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Review

An introduction to methods for analyzing thiols and disuldes: Reactions,


reagents, and practical considerations
Rosa E. Hansen
1
, Jakob R. Winther
*
Department of Biology, University of Copenhagen, DK-2200 Copenhagen, Denmark
Introduction
The majority of the thiols (SH)
2
and disuldes (SS) in cells are
found 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 the
most reactive functional groups found in proteins [1]. Protein disul-
de bonds are typically introduced and removed through a thiol
disulde exchange reaction (Fig. 1B). This mechanism of transferring
reducing equivalents between thiol and disulde pairs is central in
redox biology and is, for example, applied by cytosolic thioredoxin
with its active site in the reduced form to reduce protein disuldes
and in the endoplasmic reticulum (ER) by protein disulde isomeras-
es in their oxidized form to generate disulde bonds. The reaction is
initiated by a nucleophilic attack of a thiolate on an existing disulde
bond, leading to oxidation of the nucleophilic thiol and reduction of
the leaving group sulfur [2]. In thioldisulde exchange reactions, it
is important to consider reaction rate and the equilibrium constants
between various thiol and disulde species. Because the thiolate an-
ion is the reactive species, these properties are particularly sensitive
to thiol pK
a
values. In addition, the kinetics and thermodynamics of
thioldisulde 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]).
Cellular SH groups are implicated in the coordination of metal
ions and the defense against oxidants, and the reversible formation
of disulde bonds is involved in regulation of enzyme activity, sig-
nal 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 them
are in high demand. For example, methods for determining the
in 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 redox
regulation pathways. The reactive nature of thiols is, however, of-
ten an experimental challenge. In contrast to the extracellular
space, the cytosolic concentration of reduced thiols is much higher
than the concentration of disuldes, and the SH group easily oxi-
dizes during cell lysis and sample preparation. One should consider
that 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 dealing
with 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 highly
qualied contributions that we, within the given scope, do not
mention. The overall focus is on practical aspects, including typical
biochemical experimental conditions and caveats to consider in
interpreting results. Reagents for thiol derivatization and disulde
reduction are evaluated and compared, and we discuss how to
avoid conict between mutually cross-reactive thiol reagents. We
consider this review to be an introduction to experimental thiol
disulde biochemistry updated with selected contemporary
knowledge on the subject.
Quenching cellular thioldisulde exchange: Trapping in vivo
conditions
Probably the most critical step when working with redox biol-
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 that
perturbation 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 transfer
disuldes 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,
0003-2697/$ - see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2009.07.051
* 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, guanidinium
hydrochloride; GSH, reduced glutathione; GSSG, oxidized glutathione; NEM, N-
ethylmaleimide; DTT, dithiothreitol; EGTA, ethyleneglycoltetraacetic acid; EDTA,
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), 5,5
0
-dithiobis-
(2-nitrobenzoic acid); 4-DPS, 4,4
0
-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-glutathione
adduct; AMS, 4-acetamido-4
0
-maleimidylstilbene-2,2
0
-disulfonate; ICAT, isotope-
coded afnity tag.
Analytical Biochemistry 394 (2009) 147158
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and the other is to quench thioldisulde exchange with acid. Both
methods have advantages and pitfalls, as illustrated in Fig. 2 and
discussed 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 is
extremely rapid, with rate constants in the range of 10
9
M
1
s
1
[7]. However, in some cases redox enzymes unfold fairly slowly
even under highly acidic conditions and have very low pK
a
values.
The Escherichia coli disulde-bond-forming enzyme DsbA, for
example, has a pK
a
of 3.5 [8,9]. Because the K
ox
of the protein
thioldisulde couple is very pH dependent, its redox status may
change if acidication is not concurrent with efcient protein
denaturation. Similarly, it has been observed that the redox status
of 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
by acid also requires protein denaturation. Denaturation will en-
sure that thiols, which in enzymes can be highly activated, are
deactivated and at the same time all protein thiols are rendered
accessible to protonation.
On acid precipitation, low-molecular-weight thiol compounds
remain soluble and can easily be separated from protein thiols
by 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 sulfosalicylic
acid (SSA), are particularly potent protein denaturants (Fig. 3)
[10,11]. Oxidation of SH groups has, however, been observed in
PCA- and SSA-treated samples [1114]; therefore, TCA is probably
the 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 coprecipitation
agent such as sodium deoxycholate (0.02%, w/v) is included [16]. It
has been suggested that high concentrations of denaturants such
as guanidinium hydrochloride (GuHCl) can inhibit TCA precipita-
tion, and samples containing concentrations of GuHCl above 1 M
should be diluted before precipitation [17]. TCA quenching also
effectively limits oxidation during storage, as demonstrated by
the unchanged content of reduced glutathione (GSH) in the soluble
fraction of samples quenched with 15% TCA at 20 C for up to
7 days [12]. It is, however, important to note that acid quenching
is reversible, so if the pH is raised (e.g., to reduce disuldes or to
alkylate thiols), undesired oxidation can occur.
