Tài liệu Báo cáo khoa học: Bacitracin is not a specific inhibitor of protein disulfide isomerase pptx

Bacitracin is not a specific inhibitor of protein disulfide
isomerase
Anna-Riikka Karala and Lloyd W. Ruddock
Biocenter Oulu and Department of Biochemistry, University of Oulu, Finland
Introduction
Protein disulfide isomerase (PDI) is an endoplasmic
reticulum (ER)-resident protein catalyst that helps
newly translated polypeptide chains to fold and form
native disulfide bonds [1]. PDI can catalyze the oxida-
tion of two cysteines to form a disulfide bond, as well
as the reduction and isomerization of disulfide bonds
in peptides and proteins. PDI has four structural thior-
edoxin-like domains, a, b, b¢, and a¢, a linker region x
between the b¢ and a¢ domains, and a C-terminal acidic
extension. The a and a¢ domains contain the CGHC
active site motif, and are sufficient alone to perform
thiol–disulfide exchange reactions in simple substrates
[2]. The b¢ domain has the principal peptide and non-
native protein-binding site, and is required for isomeri-
zation reactions [2–4], whereas the b domain is of
unknown function.
PDI is one of a family of  20 PDI-like proteins
identified in the ER [1]. These proteins contain one or
more domains that are similar to the domains of PDI,
and many have been shown to catalyze thiol–disulfide
exchange reactions. However, their specific roles,
substrate specificities and mechanisms of cooperation
with other catalysts and chaperones in the cell are not
yet clear.
Besides PDI being abundant in the ER, several stud-
ies have shown non-ER locations for PDI family mem-

bers [5]. PDI inhibitors and specific antibodies have
often been used to discover the function of PDI-like
proteins, especially outside the ER. Bacitracin is a
commonly used inhibitor in these studies, and it is usu-
ally considered to be a specific inhibitor of PDI activ-
ity [6–8]. However, in vitro evidence for the action of
bacitracin as an inhibitor of PDI is scarce, and evi-
dence of its specificity for PDI is nonexistent. Bacitracin
is also used medicinally to prevent infections in small
cuts and burns and to treat gastrointestinal infections.
In addition, it is used as an animal feed additive for
disease prevention and growth promotion in farm
animals. For all of these functions, the effects are
unrelated to PDI inhibition. Commercially available
Keywords
bacitracin; chaperone; protein disulfide
isomerase; protein folding; thiol–disulfide
exchange
Correspondence
L. W. Ruddock, University of Oulu,
Department of Biochemistry, PO Box 3000,
University of Oulu, Oulu 90014, Finland
Fax: +358 8 5531141
Tel: +358 8 5531683
E-mail: lloyd.ruddock@oulu.fi
(Received 12 August 2009, revised 3 March
2010, accepted 19 March 2010)
doi:10.1111/j.1742-4658.2010.07660.x
To successfully dissect molecular pathways in vivo, there is often a need to
use specific inhibitors. Bacitracin is very widely used as an inhibitor of pro-

tein disulfide isomerase (PDI) in vivo. However, the specificity of action of
an inhibitor for a protein-folding catalyst cannot be determined in vivo.
Furthermore, in vitro evidence for the specificity of bacitracin for PDI is
scarce, and the mechanism of inhibition is unknown. Here, we present
in vitro data showing that 1 mm bacitracin has no significant effect on the
ability of PDI to introduce or isomerize disulfide bonds in a folding protein
or on its ability to act as a chaperone. Where bacitracin has an effect on
PDI activity, the effect is relatively minor and appears to be via competition
of substrate binding. Whereas 1 mm bacitracin has minimal effects on PDI,
it has significant effects on both noncatalyzed protein folding and on other
molecular chaperones. These results suggest that the use of bacitracin as a
specific inhibitor of PDI in cellular systems requires urgent re-evaluation.
Abbreviations
BPTI, bovine pancreatic trypsin inhibitor; CM, carboxymethyl; ER, endoplasmic reticulum; PDI, protein disulfide isomerase.
2454 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS
bacitracin contains at least nine different peptides, of
which bacitracin A is the most abundant, and it is
mainly used as an antibiotic against infections caused
by Gram-positive bacteria [9]. The antibiotic effect is
based on the inhibition of bacterial cell wall synthesis
by a variety of mechanisms.
Bacitracin has been used as a specific PDI inhibitor in
a very wide range of studies. These include studying the
mechanisms of virus entry [10–12], the reductive activa-
tion of diphtheria and cholera toxins [7,13], gamete
fusion [14], platelet adhesion [15,16], melanoma cell
death [17], glioma cell invasion [18], the regulation of
transcriptional activity of nuclear factor kappaB [19],
the regulation of NAD(P)H oxidase [20], the shedding
of human thyrotropin receptor ectodomain [21], the

aggregation of Cu ⁄ Zn superoxide dismutase in motor
neurons [22], the operation of the vitamin K cycle [23],
protection against stroke [24] and thrombus formation
[25], and the functions of coagulation factor XIII [26]
and tissue factor [27–29]. Although it is very commonly
used in cell biological studies, the mechanism of PDI
inhibition by bacitracin is unknown. We have recently
speculated that inhibition could arise because of one of
two effects [30]. First, bacitracin could inhibit PDI by
competing with substrate binding, especially by compet-
ing for the substrate-binding site on the b¢ domain. Sec-
ond, PDI activity could be inhibited by the metal ions
that bacitracin is known to bind. These metal ions could
be coordinated by the active site cysteines of the catalytic
domains of PDI, decreasing their activity. In addition,
other thiols present in the studied system could bind
metal ions, and their reactivity could be changed. It has
also been shown that some commercially available baci-
tracin preparations contain proteases, which could also
explain some of the inhibitory effects against PDI [31].
In the present study, we studied the effect of bacitra-
cin on PDI activity in a variety of in vitro assays. Our
results show that 1 mm bacitracin can partially inhibit
the reductive activity of PDI, but it has no significant
influence on other in vitro functions of PDI. However,
bacitracin has effects on other proteins involved in
protein folding and on noncatalyzed systems, with the
effects on these systems being larger than the maximal
effect seen on PDI. Hence, we propose that bacitracin
should not be regarded as a specific inhibitor of PDI.

