; ICAT, isotope-

Analytical Biochemistry 394 (2009) 147158

Contents lists availab

Analytical Bio

journal homepage: www.ecoded afnity tag.Cellular SH groups are implicated in the coordination of metalions and the defense against oxidants, and the reversible formationof disulde bonds is involved in regulation of enzyme activity, sig-

reduction are evaluated and compared, and we discuss how toavoid conict between mutually cross-reactive thiol reagents. Weconsider this review to be an introduction to experimental thioldisulde biochemistry updated with selected contemporaryknowledge on the subject.

Quenching cellular thioldisulde exchange: Trapping in vivoconditions

Probably the most critical step when working with redox biol-

* Corresponding author. Fax: +45 3532 1567.E-mail address: jrwinther@bio.ku.dk (J.R. Winther).

1 Present address: Novo Nordisk A/S, DK-2880 Bagsvaerd, Denmark.2 Abbreviations used: SH, thiol; SS, disulde; ER, endoplasmic reticulum; TCA,

trichloroacetic acid; PCA, perchloric acid; SSA, sulfosalicylic acid; GuHCl, guanidiniumhydrochloride; GSH, reduced glutathione; GSSG, oxidized glutathione; NEM, N-ethylmaleimide; DTT, dithiothreitol; EGTA, ethyleneglycoltetraacetic acid; EDTA,Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark

Introduction

The majority of the thiols (SH)2 and disuldes (SS) in cells arefound as the amino acid cysteine and its disulde, cystine(Fig. 1A). The thiolate anion is intrinsically one of the strongest bio-logical nucleophiles; thus, the thiol group of cysteine is one of themost reactive functional groups found in proteins [1]. Protein disul-de bonds are typically introduced and removed through a thioldisulde exchange reaction (Fig. 1B). This mechanism of transferringreducing equivalents between thiol and disulde pairs is central inredox biology and is, for example, applied by cytosolic thioredoxinwith its active site in the reduced form to reduce protein disuldesand in the endoplasmic reticulum (ER) by protein disulde isomeras-es in their oxidized form to generate disulde bonds. The reaction isinitiated by a nucleophilic attack of a thiolate on an existing disuldebond, leading to oxidation of the nucleophilic thiol and reduction ofthe leaving group sulfur [2]. In thioldisulde exchange reactions, itis important to consider reaction rate and the equilibrium constantsbetween various thiol and disulde species. Because the thiolate an-ion is the reactive species, these properties are particularly sensitiveto thiol pKa values. In addition, the kinetics and thermodynamics ofthioldisulde exchange reactions are affected by electrostatic fac-tors from neighboring charged groups as well as strain and entropy(for detailed reviews, see Refs. [3,4]).0003-2697/$ - see front matter 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2009.07.051nal transduction, transcriptional activity, and protein folding [5].Because the thiols and disuldes of proteins and low-molecular-weight compounds are involved in so many essential cellular func-tions, reliable and accurate methods to identify and quantify themare in high demand. For example, methods for determining thein vivo thiol oxidation state of specic oxidoreductases can be cru-cial for determining their functions, and the identication of pro-teins with redox-active cysteines can lead to elucidation of redoxregulation pathways. The reactive nature of thiols is, however, of-ten an experimental challenge. In contrast to the extracellularspace, the cytosolic concentration of reduced thiols is much higherthan the concentration of disuldes, and the SH group easily oxi-dizes during cell lysis and sample preparation. One should considerthat these chemical reactions can take place rapidly and spontane-ously [6], and overlooking the possibility of postlysis thioldisul-de exchange reactions can lead to mis-interpretations of data.

This review outlines the basic issues to consider when dealingwith biochemical and cellular aspects of thioldisulde chemistry.Considering the volume of literature on the subject, we cannot cov-er it comprehensively and so we apologize to the many highlyqualied contributions that we, within the given scope, do notmention. The overall focus is on practical aspects, including typicalbiochemical experimental conditions and caveats to consider ininterpreting results. Reagents for thiol derivatization and disuldehiols and disuldes: Reactions,

le at ScienceDirect

chemistry

lsevier .com/locate /yabio

Cl

Cl

Cl

OH

O

TCA

Cl O

O

OH

O

PCA

OH

S

OHO

O

O

OH

SSA

Fig. 3. Structures of commonly used protein-precipitating acids.

NH 3+

SH O

O

NH 3+

S

O

O

NH 3+

S

O

O

S R1 S S

R2

SS

R2

S R3+ +

Cysteine Cystine

A

B

148 Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158by acid also requires protein denaturation. Denaturation will en-sure that thiols, which in enzymes can be highly activated, aredeactivated and at the same time all protein thiols are renderedaccessible to protonation.

On acid precipitation, low-molecular-weight thiol compoundsand the other is to quench thioldisulde exchange with acid. Bothmethods have advantages and pitfalls, as illustrated in Fig. 2 anddiscussed below. In any case, however, one should avoid using eth-anol or acetone, neither of which acidies or denatures efciently.

Quenching by acidication

Because the thiolate anion is the reactive species in thioldisul-de exchange reactions, acidication of the sample will signi-cantly slow down the rate of disulde exchange. Protonation isextremely rapid, with rate constants in the range of 109 M1 s1

[7]. However, in some cases redox enzymes unfold fairly slowlyeven under highly acidic conditions and have very low pKa values.The Escherichia coli disulde-bond-forming enzyme DsbA, forexample, has a pKa of 3.5 [8,9]. Because the Kox of the proteinthioldisulde couple is very pH dependent, its redox status maychange if acidication is not concurrent with efcient proteindenaturation. Similarly, it has been observed that the redox statusof the glutaredoxin thioldisulde couple can change even on acid-ication to pH 2 (R. Iversen and J.R. Winther, unpublished observa-tions). Therefore, efcient quenching of protein disulde exchange

R3 R1

Thiol - disulfide exchange

Fig. 1. Structure of cysteine and cystine (A) and mechanism of thioldisuldeexchange reaction (B).remain soluble and can easily be separated from protein thiolsby centrifugation. This can be convenient if the aim is, for example,specic measurement of glutathione. Some acids, including tri-chloroacetic acid (TCA), perchloric acid (PCA), and sulfosalicylicacid (SSA), are particularly potent protein denaturants (Fig. 3)[10,11]. Oxidation of SH groups has, however, been observed in

S

S

S-

Acidification

Alkylation

S

S

H

S

S

X

Fig. 2. Common strategies for quenching the cellular thioldisulde status. Plus symrespectively.The main advantage of using cell-permeable alkylating agentsfor thioldisulde quenching is that the thiols, in general, are irre-versibly blocked under conditions where different compartmentsare not mixed. Nonetheless, this quenching procedure might per-turb the in vivo thioldisulde equilibrium. For example, theGSH and oxidized glutathione (GSSG) ratio of cytosolic glutathioneis dened in part by the enzyme glutathione reductase. If GSH isdepleted by an alkylating agent, glutathione reductase can, in the-ory, drive the redox equilibrium from GSSG toward GSH, resultingin articially low GSSG values [14]. Thus, an absolute requirementfor efcient quenching is that the alkylating agent not only com-pletely block all thiols but also inactivate all redox enzymes beforeany perturbation of the redox equilibrium can take place.