Quenching by thiol alkylation
The main advantage of using cell-permeable alkylating agents
for thioldisulde quenching is that the thiols, in general, are irre-
versibly blocked under conditions where different compartments
are not mixed. Nonetheless, this quenching procedure might per-
turb the in vivo thioldisulde equilibrium. For example, the
GSH and oxidized glutathione (GSSG) ratio of cytosolic glutathione
is dened in part by the enzyme glutathione reductase. If GSH is
depleted by an alkylating agent, glutathione reductase can, in the-
ory, drive the redox equilibrium from GSSG toward GSH, resulting
in articially low GSSG values [14]. Thus, an absolute requirement
for efcient quenching is that the alkylating agent not only com-
pletely block all thiols but also inactivate all redox enzymes before
any perturbation of the redox equilibrium can take place.
As described in detail in the following sections, choosing the
appropriate reaction conditions for alkylation reactions involves
many factors. On the one hand, a high-reaction pH will increase
the rate of thioldisulde exchange and increase the risk of un-
wanted side reactions. On the other hand, if low-reaction pH and
S
S
S
-
A
c
id
ific
a
tio
n
A
lk
y
la
tio
n
S
S
HS
+ Extremely rapid quench
+ Protein denaturation ensures that
the proton can access all thiols
- Reversible quench
S
S
XS
+ Irreversible quench
- Slow quench at pH < 7
- Buried thiols can be inaccessible
- Not completely thiol specific
Fig. 2. Common strategies for quenching the cellular thioldisulde status. Plus symbols (+) and minus symbols () denote general advantages and disadvantages,
respectively.
Cl
Cl
Cl
OH
O
TCA
Cl O
O
OH
O
PCA
OH
S
O HO
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
R3
S S
R2
R1
S S
R2
S

R3 + +
Cysteine
Thiol - disulfide exchange
Cystine
A
B
Fig. 1. Structure of cysteine and cystine (A) and mechanism of thioldisulde
exchange reaction (B).
148 Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158
short incubation times are used, the alkylation of protein thiols
buried by the three-dimensional structure can be incomplete. In
a study by Lind and coworkers [18], a 5-min incubation of intact
mammalian 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 longer
times (15 min) did not improve blocking efciency, so the authors
concluded that approximately 20% of the cellular protein thiols of
intact cells were inaccessible to NEM.
Thioldisulde quenching: A combination strategy
The two main methods for quenching the thioldisulde state
have advantages as well as drawbacks, so the optimal procedure
is often a combination of the two strategies, where samples are
rst 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, the
TCA pellet can be solubilized by sonication in strongly denaturing
buffers containing the alkylating agent [20,21]. This strategy will
render even buried protein thiols accessible to the alkylating
agent and will ensure that catalytic thiols are not active during
alkylation.
When the objective is to measure, for example, low-molecular-
weight thiols in the TCA supernatant, it is advantageous to add the
alkylating 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 of
hydroxide ions. The presence of alkylating agents will minimize
the risk of thiol oxidation during titration. It should be noted that
NEM is relatively stable in 10% TCA (Hansen and Winther, unpub-
lished observations) and that, therefore, this alkylating reagent can
conveniently 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 a
classic study by Haber and Annsen [22] where reduced ribonucle-
ase regained both native disulde bonds and enzymatic activity
within 20 h at pH 8 in the absence of oxidants except dissolved
oxygen. We now know that this process is catalyzed by trace metal
ion contaminants in reagent solutions. Intermediates in the oxida-
tion reaction include reactive oxygen species [23], which can also
induce irreversible oxidation of proteins thiols [4,5]. The reaction
is dependent on pH and temperature, with slower oxidation rates
at lower temperature and pH values [2426]. As shown by Getz
and coworkers [24], more than 80% of a 0.5-mM solution of the
small thiol dithiothreitol (DTT) was oxidized in 1 day at 25 C
and pH 7.2 in the presence of 1 lM Fe
3+
or Ni
2+
. If the metal chelat-
ing agent ethyleneglycoltetraacetic acid (EGTA) was present, the
stability greatly improved, with only 10% oxidation under the same
conditions. The ability of common metal ions to catalyze oxidation
of thiols ranks in the order Cu
2+
> Fe
3+
> Ni
2+
>> Co
2+
[26]. For these
reasons, oxygen should be eliminated from the reagent solutions
used 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 and
electrostatic properties of the low-molecular-weight thiol compo-
nent inuence the oxidation rates more strongly than does the
presence/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 reagent
should be considered. Steric hindrance or unfavorable electrostatic
interactions may block access to protein thiols or disuldes and
have considerable effects on the reaction rate. In the case of disul-
de reduction, protein structure may provide intrinsic stability to
the 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 into
generic thiols and disuldes with stabilities and reactivities similar
to those of GSH and GSSG. Typical protein denaturants are 6 M
GuHCl, 8 M urea, and 5% sodium dodecyl sulfate (SDS).
