Acceleration of disulfide-coupled protein folding using
glutathione derivatives
Masaki Okumura
1,2
, Masatoshi Saiki
2,3
, Hiroshi Yamaguchi
1
and Yuji Hidaka
2
1 School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan
2 Graduate School of Science and Engineering, Kinki University, Osaka, Japan
3 Department of Applied Chemistry, Faculty of Engineering, Tokyo University of Science, Yamaguchi, Japan
Introduction
The formation of the correct disulfide bonds and the
conversion of a protein into its native conformation
are the result of reversible thiol(SH) ⁄ disulfide(SS)
exchange reactions that occur during protein folding
and are thermodynamically and kinetically related to
the redox potential in the biological environment. Glu-
tathione (c-Glu-Cys-Gly), one of the most abundant
thiol compounds found in cells, plays a major role in
the formation of disulfide bonds in proteins in the
endoplasmic reticulum [1]. Oxidized glutathione
(GSSG) functions as an oxidant in the formation of
disulfide bonds in proteins and reduced glutathione
(GSH) functions as a reducing agent that cleaves mis-
bridged disulfide bonds in proteins, resulting in the
formation of the thermodynamically stable conforma-
tion of proteins in vivo [2]. Because of this, glutathione
Keywords

= 3.69 · 10
)3
s
)1
)
using reduced RCG ⁄ oxidized RCG was approximately threefold higher
than that using reduced glutathione ⁄ oxidized glutathione. In addition, fold-
ing experiments using only the oxidized form of RCG or glutathione indi-
cated that prouroguanylin was converted to the native conformation more
efficiently in the case of RCG, compared with glutathione. The findings
indicate that a positively charged redox molecule is preferred to accelerate
disulfide-exchange reactions and that the RCG system is effective in medi-
ating the formation of native disulfide bonds in proteins.
Abbreviation
Arg-C, arginylendopeptidase C; ECR, glutamyl-cysteinyl-arginine; ECR
ox
, oxidized ECR; ECR
red
, reduced ECR; GSH, reduced glutathione;
GSSG, oxidized glutathione; RCG, arginyl-cysteinyl-glycine; RCG
ox
, oxidized RCG; RCG
red
, reduced RCG.
FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS 1137
is widely employed in studies of folding reactions of
disulfide-containing proteins in vitro [3,4].
Generally, folding reactions of proteins that do not
involve disulfide bond formation occur within a few
minutes. However, disulfide-containing proteins require

residue of glutathione, a series of glutathione analogs
– arginyl-cysteinyl-glycine (RCG) and glutamyl-cyste-
inyl-arginine (ECR) – were prepared, and their ability
to serve as a redox reagent in disulfide-exchange reac-
tions was examined. Arginine has recently been
employed in studies of protein folding [9]. It is thought
that arginine prevents the formation of nonspecific
aggregates during the folding reaction. Folding experi-
ments using lysozyme in the presence of arginine indi-
cated that arginine promoted the formation of its
native conformation, compared with other amino acids
[10], and that arginine effectively suppressed the aggre-
gation of denatured lysozyme [11], resulting in an
increase in folding yield [12]. Arginine has also been
reported to stabilize the exposed hydrophobic area of
single-chain Fv fragments during the folding reaction
[13], and the addition of both GSSG and arginine
resulted in an increase in folding recovery [14]. To
examine this aspect further, an arginine residue was
introduced into a glutathione molecule to increase the
solubility of folding intermediates in which cross-disul-
fide bond(s) are formed between the reagents and pro-
teins. In this study, except for the cysteine residue,
each amino acid residue of glutathione was systemati-
cally replaced with an arginine residue.
RCG and ECR were chemically synthesized and
their participation in disulfide-coupled folding reac-
tions of lysozyme and prouroguanylin, as model pro-
teins containing disulfide bonds, were examined.
Substituting a glutamic acid residue for an arginine