Results
Bacitracin does not inhibit the catalysis of
disulfide bond formation and isomerization by PDI
PDI is a catalyst of thiol–disulfide exchange reactions,
including oxidation, reduction and isomerization [1].
The simplest in vitro assays for catalysis of thiol–disul-
fide exchange are based on small peptides. To examine
whether bacitracin is able to inhibit the ability of PDI
to introduce disulfide bonds into a substrate in the
absence of the concomitant formation of secondary
structure, a fluorescent decapeptide PDI substrate [32]
was used. In a glutathione buffer at pH 7.0, a time-
dependent decrease in fluorescence was observed that
could be fitted to a first-order process (Fig. 1A), con-
sistent with the formation of a disulfide bond in the
substrate [32]. The rate constant for oxidation of
3.4 lm peptide in the presence of 0.7 lm PDI was
0.85 ± 0.05 min
)1
(n = 6). Bacitracin contains a mix-
ture of peptides, with the most abundant, bacitracin A,
containing an aromatic phenylalanine moiety. Hence,
at 1 mm, there are two opposing effects on the fluores-
cence of the system in the presence of bacitracin. First,
there is a net increase in fluorescence due to the baci-
tracin. However, with excitation at 280 nm and emis-
sion at 350 nm, bacitracin is much less fluorescent on
a per molar basis than the PDI peptide substrate,
which contains a tryptophan. Second, there is a net
decrease in the fluorescence due to the inner filter

effect, whereupon if the sample absorbs strongly at the
excitation and ⁄ or emission wavelength, the fluores-
cence signal decreases. However, this effect was mini-
mized by using a cuvette with an excitation pathlength
of 4 mm. Because of these opposing effects, the fluo-
rescence of the peptide is quenched, and it contributes
a smaller proportion of the total fluorescence of the
system. However, in the presence of 1 mm bacitracin,
the catalyzed formation of a disulfide bond in the
decapeptide PDI substrate can still be observed
through a decrease in its fluorescence, and this could
be fitted to a first-order process (Fig. 1A). The rate
constant for PDI-catalyzed oxidation of the peptide
substrate with 1 mm bacitracin present was
0.73 ± 0.09 min
)1
(n = 6), or 86% ± 11% of that in
the absence of bacitracin. Higher concentrations of
bacitracin could not be used, owing to the two effects
outlined above, but these results suggest that bacitracin
has minimal effects on the catalysis of oxidation by
PDI.
The ability of PDI to introduce and isomerize disul-
fide bonds can be also be analyzed in folding proteins,
e.g. in the bovine pancreatic trypsin inhibitor (BPTI)
refolding assay. BPTI is a widely studied protein con-
taining three disulfides in the native form. In a gluta-
thione-based refolding buffer, BPTI becomes
kinetically trapped in states containing two disulfide
bonds (2S), and in order to reach the native 3S state,

BPTI has to undergo isomerization reactions. Noncat-
alyzed glutathione-based refolding of BPTI is slow,
A R. Karala and L. W. Ruddock Bacitracin is not specific for PDI
FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS 2455
with only around one-quarter of the BPTI being able
to achieve the native 3S state within 2 h [33]. However,
all the steps of BPTI refolding are catalyzed by PDI,
and within 40 min BPTI was refolded to 94% ± 3%
native 3S form (Fig. 1B). When 1 mm bacitracin is
added to the PDI-catalyzed BPTI refolding system, the
MS analysis becomes significantly less accurate, so an
additional step to remove excess bacitracin after
quenching of the reaction but prior to analysis is
required. With this, the refolding of BPTI followed
very similar kinetics in the presence or absence of baci-
tracin, and after 40 min of refolding with 1 mm baci-
tracin present, 90% ± 5% of BPTI was in the native
3S state (Fig. 1C). These results imply that bacitracin
does not inhibit the ability of PDI to introduce or
isomerize disulfide bonds in a folding protein.
Bacitracin inhibits rhodanese aggregation and
the chaperone activity of BiP
In addition to disulfide bond formation, PDI has been
shown to have chaperone-like activity [34]. As rhoda-
nese contains no disulfide bonds, and is prone to aggre-
gation during refolding, it can be used as a model with
which to study chaperone activity in folding. Analysis
of the nonassisted refolding of rhodanese showed the
expected aggregation of the folding intermediates. The
addition of PDI or the noncatalytic PDI family member