Urea has the disadvantage of containing varying amounts of
cyanate (Fig. 4A), which reacts with thiol groups to form thiocarba-
mates, as shown in Fig. 4B [28,29]. The rate constant for reaction of
cysteine with cyanate is 0.07 M
1
s
1
at pH 6 [30], and because an
8-M urea solution may contain up to 20 mM cyanate [31], this
reaction can be signicant. Thiocarbamate is unstable at a pH
above 6 with a half-time of 11 min at pH 8, according to Stark
[30]. The alternative denaturant thiourea, which is typically used
for isoelectric focusing, does not contain cyanate. A major draw-
back of this solubilizing agent is, however, that it reacts directly
with alkylating agents such as iodoacetamide (IAM) and, thereby,
competes for alkylation of thiol groups [32]. GuHCl is an inert
and extremely potent protein denaturant that, unfortunately, is
not compatible with many downstream processes such as isoelec-
tric focusing and SDSpolyacrylamide gel electrophoresis (PAGE).
Finally, although protein denaturants can be indispensable for
reactions such as alkylation of buried protein thiols, the alkylation
rate 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 of
the small thiol 2-nitro-5-thiobenzoic acid (NTB) by the alkylation
agent 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,
and 5% SDS, respectively (our unpublished observations). The
observation that SDS, in particular, decreases alkylation rates has
been suggested previously [33] and is probably due to formation
of micelles that hinder free diffusion.
pH during reduction and derivatization
As described in the previous section, preventing perturbation of
the 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 during
thiol reactions. A low pH will minimize thioldisulde rearrange-
ments, metal-catalyzed oxidation, and unwanted side reactions
such as reactions with amino acid residues other than cysteines.
Accordingly, when choosing reagents for measuring thiols and
disuldes, the pH optimum of the reaction should be considered.
NH
2
NH
2
O NH
4
+
+
Urea Cyanate
HN C O + H
+
RSH +
NH
2
SR
O HN C O
A
B
Fig. 4. Urea is in equilibrium with cyanate (A), which in turn can react with thiols
(B).
Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158 149
Measuring thioldisulde ratios: Common pitfalls
The general approach for measuring thiols and disuldes is
shown 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 incubating
with a suitable thiol-specic detection reagent (Fig. 5A). Here the
main 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 of
disulde bonds involves three steps (Fig. 5B): (i) blocking free thiol
groups, (ii) reducing disulde, and (iii) detecting newly reduced
thiols. The selection of reagents at each step is crucial for the suc-
cess of the experiment. In the rst step, the alkylating agent must
react quickly and irreversibly with thiol groups. Furthermore, any
excess alkylating agent must be removed prior to disulde reduc-
tion to avoid alkylation of newly reduced disuldes that would
lead to an underestimation of oxidized thiols. Compatibility be-
tween the reducing agent and the thiol detection reagent must also
be considered. To avoid an overestimation of oxidized thiols, the
reducing agent preferably should not cross-react with the thiol
detection reagent. Alternatively, the reducing agent must be re-
moved prior to quantication. The following sections provide an
introduction 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, alkylation
reagents are used to quench the thioldisulde status on cell lysis
(Fig. 2) and, as illustrated in Fig. 5B, are applied to mask free thiols
so as to specically detect disuldes. In this section, a general
introduction to the most commonly used thiol alkylating agents
is provided, and their individual chemical properties are reviewed
in terms of their practical advantages and drawbacks.
Iodoacetic acid and iodoacetamide
Iodoacetic acid (IAA) and IAM react irreversibly with thiols in a
nucleophilic substitution reaction to form the corresponding car-
boxymethyl or carboxamidomethyl derivatives (Fig. 6). IAA and
IAM 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 and
during 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 neutral
or alkaline pH, IAA and IAM also react slowly with the hydroxyl
group of tyrosine [34], the e-amino group of lysine [35,36], the
imidazole group of histidine [37,38], and the N terminus of pro-
teins [39]. Furthermore, IAM and IAA react with the sulfur atom
of methionine at pH values as low as 2.8 [40,41]. The extent of
the 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 any
other group in the protein. For example, the reaction rate of IAA
with histidine has been reported to be 1000-fold slower than that
with cysteine [42]. Still, to ensure that the alkylation is thiol-spe-
cic, reactions should be performed under careful consideration
of concentrations and incubation times. In other words, the ideal
condition is one where the concentration is as low as possible
and the reaction time is as short as possible to achieve complete
modication. Unfortunately, complete alkylation of protein thiols
with 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 was
not complete after 1 h.