As described in detail in the following sections, choosing theappropriate reaction conditions for alkylation reactions involvesmany factors. On the one hand, a high-reaction pH will increasethe rate of thioldisulde exchange and increase the risk of un-wanted side reactions. On the other hand, if low-reaction pH andPCA- and SSA-treated samples [1114]; therefore, TCA is probablythe most reliable quenching agent.

Protein precipitation is dependent on the TCA concentration[15], and concentrations in the range of 1020% (w/v) are recom-mended for efcient precipitation. TCA does not quantitatively pre-cipitate low levels of protein (125 lg/ml) unless a coprecipitationagent such as sodium deoxycholate (0.02%, w/v) is included [16]. Ithas been suggested that high concentrations of denaturants suchas guanidinium hydrochloride (GuHCl) can inhibit TCA precipita-tion, and samples containing concentrations of GuHCl above 1 Mshould be diluted before precipitation [17]. TCA quenching alsoeffectively limits oxidation during storage, as demonstrated bythe unchanged content of reduced glutathione (GSH) in the solublefraction of samples quenched with 15% TCA at 20 C for up to7 days [12]. It is, however, important to note that acid quenchingis reversible, so if the pH is raised (e.g., to reduce disuldes or toalkylate thiols), undesired oxidation can occur.

Quenching by thiol alkylationS+ Extremely rapid quench+ Protein denaturation ensures that the proton can access all thiols

- Reversible quench

S+ Irreversible quench

- Slow quench at pH < 7- Buried thiols can be inaccessible- Not completely thiol specific

bols (+) and minus symbols () denote general advantages and disadvantages,

weight thiols in the TCA supernatant, it is advantageous to add the

small thiol dithiothreitol (DTT) was oxidized in 1 day at 25 C

and 5% SDS, respectively (our unpublished observations). Theobservation that SDS, in particular, decreases alkylation rates hasbeen suggested previously [33] and is probably due to formationof micelles that hinder free diffusion.

pH during reduction and derivatization

As described in the previous section, preventing perturbation ofthe thioldisulde equilibria on cell lysis is essential. This also ap-plies to any thiol alkylation steps following cell lysis. For this rea-son, it is desirable to keep the pH value as low as possible duringthiol reactions. A low pH will minimize thioldisulde rearrange-ments, metal-catalyzed oxidation, and unwanted side reactions

nsenand pH 7.2 in the presence of 1 lM Fe3+ or Ni2+. If the metal chelat-ing agent ethyleneglycoltetraacetic acid (EGTA) was present, thestability greatly improved, with only 10% oxidation under the sameconditions. The ability of common metal ions to catalyze oxidationof thiols ranks in the order Cu2+ > Fe3+ > Ni2+ >> Co2+ [26]. For thesereasons, oxygen should be eliminated from the reagent solutionsused in thioldisulde reactions by bubbling with nitrogen or ar-gon, and metal chelating agents (typically 1 mM ethylenediamine-tetraacetic acid [EDTA]) should be included.

It should be noted that, in addition to metal ions, structural andalkylating agent to the sample before the pH is increased to neu-trality. Because a 10% TCA supernatant contains 0.6 M TCA, neu-tralizing the pH requires equivalently high concentrations ofhydroxide ions. The presence of alkylating agents will minimizethe risk of thiol oxidation during titration. It should be noted thatNEM is relatively stable in 10% TCA (Hansen and Winther, unpub-lished observations) and that, therefore, this alkylating reagent canconveniently be added before titration of pH.

Reaction conditions: Measuring in vivo redox status

Preventing thiol oxidation by molecular oxygen

Thiols can be oxidized by molecular oxygen, as illustrated in aclassic study by Haber and Annsen [22] where reduced ribonucle-ase regained both native disulde bonds and enzymatic activitywithin 20 h at pH 8 in the absence of oxidants except dissolvedoxygen. We now know that this process is catalyzed by trace metalion contaminants in reagent solutions. Intermediates in the oxida-tion reaction include reactive oxygen species [23], which can alsoinduce irreversible oxidation of proteins thiols [4,5]. The reactionis dependent on pH and temperature, with slower oxidation ratesat lower temperature and pH values [2426]. As shown by Getzand coworkers [24], more than 80% of a 0.5-mM solution of theshort incubation times are used, the alkylation of protein thiolsburied by the three-dimensional structure can be incomplete. Ina study by Lind and coworkers [18], a 5-min incubation of intactmammalian cells with 40 mM of the highly reactive and mem-brane-permeable alkylating agent N-ethylmaleimide (NEM) re-sulted in the blocking of only 80% of total cellular protein thiols.Incubation with higher NEM concentrations (60 mM) or for longertimes (15 min) did not improve blocking efciency, so the authorsconcluded that approximately 20% of the cellular protein thiols ofintact cells were inaccessible to NEM.

Thioldisulde quenching: A combination strategy

The two main methods for quenching the thioldisulde statehave advantages as well as drawbacks, so the optimal procedureis often a combination of the two strategies, where samples arerst quenched with TCA, followed by alkylation of thiols [19].Alkylation reactions typically occur at neutral pH, and the practi-cal handling of samples depends on the purpose of the experi-ment. If the aim is to measure protein thiols and disuldes, theTCA pellet can be solubilized by sonication in strongly denaturingbuffers containing the alkylating agent [20,21]. This strategy willrender even buried protein thiols accessible to the alkylatingagent and will ensure that catalytic thiols are not active duringalkylation.

