Tài liệu Báo cáo khoa học: Unfolding of human proinsulin Intermediates and possible role of its C-peptide in folding/unfolding pptx

Unfolding of human proinsulin
Intermediates and possible role of its C-peptide in folding/unfolding
Cheng-Yin Min, Zhi-Song Qiao and You-Min Feng
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
We have investigated the in vitro refolding process of human
proinsulin (HPI) and an artificial mini-C derivative of HPI
(porcine insulin precursor, PIP), and found that they have
significantly different disulfide-formation pathways. HPI
and PIP differ in their amino acid sequences due to the
presence of the C-peptide linker found in HPI, therefore
suggesting that the C-peptide linker may be responsible for
the observed difference in folding behaviour. However, the
manner in which the C-peptide contributes to this difference
is still unknown. We have used both the disulfide scrambling
method and a redox-equilibrium assay to assess the stability
of the disulfide bridges. The results show that disulfide
reshuffling is easier to induce in HPI than in PIP by the
addition of thiol reagent. Thus, the C-peptide may affect the
unique folding pathway of HPI by allowing the disulfide
bonds of HPI to be easily accessible. The detailed processes
of HPI unfolding by reduction of its disulfide bonds and by
disulfide scrambling methods were also investigated. In the
reductive unfolding process no accumulation of intermedi-
ates was detected. In the process of unfolding by disulfide
scrambling, HPI gradually rearranged its disulfide bonds to
form three major isomers G1, G2 and G3. The most abun-
dant isomer, G1, contains the B7-B19 disulfide bridge. Based
on far-UV CD spectra, native gel analysis and cleavage
by endoproteinase V8, the G1 isomer has been shown to
resemble the intermediate P4 found in the refolding process
of HPI. Finally, the major isomer G1 is allowed to refold to

native protein HPI by disulfide rearrangement, which indi-
cates that a similar molecular mechanism may exist for the
unfolding and refolding process of HPI.
Keywords: C-peptide; disulfide scrambling; disulfide stabil-
ity; human proinsulin; unfolding.
The protein folding process can be simply considered as a
process in which a biologically inactive amino acid sequence
becomes a uniquely structured molecule possessing a
specifically biological activity. Conversely the unfolding of
a protein can be considered as the other half of the protein
folding process which causes a protein to lose its biological
activity and become an ensemble of structurally denatured
states [1–3]. The characterization of the protein folding and
unfolding processes has become of great interest. It has been
recognized that protein unfolding is a crucial step in protein
degradation and protein translocation in vivo [4]. In
addition, it has been observed that some unfolded proteins
are capable of retaining some structural elements that may
reflect folding initiation sites or inferred intermediates in the
folding pathway [5].
Disulfide bond-containing proteins provide an advantage
to the study of both protein folding and unfolding process
due to the ability to capture intermediates contains partial
disulfide bonds. Two conventional methods are often used
in the investigation of disulfide bond-containing protein
unfolding. One such method is denaturation, which requires
the use of denaturants to unfold a protein in the absence
of reducing reagent [6,7]. The other method is reductive
unfolding, in which protein is unfolded by the additional
reducing reagents (such as dithiothreitol) in the absence

of denaturants [8–10]. The unfolding pathways of most
proteins have been studied using denaturation, in which the
disulfide bonds remain intact. Due to the cooperative and
interdependent role of the disulfide bonds in maintaining
the native conformation of the majority of proteins, the
reductive unfolding pathway always results in an Ôall-
or-noneÕ mechanism. Therefore, it is very difficult to capture
the disulfide intermediates and to complete additional
investigation of the molecular mechanism of the unfolding
pathways. Currently, the unfolding pathway of a limited
number proteins, such as bovine pancreatic trypsin inhi-
bitor, RNaseA and a-lactoalbumin, have been well charac-
terized by using the reductive unfolding method [11–14].
The recently established disulfide scrambling method of
Chang et al. may make it possible to dissect experimentally
the reductive unfolding of a disulfide-containing protein
into two distinct stages [15]. During the first stage, in
the presence of denaturant and trace thiol catalyst, native
Correspondence to Y M. Feng, Shanghai Institute of Biochemistry,
Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031,
China. Fax: + 86 021 54921011, Tel.: + 86 021 54921133,
E-mail:
Abbreviations: HPI, human proinsulin; PIP, porcine insulin precursor;
IGF-I, insulin-like growth factor-I; TAP, tick anticoagulant peptide;
PCI, potato carboxypeptidase inhibitor; LCI, leech carboxypeptidase
inhibitor; GdnHCl, guanidine hydrochloride; IAA, sodium salt of
idoacetic acid; GSH, reduced glutathione; GSSG, oxidized gluta-
thione; frdHPI, fully reduced/dentured HPI; frHPI, fully reduced HPI;
ESI-MS, electrospray ionization-mass spectrometry.
Note: C Y. Min and Z S. Qiao contributed equally to this work.