Due to the negative charge of IAA, this reagent is membrane
impermeable and is inappropriate as an in vivo quenching agent
of thioldisulde exchange. Furthermore, the negative charge of
IAA can engage in electrostatic interactions with charged groups
in 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, hinder
the access of IAA to thiols in hydrophobic environments. A possible
benecial feature of IAA is that the modication results in an in-
crease in the negative charge of the protein, and this in some cases
results in a mobility shift that can be followed with nondenaturing
PAGE (Fig. 7). In contrast, modication with IAM does not change
the electrophoretic mobility of proteins. Because of its lack of
charge, 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, for
most 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-
S
S
HS S
S
XS
Thiol
blocking
Disulfide
reduction
SH
SH
XS S*
S*
XS
Thiol
detection
S
S
HS
Thiol
detection
S
S
*S
A
B
Fig. 5. Common strategy for detection of thiols (A) and disuldes (B). Thiols destined for detection are denoted S
*
, whereas alkylated thiols are denoted SX.
RS
O

O
HI RSH + + I
O

O
IAA
HI RS
NH
O
2
RSH
+ +
I
NH
O
2
IAM
Fig. 6. Alkylation of thiols with IAA and IAM.
150 Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158
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, on
solution pH. NEM is generally thiol-specic below pH 7 and with
concentrations in the range of 120 mM [45]. However, reactions
with protein amines have been observed at pH values above 7 if
NEM is in large excess or incubation times are prolonged to more
than 2 h [45,46].
The absorption spectrum of NEM has a maximum at 305 nm
with an extinction coefcient of 620 M
1
cm
1
[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 instability
in alkaline media is caused by hydrolysis of the maleimide ring,
leading to the formation of N-ethylmaleamic acid (Fig. 8B), which
is accompanied by loss of thiol reactivity [49,50]. In addition, some
low-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 been
shown to be complete after incubation of NEMCys adducts for
36 h at room temperature and pH 9 [51]. Another crucial observa-
tion is that GSH alkylated with NEM slowly regenerates the thiol at
pH 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, at
pH 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 of
NEM compared with IAA and IAM has also been measured for
the alkylation of protein thiols [43,53]. Thus, NEM is in most cases
preferable to IAA and IAM. Finally, NEM is a small and uncharged
reagent, so it is membrane-permeable and can react with thiols
even 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 a
pH optimum between 5 and 8 [55]. Reactions are typically per-
formed at a pH between 6 and 7 with 170 mM VP and incubation
for 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 of
0.020.05 M
1
s
1
[55]. In comparison, rate constants for alkyl-
ation of GSH or cysteine by NEM in the same pH interval are in
the range of 10 to 10
3
M
1
s
1
[48,57]. Above pH 7, VP may react
with amine groups in proteins, but the reaction rate with cysteines
was reported to be approximately 300 times higher [58]. Although
VP reacts more slowly with thiols than does NEM, VP is used as the
alkylating 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 is
S-methyl methanethiosulfonate (MMTS) [60,61]. Reaction with
thiols leads to the formation of an SSCH
3
group that can be
reduced by DTT or another free thiol group in the protein. Unfortu-
nately, MMTS has been shown to induce formation of intra- and
intermolecular protein disulde bonds [62] and should be avoided
as 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 an
unwanted side reaction. Because the pK
a
values of thiols are typi-
cally lower than those of amines, the optimization of reaction pH
can ensure that the amino groups are in their NH
3
+
state and, con-
sequently, do not behave as nucleophiles.
As mentioned previously, it may be necessary to remove excess
alkylating agent prior to disulde reduction to avoid alkylation of
newly reduced thiol groups. Alkylation agents can be removed
quantitatively by techniques such as gel ltration and acid precip-
itation. Alternatively, the concentration of the reducing agent can
be optimized to consume the excess alkylating agent. This requires,
however, that the reducing agent reacts faster with the alkylating
agent than the alkylating agent reacts with newly reduced thiols.