When the objective is to measure, for example, low-molecular-

Methods for analyzing thiols and disuldes / R.E. Haelectrostatic properties of the low-molecular-weight thiol compo-nent inuence the oxidation rates more strongly than does thepresence/absence of metal ions [27].Considerations relating to bulk protein denaturation

Before protein thiol alkylation or disulde bond reduction, fac-tors that determine reactivity with the thiol-directed reagentshould be considered. Steric hindrance or unfavorable electrostaticinteractions may block access to protein thiols or disuldes andhave considerable effects on the reaction rate. In the case of disul-de reduction, protein structure may provide intrinsic stability tothe disulde, and this will affect its redox potential and, therefore,the nal ratio of reduced to oxidized species at equilibrium. Dena-turing the proteins converts buried thiols and disulde bonds intogeneric thiols and disuldes with stabilities and reactivities similarto those of GSH and GSSG. Typical protein denaturants are 6 MGuHCl, 8 M urea, and 5% sodium dodecyl sulfate (SDS).

Urea has the disadvantage of containing varying amounts ofcyanate (Fig. 4A), which reacts with thiol groups to form thiocarba-mates, as shown in Fig. 4B [28,29]. The rate constant for reaction ofcysteine with cyanate is 0.07 M1 s1 at pH 6 [30], and because an8-M urea solution may contain up to 20 mM cyanate [31], thisreaction can be signicant. Thiocarbamate is unstable at a pHabove 6 with a half-time of 11 min at pH 8, according to Stark[30]. The alternative denaturant thiourea, which is typically usedfor isoelectric focusing, does not contain cyanate. A major draw-back of this solubilizing agent is, however, that it reacts directlywith alkylating agents such as iodoacetamide (IAM) and, thereby,competes for alkylation of thiol groups [32]. GuHCl is an inertand extremely potent protein denaturant that, unfortunately, isnot compatible with many downstream processes such as isoelec-tric focusing and SDSpolyacrylamide gel electrophoresis (PAGE).

Finally, although protein denaturants can be indispensable forreactions such as alkylation of buried protein thiols, the alkylationrate of accessible thiols can be decreased by the presence of dena-turants. As described in detail in part I of the Supplementary mate-rial, these effects were studied by following the alkylation rate ofthe small thiol 2-nitro-5-thiobenzoic acid (NTB) by the alkylationagent NEM at pH 7. Two-, three-, and fourfold decrease in alkyl-ation rates were observed in the presence of 6 M GuHCl, 8 M urea,

NH2

NH2

O NH4+ +

Urea Cyanate

HN C O+H+

RSH +NH2

SROHN C O

A

B

Fig. 4. Urea is in equilibrium with cyanate (A), which in turn can react with thiols(B).

, J.R. Winther / Anal. Biochem. 394 (2009) 147158 149such as reactions with amino acid residues other than cysteines.Accordingly, when choosing reagents for measuring thiols anddisuldes, the pH optimum of the reaction should be considered.

thiols. The selection of reagents at each step is crucial for the suc-

condition is one where the concentration is as low as possibleand the reaction time is as short as possible to achieve completemodication. Unfortunately, complete alkylation of protein thiolswith IAA and IAM often requires a high concentration. This was re-cently illustrated in a study of alkylation of myobril preparations[43]. Even with 65 mM IAA and IAM at pH 8, the thiol blocking wasnot complete after 1 h.

Due to the negative charge of IAA, this reagent is membraneimpermeable and is inappropriate as an in vivo quenching agentof thioldisulde exchange. Furthermore, the negative charge ofIAA can engage in electrostatic interactions with charged groupsin the proximity of the cysteine, and this can have substantial ef-fects on the reaction rate, with thiols depending on their electro-static environment. The negative charge can, in addition, hinderthe access of IAA to thiols in hydrophobic environments. A possible

ulfiducti

des

nsencess of the experiment. In the rst step, the alkylating agent mustreact quickly and irreversibly with thiol groups. Furthermore, anyexcess alkylating agent must be removed prior to disulde reduc-tion to avoid alkylation of newly reduced disuldes that wouldlead to an underestimation of oxidized thiols. Compatibility be-tween the reducing agent and the thiol detection reagent must alsobe considered. To avoid an overestimation of oxidized thiols, thereducing agent preferably should not cross-react with the thioldetection reagent. Alternatively, the reducing agent must be re-moved prior to quantication. The following sections provide anintroduction to the most commonly used reagents for thiol alkyl-ation, disulde reduction, and thiol detection. Their optimal reac-tion conditions are discussed, as are their mutual compatibilities.

Thiol alkylating agents

Thiol alkylating agents are central to experimental redox biol-ogy and are used for a variety of applications in the characteriza-tion of thiols and disuldes. As described previously, alkylationreagents are used to quench the thioldisulde status on cell lysis(Fig. 2) and, as illustrated in Fig. 5B, are applied to mask free thiolsso as to specically detect disuldes. In this section, a generalintroduction to the most commonly used thiol alkylating agentsis provided, and their individual chemical properties are reviewedin terms of their practical advantages and drawbacks.Measuring thioldisulde ratios: Common pitfalls

The general approach for measuring thiols and disuldes isshown in Fig. 5. Although the depicted strategy initially seems sim-ple, several issues should be considered before proceeding. Re-duced sulfhydryl groups can be detected merely by incubatingwith a suitable thiol-specic detection reagent (Fig. 5A). Here themain challenge is to avoid perturbation of the thioldisulde equi-librium, that is, by applying alkylating agents that block thiols fas-ter than disulde exchange can occur. The specic measurement ofdisulde bonds involves three steps (Fig. 5B): (i) blocking free thiolgroups, (ii) reducing disulde, and (iii) detecting newly reducedS

S

HS S

S

XSThiol

blockingDisred

S

S

HSThiol

detection S

S

*SA

B

Fig. 5. Common strategy for detection of thiols (A) and disuldes (B). Thiols

150 Methods for analyzing thiols and disuldes / R.E. HaIodoacetic acid and iodoacetamide

Iodoacetic acid (IAA) and IAM react irreversibly with thiols in anucleophilic substitution reaction to form the corresponding car-boxymethyl or carboxamidomethyl derivatives (Fig. 6). IAA andIAM are water-soluble and can, at pH 8, be prepared at concentra-tions of 1 and 0.5 M, respectively [19]. The reagents are light sen-sitive and must be protected from light both during storage andduring reaction.

Because the thiolate anion is the reactive nucleophile, the reac-tion rate is dependent on thiol deprotonation, and a pH of 8 is typ-ically used for the modication of protein thiols [6,19]. At neutralor alkaline pH, IAA and IAM also react slowly with the hydroxylgroup of tyrosine [34], the e-amino group of lysine [35,36], theimidazole group of histidine [37,38], and the N terminus of pro-teins [39]. Furthermore, IAM and IAA react with the sulfur atomof methionine at pH values as low as 2.8 [40,41]. The extent ofthe side reactions (e.g., with amines) depends on the nucleophilic-ity of the specic NH groups, which can vary signicantly in pro-teins. In general, the SH group will react much faster than anyother group in the protein. For example, the reaction rate of IAAwith histidine has been reported to be 1000-fold slower than thatwith cysteine [42]. Still, to ensure that the alkylation is thiol-spe-cic, reactions should be performed under careful considerationof concentrations and incubation times. In other words, the ideal

eon SH

SH

XS S*

S*

XSThiol

detection

tined for detection are denoted S*, whereas alkylated thiols are denoted SX.