(Received 12 November 2003, revised 17 February 2004,
accepted 9 March 2004)
Eur. J. Biochem. 271, 1737–1747 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04079.x
proteins are unfolded by reshuffling their native disulfide
bonds and are thus converted into a mixture of disulfide
isomers. In the subsequent stage, the disulfide bonds of the
scrambled isomers could be readily reduced by a low
concentration of reductive reagents, and intermediates with
heterogeneous disulfide bonds could then be observed
during this process. The unfolding pathway of many
proteins, among them hirudin, tick anticoagulant peptide
(TAP), RNase A, cardiotoxin III, potato carboxypeptidase
inhibitor (PCI) and leech carboxypeptidase inhibitor (LCI)
[15–18], have been studied by this method.
Insulin is a two-chain protein hormone, designated A and
B chain, respectively, containing three disulfide bonds. Two
interchain disulfide bonds are A7Cys–B7Cys, A20Cys–
B19Cys and one intrachain disulfide bond is A6Cys–
A11Cys [19]. The disulfide linkages of insulin have been
shown to be important in maintaining its native conforma-
tion and biological activity [20–25]. The double-chain
insulin is synthesized in vivo as a single-chain precursor
(preproinsulin) and folded as proinsulin, in which a
connecting peptide of 35 residues links the C terminus of
the B chain and N terminus of the A chain. After digestion
by a specific set of protein enzymes in the B-cell granule,
proinsulin is converted into insulin and C-peptide of 31
amino acids [26].
Previous studies completed on the unfolding process of
insulin or proinsulin were often carried out with disulfide

bonds intact [27,28]. By using near- and far-UV CD, Brems
et al. have investigated the guanidine hydrochloride-
induced equilibrium denaturation of insulin and proinsulin
[29,30]. The results of previous work on insulin are
consistent with a two-state denaturation process that lack
any appreciable equilibrium intermediates. The character-
ization of the unfolding of insulin and proinsulin using the
reductive unfolding method has not been thoroughly
investigated. We have characterized the unfolding process
of an artificial porcine insulin precursor (PIP), in which a
dipeptide, AK, links the B and A chain, as shown in
Fig. 1A, in denaturants containing a thiol catalyst. We
observed that PIP reshuffled its native disulfide bonds to
form disulfide isomers, with one major disulfide isomer
present. The disulfide isomers of PIP could spontaneously
refold to native PIP in the presence of a thiol reagent, clearly
demonstrating that PIP has only one thermodynamically
stable form [31]. Recently, the in vitro refolding process of
human proinsulin (HPI) has been investigated in our
laboratory. Four scrambled disulfide isomers with three
intact disulfide bonds have been captured as intermediates
[32]. To compare the disulfide isomers that appeared during
the refolding and unfolding of HPI, we have investigated the
process of unfolding by using disulfide scrambling method
as well as the denaturation method. These results show a
striking correlation between the oxidized refolding and
unfolding of HPI by the disulfide scrambling method.
HPIisthenativein vivo precursor of human insulin in
which the B-chain and A-chain are connected by a flexible
31 residues connecting peptide (C-peptide), as shown in

Fig. 1A. PIP is an artificial mini-proinsulin in which two
amino acids, Ala, Lys, have been substituted for the
C-peptide found in HPI. Thus, the only amino acid
sequences difference between HPI and PIP are within the
connecting peptide region. As the previous studies showed
that the insulin A and B chains contain sufficient folding
information for correct disulfide pairing [33,34], one
may reasonably assume there should not be an obvious
Fig. 1. Amino acid sequences and in vitro refolding pathway of PIP and
HPI. (A) Amino acid sequences of PIP and HPI. Amino acids are
shown in the one-letter code. The numbering of the residues in HPI
and PIP are based on each chain separately. For examples, B19
denotes the nineteenth residue of B-chain and A1 denotes the first
residue in A-chain. Disulfide bonds in the native HPI and PIP are
indicated by dashed lines. For HPI, the corresponding insulin B- and
A-chain are linked by the 31-residue C-peptide and two dibasic resi-
dues, which are shown as dark circles. For PIP, the linker (KAA)
between B29-K and A1-G is indicated by an asterisk. Please note that
the B30 residue in HPI is Thr, while that in PIP is Ala. (B) Putative
disulfide formation pathway of PIP in vitro. Intermediates are named
using the disulfide bonds they contain. Arrows with dashed lines
indicate the folding pathway for the first formation of the intra-A
disulfide bonds. Another major folding pathway is indicated by the
solid arrows, it begins with the A20–B19 disulfide bond formation and
then involves the disulfide rearrangement [35]. (C) Schematic repre-
sentation of the putative disulfide folding pathway of HPI in vitro. I–III
represent the intermediates mixtures with one, two and three disulfide
bonds, respectively. P1–P4 are the HPI disulfide isomers captured
during the oxidized refolding process of HPI in vitro [32].
1738 C Y. Min et al. (Eur. J. Biochem. 271) Ó FEBS 2004