CH
3
N
CH
3
H
N
O
O
O

O
O
OH

CH
3
N
O
O
RS
CH
3
N
O
O
+ RSH
CH
3
N
S
H
2
N OH
O
NH
CH
3
O S
H
N
O
OH
O
O
O
A
B
C
D
CH
3
N
O
O
GS
CH
3
N
O
O
HO
+ GSH
H O
2
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 the
NEMCys adduct. For simplicity, not all electron movements are shown. (D)
Reaction for the formation of glutathione from NEM adducts with glutathione
(NEM-GS) [52].
N
CH
2
N SR
+ HSR
Fig. 9. Reaction of VP with thiols.
SH S-AM S-AA
Fig. 7. Protein thiol alkylation with IAA can be followed with native PAGE. Yellow
uorescent protein (3 lM) with a surface-exposed cysteine was modied with
3 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 151
This issue is discussed further in the following section. Finally, note
that all reagents described in this section are fairly toxic and
should be handled with caution.
Reducing agents
Any detection and quantication of disulde bonds requires
efcient and quantitative reduction of disuldes. As illustrated in
Fig. 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 must
be removable (e.g., by gel ltration) or unable to cross-react with
the derivatization agent in the next step. Alternatively, the product
of the reaction between the derivatization agent and the thiol of
interest can be detected independently (e.g., after separation by
high-performance liquid chromatography [HPLC]). Here we pro-
vide an introduction to the most commonly used reductants. A
strong reductant typically possesses a high degree of S-nucleophi-
licity. The disulde reductants discussed in this section have the
following order of decreasing S-nucleophilicity: hydride > phos-
phine > thiol > cyanide > sulte [63].
Thiol-containing reductants
The 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 advantages
and disadvantages of thiol exchange reductants, the main advan-
tage is the high degree of specicity, meaning that only disuldes
are chemically affected, leaving the protein ideally suited for
downstream 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, the
reagents require a reaction with pH above 7, where oxidation by
ambient molecular oxygen can occur. The reagents themselves
are particularly sensitive to oxidation, and consequently all solu-
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 pK
a
value of ME is 9.5 [64],
and alkaline pH values will signicantly increase the reaction rate
with disuldes. ME is a weak reducing agent, with an equilibrium
constant for the reduction of disuldes that is near unity [65]. Con-
sequently, ME must be used at very high concentrations to avoid
the formation of mixed disuldes. Furthermore, ME is volatile with
an unpleasant odor, and concentrated solutions should be handled
in 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 at
100 C in the presence of SDS). Notably, in this application the for-
mation of mixed disuldes with ME will not likely change mobility
signicantly. ME can diffuse into adjacent lanes during electropho-
resis; however, this can affect samples without reducing agents
that are run on the same gel.
The most commonly used reductant is the dithiol DTT, which
reduces disuldes in a reaction where DTT is converted into a sta-
ble intramolecular cyclic disulde (Fig. 10B). Disulde reduction by
DTT can be followed spectrophotometrically by the appearance of
oxidized DTT, which absorbs at 310 nm with an extinction coef-
cient of 110 M
1
cm
1
[66]. The formation of oxidized DTT is dri-
ven by favorable steric and entropic effects [4,65], and DTT is a
considerably stronger reducing agent than ME, with an equilibrium
constant for cystine reduction of 1.3 10
4
M [65]. Accordingly,
mixed disuldes between thiols and DTT do not accumulate, and
a large excess of reagent is not required for complete disulde
reduction. The thiol pK
a
values of DTT are 9.2 and 10.1 [67], and
reduction is typically carried out at or above pH 8. A quantitative
reduction 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 its
membrane permeability, and it is widely used as a reductant in cel-
lular systems, where it reversibly inhibits protein disulde bond
formation without hindering protein synthesis [69]. Typically, 5
10 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, rapid
hydrolysis releases the second thiol fragment and the phosphine
oxide (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 thioldisulde
reactions [70]. Trialkylphosphines react specically with disuldes
and do not react with other functional groups commonly found in
proteins [71]. Furthermore, phosphines are unreactive toward cer-
tain thiol alkylating and derivatization agents such as VP and the
uorescent benzofurazans (described below). This has the advan-
tage that reduction and alkylation conveniently can take place con-
comitantly [7174]. Phosphines do, however, react rapidly with
IAA, IAM, and NEM even under acidic conditions [24,7577]. In
fact, IAM reacts with tris(2-carboxyethyl)phosphine (TCEP) three
times more effectively than it does with ME at pH 7.3. The reaction
with NEM is somewhat slower, with ME reacting twice as fast as
TCEP [76]. Thus, as in the case of thiol reductants, reactions with
phosphines must be carried out in a step separate from alkylation
with IAA, IAM, NEM, and their derivatives.