RSO

O

HIRSH ++ IO

O

IAA

HIRSNH

O

2

RSH ++ INH

O

2IAM

Fig. 6. Alkylation of thiols with IAA and IAM.

, J.R. Winther / Anal. Biochem. 394 (2009) 147158benecial feature of IAA is that the modication results in an in-crease in the negative charge of the protein, and this in some casesresults in a mobility shift that can be followed with nondenaturingPAGE (Fig. 7). In contrast, modication with IAM does not changethe electrophoretic mobility of proteins. Because of its lack ofcharge, however, IAM is membrane-permeable [19] and is, in gen-eral, observed to react faster with thiols in proteins and low-molecular-weight compounds than does IAA [43,44]. Thus, formost purposes, IAM is preferable to IAA as an alkylating agent.

N-Ethylmaleimide

NEM reacts with thiols via an addition reaction across the dou-ble bond to form a thioether derivative (Fig. 8A). Here 1-M solu-

tions can be prepared in ethanol [19]. Like IAA and IAM, the reac-tion rate of NEM is dependent on the thiolate anion and, hence, onsolution pH. NEM is generally thiol-specic below pH 7 and withconcentrations in the range of 120 mM [45]. However, reactionswith protein amines have been observed at pH values above 7 ifNEM is in large excess or incubation times are prolonged to morethan 2 h [45,46].

The absorption spectrum of NEM has a maximum at 305 nmwith an extinction coefcient of 620 M1 cm1 [47] that is abol-ished when the agent decomposes or combines with a thiol com-pound. NEM has been reported to be stable at or below pH 6[48]. At pH 7 the half-time of hydrolysis is approximately 45 h,whereas at pH 9 the half-time is less than 1 h [49]. The instabilityin alkaline media is caused by hydrolysis of the maleimide ring,leading to the formation of N-ethylmaleamic acid (Fig. 8B), whichis accompanied by loss of thiol reactivity [49,50]. In addition, somelow-molecular-weight thiol adducts with NEM, such as NEMCys,can undergo intramolecular transamidation at pH values above 9

to form a cyclic compound (Fig. 8C). This conversion has beenshown to be complete after incubation of NEMCys adducts for36 h at room temperature and pH 9 [51]. Another crucial observa-tion is that GSH alkylated with NEM slowly regenerates the thiol at

pH optimum between 5 and 8 [55]. Reactions are typically per-

unwanted side reaction. Because the pKa values of thiols are typi-

SH S-AM S-AA

Fig. 7. Protein thiol alkylation with IAA can be followed with native PAGE. Yellowuorescent protein (3 lM) with a surface-exposed cysteine was modied with3 mM IAM (S-AM) and 3 mM IAA (S-AA), and samples were separated on a 10%nondenaturing PAGE (gure adapted from Hansen et al. [122]).

Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158 151CH3N

CH3HN

O

O

O

O

O

OH

CH3N

O

O

RSCH3N

O

O

+RSH

CH3H2N OH

CH3HNO

OH

OO

A

B

CN

SO

NH

O SO

DCH3

N

O

O

GS CH3N

O

O

HO

+GSH

H O2

Fig. 8. Common thiol reactions involving NEM. (A) Reaction of NEM with thiols. (B)Alkaline hydrolysis of NEM to N-ethylmaleamic acid. (C) Transamidation of theNEMCys adduct. For simplicity, not all electron movements are shown. (D)Reaction for the formation of glutathione from NEM adducts with glutathione(NEM-GS) [52].cally lower than those of amines, the optimization of reaction pHcan ensure that the amino groups are in their NH3+ state and, con-sequently, do not behave as nucleophiles.

As mentioned previously, it may be necessary to remove excessalkylating agent prior to disulde reduction to avoid alkylation ofnewly reduced thiol groups. Alkylation agents can be removedquantitatively by techniques such as gel ltration and acid precip-itation. Alternatively, the concentration of the reducing agent canbe optimized to consume the excess alkylating agent. This requires,however, that the reducing agent reacts faster with the alkylatingagent than the alkylating agent reacts with newly reduced thiols.

CH2+ HSRformed at a pH between 6 and 7 with 170 mM VP and incubationfor 60 min [56]. High VP excess and long incubation times are re-quired because the thiol alkylation rate is very slow. The rate con-stant for GSH alkylation between pH 5 and pH 7 is in the range of0.020.05 M1 s1 [55]. In comparison, rate constants for alkyl-ation of GSH or cysteine by NEM in the same pH interval are inthe range of 10 to 103 M1 s1 [48,57]. Above pH 7, VP may reactwith amine groups in proteins, but the reaction rate with cysteineswas reported to be approximately 300 times higher [58]. AlthoughVP reacts more slowly with thiols than does NEM, VP is used as thealkylating agent in the DTNBglutathione reductase recycling as-say for quantication of cellular GSSG [59]. In this assay, VP is pref-erable to NEM because VP does not inhibit glutathione reductase.

Another agent occasionally used for blocking of thiols isS-methyl methanethiosulfonate (MMTS) [60,61]. Reaction withthiols leads to the formation of an SSCH3 group that can bereduced by DTT or another free thiol group in the protein. Unfortu-nately, MMTS has been shown to induce formation of intra- andintermolecular protein disulde bonds [62] and should be avoidedas a thiol alkylating agent.

General practical considerations for alkylation reactions

In spite of the intrinsically lower nucleophilicity of amines rel-ative to thiols, alkylation of amines can sometimes take place as anpH values between 7 and 9 (Fig. 8D) [52].Although alkylation with NEM has a few signicant drawbacks,

the reagent is far more effective than IAA or IAM. For example, atpH 7 the alkylation rates of the small thiol NTB by NEM are 85-and 20-fold faster than alkylation by IAA and IAM, respectively(see part I of Supplementary material). The superior reactivity ofNEM compared with IAA and IAM has also been measured forthe alkylation of protein thiols [43,53]. Thus, NEM is in most casespreferable to IAA and IAM. Finally, NEM is a small and unchargedreagent, so it is membrane-permeable and can react with thiolseven in hydrophobic environments.