difference between PIP and HPI in the refolding pathway.
However, our studies of the oxidized refolding process of
PIP and HPI in vitro [32,35] have found that these two
proteins adopt two significantly different disulfide forming
pathways as shown in Fig. 1B (PIP) and 1C (HPI). As a
result, we can conclude that the connecting peptide in HPI
partially controls its unique folding behaviour. However,
the manner in which flexible C-peptide contributes to this
folding process is still unknown. Compared with the step-
by-step formation of the disulfide bonds in PIP (Fig. 1B),
disulfide bond formation in HPI occurs by random
formataion of intramolecular disulfide bonds at the begin-
ning of oxidized refolding, and then rearrangement from
non-native to native disulfide bonds. This different folding
behaviour indicates that the energy state of the disulfide
bonds in HPI and PIP may not be similar. During the HPI
unfolding studies here, the disulfide scrambling method and
redox equilibrium assays were used to test this hypothesis.
The results confirm that the disulfide bond stability of
HPI is lower than that of PIP, which indicates that the
C-peptide may control the folding behaviour of HPI by
making the disulfide bonds more accessible.
Experimental procedures
Materials
Recombinant HPI and PIP were of > 98% purity as
confirmed by RP-HPLC on a C8 column. Endoproteinase
Lys-C and V8 were of sequencing grade (Sigma). The
sodium salt of iodoacetic acid (IAA), reduced glutathione
(GSH) and oxidized glutathione (GSSG) were ultra pure
(Amersham Biosciences, Piscataway, NJ). Ultra pure

dithiothreitol was from Sigma. Ultra pure urea and
guanidine-HCl were from Promega. Acetonitrile and tri-
fluoroacetic acid were of HPLC grade. All other reagents
used in the experiment were of analytical grade.
Reductive unfolding of the native protein
in the absence of denaturant
Native HPI was dissolved in buffer containing 100 m
M
Tris
pH 8.7, 1 m
M
EDTA and various concentrations of
dithiothreitol (ranging between 0.5 and 100 m
M
)atafinal
protein concentration of 0.5 mgÆmL
)1
. The reduction
experiments were carried out at 25 °C for 16 h. To trap
the unfolding intermediates, reduction was carried out at
25 °C in the presence of 1 m
M
dithiothreitol. At different
time points during the reaction, 20 lL of the reaction
sample was taken out and mixed with 80 lL0.3%
trifluoroacetic acid to stop the reaction, followed by RP-
HPLC on a C4 column. Fully reduced HPI (frHPI) was
obtained by reducing the native HPI with 100 m
M
dithio-

threitol in the above buffer for 16 h at 25 °C. To confirm the
identity of the reduced protein, frHPI was modified by IAA
and then separated by native PAGE. The native PAGE
showed that there was only one single band, suggesting that
disulfide bonds in HPI were fully reduced. The fully
reduced/denatured HPI (frdHPI) was obtained by reducing
the native HPI with dithiothreitol in the presence of 6.0
M
guanidine hydrochloride (GdnHCl), as described in our
previous work [32].
Unfolding of HPI in the presence of denaturant
and thiol catalyst
The native HPI was dissolved in buffer containing 100 m
M
Tris pH 8.7, 1 m
M
EDTA, 0.2 m
M
2-mercaptoethanol and
different concentrations of GdnHCl at a final protein
concentration of 0.25 mgÆmL
)1
. The unfolding reaction was
carried out at 25 °C for 16 h. For the HPLC analysis, the
reaction was terminated by adding trifluoroacetic acid and
analysed by RP-HPLC on a C4 column. To observe the
time-dependent distribution of the unfolding intermediates
during this process, native HPI was dissolved in the
unfolding buffer (100 m
M

Tris pH 8.7, 1 m
M
EDTA,
0.2 m
M
2-mercaptoethanol, 6.0
M
GdnHCl) at a final
concentration of 0.25 mgÆmL
)1
and the reaction was
quenched by adjusting the pH to 1.0 with trifluoroacetic
acid at different unfolding time point, followed by analysis
on HPLC.
Disulfide stability of the HPI and PIP in redox buffer
HPI or PIP was dissolved in Tris buffer (0.1
M
Tris, 1 m
M
EDTA pH 8.7) containing different redox potentials at
the final concentration of 0.2 mgÆmL
)1
. In the redox
buffer, the ratio (m
M
/m
M
) of GSH to GSSG was 1 : 10,
5 : 5, 10 : 1, 20 : 1, 30 : 1 and 50 : 1, respectively. Simul-
taneously, a sample dissolved in the Tris buffer lacking