ERp27 to the refolding system decreased the aggrega-
tion rate (Table 1), with ERp27 showing a greater effect
(29% decrease in aggregation rate) than PDI (18%
decrease in rate). When 1mm bacitracin was added to
the PDI-catalyzed reaction the rate of aggregation of
rhodanese was significantly reduced. This is unexpected
as inhibition of PDI activity would be expected to
increase the rate of aggregation. However, the rate of
aggregation in the noncatalyzed refolding of rhodanese
was also decreased by bacitracin. The decrease in the
noncatalyzed rate (31%) was similar to that of the
PDI-catalyzed reaction (a 32% decrease). These results
Fig. 1. Bacitracin has minimal effects on the oxidation and isomeri-
zation reactions of PDI. (A) Representative traces showing the fluo-
rescence change associated with oxidation of the PDI substrate
peptide NRCSQGSCWN in a glutathione-based buffer at pH 7.0.
The upper trace shows the PDI-catalyzed reaction, and the lower
trace the PDI-catalyzed reaction in the presence of 1 m
M bacitracin.
The lines of best fit are to first-order reactions. (B, C) Time course
analysis of the oxidative refolding of BPTI. The refolding experi-
ments were performed in a glutathione-based buffer at pH 7.0. The
relative amounts of the folding species were analyzed by ESI-MS.
(B) Representative trace for BPTI refolding in the presence of PDI.
(C) Representative trace for BPTI refolding in the presence of PDI
and 1 m
M bacitracin. For clarity, the glutathionylated intermediates
are not shown separately. The sum of all glutathionylated interme-
diates never represents more than 10% of the total protein at any
time point.

Table 1. Analysis of the aggregation rate during rhodanese refold-
ing at pH 7.2. The rate of aggregation relative to the negative con-
trol in the absence of bacitracin is presented as mean ± standard
deviation (number of samples). Statistical significance between
each pair of samples with and without bacitracin present was
determined using Student’s t-test (two-tailed, two-sample unequal
variance). Note that the effects of bacitracin on PDI and ERp27
inhibition of aggregation are equivalent to those on the noncata-
lyzed reaction.
Sample
No
bacitracin
1m
M
bacitracin
t-test for an effect
of bacitracin
Negative control 100 ± 19 (8) 69 ± 9 (8) P < 0.05
+4.5 l
M PDI 82 ± 12 (6) 56 ± 15 (5) P < 0.05
+4.5 l
M BiP 6 ± 11 (6) 18 ± 6 (5) P < 0.05
+4.5 l
M ERp27 71 ± 11 (4) 54 ± 2 (3) P < 0.05
Bacitracin is not specific for PDI A R. Karala and L. W. Ruddock
2456 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS
imply that bacitracin interacts with rhodanese, decreas-
ing the aggregation of its folding intermediates, and that
it has no observable effect on the chaperone activity of
PDI family members. In parallel studies, the aggrega-

tion rate of rhodanese was reduced, on average, by 95%
by the addition of the ER-resident molecular chaperone
BiP (Table 1). However, the addition of 1 mm bacitra-
cin to the BiP-assisted refolding reaction did not
decrease the aggregation rate, as would have been
expected from the previous results, but increased the
rate of aggregation. This implies that bacitracin signifi-
cantly inhibits the chaperone activity of BiP.
Bacitracin inhibits the reductive activity of PDI by
competing with substrate binding
As well as having oxidase, isomerase and chaperone-like
activity, PDI is also able to catalyze the reduction of
disulfide bonds. The effects of bacitracin on this activity
were examined using the insulin precipitation assay. In
the noncatalyzed assay, the disulfides of insulin are
reduced by dithiothreitol, causing the aggregation and
precipitation of the B-chain of insulin, resulting in an
increase in light scattering that can be monitored by
increased absorbance, e.g. at 540 nm (Fig. 2A). Like
many in vitro PDI assays, the assay is indirect, with
complex kinetics. For this reason, we decided to mea-
sure the lag-phase of the reaction, i.e. the time before an
apparent increase in absorbance of 0.1 was recorded.
The addition of PDI to the assay accelerated the reduc-
tion and precipitation of the B-chain significantly,
decreasing the lag-phase (Fig. 2A). When bacitracin was
included with PDI, the lag-phase of the insulin precipita-
tion increased (Fig. 2A), with the effects increasing with
increasing concentration of bacitracin (Fig. 2B). With
Student’s t-test (two-tailed, two-sample unequal vari-

ance), this effect was found to be significant (P < 0.05),
even with the addition of 0.1 mm bacitracin. Unlike in
the rhodanese assay, in this assay 1 mm bacitracin had
no significant effect on the lag-phase of the reaction or
on the subsequent gradient for aggregation (Fig. 2A),
implying that the effects of bacitracin addition observed
on PDI were due directly to inhibition of PDI-catalyzed
insulin reduction.
Fig. 2. Effects of bacitracin and other compounds on the relative rate of reduction of the B-chain of bovine insulin. Insulin was reduced at
1mgÆmL
)1
in the presence of 10 mM dithiothreitol and 1 mM EDTA at pH 7. When present, PDI, PDI a domain (PDIa), DsbA and DsbC were
used at 1 l
M. Bacitracin (Bac) was used at 1 mM, if not indicated otherwise in the figure. Triton X-100 (TX) was used at 0.05% (v ⁄ v) and 2-
propylphenol (2PP) at 1 m
M. The reduction of the B-chain of insulin causes precipitation that can be followed as an absorbance increase at
540 nm. (A) Representative changes in absorbance as a function of time. From left to right, the traces are: PDI, PDI + bacitracin, noncata-
lyzed reaction, noncatalyzed reaction + bacitracin. (B–D) Lag times for precipitation of insulin under different conditions. The relative activity
is presented as mean ± standard deviation; n = 2–7, with the value given in parentheses. (B) With PDI present. (C) Noncatalyzed reactions.
(D) With PDI a domain, DsbA or DsbC present.
A R. Karala and L. W. Ruddock Bacitracin is not specific for PDI
FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS 2457
To study the potential mechanism of action of baci-
tracin, the insulin reduction assay was also performed
in the presence of other two other thiol–disulfide
exchange enzymes, Escherichia coli DsbA and DsbC,
as well as the isolated catalytic a domain of PDI. Both
the PDI a domain and DsbA have a catalytic site, with
an associated substrate-binding site, but lack an inde-
pendent substrate-binding site, which is present in full-