Other alkylating agents

Thiol groups can add to the double bond of 2-vinylpyridine (VP)[54], as shown in Fig. 9. The reaction between VP and thiols has aN N SR

Fig. 9. Reaction of VP with thiols.

This issue is discussed further in the following section. Finally, notethat all reagents described in this section are fairly toxic andshould be handled with caution.

Reducing agents

Any detection and quantication of disulde bonds requiresefcient and quantitative reduction of disuldes. As illustrated inFig. 5, disulde reduction is typically anked by two thiol derivati-zation steps, rendering the reduction step particularly complicated.In addition to being an efcient reductant, the reducing agent mustbe removable (e.g., by gel ltration) or unable to cross-react withthe derivatization agent in the next step. Alternatively, the productof the reaction between the derivatization agent and the thiol ofinterest can be detected independently (e.g., after separation byhigh-performance liquid chromatography [HPLC]). Here we pro-vide an introduction to the most commonly used reductants. Astrong reductant typically possesses a high degree of S-nucleophi-licity. The disulde reductants discussed in this section have thefollowing order of decreasing S-nucleophilicity: hydride > phos-phine > thiol > cyanide > sulte [63].

Thiol-containing reductants

SH S R

S

A

152 Methods for analyzing thiols and disuldes / R.E. HansenS

OH

+ 2 RSHHO R S+2

HOS

SOH

RSH+ 2

SHHS

OH

OH

RS

SR+

OH

BThe most commonly used disulde reductants are thiols them-selves. The reaction follows the exchange mechanism of the thio-late anion described previously (Fig. 1B). Among the advantagesand disadvantages of thiol exchange reductants, the main advan-tage is the high degree of specicity, meaning that only disuldesare chemically affected, leaving the protein ideally suited fordownstream processing. The main disadvantage of thiol-contain-ing reductants is that the SH groups compete directly with the pro-tein thiols for attachment of thiol reactive reagents and cross-reactivity with thiol detection agents is an issue. In addition, thereagents require a reaction with pH above 7, where oxidation byambient molecular oxygen can occur. The reagents themselvesare particularly sensitive to oxidation, and consequently all solu-Fig. 10. Reduction of disuldes with the thiol-containing reductants ME (A) andDTT (B).tions should be maintained in an oxygen-free state whenever pos-sible and should contain metal chelating agents such as EDTA.

The simplest water-soluble thiol is the monothiol b-mercap-toethanol (ME) (Fig. 10A). The thiol pKa value of ME is 9.5 [64],and alkaline pH values will signicantly increase the reaction ratewith disuldes. ME is a weak reducing agent, with an equilibriumconstant for the reduction of disuldes that is near unity [65]. Con-sequently, ME must be used at very high concentrations to avoidthe formation of mixed disuldes. Furthermore, ME is volatile withan unpleasant odor, and concentrated solutions should be handledin a hood. Today the main use of ME is reduction of protein disul-des prior to SDSPAGE (typically 0.7 M at pH 6.8 for 2 min at100 C in the presence of SDS). Notably, in this application the for-mation of mixed disuldes with ME will not likely change mobilitysignicantly. ME can diffuse into adjacent lanes during electropho-resis; however, this can affect samples without reducing agentsthat are run on the same gel.

The most commonly used reductant is the dithiol DTT, whichreduces disuldes in a reaction where DTT is converted into a sta-ble intramolecular cyclic disulde (Fig. 10B). Disulde reduction byDTT can be followed spectrophotometrically by the appearance ofoxidized DTT, which absorbs at 310 nm with an extinction coef-cient of 110 M1 cm1 [66]. The formation of oxidized DTT is dri-ven by favorable steric and entropic effects [4,65], and DTT is aconsiderably stronger reducing agent than ME, with an equilibriumconstant for cystine reduction of 1.3 104 M [65]. Accordingly,mixed disuldes between thiols and DTT do not accumulate, anda large excess of reagent is not required for complete disuldereduction. The thiol pKa values of DTT are 9.2 and 10.1 [67], andreduction is typically carried out at or above pH 8. A quantitativereduction of protein disuldes in lysozyme or bovine serum albu-min can be obtained in 1 h with 25 mM DTT at pH 8.4 in the pres-ence of 6 M urea [68]. An important advantage of DTT is itsmembrane permeability, and it is widely used as a reductant in cel-lular systems, where it reversibly inhibits protein disulde bondformation without hindering protein synthesis [69]. Typically, 510 mM DTT for 510 min is used for the reduction of whole cells.

Phosphines

Trialkylphosphines quantitatively reduce disuldes to thiols.The rst and rate-limiting step is attack of the SS bond by the phos-phine nucleophile, forming a thiophosphonium salt. Next, rapidhydrolysis releases the second thiol fragment and the phosphineoxide (Fig. 11A). Importantly, the hydrolysis step renders the reac-tion essentially irreversible. Thus, contrary to ME and DTT, the oxi-dized phosphine cannot participate in further thioldisuldereactions [70]. Trialkylphosphines react specically with disuldesand do not react with other functional groups commonly found inproteins [71]. Furthermore, phosphines are unreactive toward cer-tain thiol alkylating and derivatization agents such as VP and theuorescent benzofurazans (described below). This has the advan-tage that reduction and alkylation conveniently can take place con-comitantly [7174]. Phosphines do, however, react rapidly withIAA, IAM, and NEM even under acidic conditions [24,7577]. Infact, IAM reacts with tris(2-carboxyethyl)phosphine (TCEP) threetimes more effectively than it does with ME at pH 7.3. The reactionwith NEM is somewhat slower, with ME reacting twice as fast asTCEP [76]. Thus, as in the case of thiol reductants, reactions withphosphines must be carried out in a step separate from alkylationwith IAA, IAM, NEM, and their derivatives.

TCEP is a highly water-soluble reagent and the most commonlyused phosphine for disulde bond reduction (Fig. 11B). The pKa va-

, J.R. Winther / Anal. Biochem. 394 (2009) 147158lue of the phosphorus is 7.6 [78], and TCEP is signicantly moreeffective in reducing low-molecular-weight disuldes and sur-face-exposed protein disuldes than is DTT (Table 1), particularly

nsenP+ R2

R2

R2

S

R1R1

S

R1S

SR1

H2O

P

R2

R2

R2 +

+

A

Methods for analyzing thiols and disuldes / R.E. Haat pH below 7 [79,80]. DTT has been observed to reduce certainprotein disuldes, such as the active site of thioredoxin, faster thanTCEP, probably because of the greater steric hindrance of thenucleophilic phosphorus atom of TCEP compared with the DTTthiolate [80]. This disadvantage can obviously be eliminated byprotein denaturation prior to TCEP reduction.