both GSH and GSSG was used as a negative control. The
reaction was carried out at 4 °C overnight. After incuba-
tion, one-fifth of the volume of freshly prepared 0.5
M
sodium iodoacetate solution was added to carboxymethy-
late the free thiol groups of proteins. The carboxymehy-
lation reaction was carried out at room temperature for
5 min. The modified mixture was then analysed by native
PAGE.
Isolation and purification of the scrambled disulfide
isomers of HPI
In the presence of denaturant and thiol catalyst as
indicated above, HPI was converted into the mixture of
native and scrambled disulfide isomers, which existed in a
state of equilibrium. The mixture was adjusted to pH 1.0
with trifluoroacetic acid and separated using RP-HPLC
on a C4 column (Sephasil peptide, ST 4.6/250 mm,
Pharmacia). Unless otherwise indicated, the solvent A
was 0.15% trifluoroacetic acid in water and solvent B
was 60% acetonitrile containing 0.125% trifluoroacetic
acid. The linear elution gradient was 50% B to 80% B
in 30 min with a flow rate of 0.5 mLÆmin
)1
. The
detection wavelength was 280 nm. The partially isolated
disulfide isomers of HPI were further purified by HPLC
on a C8 column (Sephasil peptide, ST 4.6/250 mm,
Pharmacia). The corresponding fraction was collected
and lyophilized.
Disulfide linkage analysis of the intermediates

by enzyme digestion
The endoproteinase V8 that cleaves at the C terminus of Glu
residues was used to digest the disulfide isomers of HPI
in order to elucidate their disulfide linkage patterns. Ten
Ó FEBS 2004 Possible role of C-peptide in the folding/unfolding of HPI (Eur. J. Biochem. 271) 1739
micrograms of the isomer was dissolved in 10 lL 100 m
M
NH
4
HCO
3
(pH 8.0) and 0.5 lg endoproteinase V8 was
added. HPI was used as a positive control in each enzyme
digestion. The reaction was carried out at 25 °Cfor16h
and quenched by addition of 90 lL of 0.3% trifluoroacetic
acid. The digestion mixture was then immediately analysed
by RP-HPLC on a C8 column (ZORBAX SB-C8, 5l,
4.6/150 mm; DuPont, San Diego, CA). The elution gradient
was 25% B to 65% B linear in 35 min. The flow rate was
0.5 mLÆmin
)1
and the detection wavelength was 210 nm.
The characteristic peaks on HPLC were manually collected,
lyophilized and their molecular masses measured by ESI
MS.
Refolding of scrambled disulfide isomer G1
To initiate the refolding process, the HPLC purified isomer
G1 of HPI was dissolved in buffer containing 100 m
M
Tris

pH 8.7, 1 m
M
EDTA and 0.2 m
M
2-mercaptoethanol at
final concentration of 0.1 mgÆmL
)1
. The refolding reaction
was carried out at 4 °C. Aliquots of the folding solution
were removed at time intervals and mixed with an equal
volume of 2% trifluoroacetic acid to stop the folding
process. The mixture was then analysed by RP-HPLC on a
C4 column (Sephasil peptide, ST 4.6/250 mm, Pharmacia)
with a linear gradient of 50% B to 80% B in 30 min. The
flow rate was 0.5 mLÆmin
)1
and the detector wavelength
was 230 nm.
Protein analysis
The protein concentration of HPI and the disulfide isomers
were calculated by UV spectroscope using an absorption
constant A
276
(1 cm, 1.0 mg mL
)1
) ¼ 0.65 according to the
reference [36]. The molecular mass of the disulfide isomers
of HPI and the enzyme-digested fragments were measured
by ESI MS. The molecular mass of the mixture of
scrambled isomers was measured by MALDI-TOF MS.

CD studies
CD measurements were performed on a Jasco-700 CD
spectropolarimeter at 25 °C. The protein samples of disul-
fide isomers and HPI were dissolved in 5 m
M
HCl at a
concentration of 0.25 mgÆmL
)1
. Samples were scanned
from 190 nm to 250 nm and accumulated twice at the
resolution of 1.0 nm with the scanning speed of 50 nmÆ
min
)1
. The cell length was 0.1 cm and the stepwise
increment was 0.1 nm.
Results
Reductive unfolding of the native HPI in the
absence of denaturant showed no obviously
accumulated intermediates
Native HPI was reduced by varies the concentration of
dithiothreitol (0.1, 0.5, 1, 5, 10, 50 m
M
)at25°Cfor16h
and then the reaction was stopped by addition of trifluoro-
acetic acid. RP-HPLC was used to measure the amount of
HPI that had been reduced. We found that the lowest
concentration of dithiothreitol capable of completely redu-
cing HPI is 1.0 m
M
. At concentrations less than 1.0 m

M
dithiothreitol, most of the HPI accumulated as disulfide-
linked aggregates with only a small portion being reduced.
The reduction of HPI by 1 m
M
dithiothreitol is shown in
Fig. 2. The native disulfide bonds of HPI were rapidly
reduced in a collective manner. After 20 min,  95% of the
native HPI had been converted into frHPI. At early
points (2 or 5 min) only a small fraction of intermediates
existed between HPI and frHPI as measured by HPLC. At
10 s or 20 min during the reaction, we could not detect any
visible unfolding intermediates by HPLC. Since there are no
obviously accumulated intermediates during this reducing
process, it is very difficult to study the reductive unfolding
pathway by analysis of intermediates.
Fig. 2. Reductive unfolding of HPI by the addition of 1 m
M
dithio-
threitol in alkaline buffer. The reducing reaction was quenched at dif-
ferent time points, as indicated at the right side of each HPLC
chromatograph. The corresponding peaks of native HPI and reduced
(frHPI) are indicted at the top of the peaks. HPLC conditions are
described in Experimental procedures.
1740 C Y. Min et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Native HPI unfolds more readily than PIP using
the disulfide scrambling method
Because it is difficult to study the reductive unfolding of HPI
in the absence of denaturant, we used the disulfide
scrambling method to monitor the HPI unfolding process