length PDI [4,35] and DsbC [36]. DsbC substantially
increased the rate of insulin reduction, although to a
lower level than that of PDI, and as with PDI, this
catalysis was inhibited by the presence of bacitracin
(Fig. 2D; P < 0.1). In contrast, DsbA and the PDI
a domain exhibited significantly lower rates of cataly-
sis, and neither showed any change in activity upon
the addition of bacitracin (P > 0.5).
For further study of the potential mechanism of
action, the reaction conditions of the assay were var-
ied. The removal of EDTA from the reaction signifi-
cantly reduced the noncatalyzed rate (Fig. 2C;
P < 0.05) and the catalysis of insulin reduction ⁄ aggre-
gation by PDI (Fig. 2B; P < 0.05), presumably owing
to the presence of trace amounts of metal ions in the
buffer that would be able to coordinate to thiol groups
(for example, see [37] for the effects of zinc on PDI
activity). However, the effects of removal of EDTA
and addition of bacitracin appeared to be independent
of each other (Fig. 2B), implying that any metal ions
bound by bacitracin and taken into the reaction are
not causing the inhibition.
It has previously been shown that PDI binds its sub-
strates via hydrophobic interactions, and that substrate
binding to the noncatalytic b¢ domain can be inhibited
by low molecular mass compounds such as Triton
X-100 and 2-propylphenol [3,38]. To study the effect
of inhibitors of substrate binding on the reductive
activity of PDI, the precipitation of insulin was
followed in the presence of PDI and 0.05% (v ⁄ v)

Triton X-100 or 1 mm 2-propylphenol. In the noncata-
lyzed reaction, the addition of Triton X-100 had a
minimal effect on the system, whereas the addition of
2-propylphenol increased the rate of insulin aggre-
gation (Fig. 2C; P < 0.05). In contrast, the addition
of either Triton X-100 or 2-propylphenol decreased the
rate of PDI-catalyzed insulin aggregation (Fig. 2B;
P < 0.05), showing that inhibition of the primary
substrate-binding site in the b¢ domain decreases the
insulin-reducing activity of PDI.
Discussion
Inhibitors are widely used to study the physiological
functions of proteins in vivo. Bacitracin is a metallo-
peptide antibiotic that has been widely used as a spe-
cific PDI inhibitor [7,10–29]. However, neither the
specificity of bacitracin for PDI nor the detailed mech-
anisms of inhibition of PDI have been investigated.
Furthermore, since the original reporting of the inhibi-
tion of PDI by bacitracin [8], concerns have been
raised about protease contamination of some commer-
cially available bacitracin preparations [31].
Here, we have tested the effect of bacitracin in a
variety of in vitro assays for PDI activity. On the basis
of the BPTI refolding assay and peptide oxidation
assay 1 mm bacitracin does not have a significant
effect on the oxidative or isomerization activity of
PDI. In addition, the chaperone activity of PDI in the
rhodanese-refolding assay was not significantly chan-
ged in the presence of bacitracin. In the insulin reduc-
tion assay, bacitracin was able to decrease the activity

of PDI in a concentration-dependent manner, but this
effect was small, such that, in the presence of 1 mm
bacitracin with 1 lm PDI, there was a decrease in the
contribution of PDI to the lag-phase of the reaction
by 30%. It is unclear why bacitracin had a significant
effect on PDI activity in only one of the four assays
examined. However, three of these assays are indirect
measures of complex multistep kinetic processes. Fur-
thermore, PDI has two active sites with concomitant
substrate-binding ability and an additional substrate-
binding site located in a noncatalytic domain [1], and
the relative contributions of these to each of the assays
is unclear. It should also be noted that the starting
states of the proteins ⁄ peptides are very different, with
insulin starting in the disulfide-linked folded state, and
rhodanese and BPTI starting in the reduced and
unfolded state. Despite this complication, these results
imply that bacitracin, even at high concentrations, is
ineffective at inhibiting the majority of the functions of
PDI usually analyzed in vitro and that are thought to
be representative of its in vivo functions [1].
Although 1 mm bacitracin had only a minor effect
on PDI, it had more significant effects on other com-
ponents in our assays. In the noncatalyzed refolding of
rhodanese, bacitracin alone was able to decrease the
aggregation of the folding intermediates more signifi-
cantly than PDI itself. In addition, bacitracin signifi-
cantly reduced the chaperone activity of BiP in this
assay. Furthermore, in the insulin reduction assay,
bacitracin inhibited the activity of DsbC. These results

strongly imply that bacitracin is not a selective inhibi-
tor of PDI. Instead, bacitracin can also interact with
folding polypeptide chains and other molecular chaper-
ones and folding catalysts. Bacitracin probably inter-
acts with these, and with PDI, via its hydrophobic side
chains, which could interact with exposed hydrophobic
Bacitracin is not specific for PDI A R. Karala and L. W. Ruddock
2458 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS
side chains of a folding protein or with the hydropho-
bic binding site of molecular chaperones such as BiP,
PDI and DsbC.
To further study the mechanism of action of bacitra-
cin, the reduction of insulin was analyzed in the pres-
ence of the PDI a domain and DsbA, which are
capable of catalyzing the oxidation and reduction reac-
tions, but lack the independent substrate-binding site
that is present in PDI and DsbC. Bacitracin had no
effect on the activity of the PDI a domain or DsbA,
implying that the active site is probably not the target
of inhibition by bacitracin. This result was confirmed
by assays in which EDTA was omitted from the assay.
Molecules that are known to interfere with substrate
binding by PDI were also used in the insulin reduction
assay. Triton X-100 at 0.05% (v ⁄ v) (equivalent to
0.8 mm), which is known to affect substrate binding by
the noncatalytic b¢ domain [3], reduced PDI activity
in the assay to a slightly greater extent than 1 mm
bacitracin.
Although pathways for cellular metabolism are
unknown, and it is possible that in vivo processing of