TCEP is more resistant toward metal-catalyzed oxidation thanthe thiol-containing reductants [24,78]. Oxidation of TCEP appearsto be catalyzed by chelating agents such as EGTA [24], however,and TCEP has been reported to be unstable in phosphate buffer,especially between pH 7 and pH 8 [70,79]. The basis for the desta-bilizing effect of phosphate is not known. TCEP is highly acidicwhen dissolved in water (four protons accompany each TCEP mol-ecule), so samples must be adequately buffered after the additionof this reagent.

Besides TCEP, another water-soluble reagent, tris(hydroxypro-pyl)phosphine (THP), is commercially available (Fig. 11B). THPhas a phosphorus pKa value of 7.22, and it is even more reactive

R1SHP R2

R2

R2

O + 2

PH

O

OH

HO

O

OH

O

TCEP

P

OH

OHHO

THP

+

B

Cl -

Fig. 11. Reduction of disuldes with trialkylphosphines. (A) Reaction mechanism.(B) Structures of TCEP and THP. TCEP is shown as the commercially availablephosphonium chloride.toward small molecule disuldes and surface-exposed proteindisuldes than is TCEP (Table 1) [80]. In the presence of 6 M urea,a quantitative reduction of lysozyme disuldes is completed with-in 5 min using 10 mM THP at pH 7.3 (Hansen et al., unpublished re-sults). Finally, whereas the negative charge of TCEP at neutral pHmakes this reagent impermeable to membranes, THP is neutraland is as effective as DTT at traversing membranes [80].

As a complement to commercially available phosphines, Clineand coworkers [80] devised a simple method for synthesis ofmethyl ester derivatives of TCEP. These water-soluble phosphinesshow enhanced reactivity at low pH compared with TCEP. In addi-tion, esterication increases membrane permeability, allowing theTCEP esters to penetrate phospholipid bilayers 30 times faster thanDTT.

Sodium borohydride

Sodium borohydride (BH) is an extremely reactive and strongreducing agent due to the high S-nucleophilicity of the borohy-dride ion [81]. This reagent is highly reactive, and BH solutionsshould be prepared immediately before use to avoid decomposi-tion. Several studies have shown that BH is able to quantitativelyreduce protein disuldes with or without the presence of denatur-ing agents [68,8284], and BH has been used for the reduction ofprotein disuldes in whole cell extracts [21]. In general, reductionof protein disuldes with 1 M BH at pH 12 is complete after 30 minat 50 C. Because hydrogen is formed during the reaction, proteinsolutions tend to foam, but this can easily be avoided by addinga small volume of, for example, octanol or hexanol.

The main benet of BH is that excess reagent can easily be re-moved by the addition of acid or acetone. Removal of BH by acid-ication has the advantage that the released thiol groups areprotected from oxidation even when the reducing agent has been

Table 1Second-order rate constants (M1 s1) for reduction of disuldes by DTT, TCEP, andTHP.

DTT TCEP THP

DTNB (pH 7.5)a 2900 18,000 ndDTNB (pH 4.5)b 3.5 640 1600rxYFP (pH 7.0)c 0.4 7.8 28.9

Note. nd, not determined.a Data were obtained from Cline et al. [80].b Data were obtained in 100 mM acetate and 1 mM EDTA (pH 4.5) at 25 C, as

described in part II of the Supplementary material.c Redox-sensitive yellow uorescent protein (rxYFP) was used as a model for a

protein with a surface-exposed disulde. DTT data were obtained from Ostergaardet al. [123]. THP and TCEP data were obtained in 100 mM phosphate and 1 mMEDTA (pH 7.0) at 30 C, as described in part III of the Supplementary material.

, J.R. Winther / Anal. Biochem. 394 (2009) 147158 153destroyed. In addition, NEM is rapidly inactivated by the additionof BH without regenerating the SH from NEMthiol adducts [85].This can be an advantage if alkylation of free thiols is required be-fore disulde bond reduction. Although BH may be a highly usefulreducing agent for a number of applications, it can reduce ketogroups to alcohols. Furthermore, the high pH at which the reactionis carried out may catalyze hydrolysis of Asn and Gln amides aswell as the peptide bond. Therefore, BH is not applicable to proteinsamples destined for, for example, mass spectrometry.

Alternative reducing agents

Disulde bonds can be reduced by other nucleophiles such assulte (SO3) and cyanide (CN). However, reduction by theseagents is rarely quantitative unless catalysts are present to ensureefcient reduction [86]. The enzyme glutaredoxin has been used asa specic reductant of proteinGSH mixed disulde (PSSG) [18]. Inthe presence of GSH, however, glutaredoxins can also reduce intra-

tion or acid precipitation of proteins. However, a quantitative elim-

agents and their applicability. When using derivatives of alkylating

Aromatic disulde reagents

5,50-Dithiobis-(2-nitrobenzoic acid) (DTNB or Ellmans reagent)is the most common reagent for the quantication of thiols [91].DTNB reacts with the thiolate anion in a thioldisulde exchangereaction, resulting in the stoichiometric formation of the yellowNTB (Fig. 12A), which absorbs at 412 nm (e = 14,150 M1 cm1 atpH 7.3) [92]. The NTB anion is an excellent leaving group becauseof the electronegative character of the nitro group combined withits conjugated nature. The reaction is essentially irreversible, andthe absorbance of the generated NTB serves as a measure of thenumber of reacted SH groups [93]. The molar extinction coefcientof the NTB anion is stable from pH 6 to pH 9.5. Below pH 6, NTBbecomes protonated, and this abolishes the absorbance [68]. Fur-thermore, alkylation of the NTB thiolate also eliminates absorption(part I of Supplementary material). This can be exploited as amethod for quantifying concentrations of alkylating agents.