by RP-HPLC. In the presence of denaturant and a low
concentration of thiol reagent (0.2 m
M
2-mercaptoethanol),
HPI will unfold by reshuffling the native disulfide bonds,
which leads to the formation of scrambled disulfide isomers.
The scrambled isomers each contain three disulfide bonds,
of which at least two are non-native. The denatured states of
HPI under varying concentrations of urea and GdnHCl are
shown in Fig. 3. With increasing concentration of denatu-
rant, an increasing amount of native HPI becomes conver-
ted into disulfide isomers that accumulate as three major
peaks designated G1, G2 and G3, respectively, on HPLC.
The G1, G2 and G3 were collected, lyophilized and
modified by iodoacetic acid, followed by molecular mass
measurement by MALDI-TOF. The results show that the
isomers all have a molecular weight of 9388, identical with
native HPI. This indicates that there are no free cysteines
in G1, G2 or G3. The scrambled isomers of HPI always
equilibrated with native HPI after 1–2 h of unfolding. The
denaturation curves, calculated from the fraction of native
HPI retained during the unfolding process, are shown in
Fig. 4. As a control, PIP was also unfolded using the same
disulfide scrambling method with that of HPI and the
denaturation curves are also shown in Fig. 4. Comparison
of the denaturation curves of PIP with those of HPI show
that PIP is significantly more stable than HPI, regardless
of whether urea or GdnHCl is used as the denaturant.
The native HPI fraction decreased rapidly even at the
lowest concentration of denaturant, such that  30% of the

native HPI fraction was retained when the concentration
of urea or GdnHCl was 1.0
M
. In contrast, almost 4.0
M
GdnHCl or 8.0
M
urea was needed to reduce the native PIP
fraction to 30%, indicating that PIP is much more able to
maintain its native structure and disulfide bonds than HPI.
Moreover, PIP showed a cooperative unfolding process
with increasing denaturant conditions, while HPI rapidly
lost its native structure even at the lowest concentration of
denaturant.
Fig. 3. Unfolding of HPI in the presence of denaturant and thiol cata-
lyst. Controls for the disulfide scrambling method include the lack of
thiol catalyst and lack of denaturant in the buffer. As both controls
give the same results, only one is shown. Native HPI exists stably in
both control experiments after incubation at 16 °C for 16 h. The three
major peaks containing scrambled disulfide isomers of HPI are desi-
gnated G1, G2 and G3 separately, based on their elution sequence on
HPLC.
Fig. 4. Denaturation curve of HPI and PIP by disulfide scrambling
method. The native fraction retained is the percentage of native HPI
that is not converted into the scrambled isomers. Denaturation was
carried out at 16 °C for 16 h in denaturing buffer containing
0.2 m
M
2-mercaptoethanol and the indicated concentration of
denaturant.

Ó FEBS 2004 Possible role of C-peptide in the folding/unfolding of HPI (Eur. J. Biochem. 271) 1741
Redox-equilibrium assay shows that HPI has a lower
disulfide stability than PIP in redox buffer
To further address differences in the disulfide stability
between HPI and PIP, we used a redox-equilibrium assay,
which has been routinely used to compare the disulfide
stability of different disulfide-containing proteins [20,37–39].
The redox-equilibrium assay involves dissolving the protein
in a redox buffer that contains different ratios of GSH/
GSSG. The disulfide bonds of the proteins remain stable
when the ratio of GSH/GSSG is lower than a fixed redox
potential point, whereas if the GSH/GSSG ratio is above
the redox potential point, an increasing amount of native
disulfide bonds will be reshuffled or reduced with increases
in GSH relative to GSSG (or with an increase in the
reductive potency), until the disulfide bonds reasch an
equilibrium. Thus, proteins with different disulfide stability
will have a different redox potential point. The redox
equilibrium assay results of PIP and HPI are shown in
Fig. 5. For PIP, part of the protein began to form high
molecular mass aggregates when the ratio of GSH/GSSG
was 20 : 1, this indicates that the disulfide bonds are
disrupted by the redox potency used. Whereas for HPI, the
disulfide bonds begin to be disrupted when the ratio of
GSH/GSSG was only 5 : 5. These results show that
disulfide bonds in HPI are more sensitive to changes in
the reduction potential of the redox buffer compared with
that of PIP, hence the disulfide stability of HPI is lower than
that of PIP.
Physical and chemical properties of the HPI disulfide