bacitracin may yield a product that can inhibit PDI
activity, there is no published evidence for the in vivo
specificity of bacitracin (or potential products) for PDI
inhibition. Our results clearly show that bacitracin
in vitro is not a specific inhibitor of PDI, but that it
interacts with many other molecules present in the cell,
including nonfolded proteins and other molecular
chaperones, probably via hydrophobic interactions. As
bacitracin, a peptide with a molecular mass of
 1400 Da, is often used at millimolar concentrations
to inhibit PDI in vivo, the risk of nonspecific effects on
other systems increases even more. Furthermore, the
mechanism of action of bacitracin is not very effective
in inhibiting PDI in vitro, even at millimolar concen-
trations, with only a 30% reduction in the insulin
reduction assay and no significant changes in the ability
of PDI to introduce or isomerize disulfide bonds or to
act as a chaperone. Hence, the use of bacitracin as a
specific inhibitor for studying the role and function of
PDI in cellular systems requires urgent re-evaluation.
Experimental procedures
Bacitracin
The bacitracin used in this study was from Sigma-Aldrich
(Steinheim, Germany). Although there have been reports
that there is protease contamination of some commercially
available bacitracin preparations [31], we were loathe to
fractionate the material to ensure that there was no prote-
ase contamination, as bacitracin is a complex mixture of
peptides, and we did not want to lose a potentially inhibi-
tory subpopulation. To confirm that there was no signifi-

cant protease activity in the material that we used, reduced,
denatured BPTI was incubated with 1 mm bacitracin for
1 h at room temperature in 0.1 m sodium phosphate buffer
(pH 7.0) containing 1 mm EDTA. Analysis by SDS ⁄ PAGE
showed no evidence of degradation of the denatured BPTI
over this time period. In addition, ESI-MS analysis of BPTI
refolding (see below) showed no evidence of BPTI degrada-
tion products.
Generation of expression vectors
N-terminally histidine-tagged mature PDI, PDI a domain
and mature BPTI with an additional initiating methionine
expression vector were generated for previous studies
[39,40]. Mature human BiP (Glu19–Leu653) was generated
by PCR from a human liver cDNA library (Clontech,
Mountain View, CA, USA) in two parts. BiP Glu19–
Arg323 was constructed as an NdeI–SacI fragment, and
BiP Ala324–Leu653 as a SacI–XhoI fragment. Mature
human ERp27 (Glu26–Leu273) was generated by PCR
from IMAGE clone 5207225 as an NdeI–BamHI fragment.
Mature E. coli DsbA (Ala20–Leu208) and mature E. coli
DsbC (Asp21–Lys236) were constructed as NdeI–BamHI
fragments by PCR from an E. coli colony. All inserts were
cloned into a modified pET23b (Novagen, Madison, WI,
USA), which codes for an N-terminal hexahistidine tag
before the first amino acid of the protein sequence.
Protein expression and purification
PDI (EC 5.3.4.1; UniProt ID P07237), PDI a domain, BiP
(EC 3.6.4.10; UniProt ID P11021), ERp27 (Uni-
Prot ID Q96DN0), DsbA (UniProt ID P0AEG4) and
DsbC (UniProt ID P0AEG6) were expressed in E. coli

strain BL21(DE3) pLysS grown in LB medium at 37 °C
and induced at a D
600 nm
of 0.3 for 3 h with 1 mm isopro-
pyl thio-b-d-galactoside. Lysis of bacteria was performed
by freeze–thawing the samples twice. PDI, PDI a domain,
BiP and DsbA were purified by immobilized metal affinity
chromatography and anion exchange chromatography, as
previously described for PDIs from Caenorhabditis elegans
[40], except that for PDI a domain and DsbA, the anion
exchange column was run in 20 mm Tris buffer (pH 8.6)
instead of 20 mm sodium phosphate buffer (pH 7.2). DsbC
was purified in the same way as PDIs from C. elegans [40],
except that DsbC was applied to a Resource S (Amersham
Biosciences, Uppsala, Sweden) cation exchanger instead of
a Resource Q anion exchanger, and the column was run in
20 mm citric acid trisodium buffer (pH 5.5). ERp27 was
purified in the same way as PDIs from C. elegans [40],
except that ERp27 was eluted from the anion exchange
column with a tripartite gradient (0–45% over one column
volume, 45–70% over seven column volumes, and 60–100%
A R. Karala and L. W. Ruddock Bacitracin is not specific for PDI
FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS 2459
over two column volumes). BPTI (UniProt ID P00974)
was expressed and purified as described previously [40].
Pure reduced BPTI was lyophilized and resuspended
in 10 mm HCl (pH 2.0) to prevent oxidative refolding. The
concentrations of proteins were determined spectrophoto-
metrically, using a calculated absorption at 280 nm (PDI,
45 040 m

)1
Æcm
)1
, M
r
= 56386; BPTI, 5680 m
)1
Æcm
)1
,
M
r
= 6648; BiP, 29 660 m
)1
Æcm
)1
, M
r
= 71 356; ERp27,
18 450 m
)1
Æcm
)1
, M
r
= 28 837; DsbA, 22 560 m
)1
Æcm
)1
,