DTNB is particularly useful for the quantication of low-molec-ular-weight thiols and is convenient for determining the thiol con-centration of stock solutions of, for example, GSH, ME, DTT, andphosphines [79]. Using a high molar excess of DTNB (typically1 mM), the reaction is, for practical purposes, complete within1 min at pH 7.3. In addition, DTNB is widely used to quantify cellu-lar GSH and GSSG via the DTNBglutathione reductase recyclingassay [94]. Although DTNB works well for small water-soluble thiolcompounds, the reaction with protein SH is frequently hindered byits high polarity and negative charge [95]. The DTNB assay, further-more, suffers from a rather limited sensitivity, with a thiol detec-tion limit in the nanomolar range, and this can be a problem

nsen, J.R. Winther / Anal. Biochem. 394 (2009) 147158agents, the experimental considerations described in the previoussections for reaction conditions, reactivity, specicity, and stabilityapply. After labeling cellular thiols with suitable reagents, the dif-ferent species can be separated and characterized using a variety oftechniques, including HPLC, afnity purication, variations of two-ination of reducing agent can be difcult to obtain with acidprecipitation [68]. Although gel ltration can effectively removereducing agents, regeneration of disuldes might occur during l-tration unless the column equilibration buffer is kept at low pH.Furthermore, when working with complex mixtures that containpartially aggregatedmaterial, gel ltration is not an option. In thesecases, BH is the reagent of choice because the excess reagent is sim-ply removed by the addition of acid. If BH is not applicable becauseof side reactions with other protein functional groups, one of thephosphines could be used because their reducing capacity and reac-tivity at low pH are superior to those of the thiol reductants. Werecommend THP because it has proven to be the most reactivephosphine in the reduction of both low-molecular disuldes andprotein disuldes. In addition, only a small excess is needed for ef-cient reduction. This is a crucial advantage when the thiol detectionagents cross-react with the phosphines because this limits con-sumption of the detection agent. Furthermore, in contrast to TCEP,THP solutions are not acidic and do not require titration.

As mentioned previously, reducing agents can be used to con-sume excess alkylating agent. This requires that the reaction be-tween the reducing agent and the alkylating agent be faster thanthe reaction between the alkylating agent and the newly reducedthiols. The addition of a small excess of ME relative to alkylatingagents can be particularly suitable for this purpose due to thelow reducing capacity of ME. Thus, the alkylating agent can reactrapidly with the ME thiol essentially without any reduction ofdisuldes.

All of the reagents discussed in this section are susceptible tooxidation. Preferably, reducing agents should be prepared freshlyon a daily basis. Stock solutions of 0.1 M DTT or TCEP, for example,lose approximately 20% reducing capacity after 2 weeks at 20 Cand pH 7 (Hansen, personal observation). If stock solutions areused, they should be as concentrated as possible and kept at lowpH, and the exact thiol concentration should be determined beforeuse using one of the aromatic disulde reagents described in thefollowing section.

Thiol detection agents

A wide variety of reagents is available for detection of thiols.These include active aromatic disulde reagents with useful spec-trophotometric properties and alkylating reagents modied withdetection labels. This section provides examples of thiol detectionmolecular protein disuldes and, thus, cannot be considered as aspecic reductant of PSSG. Finally, reducing agents for thioloxidation states other than disuldes exist. These include sodiumarsenite to reduce sulfenic acids and ascorbate to reduce nitroso-thiols [29,60,87].

Reducing agents: Practical considerations

For many analytical purposes, it is necessary to completely re-move the reducing agent before further modication or analysisof protein thiol groups. For the thiol-containing reductants andthe phosphines, this can be accomplished by, for example, gel ltra-

154 Methods for analyzing thiols and disuldes / R.E. Hadimensional PAGE, and mass spectrometry. These methodologieshave been reviewed elsewhere and are not discussed in detail here[8890].when measuring thiol concentrations in dilute protein solutions.An alternative to DTNB is 4,40-dithiodipyridine (4-DPS), which

reacts with thiols in an exchange reaction to form 4-thiopyridone(4-TP), absorbing at 324 nm (Fig. 12B) [96]. At pH 7, 4-DPS hasan extinction coefcient of 21,400 M1 cm1 and, therefore, is a

+2 RSH

2 S

N S S N

+RSSR NH

4-DPS

4-TP

S S NO2O2N

COO

COO

+2 RS

S2 O2N

COO

+RSSR

DTNB

NTB

A

B

Fig. 12. Activated disuldes used for quantication of thiols. (A) Reaction of thiols

with DTNB results in the stoichiometric formation of the chromogenic NTB. (B)Reaction of thiols with 4-DPS results in the stoichiometric formation of thechromogenic 4-TP.

pH and temperature (pH 8.5 and 60 C) as well as long incubationtimes (1 h). Under these conditions, there is a risk of regeneratingthiols from NEMGS adducts; accordingly, the combination of NEMand SBD-F should be avoided during quantication of GSSG. ABD-Fis more reactive, and the derivatization of thiols at pH 8 and 60 Cis completed in 10 min. ABD-F and SBD-F have been used to quan-tify low-molecular-weight thiols as well as PSSG [21,73,110].

Thiol reagents that signicantly increase molecular mass

Thiol-specic reagents that change the electrophoretic mobilityof a protein can be used to estimate the redox state of a thioldisul-de couple. After thiol blocking and disulde reduction, thiolderivatization with these reagents results in proteins with a signif-icantly higher molecular weight. Thus, proteins with oxidized thi-ols run more slowly on SDSPAGE than do proteins with reducedthiols. The advantage of this technique is that it can be used incombination with Western blot analysis, where specic cellularproteins can be detected without the need for purication. Exam-ples of these reagents include polyethylene glycol derivatives ofNEM with molecular masses of 2 and 5 kDa that have been usedto determine the in vivo redox state of p53 and protein disuldeisomerase [111,112]. In addition, the NEM derivative 4-acetam-ido-40-maleimidylstilbene-2,20-disulfonate (AMS), with a molecu-lar mass of 536 Da, has been applied extensively in a similar

H3C CH3+HBr

N

N

O

F

SO2NH2

N

N

O

F

SO3 NH4+-

SBD-F ABD-F

Fig. 14. Structure of SBD-F and ABD-F.

nsenmore sensitive thiol detection agent than DTNB [95]. In addition,the extinction coefcient is stable in the pH range of 37 [68], en-abling detection at low pH. Thus, detection of 4-TP can be obtainedwith HPLC, which is usually carried out at acidic pH, increasing thesensitivity of thiol quantication to the picomolar range [68].

4-DPS has the major advantage of reacting with thiols at pH 4.5.At low pH, the decrease in concentration of the thiolate anion ispartly compensated for by an increase in the protonation of thenitrogen in the pyridine ring, rendering 4-DPS 1000-fold morereactive than the neutral molecule [97,98]. An additional advan-tage of the low-reaction pH of 4-DPS over DTNB is the slow rateof hydrolysis. The hydrolysis of 4-DPS and DNTB results in genera-tion of 4-TP and NTB, respectively, and this factor should be con-sidered when using these reagents. At pH 4.5, the rate of 4-DPShydrolysis is 10-fold slower than the hydrolysis of DTNB at pH 8[95].