zyme decreases at high protein concentrations (> 0.1
mgÆmL
)1
) [17]. To estimate the ability of RCG [both
reduced (RCG
red
) and oxidized (RCG
ox
) forms] and
ECR [both reduced (ECR
red
) and oxidized (ECR
ox
)
forms] to permit the disulfide-coupled folding of
proteins, lysozyme was folded at high concentrations
(0.1–1.6 mgÆmL
)1
). The molar ratios of the reduced
and oxidized form of the reagents were adjusted to
2 : 1 in these experiments, because the molar ratio of
Acceleration of disulfide-coupled protein folding M. Okumura et al.
1138 FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS
GSH to GSSG in the endoplasmic reticulum ranges
from 1 : 1 to 3 : 1 [2]. The fully reduced and the native
form of lysozyme were eluted at positions correspond-
ing to R and N, respectively, in Fig. 1. The HPLC
peak for lysozyme folded in the correct conformation
was assigned based on a co-elution experiment using
native lysozyme [17,18]. The folding yields of lysozyme

due introduced into the glutathione molecule prevented
nonspecific aggregation of the folding intermediates at
higher protein concentrations. However, the mecha-
nism of the RCG-mediated folding remains to be
examined.
In order to further examine the effect of RCG
red
⁄
RCG
ox
in protein folding, recombinant human prou-
roguanylin was also examined as a model protein.
Prouroguanylin contains six cysteine residues that par-
ticipate in three intramolecular disulfide bonds in the
native state (Cys41–Cys45, Cys74–Cys82 and Cys77–
Cys85) and formation of the correct disulfide pairs is
important for the complete folding recovery of the
protein [19]. The folding reactions of prouroguanylin
were examined using RCG
red
⁄ RCG
ox
or GSH ⁄ GSSG,
and the folding yields were estimated by HPLC analy-
ses. The folding recovery of prouroguanylin using the
typical GSH system was decreased at higher protein
concentrations, as reported previously [19]. The RCG
redox system resulted in improved folding yields at
higher protein concentrations, compared with the GSH
system, as shown in Fig. 3. When the RCG

native conformation (%)
Protein concentration (mg·mL
–1
)
100
80
60
40
20
0
0.1
0.2 0.4 0.8
1.6
Fig. 2. Folding yields of lysozyme in the presence of GSH ⁄ GSSG
(open bars), reduced (ECR
red
) and oxidized (ECR
ox
) forms of ECR
(cross-hatched bars), and reduced (RCG
red
) and oxidized (RCG
ox
)
forms of RCG (shaded bars).
0.1 0.2 0.4
Recovery of
native conformation (%)
100
80

molar concentrations of Arg. Therefore, it is likely that
the efficient folding recovery using RCG is not the
result of direct effects, such as stabilization of the
hydrophobic surface of the denatured proteins.
To address the question of how the RCG redox sys-
tem increases the folding recovery of proteins and the
nature of the mechanism responsible for this, it is nec-
essary to characterize the folding intermediates. We
hypothesized that RCG plays a role in the solubility of
the denatured protein when the RCG is linked to fold-
ing intermediates via disulfide bonds, and that the Arg
residue in the cross-disulfide-linked RCG is important
for the accumulation of proper folding intermediates
in the native conformation. Generally, it is difficult to
analyze folding intermediates of disulfide-containing
proteins at each step of the conformational transition
[20]. However, the folding intermediates of prourogu-
anylin can be separated by HPLC. We therefore
employed prouroguanylin as a model protein to ana-
lyze the folding mechanism using RCG
red
⁄ RCG
ox
. The
distribution of folding intermediates of prouroguanylin
produced using RCG
red
⁄ RCG
ox
was compared with