M
r
= 22 217; DsbC, 16 960 m
)1
Æcm
)1
, M
r
= 24 545). All
proteins were stored in aliquots at – 20 °C. All purified
proteins were analyzed for authenticity by MS, and all
experimentally determined masses were the same as
the expected masses (within the mass accuracy limit of the
spectrometer).
Peptide oxidase assay
The method of Ruddock et al. [32], using a fluorescent
decapeptide, was used to determine the oxidase activity of
PDI. In brief, disulfide bond formation (oxidation) is moni-
tored by the quenching of the intrinsic fluorescence of the
single tryptophan in the peptide as the arginine is brought
into close proximity upon disulfide formation. McIlvaine
buffer (0.2 m disodium hydrogen phosphate, 0.1 m citric
acid, pH 7.0), to give a final assay volume of 1 mL, was
placed in a fluorescence cuvette. Except where noted in the
text, to this was added 10 lL of oxidized glutathione
(50 mm stock solution in 20 m m sodium phosphate buffer,
pH 7.2), 20 lL of reduced glutathione (100 mm stock solu-
tion in 20 mm sodium phosphate buffer, pH 7.2) and
0.7 lm enzyme. After mixing, the cuvette was placed in a
Perkin-Elmer LS50 spectrophotometer for 5 min to allow

thermal equilibration of the solution; 6.3 lL of substrate
peptide (539 lm stock solution in 30% acetonitrile ⁄ 0.1%
trifluoroacetic acid) was added and mixed, and the change
in fluorescence intensity (excitation at 280 nm, emission at
350 nm, slits of 5 ⁄ 5 nm) was monitored over an appropri-
ate time period (15 min–1 h), with 600–1800 data points
being collected.
Refolding of reduced and denatured BPTI
A modified version of the methods that we have previously
reported [40] was used to analyze BPTI refolding. In partic-
ular, this method has additional steps to remove excess bac-
itracin prior to the MS analysis. The refolding of BPTI was
initiated by the addition of denatured reduced protein to
the refolding buffer (2 mm reduced glutathione, 0.5 mm
oxidized glutathione, 0.1 m sodium phosphate, 1 mm
EDTA, pH 7.0). In the catalyzed refolding, PDI was pre-
equilibrated in the refolding buffer for 5 min before BPTI
was added. BPTI was used at 50 lm and, when present in
the refolding reaction, PDI was used at 7 lm and bacitracin
at 1 mm. The folding reaction was stopped by the addition
of 1.1 m iodoacetamide (Sigma-Aldrich), and BPTI and its
folding intermediates were purified with a PepClean C-18
spin column (Pierce, Rockford, IL, USA) before ESI-MS
analysis (Micromass, Manchester, UK). Bacitracin-contain-
ing BPTI samples were additionally purified by cation
exchange chromatography and with a PepClean C-18 spin
column. The cation exchange resin carboxymethyl (CM)
cellulose 32 (Whatman, Maidstone, UK) was first pretreat-
ed by suspending 3 g of resin in 30 mL of 0.5 m sodium
hydroxide and stirring for 30 min. The cellulose was then

allowed to settle, and washed with double-distilled water.
After being washed with water, the cellulose was washed
with 0.5 m HCl for 30 min, and then with double-distilled
water until neutral pH was achieved. Before use, the cellu-
lose was washed with 10 mm EDTA to remove metal ions.
The eluants from the PepClean C-18 spin column were
diluted nine-fold with equilibration buffer (50 mm Tris buf-
fer, pH 8) before being mixed with pre-equilibrated CM cel-
lulose (250 lL) and stirred for 30 min. The unbound
sample was discarded by centrifugation (1500 g for 1 min).
After washing of the CM cellulose three times with the
equilibration buffer, BPTI and its folding intermediates
were eluted with elution buffer (50 mm Tris, 1 m NaCl,
pH 8). Before the ESI-MS analysis, eluted proteins were
purified with a PepClean C-18 spin column. The kinetic
traces with and without bacitracin were repeated in tripli-
cate. It should be noted that different species may bias their
detection by ESI-MS, and the results are therefore only
semiquantitative.
Inhibition of aggregation of denatured rhodanese
The molecular chaperone-like activity of PDI was moni-
tored using a slightly modified version of the rhodanese
assay used by Song and Wang [34]. Rhodanese from bovine
liver (Sigma-Aldrich) was denatured to a final concentra-
tion of 45 lm in 0.2 m sodium phosphate buffer (pH 7.2)
containing 6 m guanidine hydrochloride and 10 mm dith-
iothreitol for 45 min at room temperature. The refolding
was started by diluting denatured rhodanese to a final con-
centration of 0.9 lm in the refolding buffer (0.1 m sodium
phosphate, pH 7.2, 5 mm dithiothreitol, 50 mm sodium

thiosulfate). The aggregation of rhodanese during refolding
was followed spectrophotometrically at 320 nm over 5 min.
PDI, BiP and ERp27 were used at 4.5 lm, and bacitracin
at 1 mm, when present. ATP was used at 2 mm when BiP
was present. Proteins were equilibrated in the refolding
buffer for 3 min before the addition of rhodanese.
Insulin precipitation assay
A modified version of the insulin turbidity assay reported
by Holmgren [41] was used. The precipitation reaction of
the B-chain of bovine insulin (Sigma-Aldrich) was initiated
by adding the insulin to 0.1 m sodium phosphate buffer
Bacitracin is not specific for PDI A R. Karala and L. W. Ruddock
2460 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS
(pH 7.0) containing 1 mm EDTA and 10 mm dithiothreitol.
PDI, DsbC, PDI a domain and DsbA were used at 1 lm,
and bacitracin and 2-propylphenol at 1 mm and Triton X-
100 at 0.05% (v ⁄ v), if included in the reaction. Insulin was
used at 1 mgÆmL
)1
. Before the insulin addition, protein cat-
alysts and bacitracin were equilibrated in the reaction buf-
fer for 5 min. The precipitation of the B-chain of bovine
insulin was monitored spectrophotometrically at 540 nm.
Acknowledgements
This work was supported by the University of Oulu
and Biocenter Oulu.
References
1 Hatahet F & Ruddock LW (2009) Protein disulfide
isomerase: a critical evaluation of its function in disul-
fide bond formation. Antioxid Redox Signal 11, 2807–