The amphiphilic nature and small size of 4-DPS provide accessto thiols in hydrophobic environments, and 4-DPS is generallypreferable to DTNB for quantication of protein thiols [68,95].However, the low detection wavelength of 324 nm may pose aproblem and necessitates correction for background absorbanceunless HPLC is used to separate 4-TP from other components.

One of the principal advantages of DTNB and 4-DPS, beingthemselves disuldes, is their high specicity toward thiols, butas a consequence they will also rapidly react with any reductantspresent in the sample. Thus, when applying DTNB or 4-DPS toquantify protein disuldes using the strategy depicted in Fig. 5, itis extremely important that the reducing agent be completelyeliminated to avoid cross-reactivity. Furthermore, the extinctioncoefcients of NTB and 4-TP decrease in the presence of denatur-ants, most notably GuHCl, so a standard curve with known concen-trations of thiols should be included to correct for this [92,95].

Fluorescent thiol reagents

Fluorescent labeling of thiol compounds, followed by chromato-graphic or electrophoretic separation, is a sensitive method for thedetection of thiols. Fluorescent N-substituted derivatives of NEMand IAM are applied extensively for thiol detection. A wide varietyof reagents exist (for a review, see Ref. [90]), including N-(1-pyre-nyl)maleimide, used in HPLC assays for quantication of GSH andGSSG [99]. Other examples include the NEM derivative Cy5 malei-mide [100] and the IAMderivative BODIPY FL C1IA [77],whichhavebeen used to detect thiols by two-dimensional PAGE [101,102].

Alternatively, uorescent reactive halides, not directly derivedfrom IAM but with similar chemical properties, are also widely ap-plied for thiol detection. One example is the weakly uorescent re-agent monobromobimane (mBBr) (Fig. 13), which reacts with thiolgroups to form a highly uorescent thioether [103]. mBBr has beenused in one- and two-dimensional PAGE for detection of thiol-con-taining proteins [43,104106] and in combination with HPLC toquantify low-molecular-weight thiols [107]. mBBr is susceptibleto photodegradation, yielding uorescent products that interferewith thiol determination, so reactions should be carried out inthe dark. Like IAA and IAM, mBBr cross-reacts with thiol-basedreductants and phosphines [108].

The benzofurazans ammonium 7-uoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) and 4-aminosulfonyl-7-uoro-2,1,3-benzox-adiazole (ABD-F) (Fig. 14) are also uorescent reactive halides usedas thiol detection agents. The reagents are nonuorescent untilthey form derivatives with thiols and have no interfering back-ground uorescence, in contrast to most of the other uorescentthiol detection agents. These reagents are interesting because they

Methods for analyzing thiols and disuldes / R.E. Hado not cross-react with phosphines; thus, disulde reduction andthiol derivatization can take place in the same step [73,109]. Unfor-tunately, thiol derivatization with SBD-F requires elevated reactionN

H3C SR

Fig. 13. Reaction of mBBr with thiol.+RSHN

N

OO

H3C CH3

H3C Br

N

OO

mBBr

, J.R. Winther / Anal. Biochem. 394 (2009) 147158 155manner to measure the in vivo redox state of Hsp33 and variousER oxidoreductases [113,114]. AMS is light sensitive, so all reac-tions should be carried out in the dark [19].

nsenBiotin and isotope reagents for thiol detection

Protein thiols can be modied with biotin linked to IAM or NEM,followed by avidin afnity purication [18] or detection by immu-noblotting [115]. Another approach uses radioactive derivatives ofIAM and NEM. This has been successfully applied in proteomicstudies for monitoring protein thiols in combination with two-dimensional PAGE [116,117]. A combination of these two tech-niques is found in a reagent known as isotope-coded afnity tag(ICAT), where stable isotope derivatives of IAM are linked to biotin.Among other applications, this reagent has been used in combina-tion with mass spectrometry to measure the oxidation state of pro-tein thiols and disuldes [118,119].

Electrochemical detection of redox-active compounds

Recently, it has been shown that it is possible to detect thepresence of not only thiol compounds but also other redox-activespecies in complex mixtures following HPLC separation andcoulometric detection [120,121]. The detection is based on themeasurement of electrochemical reaction of redox-active com-pounds on the surface of electrodes in the detector. A particularlyuseful variant of this system uses an array of electrodes with differ-ent potentials, allowing for separation not only based on retentiontime on the column but also based on different redox potentials.

Concluding remarks

The past decade has been an exciting time in the eld of cellularthioldisulde biochemistry. Considerable insight into many as-pects of cellular redox regulation has been obtained, and the eldis gaining the interest of a wider audience. Nevertheless, the reac-tive character of the SH group provides researchers in the eldwith quite a challenge. Reliable measurements of thiols and disul-des are largely dependent on proper sample treatment, and therelevant controls should always be included to verify the reliabilityof the method. Suitable controls, of course, depend on the nature ofthe experiment. The aim of this review was to list some of the typ-ical challenges in experimental redox biology and to offer somesolutions. We hope to have provided the reader with basic infor-mation about the most commonly used thioldisulde reagents,and their advantages and disadvantages, for use in the design of fu-ture experiments.

Acknowledgments

Colin Thorpe, Kristine Steen Jensen, Jonas Nielsen, and ChristineTachibana are thanked for critically reading the manuscript. Thetechnical assistance of Svetlana Hansen and Berit Schultz is grate-fully acknowledged. This work was supported by the Danish Natu-ral Science Research Council and a generous donation from IbHenriksens Fond for HPLC equipment.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ab.2009.07.051.

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158 Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158

An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerationsIntroductionQuenching cellular thioldisulfide exchange: TraQuenching by acidificationQuenching by thiol alkylationThioldisulfide quenching: A combination strategy

Reaction conditions: Measuring in?vivo redox staPreventing thiol oxidation by molecular oxygenConsiderations relating to bulk protein denaturationpH during reduction and derivatization

Measuring thioldisulfide ratios: Common pitfallsThiol alkylating agentsIodoacetic acid and iodoacetamideN-EthylmaleimideOther alkylating agentsGeneral practical considerations for alkylation reactions

Reducing agentsThiol-containing reductantsPhosphinesSodium borohydrideAlternative reducing agentsReducing agents: Practical considerations

Thiol detection agentsAromatic disulfide reagentsFluorescent thiol reagentsThiol reagents that significantly increase molecular massBiotin and isotope reagents for thiol detectionElectrochemical detection of redox-active compounds

Concluding remarksAcknowledgmentsSupplementary dataReferences