TCEP is a highly water-soluble reagent and the most commonly
used phosphine for disulde bond reduction (Fig. 11B). The pK
a
va-
lue of the phosphorus is 7.6 [78], and TCEP is signicantly more
effective in reducing low-molecular-weight disuldes and sur-
face-exposed protein disuldes than is DTT (Table 1), particularly
HO
SH
R
S
S
R
+ 2
HO
S
S
OH
R
SH
+ 2
SH HS
OH
OH
R
S
S
R
+
S
S
OH
OH
+ 2 RSH
A
B
Fig. 10. Reduction of disuldes with the thiol-containing reductants ME (A) and
DTT (B).
152 Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158
at pH below 7 [79,80]. DTT has been observed to reduce certain
protein disuldes, such as the active site of thioredoxin, faster than
TCEP, probably because of the greater steric hindrance of the
nucleophilic phosphorus atom of TCEP compared with the DTT
thiolate [80]. This disadvantage can obviously be eliminated by
protein denaturation prior to TCEP reduction.
TCEP is more resistant toward metal-catalyzed oxidation than
the thiol-containing reductants [24,78]. Oxidation of TCEP appears
to 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 acidic
when dissolved in water (four protons accompany each TCEP mol-
ecule), so samples must be adequately buffered after the addition
of this reagent.
Besides TCEP, another water-soluble reagent, tris(hydroxypro-
pyl)phosphine (THP), is commercially available (Fig. 11B). THP
has a phosphorus pK
a
value of 7.22, and it is even more reactive
toward small molecule disuldes and surface-exposed protein
disuldes 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 pH
makes this reagent impermeable to membranes, THP is neutral
and is as effective as DTT at traversing membranes [80].
As a complement to commercially available phosphines, Cline
and coworkers [80] devised a simple method for synthesis of
methyl ester derivatives of TCEP. These water-soluble phosphines
show enhanced reactivity at low pH compared with TCEP. In addi-
tion, esterication increases membrane permeability, allowing the
TCEP esters to penetrate phospholipid bilayers 30 times faster than
DTT.
Sodium borohydride
Sodium borohydride (BH) is an extremely reactive and strong
reducing agent due to the high S-nucleophilicity of the borohy-
dride ion [81]. This reagent is highly reactive, and BH solutions
should be prepared immediately before use to avoid decomposi-
tion. Several studies have shown that BH is able to quantitatively
reduce protein disuldes with or without the presence of denatur-
ing agents [68,8284], and BH has been used for the reduction of
protein disuldes in whole cell extracts [21]. In general, reduction
of protein disuldes with 1 M BH at pH 12 is complete after 30 min
at 50 C. Because hydrogen is formed during the reaction, protein
solutions tend to foam, but this can easily be avoided by adding
a 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 are
protected from oxidation even when the reducing agent has been
destroyed. In addition, NEM is rapidly inactivated by the addition
of 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 useful
reducing agent for a number of applications, it can reduce keto
groups to alcohols. Furthermore, the high pH at which the reaction
is carried out may catalyze hydrolysis of Asn and Gln amides as
well as the peptide bond. Therefore, BH is not applicable to protein
samples destined for, for example, mass spectrometry.
Alternative reducing agents
Disulde bonds can be reduced by other nucleophiles such as
sulte (SO
3

) and cyanide (CN

). However, reduction by these


agents is rarely quantitative unless catalysts are present to ensure
efcient reduction [86]. The enzyme glutaredoxin has been used as
a specic reductant of proteinGSH mixed disulde (PSSG) [18]. In
the presence of GSH, however, glutaredoxins can also reduce intra-
P
+
R2
R2
R2
S
R1
R1
S

R1
SH
R1
S
S
R1
P R2
R2
R2
O
H
2
O
P
R2
R2
R2
+
+
+ 2
PH
O
OH
HO
O
OH
O
TCEP
P
OH
OH HO
THP
+
A
B
Cl
-
Fig. 11. Reduction of disuldes with trialkylphosphines. (A) Reaction mechanism.
(B) Structures of TCEP and THP. TCEP is shown as the commercially available
phosphonium chloride.
Table 1
Second-order rate constants (M
1
s
1
) for reduction of disuldes by DTT, TCEP, and
THP.
DTT TCEP THP
DTNB (pH 7.5)
a
2900 18,000 nd
DTNB (pH 4.5)
b
3.5 640 1600
rxYFP (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 Ostergaard
et al. [123]. THP and TCEP data were obtained in 100 mM phosphate and 1 mM
EDTA (pH 7.0) at 30 C, as described in part III of the Supplementary material.
Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158 153
molecular protein disuldes and, thus, cannot be considered as a
specic reductant of PSSG. Finally, reducing agents for thiol
oxidation states other than disuldes exist. These include sodium
arsenite 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 analysis
of protein thiol groups. For the thiol-containing reductants and
the phosphines, this can be accomplished by, for example, gel ltra-
tion or acid precipitation of proteins. However, a quantitative elim-
ination of reducing agent can be difcult to obtain with acid
precipitation [68]. Although gel ltration can effectively remove
reducing 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 contain
partially aggregated material, gel ltration is not an option. In these
cases, BH is the reagent of choice because the excess reagent is sim-
ply removed by the addition of acid. If BH is not applicable because
of side reactions with other protein functional groups, one of the
phosphines could be used because their reducing capacity and reac-
tivity at low pH are superior to those of the thiol reductants. We
recommend THP because it has proven to be the most reactive
phosphine in the reduction of both low-molecular disuldes and
protein disuldes. In addition, only a small excess is needed for ef-
cient reduction. This is a crucial advantage when the thiol detection
agents 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 than
the reaction between the alkylating agent and the newly reduced
thiols. The addition of a small excess of ME relative to alkylating
agents can be particularly suitable for this purpose due to the
low reducing capacity of ME. Thus, the alkylating agent can react
rapidly with the ME thiol essentially without any reduction of
disuldes.
All of the reagents discussed in this section are susceptible to
oxidation. Preferably, reducing agents should be prepared freshly
on a daily basis. Stock solutions of 0.1 M DTT or TCEP, for example,
lose approximately 20% reducing capacity after 2 weeks at 20 C
and pH 7 (Hansen, personal observation). If stock solutions are
used, they should be as concentrated as possible and kept at low
pH, and the exact thiol concentration should be determined before
use using one of the aromatic disulde reagents described in the
following 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 with
detection labels. This section provides examples of thiol detection
agents and their applicability. When using derivatives of alkylating
agents, the experimental considerations described in the previous
sections for reaction conditions, reactivity, specicity, and stability
apply. After labeling cellular thiols with suitable reagents, the dif-
ferent species can be separated and characterized using a variety of
techniques, including HPLC, afnity purication, variations of two-
dimensional PAGE, and mass spectrometry. These methodologies
have been reviewed elsewhere and are not discussed in detail here
[8890].
Aromatic disulde reagents
5,5
0
-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 exchange
reaction, resulting in the stoichiometric formation of the yellow
NTB (Fig. 12A), which absorbs at 412 nm (e = 14,150 M
1
cm
1
at
pH 7.3) [92]. The NTB anion is an excellent leaving group because
of the electronegative character of the nitro group combined with
its conjugated nature. The reaction is essentially irreversible, and
the absorbance of the generated NTB serves as a measure of the
number of reacted SH groups [93]. The molar extinction coefcient
of the NTB anion is stable from pH 6 to pH 9.5. Below pH 6, NTB
becomes 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 a
method 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, and
phosphines [79]. Using a high molar excess of DTNB (typically
1 mM), the reaction is, for practical purposes, complete within
1 min at pH 7.3. In addition, DTNB is widely used to quantify cellu-
lar GSH and GSSG via the DTNBglutathione reductase recycling
assay [94]. Although DTNB works well for small water-soluble thiol
compounds, the reaction with protein SH is frequently hindered by
its 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
when measuring thiol concentrations in dilute protein solutions.
An alternative to DTNB is 4,4
0
-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 has
an extinction coefcient of 21,400 M
1
cm
1
and, therefore, is a
+ 2 RSH
2 S
N S S N
+ RSSR NH
4-DPS
4-TP

S S NO
2
O
2
N
COO
COO
+ 2 RS
S

2 O
2
N
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 the
chromogenic 4-TP.
154 Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158
more 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 obtained
with HPLC, which is usually carried out at acidic pH, increasing the
sensitivity 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 is
partly compensated for by an increase in the protonation of the
nitrogen in the pyridine ring, rendering 4-DPS 1000-fold more
reactive than the neutral molecule [97,98]. An additional advan-
tage of the low-reaction pH of 4-DPS over DTNB is the slow rate
of 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-DPS
hydrolysis is 10-fold slower than the hydrolysis of DTNB at pH 8
[95].
The amphiphilic nature and small size of 4-DPS provide access
to thiols in hydrophobic environments, and 4-DPS is generally
preferable to DTNB for quantication of protein thiols [68,95].
However, the low detection wavelength of 324 nm may pose a
problem and necessitates correction for background absorbance
unless HPLC is used to separate 4-TP from other components.