then becomes predominant at the later stage in folding
because the native disulfide bond is shielded by virtue
of the fact that it is located in the interior of a protein
molecule. In our experiment, the reduced form of
prouroguanylin disappeared within 5 min (Fig. 4B) in
the reaction mixture using RCG
red
⁄ RCG
ox
but detect-
able amounts were still present in the reaction mixture
using GSH ⁄ GSSG (Fig. 4A) at that time-point. There-
fore, these results indicate that the formation of intra-
molecular disulfide bonds is rapid in the presence of
RCG
red
⁄ RCG
ox
and that the half life of folding inter-
mediates with a low solubility becomes small, resulting
in an improved folding recovery.
In order to better understand the folding mechanism
in the presence of RCG
red
⁄ RCG
ox
, the folding inter-
mediates were further analyzed in detail. The disulfide
pairing of prouroguanylin was determined using a pre-
viously reported method [21]. The intermediates 0SH

2 h
24 h
Retention time
R
N
2SH
0SH
20 min
N
N
N
Refolding time
Fig. 4. HPLC profiles of the reaction mix-
tures of prouroguanylin in the presence of
2m
M GSH ⁄ 1mM GSSG (A) and 2 mM
RCG
red
⁄ 1mM RCG
ox
(B). N, 2SH, 0SH and
R, represent the positions of prouroguanylin
with native disulfide pairing, with two thiols
and two disulfide bonds, with three disulfide
bonds and no thiol groups, and the fully
reduced form of prouroguanylin, respec-
tively.
Acceleration of disulfide-coupled protein folding M. Okumura et al.
1140 FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS
pairings using HPLC (data not shown), as described

reaction in vitro. The mis-bridged disulfide species then
disappeared, followed by the formation of native disul-
fide bonds. Therefore, the velocity of the disulfide-
exchange reaction is important in achieving correct
folding and folding recovery. The folded prouroguany-
lin with the correct disulfide bonds was observed
within 5 min in the folding reaction carried out in the
presence of RCG
red
⁄ RCG
ox
. This result indicates that
RCG
red
⁄ RCG
ox
accelerates the exchange reaction of
disulfide bonds and effectively permits prouroguanylin
to be converted into the native conformation.
To further estimate the ability of the RCG redox sys-
tem in the disulfide-coupled folding of proteins, the
folding reaction was performed under anaerobic condi-
tions using only RCG
ox
or GSSG. Under anaerobic
conditions, RCG
ox
first oxidatively reacts with the thiol
groups of proteins to form cross-disulfide bonds
between RCG and proteins. The intramolecular disul-

ox
, respectively. This indicates that RCG
ox
promotes the formation of the native conformation of
prouroguanylin faster than that of GSSG and that this
occurs via intramolecular disulfide-exchange reactions.
Therefore, we conclude that the native conformation of
proteins can be achieved effectively using RCG
red
⁄ RC-
G
ox
, which accelerates intramolecular disulfide-
exchange reactions.
To determine the kinetic parameters for the folding
reaction using the RCG
red
⁄ RCG
ox
system, CD mea-
surements were carried out at 222 nm. The CD analysis
Relative absorbance at 220 nm
Retention time (min)
N
N
I
I
a
b
0 10 20 30 0 10 20 30 0 10 20 30 0 10 20 30

kinetic parameters, and the results indicated that the
reaction followed single exponential kinetics. Therefore,
the rate constants for the folding of prouroguanylin
were calculated to be K
gsh
= 1.20 · 10
)3
s
)1
and
K
rcg
= 3.69 · 10
)3
s
)1
(calculation using Kaleida
Graph version 3.5) (i.e. the reaction rate in the case of
RCG
red
⁄ RCG
ox
was approximately three times faster
than that of GSH ⁄ GSSG).
Arginine has been employed in folding reactions of
proteins to prevent the formation of nonspecific aggre-
gates [9–12] and to improve folding recovery. On the
other hand, arginine has also been reported to stabilize
the exposed hydrophobic area of single-chain Fv frag-
ments [13] and to increase the folding recovery of pro-