2850.
2 Darby NJ, Penka E & Vincentelli R (1998) The multi-
domain structure of protein disulfide isomerase is essen-
tial for high catalytic efficiency. J Mol Biol 276, 239–
247.
3 Klappa P, Ruddock LW, Darby NJ & Freedman RB
(1998) The b¢ domain provides the principal peptide-
binding site of protein disulfide isomerase but all
domains contribute to binding of misfolded proteins.
EMBO J 17, 927–935.
4 Pirneskoski A, Klappa P, Lobell M, Williamson RA,
Byrne L, Alanen HI, Salo KE, Kivirikko KI, Freedman
RB & Ruddock LW (2004) Molecular characterization
of the principal substrate binding site of the ubiquitous
folding catalyst protein disulfide isomerase. J Biol Chem
279, 10374–10381.
5 Turano C, Coppari S, Altieri F & Ferraro A (2002)
Proteins of the PDI family: unpredicted non-ER loca-
tions and functions. J Cell Physiol 193, 154–163.
6 Essex DW, Chen K & Swiatkowska M (1995) Localiza-
tion of protein disulfide isomerase to the external surface
of the platelet plasma membrane. Blood 86, 2168–2173.
7 Mandel R, Ryser HJ, Ghani F, Wu M & Peak D
(1993) Inhibition of a reductive function of the plasma
membrane by bacitracin and antibodies against protein
disulfide-isomerase. Proc Natl Acad Sci USA 90, 4112–
4116.
8 Roth RA (1981) Bacitracin: an inhibitor of the insulin
degrading activity of glutathione-insulin transhydrogen-
ase. Biochem Biophys Res Commun 98, 431–438.

9 Ming LJ & Epperson JD (2002) Metal binding and
structure–activity relationship of the metalloantibiotic
peptide bacitracin. J Inorg Biochem 91, 46–58.
10 Markovic I, Stantchev TS, Fields KH, Tiffany LJ,
Tomic M, Weiss CD, Broder CC, Strebel K & Clouse
KA (2004) Thiol ⁄ disulfide exchange is a prerequisite for
CXCR4-tropic HIV-1 envelope-mediated T-cell fusion
during viral entry. Blood 103, 1586–1594.
11 Ryser HJ, Levy EM, Mandel R & DiSciullo GJ (1994)
Inhibition of human immunodeficiency virus infection
by agents that interfere with thiol–disulfide interchange
upon virus-receptor interaction. Proc Natl Acad Sci
USA 91, 4559–4563.
12 Jain S, McGinnes LW & Morrison TG (2007)
Thiol ⁄ disulfide exchange is required for membrane
fusion directed by the newcastle disease virus fusion
protein. J Virol 81, 2328–2339.
13 Orlandi PA (1997) Protein-disulfide isomerase-mediated
reduction of the A subunit of cholera toxin in a human
intestinal cell line. J Biol Chem 272, 4591–4599.
14 Ellerman DA, Myles DG & Primakoff P (2006) A role
for sperm surface protein disulfide isomerase activity in
gamete fusion: evidence for the participation of ERp57.
Dev Cell 10, 831–837.
15 Lahav J, Wijnen EM, Hess O, Hamaia SW, Griffiths D,
Makris M, Knight CG, Essex DW & Farndale RW
(2003) Enzymatically catalyzed disulfide exchange is
required for platelet adhesion to collagen via integrin
alpha2beta1. Blood 102, 2085–2092.
16 Essex DW & Li M (1999) Protein disulphide isomerase

mediates platelet aggregation and secretion. Br J
Haematol 104, 448–454.
17 Lovat PE, Corazzari M, Armstrong JL, Martin S,
Pagliarini V, Hill D, Brown AM, Piacentini M,
Birch-Machin MA & Redfern CP (2008) Increasing
melanoma cell death using inhibitors of protein disulfide
isomerases to abrogate survival responses to endoplas-
mic reticulum stress. Cancer Res
68, 5363–5369.
18 Goplen D, Wang J, Enger PO, Tysnes BB, Terzis AJ,
Laerum OD & Bjerkvig R (2006) Protein disulfide isom-
erase expression is related to the invasive properties of
malignant glioma. Cancer Res 66, 9895–9902.
19 Higuchi T, Watanabe Y & Waga I (2004) Protein disul-
fide isomerase suppresses the transcriptional activity of
NF-kappaB. Biochem Biophys Res Commun 318, 46–52.
20 Janiszewski M, Lopes LR, Carmo AO, Pedro MA,
Brandes RP, Santos CX & Laurindo FR (2005) Regula-
tion of NAD(P)H oxidase by associated protein
disulfide isomerase in vascular smooth muscle cells.
J Biol Chem 280, 40813–40819.
21 Couet J, de Bernard S, Loosfelt H, Saunier B, Milgrom
E & Misrahi M (1996) Cell surface protein disulfide-
isomerase is involved in the shedding of human thyrotro-
pin receptor ectodomain. Biochemistry 35, 14800–14805.
22 Atkin JD, Farg MA, Turner BJ, Tomas D, Lysaght JA,
Nunan J, Rembach A, Nagley P, Beart PM, Cheema
SS et al. (2006) Induction of the unfolded protein
response in familial amyotrophic lateral sclerosis
and association of protein-disulfide isomerase with