One of the principal advantages of DTNB and 4-DPS, being
themselves disuldes, is their high specicity toward thiols, but
as a consequence they will also rapidly react with any reductants
present in the sample. Thus, when applying DTNB or 4-DPS to
quantify protein disuldes using the strategy depicted in Fig. 5, it
is extremely important that the reducing agent be completely
eliminated to avoid cross-reactivity. Furthermore, the extinction
coefcients 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 the
detection of thiols. Fluorescent N-substituted derivatives of NEM
and IAM are applied extensively for thiol detection. A wide variety
of reagents exist (for a review, see Ref. [90]), including N-(1-pyre-
nyl)maleimide, used in HPLC assays for quantication of GSH and
GSSG [99]. Other examples include the NEM derivative Cy5 malei-
mide [100] andthe IAMderivative BODIPYFL C
1
IA[77], whichhave
been used to detect thiols by two-dimensional PAGE [101,102].
Alternatively, uorescent reactive halides, not directly derived
from 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 thiol
groups to form a highly uorescent thioether [103]. mBBr has been
used in one- and two-dimensional PAGE for detection of thiol-con-
taining proteins [43,104106] and in combination with HPLC to
quantify low-molecular-weight thiols [107]. mBBr is susceptible
to photodegradation, yielding uorescent products that interfere
with thiol determination, so reactions should be carried out in
the dark. Like IAA and IAM, mBBr cross-reacts with thiol-based
reductants 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 used
as thiol detection agents. The reagents are nonuorescent until
they form derivatives with thiols and have no interfering back-
ground uorescence, in contrast to most of the other uorescent
thiol detection agents. These reagents are interesting because they
do not cross-react with phosphines; thus, disulde reduction and
thiol derivatization can take place in the same step [73,109]. Unfor-
tunately, thiol derivatization with SBD-F requires elevated reaction
pH and temperature (pH 8.5 and 60 C) as well as long incubation
times (1 h). Under these conditions, there is a risk of regenerating
thiols from NEMGS adducts; accordingly, the combination of NEM
and SBD-F should be avoided during quantication of GSSG. ABD-F
is more reactive, and the derivatization of thiols at pH 8 and 60 C
is 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 mobility
of a protein can be used to estimate the redox state of a thioldisul-
de couple. After thiol blocking and disulde reduction, thiol
derivatization 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 reduced
thiols. The advantage of this technique is that it can be used in
combination with Western blot analysis, where specic cellular
proteins can be detected without the need for purication. Exam-
ples of these reagents include polyethylene glycol derivatives of
NEM with molecular masses of 2 and 5 kDa that have been used
to determine the in vivo redox state of p53 and protein disulde
isomerase [111,112]. In addition, the NEM derivative 4-acetam-
ido-4
0
-maleimidylstilbene-2,2
0
-disulfonate (AMS), with a molecu-
lar mass of 536 Da, has been applied extensively in a similar
manner to measure the in vivo redox state of Hsp33 and various
ER oxidoreductases [113,114]. AMS is light sensitive, so all reac-
tions should be carried out in the dark [19].
+ RSH
N
N
O O
H
3
C CH
3
H
3
C
Br
N
N
O O
H
3
C CH
3
H
3
C
SR
+ HBr
mBBr
Fig. 13. Reaction of mBBr with thiol.
N
N
O
F
SO
2
NH
2
N
N
O
F
SO
3
NH
4
+ -
SBD-F ABD-F
Fig. 14. Structure of SBD-F and ABD-F.
Methods for analyzing thiols and disuldes / R.E. Hansen, J.R. Winther / Anal. Biochem. 394 (2009) 147158 155
Biotin 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 of
IAM and NEM. This has been successfully applied in proteomic
studies 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 the
presence of not only thiol compounds but also other redox-active
species in complex mixtures following HPLC separation and
coulometric detection [120,121]. The detection is based on the
measurement of electrochemical reaction of redox-active com-
pounds on the surface of electrodes in the detector. A particularly
useful variant of this system uses an array of electrodes with differ-
ent potentials, allowing for separation not only based on retention
time on the column but also based on different redox potentials.
Concluding remarks
The past decade has been an exciting time in the eld of cellular
thioldisulde biochemistry. Considerable insight into many as-
pects of cellular redox regulation has been obtained, and the eld
is gaining the interest of a wider audience. Nevertheless, the reac-
tive character of the SH group provides researchers in the eld
with quite a challenge. Reliable measurements of thiols and disul-
des are largely dependent on proper sample treatment, and the
relevant controls should always be included to verify the reliability
of the method. Suitable controls, of course, depend on the nature of
the experiment. The aim of this review was to list some of the typ-
ical challenges in experimental redox biology and to offer some
solutions. 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 Christine
Tachibana are thanked for critically reading the manuscript. The
technical 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 Ib
Henriksens Fond for HPLC equipment.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ab.2009.07.051.
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