proteins were used in this study, based on electrostatic
effects. The pI values for prouroguanylin and lysozyme
are 5.5 and 11.0, respectively, indicating that prourogu-
anylin and lysozyme exist in solution as a negatively
charged protein and a positively charged protein,
respectively. Therefore, the RCG system accelerates
disulfide-exchange reactions, regardless of the net
charge of the protein being studied.
In addition, the ECR results were similar to that of
GSH. ECR (net charge zero) possesses a negative
charge at the side chain of the glutamic acid residue,
and the arginine residue is positively charged. There-
fore, it is possible that the negative charge of the glu-
tamic acid residue offsets the acceleration effect of the
positive charge of the arginine residue on disulfide-cou-
pled folding.
Arginine has been extensively studied as a protein-
folding reagent because of its ability to suppress aggre-
gation during the folding of proteins [10,12,24,25]. It is
generally thought that arginine provides stability for
the hydrophobic surfaces of folding intermediates. The
findings herein indicate an alternative role of the argi-
nine residue. The arginine residue in the glutathione
analog efficiently improved folding recovery and accel-
erated the disulfide-coupled folding of proteins. There
are only a few examples of reagents for the disulfide-
coupled folding of proteins. The disulfide-exchange
reagent, RCG, and related analogs show promise for
serving as a powerful tool for studies, not only of pro-
tein folding but also for the effective recovery of the

proximity to the cysteine residue of the redox molecule
effectively accelerates disulfide-exchange reactions.
Materials and methods
Materials
Glutathione, lysozyme and endoproteinase Arg-C were pur-
chased from the Peptide Institute, Inc. (Osaka, Japan), Sei-
kagaku Corporation (Tokyo, Japan) and Takara Bio Inc.
(Kyoto, Japan), respectively. RCG and ECR were synthe-
sized by Greiner Bio-One Co., Ltd. (Tokyo, Japan). All
other chemicals and solvents used were of reagent grade.
RP-HPLC
The HPLC apparatus comprised an ELITE system (Hitachi
High-Technologies Corporation, Tokyo, Japan) equipped
with an L-2400 detector and a D-2500 chromato-integrator.
Proteins were separated by RP-HPLC using a Cosmosil
5C
18
-AR-II column (8 · 250 mm; Nacalai Tesque, Inc.,
Kyoto, Japan) or a Develosil UG-5 column (4.6 · 150 mm;
Nomura Chemical Co., Ltd., Aichi, Japan). Folding yields
were estimated by the HPLC peak area at 220 nm.
MALDI-TOF
⁄
MS
The molecular mass values of proteins were determined
using a DALTONICS ultraflex spectrometer (Bruker
Japan Co., Ltd) in the positive ion mode. Mass spectro-
metric analyses of proteins and peptides were carried out
in the linear or reflector modes using 3,5-dimethoxy-4-hy-
droxycinnamic acid (Tokyo Chemical Industry Co., Ltd.,

40 °C. The reaction mixture was dialyzed against 10 mm
HCl, lyophilized and stored at )20 °C until used.
Folding reactions were carried out at several protein con-
centrations (0.1–1.6 mgÆmL
)1
of lysozyme; 0.1–0.4 mgÆmL
)1
of prouroguanylin). The denatured ⁄ reduced proteins were
dissolved in 0.1 m Tris ⁄ HCl (pH 8.0) and allowed to
undergo folding in the presence of 2 mm reductant (GSH,
RCG
red
or ECR
red
) and 1 mm oxidant (GSSG, RCG
ox
or
ECR
ox
) at room temperature for 48 h, as described previ-
ously [21]. All solutions used in the refolding experiments
were flushed with N
2
gas, and the reactions were carried
out in a sealed vial under an atmosphere of N
2
.
The kinetic experiments were performed in the same buf-
fer as described above. The reaction mixture (100-lL aliqu-
ots) was removed at several time-points, quenched with an

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