isomers during the disulfide scrambling process
HPI was unfolded by disulfide scrambling in the presence of
6.0
M
GdnHCl and 0.2 m
M
2-mercaptoethanol. The reac-
tion was quenched in a time-course dependent manner by
removing aliquots of the reaction mixture and adjusting the
pH to 1.0 with trifluoroacetic acid, the samples were then
analysed immediately by HPLC (Fig. 6). There were three
main intermediates during the unfolding process of HPI,
designated G1, G2 and G3. There were no other significant
intermediates observable that resembled the partially struc-
tured isomers, such as P1, P2 or P3, found during the
refolding study of HPI [32].The HPLC peaks corresponding
to G1, G2 and G3 were collected, partially purified and
analysed by native PAGE (Fig. 7A). The native gel shows
that the proteins corresponding to peak G1 are much more
homogeneous than those in peaks corresponding to G2 or
G3. To compare the intermediates found here with the
intermediates found during the refolding studies of HPI, a
mixture of intermediates P3 and P4 captured during the
refolding process were used as a marker. Intermediate P3 is
a scrambled disulfide isomer that contains a disulfide bond
B7-A20 and retains a few secondary structure elements,
while intermediate P4 is an unstructured isomer with a
disulfide bond B7-B19 [32]. G1 is similar to P4 in the
mobility on native PAGE. The G2 and G3 isomers contain
mainly the protein fraction similar to G1 plus some

additional proteins similar to intermediates P3. As none of
the intermediates and isomers contain additional charges
relative to native HPI, their mobility on native PAGE may
indirectly reflect their conformation, such that the more
flexible conformation will result in a slower mobility. The
similar mobility of G1 and P4 indicates that they both
possess a more flexible conformation. Some of the G2 and
G3 fractions migrate slower than G1, suggesting that these
fractions have a more flexible conformation than G1.
The far-UV CD spectra of G1 and G3 are shown in
Fig. 7B, G2 has been omitted due to the high degree of
similarity with G3. Compared with the native HPI and
frdHPI, both G1 and G3 retained little secondary structure.
At the helix-sensitive wavelength of 222 nm, the molar
ellipticity value of G3 is not as negative as that of frdHPI,
indicating a lower helix content of G3 than frdHPI.
The predominant G1 unfolding intermediates were
collected and purified by HPLC. V8 proteinase digestion
was used to characterize the disulfide-linkage pattern of G1
as described previously [32]. Briefly, there are in total seven
Glu residues (V8 cleavage site) in the sequence of HPI,
hence eight fragments, designated F1–F8 from N to C
terminus. Due to the presence of disulfide bonds, the V8-
cleaved native HPI will generate fragments F1 and F7
linked by A7-B7 as well as F2 and F8 linked by A20-B19.
In the disulfide isomers, peptide fragments are linked by
different disulfide bridges, therefore peptide mapping by V8
digestion and HPLC may also be different from that of
native HPI. The peptide mixture of V8-digested G1 and
native HPI were separated by HPLC as shown in Fig. 7C.

Fig. 5. Disulfide stability of PIP (A) and HPI (B) in redox buffer. Lane
1 is the native protein marker. Lanes 2–8 represent that the ratio of
GSHtoGSSG(m
M
/m
M
). When the disulfide reshuffling reached
equilibrium, the reaction was terminated by addition of IAA to car-
boxymethylate the free thiols. The samples were analysed on 15%
native PAGE and the gel was stained by Coomassie brilliant blue
R250.
1742 C Y. Min et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Fig. 7. Physiochemical properties and disulfide-linkage patterns of the
intermediates. (A)NativePAGE(15%acrylamide)ofthedisulfide
isomers G1, G2 and G3. P34 represents the mixture of intermediates
P3 (upper) and P4 (lower) captured during the oxidized refolding
process of HPI. The frdHPI is the IAA modified reduced/denatured
HPI. (B) Far-UV CD spectra of the disulfide isomers of HPI. G2 is not
shown due to the high degree of similarity with G3. The protein
concentration used was 0.25 mgÆmL
)1
for all the samples. (C) Peptide
mapping of HPI and G1 after digestion by endoproteinase V8. HPI
and isomer G1 were digested with the endoproteinase V8, and the
mixtures were analysed by HPLC on a reversed phase C8 column. The
peptide in peak f5 of G1 has a molecular mass of 2347, which shows
that G1 contains an intra-B chain disulfide bond.
Fig. 6. Time-course unfolding of HPI in the denaturing buffer containing
6.0
M

GdnHCl and 0.2 m
M
2-mercaptoethanol. The unfolding reaction
was quenched at different time points, as indicated to the right of each
HPLC chromatograph, by adjusting the pH to 1.0 with trifluoroacetic
acid; samples were then analysed by HPLC using the conditions des-
cribed in Experimental procedures. The disulfide scrambling process of
HPI under these conditions always reaches equilibrium after a reaction
time of 2 h.
Ó FEBS 2004 Possible role of C-peptide in the folding/unfolding of HPI (Eur. J. Biochem. 271) 1743
The fractions from each peak were collected and the
peptides were identified by ESI-MS. Compared with native
HPI, a remarkable peak designated f5 could be observed in
the digestion mixture of G1. The molecular weight of the
fragment in peak f5 was 2347.0, suggesting that the peptide
in f5 corresponded to the fragments F1 and F2 linked
by disulfide B7-B19. The profile of the enzyme digestion
pattern of the G1 intermediates is almost identical with the
intermediate P4 captured during the HPI refolding process
[32], which indicates that G1 may have the same disulfide
linkage as the P4 intermediate.
Reverse refolding of disulfide isomer G1 to native HPI
Given that the G1 isomer in the unfolding process may be
the same intermediate as the P4 refolding intermediate, we
questioned if the reverse refolding of the G1 isomer occurs
by the same process as that of P4. To initiate the refolding of
the scrambled G1 disulfide isomer, a low concentration of
2-mercaptoethanol was used as the thiol catalyst. In the
presence of 0.25 m
M

2-mercaptoethanol in alkaline buffer,
G1 was able to spontaneously reshuffle its non-native
disulfide bonds until native configuration of HPI was
adopted, as shown in Fig. 8. During the refolding process of
G1, only a few accumulated intermediates were observed by
HPLC, among which one major peak corresponded, in
elution time, to the refolding intermediates P2 of HPI. This
indicates that possession of the A20-B19 disulfide bond in
P2 is an important intermediate step during the disulfide
reshuffling of G1 into native HPI. Taken together, the
refolding process of the G1 isomer in Fig. 8 is very similar to
that of P4 as reported previously, further indicating that G1
and P4 are the same intermediates during the unfolding and
refolding process of HPI, respectively. The same inter-
mediate captured in the unfolding and refolding process
suggests that a correlation exists in the molecular mechan-
ism of the unfolding or refolding of HPI.
Discussion
Although the insulin A- and B-chains contain sufficient
structural information for the correct pairing of the disulfide
bonds [33,34], our refolding studies of HPI have shown that
both the A and B chain as well as the C-peptide contain the
information necessary for proper protein folding. Com-
pared with the cooperative, step-by-step formation of
disulfide bonds and native conformation in the folding
pathway of a mini-proinsulin (PIP) which lacks the
C-peptide [35], HPI rapidly adopts a random formation of
all the intramolecular disulfide bonds during an early stage
of the oxidized folding process. As a result of the observed
differences in the molecular folding process of PIP and HPI,

as shown in Fig. 1B and C, we can conclude that the main
function of the C-peptide in the folding process is to provide
the necessary flexibility for the formation of intramolecular
disulfide bonds. However, the manner in which the
C-peptide is able to provide this flexibility is unknown.
Although the three-dimensional structure of HPI and PIP
has not been completed, many physicochemical data
support that their core structure is similar to that of insulin
[40–42], therefore the presence of the C-peptide should not
influence dramatically the conformation of the A- and
Fig. 8. Refolding of scrambled disulfide isomer G1 to native HPI. The
isomer G1 was reconstituted in alkaline buffer containing trace
amounts of 2-mercaptoethanol to initiate refolding. The refolding
reaction was quenched at different time points and analysed on HPLC
by using the conditions described in the Experimental procedures. The
intermediates formed during the refolding reaction were identified
based on similar elution time with the purified refolding intermediates
such as P1, P2, P3 and P4.
1744 C Y. Min et al. (Eur. J. Biochem. 271) Ó FEBS 2004
B-chains. During this unfolding study, we compared the
disulfide stability of HPI with PIP using disulfide scrambling
methods and the redox-equilibrium assay. Our results show
that the disulfide bonds in HPI are more easily disrupted by
the addition of thiol reagents than those of PIP, indicating
that the C-peptide of HPI reduces the stability of the
disulfide bonds more than that of PIP. The reduced disulfide
bond stability of HPI may explain why HPI can randomly
form all of the intramolecular disulfide bonds at the
beginning of the refolding process. We can therefore deduce
that the C-peptide affects the HPI refolding process by

influencing the stability of its disulfide bonds. Due to the
absence of structural information for the C-peptide, we are
not able to determine how the C-peptide interacts with the
insulin A- or B-chains to make the disulfide bonds more
accessible than that PIP. It’s possible that the longer linker
between the B- and A-chains may make the C terminus of
the B-chain more flexible.
There are examples that conformational stability of a
protein can be modulated by changing the lengths of loop or
linker segments. For example, a four-helix bundle protein
Rop has been shown to have inverse correlation between
loop length and stability [43]. The effects of the linkers have
generally been attributed to the increased entropic penalty
associated with fixing the end positions of longer linkers.
Considering the passive role of the linker in proteins like
Rop, we may question whether the role of C-peptide in HPI
refolding is also passive and simply a flexible longer linker.
However, there are at least three examples that have shown
that the 31-amino acid C-peptide does not act as a simple
linker. First, replacing the native C-peptide of HPI with
different short linkers always resulted in lower expression
level and higher disulfide isomers formation in the mam-
malian cells [44], thus the native C-peptide of HPI is
important for its refolding in vivo. Secondly, either alanine
scanning mutagenesis or deletion of three highly conserved
acidic residues (EAED) at the N terminus of the C-peptide
resulted in severe HPI aggregation during refolding [45].
This suggests that the amino acid composition of the
C-peptide is also an important factor for its function.
Finally, the in vitro refolding yield of HPI could easily be

optimized, whereas it is difficult to efficiently refold PIP
under the same conditions [32]. In summary, we may deduce
that the C-peptide of HPI contains important folding
information necessary for the correct pairing of disulfide
bonds.
Our work shows that HPI can be denatured and reshuffle
its disulfide bonds to form a series of disulfide isomers in
the presence of denaturant and a trace thiol catalyst, with
isomer G1 being the most abundant isomers identified. The
CD spectrum and native PAGE of G1 showed that it
retained little secondary structure and adopts a flexible
conformation, as observed for frdHPI. Together with the
result that more than 95% of native HPI can be converted
into the isomer G1 in the presence of strong denaturant
(6.0
M
GdnHCl) and thiol reagent, we may suggest that the
G1 is the predominant fraction of unfolded HPI with three
disulfide bonds. The disulfide linkage analysis of G1 shows
that it contains the intra-B chain disulfide bond, B7-B19,
and two intra-A chain disulfide bonds. Due to the absence
of peptide sequencing analysis, we were not able to
determine the disulfide linkage pattern in the intra-A chain.
Insulin-like growth factor-I (IGF-I), which is homologous
to HPI, has been investigated by using disulfide scrambling
with the similar condition used for disulfide scrambling.
Three major disulfide isomers of IGF-I, namely IGF-a,
IGF-b1 and GF-b2, respectively, were identified and their
disulfide linkage patterns were analysed by Edman sequen-
cing and peptide mapping [46]. Comparison of the results of

IGF-I with that of HPI, considering the high primary
sequence homology and 3D structure of the insulin
superfamily, we can deduce that the disulfide linkage
pattern of G1 corresponds to the predominant disulfide
isomer IGF-b1 or IGF-b2, which should be [B7-B19,
A6-A11, A7-A20] or [B7-B19, A6-A20, A7-A11]. During
the disulfide scrambling process of proteins, such as PCI
[16], TAP [47], and hirudin [48], the predominant isomer
always contains the disulfide linkage pattern in which the
nearest cysteines in primary sequence pair and form the
beads-form disulfide bonds. Although the isomer IGF-a,
with a Cys47-Cys48 disulfide bond, is adopted to the pattern
of consecutive disulfide linkage, it may be absent from the
folding pathway of fully reduced IGF due to its poor
solubility [49]. There may not be IGFa-like isomers during
the unfolding of HPI because all the isomers are highly
soluble. Maybe this is one of the reasons why IGF-I has a
swap form while HPI/insulin has not.
We have studied the oxidized refolding pathway of HPI
and captured four disulfide isomers as intermediates. P4 was
identified as the most unstructured intermediate and
contains the disulfide bond B7-B19 [32]. In this study, we
found that HPI was converted mainly into a disulfide
isomer G1 during its unfolding in the presence of denaturant
and a trace thiol reagent. The native electrophoresis, CD
spectrum, disulfide linkage analysis and the HPLC beha-
viour of G1 strongly suggest that it is identical to P4. The
identical intermediate captured during the oxidized refold-
ing and disulfide-scrambling unfolding suggests that the
pathway of unfolding and refolding of HPI might be similar

but in the reverse direction, which is consistent with the
underlying mechanism of protein folding proposed by
Chang [46]. This also indicates that disulfide-scrambling
unfolding may be used as a reversible step to investigate the
folding pathway of proteins.
A key question in protein folding is how the folding
initiates from a random-coiled peptide chain [50]. In order
to solve this question, it is necessary to determine the 3D
structure of the unfolded peptide. However, since the
reduced/denatured protein adopts numerous conforma-
tional isomers, it is very difficult to complete a 3D structure
analysis. The isomer G1 obtained here may provide a
proper reduced/denatured HPI state for 3D structure
analysis by NMR. The reasons are as follows: (a) G1 is a
major scrambled isomer with three stable intradisulfide
bonds; (b) CD spectra show that G1 retains little secondary
structure; (c) G1 can reshuffle its disulfide bonds until
adopting the native conformation, which indicates that G1
may be function as the early intermediate in the oxidized
refolding of HPI; (d) the 3D structure of insulin and several
of its analogues have been well studied, which will provide a
comparison for the G1 3D structure. The elucidation of G1
structure will help us to partially understand which amino
acids in the random-coiled peptide have the potential to
participate in the formation of folding initiation sites in
Ó FEBS 2004 Possible role of C-peptide in the folding/unfolding of HPI (Eur. J. Biochem. 271) 1745
HPI, and to further learn the molecular mechanism of the
initiation of HPI folding.
Acknowledgements
We thank Profs M. A. Weiss and Q X. Hua for providing human

proinsulin and helpful discussion. We are grateful to K. Brazine at the
Dana-Farber Cancer Institute for critical reading of this manuscript.
This work was supported by the grants from the National
Foundation of Natural Science (No.39670179) and Chinese Academy
of Sciences (KJ951-B1-606).
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