A R. Karala and L. W. Ruddock Bacitracin is not specific for PDI
FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS 2461
superoxide dismutase 1. J Biol Chem 281, 30152–30165.
23 Wajih N, Hutson SM & Wallin R (2007) Disulfide-
dependent protein folding is linked to operation of the
vitamin K cycle in the endoplasmic reticulum. A protein
disulfide isomerase–VKORC1 redox enzyme complex
appears to be responsible for vitamin K1 2,3-epoxide
reduction. J Biol Chem 282, 2626–2635.
24 Descamps E, Petrault-Laprais M, Maurois P, Pages N,
Bac P, Bordet R & Vamecq J (2009) Experimental
stroke protection induced by 4-hydroxybenzyl alcohol is
cancelled by bacitracin. Neurosci Res 64, 137–142.
25 Cho J, Furie BC, Coughlin SR & Furie B (2008) A crit-
ical role for extracellular protein disulfide isomerase
during thrombus formation in mice. J Clin Invest 118 ,
1123–1131.
26 Lahav J, Karniel E, Bagoly Z, Sheptovitsky V, Dardik
R & Inbal A (2009) Coagulation factor XIII serves as
protein disulfide isomerase. Thromb Haemost 101,
840–844.
27 Ahamed J, Versteeg HH, Kerver M, Chen VM, Mueller
BM, Hogg PJ & Ruf W (2006) Disulfide isomerization
switches tissue factor from coagulation to cell signaling.
Proc Natl Acad Sci USA 103, 13932–13937.
28 Reinhardt C, von Bruhl ML, Manukyan D, Grahl L,
Lorenz M, Altmann B, Dlugai S, Hess S, Konrad I,
Orschiedt L et al. (2008) Protein disulfide isomerase acts
as an injury response signal that enhances fibrin genera-
tion via tissue factor activation. J Clin Invest 118,

1110–1122.
29 Versteeg HH & Ruf W (2007) Tissue factor coagulant
function is enhanced by protein-disulfide isomerase
independent of oxidoreductase activity. J Biol Chem
282, 25416–25424.
30 Hatahet F & Ruddock LW (2009) Modulating proteo-
stasis: peptidomimetic inhibitors and activators of pro-
tein folding. Curr Pharm Des 15, 2488–2507.
31 Rogelj S, Reiter KJ, Kesner L, Li M & Essex D (2000)
Enzyme destruction by a protease contaminant in baci-
tracin. Biochem Biophys Res Commun 273, 829–832.
32 Ruddock LW, Hirst TR & Freedman RB (1996)
pH-dependence of the dithiol-oxidizing activity of
DsbA (a periplasmic protein thiol:disulphide oxidore-
ductase) and protein disulphide-isomerase: studies with
a novel simple peptide substrate. Biochem J 315,
1001–1005.
33 Karala AR, Lappi AK, Saaranen M & Ruddock LW
(2009) Efficient peroxide mediated oxidative refolding
of a protein at physiological pH and implications for
oxidative folding in the endoplasmic reticulum. Antioxid
Redox Signal 11, 963–970.
34 Song JL & Wang CC (1995) Chaperone-like activity of
protein disulfide-isomerase in the refolding of rhoda-
nese. Eur J Biochem 231, 312–316.
35 Nguyen VD, Wallis K, Howard MJ, Haapalainen AM,
Salo KE, Saaranen MJ, Sidhu A, Wierenga RK,
Freedman RB, Ruddock LW et al. (2008) Alternative
conformations of the x region of human protein
disulphide-isomerase modulate exposure of the substrate

binding b¢ domain. J Mol Biol 383, 1144–1155.
36 McCarthy AA, Haebel PW, Torronen A, Rybin V,
Baker EN & Metcalf P (2000) Crystal structure of the
protein disulfide bond isomerase, DsbC, from Escheri-
chia coli. Nat Struct Biol 7, 196–199.
37 Solovyov A & Gilbert HF (2004) Zinc-dependent
dimerization of the folding catalyst, protein disulfide
isomerase. Protein Sci 13, 1902–1907.
38 Klappa P, Freedman RB, Langenbuch M, Lan MS,
Robinson GK & Ruddock LW (2001) The pancreas-
specific protein disulphide-isomerase PDIp interacts
with a hydroxyaryl group in ligands. Biochem J 354,
553–559.
39 Alanen HI, Salo KE, Pekkala M, Siekkinen HM,
Pirneskoski A & Ruddock LW (2003) Defining the
domain boundaries of the human protein disulfide
isomerases. Antioxid Redox Signal 5, 367–374.
40 Karala AR, Psarrakos P, Ruddock LW & Klappa P
(2007) Protein disulfide isomerases from C. elegans are
equally efficient at thiol–disulfide exchange in simple
peptide-based systems but show differences in reactivity
towards protein substrates. Antioxid Redox Signal 9,
1815–1823.
41 Holmgren A (1979) Thioredoxin catalyzes the reduction
of insulin disulfides by dithiothreitol and dihydrolipoa-
mide. J Biol Chem 254 , 9627–9632.
Bacitracin is not specific for PDI A R. Karala and L. W. Ruddock
2462 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS