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Reductive unfolding and oxidative refolding of a Bowman–Birk inhibitor
from horsegram seeds (Dolichos bif lorus): evidence for ‘hyperreactive’
disulf ide bonds and rate-limiting nature of disulf ide isomerization
in folding
R. Rajesh Singh, A.G. Appu Rao*
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570013, India
Received 2 October 2001; received in revised form 20 March 2002; accepted 20 March 2002
Abstract
Horsegram protease inhibitor belongs to the Bowman–Birk class (BBIs) of low molecular weight (8–10 kDa), disulfide-rich, ‘dual’
inhibitors, which can bind and inhibit trypsin and chymotrypsin either independently or simultaneously. They have seven conserved disulfide
bonds. Horsegram BBI exhibits remarkable stability against denaturants like urea, guanidine hydrochloride (GdmCl) and heat, which can be
attributed to these conserved disulfide bonds. On reductive denaturation, horsegram BBI follows the ‘two-state’ mode of unfolding where all
the disulfide bonds are reduced simultaneously resulting in the fully reduced protein without any accumulation of partially reduced
intermediates. Reduction with dithiothreitol (DTT) followed apparent first-order kinetics and the rate constants (kr) indicated that the disulfide
bonds were ‘hyperreactive’ in nature. Oxidative refolding of the fully reduced and denatured inhibitor was possible at very low protein
concentration in the presence of ‘redox’ combination of reduced and oxidized glutathiones. Simultaneous recovery of trypsin and
chymotryptic inhibitory activities indicated the concomitant folding of both the inhibitory subdomains. Folding efficiency decreased in the
absence of the glutathiones and in the presence of denaturants (6 M urea and 4 M GdmCl), indicating the importance of disulfide shuffling
and the formation of noncovalent interactions and secondary structural elements, respectively, for folding efficiency. Folding rate was
significantly improved in the presence of protein disulfide isomerase (PDI). A 3-fold enhancement of rate was observed in the presence of
PDI at molar ratio of 1:20 (PDI/inhibitor), indicating that disulfide bond formation and isomerization to be rate limiting in folding. Peptide
prolyl cis– trans isomerase (PPI) did not affect rate at low concentrations, but at molar ratios of 1:1.5 (PPI/inhibitor), there was 1.4-fold
enhancement of the folding rate, indicating that the prolyl imidic bond isomerizations may be slowing down the folding reaction but were not
rate limiting. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Bowman–Birk inhibitor; Disulfide bond; Reductive unfolding; Oxidative refolding
1. Introduction
The Bowman–Birk type of protease inhibitors (BBIs) are
low molecular weight, disulfide-rich, double-headed inhib-
itors, which can bind and inhibit trypsin and chymotrypsin
either independently or simultaneously. They have seven
conserved disulfide bonds and share a profound sequence
homology (up to 87% among different species) [1]. Struc-
tural elucidations of BBIs from several legume sources have
been carried out by X-ray crystallographic studies [2–6].
Detailed analysis of the secondary and tertiary structures of
soybean BBI by NMR studies have been reported [7,8].
These studies revealed the presence of two closely aligned
symmetrical subdomains with tandem homology resulting
in the so-called ‘bow-tie’ motif. The binding sites to trypsin
and chymotrypsin are located in the external loops of the
bow-tie motif. The seven conserved disulfide bonds main-
tain the tertiary structure (Fig. 1).
0167-4838/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S0167 -4838 (02 )00301 -1
Abbreviations: BBI, Bowman–Birk inhibitor; TIA, trypsin inhibitory
activity; CTIA, chymotrypsin inhibitory activity; DTNB, 5,5Vdithiobis (2-nitrobenzoic acid); BAEE, N-benzoyl-L-arginine ethyl ester; BTEE, N-
benzoyl-L-tyrosine ethyl ester; APNE, N-acetyl phenylanalyl napthyl ester;
TFA, trifluoroacetic acid; PAGE, polyacrylamide gel electrophoresis; RP-
HPLC, reverse phase-high performance liquid chromatography; CD,
circular dichroism; GdmCl, guanidine hydrochloride; PDI, protein disulfide
isomerase; PPI, peptidyl prolyl cis– trans isomerase; t1/2, half life* Corresponding author. Tel.: +91-821-515331; fax: +91-821-517233.
E-mail address: [email protected] (A.G. Appu Rao).
www.bba-direct.com
Biochimica et Biophysica Acta 1597 (2002) 280–291
Secondary structure is dominantly aperiodic with rela-
tively small degree of ordered structure in the form of two
regions of antiparallel h-sheet one in each of the subdo-
mains. The detailed structural analysis of soybean BBI
indicated the presence of structural peculiarities like
exposed hydrophobic patches, buried charged residues
and water molecules [2]. Apart from soybean BBI, the
internal water molecules were found in pea, peanut and
mungbean BBIs, indicating that they are an invariant part
of the tertiary structures of these inhibitors. The most
significant feature of BBIs is the absence of a ‘hydro-
phobic core’, which is the characteristically present in
majority of the globular proteins and a major force in
folding and stability [10]. These structural peculiarities in
BBIs result in a ‘constrained’ conformation held together
to a large extent by covalent ‘locks’ in the form of the
conserved disulfide bonds. In the absence of a ‘hydro-
phobic core’ and elaborate secondary structural elements,
the remarkable stability exhibited by the BBIs is by the
virtue of the conserved disulfide bonds. They are good
models for folding studies specifically to deduce the
efficiency of folding in the absence of the ‘hydrophobic
effect’ and the importance of the disulfide bond formation
and breakage during folding. Preliminary reduction and
refolding studies of soybean BBI have been reported
earlier [11]. Refolding of the mutational variants of soy-
bean BBI has been shown to be possible on trypsin–
sepharose matrix [12]. Mutational analysis of the disulfide
bonds in the trypsin reactive subdomain of recombinant
soybean BBI and the consequences on the activity and
refolding of the two subdomains have been studied [13].
This helped in understanding the cooperative nature of
folding of the subdomains and the importance of the polar
domain interface in the folding of the inhibitor.
Soybean BBI and soybean BBI concentrate (BBIC) have
been implicated as cancer chemopreventive agents and FDA
has awarded BBIC the ‘investigational new drug status’
[14]. Dietary BBI on reaching the blood stream and when
excreted in urine has the same molecular weight and
comparable trypsin inhibitory activity (TIA) and chymo-
trypsin inhibitory activity (CTIA) as native BBI but cannot
be detected by antibodies prepared against the native protein
[15]. This indicates the possibility of it existing in alkylated
or partially reduced active form. In light of such a finding,
we have selected horsegram BBI as model and studied the
reductive unfolding to investigate whether it unfolds by the
‘sequential’ mechanism (with distinct partially reduced
intermediates) or by the ‘two-state’ mechanism (without
any intermediates). Horsegram seeds have multiple isoforms
of these inhibitors. Isolation and purification of these iso-
forms has been reported earlier [16,17]. Preliminary studies
indicated the importance of the disulfide bonds in maintain-
ing the structure and activity [18]. Identification of the
reactive site peptide bonds and the antigenic determinants
of this inhibitor have also been elucidated [19].
An extensive study of the oxidative refolding of horse-
gram BBI under different conditions was undertaken in our
study to elucidate the importance of various factors like
disulfide bond formation, shuffling, presence of residual
secondary structure and the formation of the noncovalent
interactions in deciding the efficiency of refolding. Refold-
ing was studied in the presence of protein disulfide isomer-
ase (PDI), which is known to catalyze the oxidation and
isomerization of disulfide bonds. In addition to the disulfide
bonds, the comparison of the reported amino acid sequences
of BBIs from legumes indicated the presence of a minimum
of four and some as high as seven proline residues [20]. The
cis-prolines in the reactive site loops are conserved in most
of the BBIs from legumes. Refolding was done in the
presence of peptidyl-prolyl cis– trans isomerase (PPI),
which is known to catalyze the cis– trans isomerization of
the prolyl imidic peptide bonds.
Fig. 1. Schematic representation of soybean BBI [9]. Seven conserved disulfide bonds are represented by thick lines connecting the cysteine residues. Each of
the two symmetrical subdomains has an inhibitory loop, which binds to the active site of the respective target protease. ‘Tr’ is the trypsin-binding loop and ‘Ch’
is the chymotrypsin-binding loop. Inhibitor has six proline residues out of which four have trans conformation to their imidic peptide bonds. The two prolines
in the inhibitory loops with cis imidic bonds are conserved in BBIs from different legume sources. Residues Q11–T15 and residues Q21–S25 form a short
region of antiparallel h-sheet in the trypsin inhibitory subdomain with an analogous region of antiparallel h-sheet also present in the chymotrypsin inhibitory
subdomain between residues S38–A42 to residues Q48–V52.
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291 281
2. Materials and methods
2.1. Materials
BBI from horsegram seeds was isolated and purified
according to the reported method [16] avoiding the micro-
wave treatment of the seeds. The major isoinhibitor was used
in the present studies. Purity of the isoinhibitor was ascer-
tained by reductive SDS-polyacrylamide gel electrophoresis
(PAGE), nondenaturing PAGE and reverse phase-high per-
formance liquid chromatography (RP-HPLC). Concentration
of the pure inhibitor was measured by absorbance at 280
nm (E1% 4.0). Sodium borohydride (99%), 5,5Vdithiobis(2-nitrobenzoic acid) (DTNB), DTT, trypsin, a-chymotryp-
sin (three times crystallized), N-benzoyl-L-arginine ethyl
ester (BAEE), N-benzoyl-L-tyrosine ethyl ester (BTEE), 2-
mercapoethanol, guanidine hydrochloride (GdmCl), urea,
oxidized and reduced glutathiones, N-acetyl phenylana-
lylnapthyl ester (APNE) and O-dianisidine (tetrazotized)
were purchased from Sigma Chemical (USA). PDI (E.C.
5.3.4.1) from bovine liver was purchased from Calbiochem
(USA) and cyclophilin (peptidyl-prolyl cis– trans isomerase)
(E.C. 5.2.1.8) from calf thymus was purchased from Sigma.
All the other chemicals and reagents were of analytical
grade.
2.2. Methods
2.2.1. Reduction with sodium borohydride
Reduction was carried out by incubating a fixed concen-
tration of the inhibitor (1.5 mg/ml in 20 mM Tris–HCl
buffer, pH 9.0 with 0.02% EDTA) with different low
concentrations of sodium borohydride (0.025, 0.05, 0.1
and 0.2 M). Buffer solution was degassed and nitrogen
gas was bubbled through it before use. Reduction was
carried under a slow stream of nitrogen gas in screw-capped
glass vials placed in a thermostatically controlled water bath
set at 30 jC. A fresh stock solution of 1 M sodium
borohydride was prepared in the same buffer and appropri-
ate quantities were added to the inhibitor solution to give the
final required concentrations. Octanol (0.01 ml/ml reduction
mixture) was added to control foaming. Aliquots were
removed at various intervals of time and following treatment
with acetone to decompose excess of sodium borohydride,
they were assayed for free thiols with Ellman’s reagent [21].
Reduction with 0.025 and 0.05 M borohydride was also
carried out in the presence of 6 M urea. Stock solution of
urea (10 M) was prepared in the buffer mentioned above and
measured volumes were added to give a final concentration
of 6 M. Conditions of reduction and measurement of free
thiols during reduction were same as mentioned above.
2.2.2. Circular dichroism (CD) analysis of reductive and
nonreductive denaturation
Reductive denaturation of the inhibitor protein was
carried out at different concentrations of sodium borohy-
dride under conditions mentioned above. Reduced inhibitor
was trapped at the end of course of reduction by alkylation
of the resultant cysteines by treatment with 2-fold molar
excess of iodoacetamide. Alkylated protein was dialyzed
against 0.1% ammonium hydroxide solution to prevent
aggregation and the near UV CD spectra were recorded.
To study the changes in the secondary structure after
reduction, the inhibitor at 1 mg/ml was reduced with 5
mM DTT in 0.05 M Tris–HCl buffer, pH 8.0 and aliquots
were tapped at different intervals of time (2, 5, 10, 15, 20,
25 and 30 min) in acidic buffer (0.2 M KCl–HCl buffer pH
2.5) so as to get a final concentration of 0.1 mg/ml and far
UV CD spectra were recorded. To study the nonreductive
denaturation, the inhibitor protein (1 mg/ml in 50 mM Tris–
HCl buffer pH 8.0) was incubated with urea (8 M) and
Gdmcl (6 M) overnight at room temperature (25 jC). NearUV CD spectra were recorded at a protein concentration of
1 mg/ml and far UV CD spectra at 0.1 mg/ml. CD measure-
ments were made with JASCO J20C automatic recording
spectropolarimeter calibrated with D-10-camphorsulfonic
acid. Dry nitrogen gas was purged into the instrument before
and during measurements. Slits were programmed to yield
10 A band width at each wavelength. Near UV and far UV
CD spectra were recorded using quartz cells with 1 and 0.1
cm path lengths, respectively. The mean residue ellipticity
values were calculated using a value of 110 for mean residue
weight.
2.2.3. Measurements of TIA and CTIA
At regular intervals of time, aliquots of the reduction
mixture were removed and trapped in acidic buffer (0.05 M
KCl–HCl buffer, pH 2.0) to immediately arrest the reduc-
tion and thwart the disulfide formation among the free
cysteines. A required amount was incubated with trypsin
or chymotrypsin (present to provide a 2-fold molar excess of
the enzyme to the inhibitor). Trypsin and chymotrypsin
activities were measured spectrophotometrically using
BAEE [22] and BTEE [23] as substrates, respectively. A
comparison of the residual inhibitory activities with respect
to those of the native inhibitor (measured under similar
conditions) was used to calculate the percent remaining
activity of the inhibitor at various stages of reduction.
2.2.4. Nondenaturing PAGE and activity staining
At the end of the course of reduction with each concen-
tration of borohydride, the reduced inhibitor was trapped by
alkylation of the free thiols. One milliliter of the reduction
mixture was treated with sufficient iodoacetate (in 1 ml of a
solution of 0.5 M Tris–HCl buffer pH 8.0) to provide a 2-
fold molar excess over the total possible free thiols. After
allowing the reaction to take place for 30 min in dark,
samples were dialyzed against distilled water, followed by
dialysis against 0.1% ammonium hydroxide, which pre-
vented the precipitation of the alkylated protein. Native
inhibitor, S-carboxymethylated inhibitor samples, and fully
reduced and S-carboxymethylated inhibitor samples were
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291282
analyzed by nonreductive and nondenaturing Laemmli
PAGE [24]. Stacking and separating gels were prepared
with 5% and 20% acrylamide concentrations, respectively.
Twenty micrograms of each sample was loaded. After
electrophoresis, the gel was stained with coomassie blue
(R 250) to visualize the protein bands. For activity staining,
nondenaturing PAGE was carried out with 1.5 Ag of each
sample as mentioned above and the staining to detect the
inhibitory activity was done by the method of Filho and
Moriera [25].
2.2.5. Reductive unfolding with DTT
Reduction was done with a protein concentration of 1
mg/ml in Tris–HCl buffer (0.1 M, pH 8.0) in a water bath
maintained at 23 jC. Reduction was started by adding
required volumes of 50 mM DTT stock to give the final
desirable concentrations of DTT. To monitor the kinetics of
unfolding, aliquots of the sample were taken out at various
time intervals, quenched with equal volumes of 4% tri-
fluoroacetic acid (TFA) and analyzed by RP-HPLC. Sam-
ples were stored at � 20 jC. To test the effect of the ionic
strength on the reduction rate, reduction with 10 mM DTT
was repeated in the presence of different ionic strengths of
KCl. Required aliquots of 2 M KCl stock was added to the
reduction mixture to achieve the required ionic strength.
Conditions were maintained as mentioned above except that
reduction was done in 20 mM Tris–HCl buffer, pH 8.0.
2.2.6. Refolding kinetics
Completely unfolded inhibitor was prepared by incubat-
ing 1 mg of protein in Tris–HCl buffer (2 M, pH 8.0) with 6
M GdmCl and 2 M 2-mercaptoethanol. After incubation at
37 jC for 24 h, the fully unfolded inhibitor was separated
from the reagents by ‘desalting’ through a Sephadex G-15
gel filtration column using KCl–HCl buffer (50 mM, pH
2.0 with 0.05% EDTA) as the elution buffer. All refolding
experiments were carried out at 37 jC with a final protein
concentration of 0.01 mg/ml in ‘disulfide-thiol’ containing
‘refolding buffer (50 mM Tris–HCl, pH 8.0 with 0.01%
EDTA, 0.2 mM oxidized glutathione and 2 mM reduced
glutathione). Refolding was initiated by diluting the
unfolded inhibitor in the refolding buffer to get a final
concentration of 0.01 mg/ml. At regular intervals of time,
aliquots were removed and incubated with 2-fold molar
excess of trypsin over the inhibitor and regain of TIA was
measured. BAEE was used as substrate for trypsin. A
similar measurement of the regain of CTIAwas not possible
as the levels of free thiols created anomalies in the chymo-
trypsin assay. To follow the kinetics of regain of CTIA,
aliquots of the refolding inhibitor were removed at regular
intervals and treated with enough iodoacetamide to provide
a 2-fold molar excess to the free thiols present, which
resulted in alkylation of all the thiols. These samples were
then measured for the CTIA activity. BTEE was used as
substrate for chymotrypsin. Regain of TIA was the param-
eter used to follow the refolding kinetics in all the refolding
experiments. Effect of increased protein concentration on
refolding was studied by conducting refolding at a protein
concentration of 0.025 mg/ml. To study the refolding of the
inhibitor with all the disulfides reduced but with intact
secondary structure, the inhibitor was incubated for 24 h
at 37 jC with 2 M 2-mercaptoethanol without GdmCl to
ensure that the secondary structure was undisturbed. Excess
mercaptoethanol was removed by passing through Sephadex
G-15 column and the inhibitor was refolded in the refolding
buffer. Refolding efficiency in the absence of the redox
mixture of glutathiones was studied by allowing the
unfolded inhibitor to refold in the refolding buffer without
the combination of the reduced and oxidized glutathiones.
Refolding in the presence of high concentrations of the
denaturants like urea and GdmCl was carried out by adding
measured amounts of denaturants to the refolding buffer to
get the required concentrations (6 M urea and 4 M GdmCl).
2.2.7. Enzyme-aided refolding
Refolding of the inhibitor was done in the presence of
two folding catalysts, namely PDI and cyclophilin (peptidyl
prolyl cis– trans isomerase). Refolding was carried out in
the refolding buffer at 37 jC and required quantity of the
enzymes were added to the refolding buffer after which the
fully reduced inhibitor was added to give a final concen-
tration of 0.01 mg/ml. Refolding kinetics was followed by
measuring the recovery of TIA. Refolding was studied with
increasing concentrations of these enzymes. Different molar
ratios (enzyme/inhibitor) with which refolding was carried
out with PDI were 1:60, 1:35 and 1:20 whereas for PPI it
was 1:7 and 1:15. Refolding was also studied in the
presence of both enzymes, PDI (1:20) and PPI (1:15) to
check the synergistic effect of both the enzymes on the
refolding efficiency of the inhibitor.
2.2.8. RP-HPLC analysis of folding intermediates
Fully reduced and denatured inhibitor protein was
allowed to refold in the ‘refolding buffer’ at 37 jC and
protein concentration of 0.01 mg/ml. At regular intervals of
time, aliquots of the refolding inhibitor were removed and
the folding intermediates were ‘trapped’ in acid by adding
concentrated TFA to give a final concentration of 2% acid.
The trapped aliquots were concentrated five times in cen-
tricons (3 K cutoff). Centrifugation was done at 3 jC and
concentrated samples were stored at � 20 jC and analyzed
by RP-HPLC.
3. Results
3.1. CD analysis of the reductive and nonreductive
denaturation
Changes in the near UV CD spectra due to the reduction
are shown in Fig. 2A. The native inhibitor exhibited a
minimum at 283 nm, which is the contribution of both
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291 283
disulfide bonds and tyrosine residue and a maximum at 250
nm, due to the disulfide bonds [26]. Reduction is accom-
panied with a drastic decrease in the overall intensity of the
near UV CD spectra, indicating the loss of the tertiary
structure by the reduction of the disulfide bonds. Far UV
CD analysis of the reduced inhibitor (Fig. 2B) indicates
that even after reduction, the inhibitor retains considerable
amount of secondary structure. Very little disruption of the
tertiary structure of the inhibitor protein was observed on
denaturation with high concentration of denaturants like
urea and GdmCl (Fig. 2C). Far UV CD spectra (Fig. 2D)
indicate a noticeable disturbance of the secondary structure
by 6 M GdmCl whereas it was less drastic with 8 M urea.
There was retention of 90% and 80% of the inhibitory
activities after denaturation with 8 M urea and 6 M GdmCl,
respectively, indicating that the tertiary fold and the
remarkable stability of the horsegram BBI towards dena-
turants could be attributed to seven disulfide bonds in the
protein.
3.2. Reduction of the inhibitor
The inhibitor had seven disulfides with no free cysteine
residues. Reduction with 0.025 and 0.05 M borohydride
resulted in an average of 2 and 4 thiols per mol of protein,
respectively. Reduction with 0.1 and 0.2 M borohydride
resulted in 8–9 mol of thiols and 13–14 thiols per mol of
protein. Reduction with 0.025 and 0.05 M borohydride in
Fig. 2. Effect of reductive and nonreductive denaturation on structure. (A) After reduction with different concentrations of sodium borohydride (as mentioned in
Methods), the reduced inhibitor was trapped by S-carboxamidomethylation and near UV CD spectra were recorded. Native inhibitor ( ), after reduction
with 0.025 M (—), 0.05 M (o) and 0.1 M (. . .) sodium borohydride. Change in the secondary structure on reduction was analyzed by reducing the inhibitor
with 5 mM DTT. Aliquots of the reduced inhibitor were trapped at regular intervals (2, 5, 10, 15, 20, 25 and 30 min) in 0.2 M KCl–HC1 buffer pH 2.5 and far
UV spectra were recorded (B). The spectra of native inhibitor are represented in thick lines whereas the rest of the spectra on reduction after regular intervals
are depicted in thin lines. To study the nonreductive denaturation, inhibitor protein (1 mg/ml in 50 mM Tris–HC1 buffer pH 8.0) was incubated with urea (8 M)
and GdmC1 (6 M) overnight at room temperature (25 jC). Near UV CD spectra (C) were recorded at protein concentration of 1 mg/ml and far UV CD spectra
(D) at 0.1 mg/ml respectively. Native ( ), with 8 M urea (—) and 6 M GdmC1 (. . .).
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291284
the presence of 6 M urea resulted in an average of 5 and 8
mol of thiols per mol of protein as compared to 2 and 4 in
the absence of urea (Fig. 3A). The disruption of the
stabilizing, noncovalent interactions by a denaturant like
urea increased the susceptibility of the disulfides to the
reduction.
3.3. Loss of the inhibitory activities
Simultaneous losses of tryptic and chymotryptic inhib-
itory activities were observed at very early stages of
reduction. There were equal losses of both the activities
during the entire course of reduction. Reduction with 0.025
and 0.05 M borohydride resulted in the loss of 25–30% and
50–55% of both the inhibitory activities, respectively.
Reduction with 0.1 and 0.2 M borohydride resulted in
complete loss of both the inhibitory activities (Fig. 3B).
3.4. Nondenaturing PAGE and activity staining
PAGE under nondenaturing conditions of the S-carbox-
ymethylated inhibitor showed that the reduction product
with low concentrations of borohydride (0.025 and 0.05 M)
consisted of native intact inhibitor and the fully reduced
inhibitor. This was evident by the two protein bands (Fig.
4A, lanes 2 and 3), one corresponding to the position of the
native inhibitor and the other with increased mobility
corresponding to that of the fully reduced carboxymethy-
lated inhibitor. No additional bands were seen between these
two prominent bands, pointing towards a ‘two-state’ mech-
anism of reductive unfolding without any partially reduced
intermediates. The amount of the native inhibitor decreased
with increase in the borohydride concentration. Little or no
Fig. 3. Reduction with sodium borohydride. (A) Inhibitor was reduced with
different concentrations of sodium borohydride as described under
Methods. Reduction was carried at 30 jC in Tris–HC1 buffer (20 mM
pH 9.0 with 0.02% EDTA) at a protein concentration of 1.5 mg/ml. At
regular intervals of time, aliquots were drawn, treated with acetone to
decompose excess borohydride and assayed for thiols with Ellman’s
reagent. When reduction was done in the presence of urea, measured
volumes of buffered urea stock (10 M) was added to the reduction mixture
to get a final concentration of 6 M. Reductions with different concentrations
of borohydride are shown: 0.025 M (.), 0.05 M (E), 0.1 M (z) and 0.2 M
(n). With 6 M urea and 0.025 M (o) and 0.05 M (D) borohydride. (B) To
measure the loss of inhibitory activities during reduction, measured aliquots
of the reduction mixture were removed at regular intervals of time and
trapped in acidic buffer (0.05 M, KCl–HC1 buffer, pH 2.0 with 0.05%
EDTA). Aliquots of the ‘trapped’ inhibitor were incubated with 2-fold
molar excess of trypsin or chymotrypsin and residual inhibitory activities
were determined. BAEE and BTEE were used as substrates for trypsin and
chymotrypsin, respectively. Loss of the antitryptic activity (solid markers)
and antichymotryptic activity (hollow markers) with different concentration
of borohydride is shown.
Fig. 4. Gel electrophoresis. After reduction with each concentration of
borohydride, the reduced inhibitor samples were trapped by treatment with
a 2-fold molar excess of iodoacetate and alkylation of the free thiols. After
allowing to stay in dark for 30 min, excess of iodoacetate was removed by
dialysis against distilled water followed by dialysis against 0.01%
ammonium hydroxide to prevent aggregation of the alkylated protein.
Samples were analyzed by nonreducing, nondenaturing Laemmli PAGE. 20
Ag of protein sample was loaded in each well and stained for protein (A). A
similar gel electrophoresis was done by loading 1.5 Ag of each sample and
stained for activity (B). Lanes 1 to 6 have native inhibitor, S-carboxy-
methylated inhibitor after reduction with 0.025, 0.05, 0.1 and 0.2 M
borohydride and fully reduced and S-carboxymethylated form, respectively.
‘N’ refers to the native and ‘R’ to the fully reduced and S-carboxymethy-
lated form of the inhibitor.
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291 285
native form was seen after reduction with 0.1 and 0.2 M
borohydride (Fig. 4A, lanes 4 and 5), which would explain
the complete losses of TIA and CTIA. The activity staining
of the gels used was very sensitive and capable of detecting
0.1 Ag of inhibitor, which would have detected the presence
of any partially reduced intermediates with intact inhibitory
activities. Translucent bands indicating activities were seen
in the region corresponding to the native inhibitor (Fig. 4B,
lanes 2 and 3) without any such bands corresponding to the
lower protein band of fully reduced protein. No activity
bands were seen in between the native and fully reduced
inhibitor, confirming that there were no intermediates with
inhibitory activity. This indicated that the residual activity
after reduction (70–75% and 50% residual activity after
reduction with 0.025 and 0.05 M borohydride) were due to
the residual unreduced inhibitor and not due to partially
reduced forms with intact inhibitor activities.
Appearance of thiols (Fig. 3A) and the extent of loss of
activities (Fig. 3B) was not proportional, indicating a
possible deviation from the ‘two-state’ mode of unfolding.
Based on the proportionate loss of activities and tertiary
structure (Fig. 2A) along with the native-PAGE (Fig. 4A)
and activity staining experiments (Fig. 4B), we conclude
that unfolding was ‘two-state’.
3.5. Reductive unfolding with DTT
Horsegram BBI was reduced with different concentra-
tions of DTT as the reducing agent. Unfolded protein was
trapped in a time course manner by mixing the aliquots of
sample with an equal volume of aqueous TFA (4%) and
subsequently analyzed by RP-HPLC (see Methods). It was
evident that the reduction is by the ‘two-state’ mechanism
where the native inhibitor (N) was directly converted to the
fully reduced form (R) without any accumulation of parti-
ally reduced intermediates. This phenomenon of ‘two-state’
Fig. 5. Mechanism of unfolding of horsegram BBI. Native protein (1 mg/ml in 0.1 M Tris–HC1 buffer, pH 8.0) was treated with indicated concentrations of
DTT. At different time intervals, the unfolding inhibitor was trapped by adding equal volumes of 4% trifluoroacetic acid and analyzed by RP-HPLC. Solvent A
for HPLC was water containing 0.1% triflouroacetic acid. Solvent B was acetonitrile/water containing 0.1% trifluoroacetic acid (9:1 by volume). Gradient was
25–50% linear in 45 min. Column was Shimpack, C-18 ODS, 15 cm� 4.6 mm (10 Am). Detector wavelength was 230 nm. (A) HPLC analysis of the inhibitor,
which was reduced with 5 mM DTT and trapped at different intervals of time. (B) HPLC analysis of the inhibitor with different concentrations of DTT and
trapped after 10 min of reduction. ‘N’ and ‘R’ refer to the native and fully reduced forms of the protein, respectively. Retention times in minutes have been
indicated near the respective peaks. (C) During the course of reduction with different concentrations of DTT, remaining percentage of the native form at various
interval of time was quantitated by peak integration of the HPLC data. It was replotted and fitted as first-order linear regression using Microcal origin software
version 4.1. First-order rate constants (kr) were calculated from the linear fit data. The observed kr values displayed a linear dependence on the concentrations of
DTT.
Fig. 6. Effect of ionic strength on reduction rate. The inhibitor protein
(1 mg/ml in 0.02 M Tris–HC1 buffer, pH 8.0) was reduced with 10 mM
DTT at different ionic strengths determined by the presence of different
concentrations of KCl. During each experiment, samples were trapped,
analyzed by HPLC and the rate constants determined as mentioned in the
legend of Fig. 5C. Data plotted is an average of two separate estimations.
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291286
reduction was observed with different concentrations of
DTT (2–15 mM) (Fig. 5A,B). On reduction with DTT the
conversion from ‘N’ to ‘R’ followed apparent first-order
reaction kinetics and the rate constants (kr) displayed a linear
dependence on the concentrations of DTT (Fig. 5C). There
was noticeable increase in the rate of reduction when
performed with the presence of KCl at ionic strengths of
0.05 and 0.1. On further increase of the ionic strength, the
rate was relatively less than that observed at 0.05 and 0.1 M.
At high ionic strengths of 0.6 and 0.8, the rate was
comparable to the reduction rate in the absence of KCl
(Fig. 6).
The ‘two-state’ mode of reductive denaturation has been
observed and reported in case of several proteins like tick
anticoagulant protein (TAP), ribonuclease A (RNase A),
hirudin (Hir) and potato carboxypeptidase inhibitor (PCI)
[27]. The stability and reactivity of the disulfide bonds in
these proteins is reflected in the rate constants (kr) for the
conversion from native to fully reduced form. The order of
the disulfide reactivity was found to be TAP>Hir>RNase A.
It was estimated that the native disulfide bonds of TAP and
Hir were 64- and 8-fold more reactive than those of RNase
A, respectively [28]. The first-order rate constant (kr) for the
reduction of horsegram BBI with 10 mM DTT was 0.2
min � 1 whereas the reported kr with 10 mM DTT for TAP
and Hir were 0.025 and 0.005 min� 1, respectively [27]. It
can be deduced that the reactivity of the native disulfide
bonds of horsegram BBI was 8- and 40-fold more than that
of TAP and Hir. By extrapolation, it can be estimated that it
is 500-fold more than the reactivity of disulfide bonds of
RNase A. This indicated that the disulfide bonds of horse-
gram BBI had remarkably high reactivity.
3.6. Refolding kinetics
Refolding kinetics of the completely reduced and dena-
tured inhibitor under various conditions are shown in Fig.
7A. When allowed to refold at optimum conditions (at 0.01
mg/ml, 37 jC in the refolding buffer) folding was complete
Fig. 7. Oxidative refolding. (A) Refolding of the fully unfolded inhibitor
was initiated by diluting the reduced inhibitor to a final concentration of
0.01 mg/ml in the ‘refolding’ buffer, (50 mM Tris–HC1, pH 8.0, 0.01%
EDTA, 0.2 mM oxidized glutathione and 2 mM reduced glutathione).
Refolding was done at 37 jC. At regular intervals, aliquots were removed
and incubated with 2-fold molar excess of trypsin and region of TIA was
measured to assess the extent of refolding. BAEE was used as the substrates
for trypsin. To measure the recovery of CTIA during refolding, aliquots
were removed and treated with 2-fold molar excess of iodoacetamide over
the total free thiols. These samples were assayed for CTIA. BTEE was used
as substrates for chymotrypsin. Refolding was done at a protein
concentration of 0.025 mg/ml to assess the effect of increasing protein
concentration on refolding efficiency. Study of the refolding efficiency in
the absence of redox mixture was done in the refolding buffer, without the
combination of oxidized and reduced glutathiones. Fully reduced inhibitor
with intact secondary structure was prepared by treating the inhibitor with
2-mercaptoethanol (2 M) in the absence of any denaturants. Excess mer-
captoethanol was removed by ‘desalting’ through Sephadex G-15 column
and the inhibitor was allowed to refold. Refolding ability in the presence of
high concentrations of denaturants was studied by adding measured
quantities of denaturants to the refolding buffer to give the final required
concentrations (6 M urea and 4 M GdmC1). Recovery of TIA at inhibitor
concentrations of 0.01 mg/ml (.) and 0.025 mg/ml (n), with undisturbed
secondary structure (E), absence of redox mixture of glutathiones (w ), in
the presence of 6 M urea (y), 4 M GdmC1 (q) and recovery at CTIA at
0.01 mg/ml (o) are shown. The data plotted in each curve is an average of
two different estimations. Error bars are shown for the recovery of TIA and
CTIA under optimum folding conditions. The same amount of deviation
can be expected for other curves. (B) Fully reduced and denatured inhibitor
was allowed to refold at a concentration of 0.01 mg/ml in the ‘refolding
buffer’. At regular intervals of time, aliquots were removed and ‘trapped’
by adding concentrated TFA to get a final concentration of 2%. Samples
were concentrated five times in centricons (3 K molecular weight cutoff).
Centrifugation was done at 3 jC and concentrated samples were analyzed
by HPLC. Solvent for HPLC was water containing 0.1% TFA and solvent B
was acetonitrile/water containing 0.1% TFA (9:1 by volume). Gradient was
10–60% solvent B linear in 45 minutes. Column was Shimpack, C-18
ODS, 15 cm� 4.6 mm (10 Am). Detector wavelength was 230 nm. ‘N’
refers to the native protein and ‘R’ indicates the expected elution time of the
fully reduced protein. Retention times in minutes are indicated near the
peaks.
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291 287
within 4 h. There was an equal recovery of both TIA and
CTIA throughout the course of refolding, indicating that the
inhibitor folded as a ‘unit’ where both the subdomains
folded simultaneously. Refolding kinetics exhibited a multi-
phasic nature with a distinguishable plateau between 90 and
150 min of folding, which was detected in every repetition
of refolding. This was also reported for the folding of
soybean BBI [11]. This indicated the possibility of existence
of multiple folding populations with different refolding
propensities. The population, which started folding with
the right combination of the secondary structural elements
and correct disulfide bonds, refolded early to yield the fully
folded active form. The remaining population that had
folded improperly with incorrect disulfide bonds must break
and reform them with the aid of the combination of the
reduced and oxidized glutathiones, leading to their delayed
folding. RP-HPLC analysis of the acid-trapped folding
intermediates (Fig. 7B) revealed several fractions (labelled
from a– j) of which fraction ‘f’ constituted the major
fraction, which formed around 60% of the total folding
inhibitor. An additional proof for the existence of multiple
folding populations was evident in the RP-HPLC analysis.
Even at very early stages of folding (5–15 min), there was
an appearance of the fully folded form with retention time
corresponding to the native protein, indicating that a portion
of the total folding inhibitor had completed folding whereas
the rest of the inhibitor population was in the process of
folding. This also indicated that the initial recovery of
activity observed during folding (Fig. 7A) was not due to
the partially folded forms with inhibitory activity, but was
due to a portion of the total inhibitor that had high folding
propensity and has completed folding. There was a pro-
gressive appearance of the fully folded form throughout the
course of folding, which indicated the progressive comple-
tion of folding by the different folding populations.
Refolding in the absence of the redox combination of
glutathiones resulted in the recovery of 30–35% of activity.
Folding efficiency was also markedly decreased on increas-
ing the protein concentration. When folding was conducted
at 0.025 mg/ml, the recovery of activity was 50% at the end
of 4 h. An increase of the protein concentration greatly
enhanced the possibility of the formation of the undesirable
intermolecular disulfide bonds, which had to be reduced and
reformed to yield the properly folded form, resulting in the
reduced efficiency of folding. Refolding of the fully reduced
form with undisturbed secondary structure was studied to
check whether the presence of any residual ordered structure
would enhance the possibility of the formation of the correct
disulfide bonds and hence the refolding efficiency. A
recovery of 70% of the activity was observed after 4 h.
The residual structure may affect the freedom of movement
of the polypeptide chain, which is necessary for the efficient
shuffling of the disulfide bonds, thereby affecting the
folding efficiency. Folding was drastically hindered in the
presence of high concentrations of denaturants. There was a
recovery of 40–45% of activity both in the presence of 6 M
urea and 4 M GdmCl after 4 h of folding. High concen-
trations of denaturants hindered folding by disrupting the
formation of the secondary structural elements, which in
turn act as ‘scaffolding’ and helped in bringing the right
cysteine residues in the vicinity of each other and help in the
formation of the correct disulfide bonds. On further incu-
bation of the same samples for 24 h, there was a recovery of
80% of the activity in the presence of 6 M urea whereas
there was no similar recovery beyond 40% in the presence
of 4 M GdmCl. This indicated that urea was a milder
denaturant in the presence of which the forces aiding in
folding are not abrogated.
Fig. 8. Enzyme-aided refolding. (A) Kinetics of recovery of TIA on re-
folding of the inhibitor in the presence of two enzymes, namely PDI and
PPI, is shown here. Refolding was done at 37 jC at a protein concentration
of 0.01 mg/ml in the refolding buffer. The effect of the individual enzymes
on the refolding efficiency was studied with increasing concentrations of
PDI and PPI. Refolding was also studied in the presence of optimum
concentrations of both the enzymes to check the synergistic effect on
refolding. In absence of enzymes (.). Recovery of TIA at different molar
ratios of PDI/unfolded inhibitor, 1:60 (o), 1:35 (D) and 1:20 (E).
Kinetics of recovery of TIA at different molar ratios of PPI/inhibitor 1:7 (w )
and 1:1.5 (y). Kinetics of recovery of TIA with the presence of both PDI
(1:20) and PPI (1:1.5) (n). Inset: The primary data of folding was replotted
and fitted as a first-order linear regression using Microcal Origin software
version 4.1. First-order rate constants and half-life (t1/2) were calculated
from the linear fit data. (B) Folding was conducted in the presence of PDI at
a ratio of 1:20 (PDI/inhibitor). At different indicated time intervals, the
intermediates were trapped, concentrated and analyzed by HPLC as
mentioned in the legend to Fig. 7B.
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291288
When folding was conducted under various nonoptimal
conditions as mentioned above, the yield of the fully folded
active inhibitor after 1 h of folding varied from 25–40%,
which is comparable to the yield after 1 h when folding was
conducted under optimal conditions in the refolding buffer.
This strengthened the hypothesis of existence of multiple
folding populations, out of which 25–40% possess a high
folding propensity and could fold efficiently to yield the
active inhibitor even under nonoptimal conditions.
3.7. Enzyme-catalyzed folding
The folding rate of the fully unfolded inhibitor was
markedly enhanced in the presence of PDI and marginally
in the presence of PPI (cyclophilin) (Fig. 8A). The effect of
each enzyme was checked by performing folding experi-
ments with increasing concentrations of these enzymes.
Folding kinetics was well approximated by the first order
reaction (inset Fig. 8A). The t1/2 of unaided refolding was
5065 s. When PDI was used at a molar ratio of 1:60 (PDI/
unfolded inhibitor), there was no marked enhancement of
folding (t1/2 5453 s). There was a considerable improvement
in the folding rate when increased concentrations of PDI
were used and this was reflected in decrease in the half-life
of folding. At molar ratios 1:35 and 1:20, t1/2 were 3276 and
1768 s, respectively, indicating a 1.5- and 2.9-fold increase
of the folding rates. PPI was effective in improving the
folding rate only when used at high concentrations. When
used at a molar ratio of 1:7 (PPI/inhibitor), there was very
little improvement of the folding rate (t1/2 5392 s) but there
was an improvement when it was almost equimolar ratio
(1:15) (t1/2 3616 s). The presence of optimum concentration
of PDI (1:20) and PPI (1:1.5) during refolding marginally
enhanced the folding rate (t1/2 1468 s) as compared to the
rate when the enzymes were used independently. The 3-fold
enhancement of the folding rate in the presence of PDI
indicated that the disulfide bond isomerization was the rate-
limiting factor, which mainly decided the folding rate of the
inhibitor protein. The cis– trans isomerization of the prolyl
imidic bonds made folding a slow reaction but were not rate
limiting.
4. Discussion
The remarkable stability of BBIs can be attributed to the
array of strategically placed seven disulfide bonds. Horse-
gram BBI exhibits extraordinary stability under extreme pH
(pH 2–12), high temperature (95jC) and in the presence of
denaturants like 6 M GdmCl and 8 M urea. (Fig. 2C). The
seven disulfides are responsible for the maintenance of
the tertiary fold and activity. This is evident by the drastic
changes in the CD spectra (Fig. 2A) and loss of activity
(Fig. 3B) on subjecting to reductive denaturation.
Reductive denaturation of horsegram BBI with different
concentrations of sodium borohydride and DTT and further
analysis by gel electrophoresis (Fig. 4) and RP-HLPC (Fig.
5) proved that it followed the ‘two-state’ mode of reductive
denaturation where the native protein on reduction is con-
verted to the fully reduced form without any accumulation
of partially reduces intermediates. This tendency of ‘two-
state’ mode of denaturation has been reported in many small
disulfide-containing proteins like Hir, PCI and TAP [27].
Several small proteins like epidermal growth factor, bovine
pancreatic trypsin inhibitor and soybean trypsin inhibitor
(kunitz type) follow the ‘sequential’ mode of reductive
denaturation with distinct partially reduced intermediates
[28–30]. The mode of reductive denaturation is decided by
the relative reactivities and accessibilities of the individual
disulfide bonds in the protein molecule. In horsegram BBI,
the two subdomains have tandem homology and symmetry.
There is no presence of elaborate secondary structural
elements. This results in them having equal accessibilities
and susceptibilities for reduction. When treated with the
reducing agent any one of the disulfide bond may be
reduced first, which is the rate-limiting step, followed by
the reduction of the remaining disulfide bonds resulting in
the fully reduced inhibitor protein. The ‘‘two-state’’ reduc-
tion of the horsegram BBI with DTT followed apparent
first-order reaction kinetics. Comparison of the first-order
rate constants for the conversion of the native to fully
reduced form (kr) to similar reported values for the ‘‘two-
state’’ reduction of other disulfide-containing protein indi-
cated that the native disulfide bonds of the horsegram BBI
exhibited remarkably high activities (8-fold, 40- and 500-
fold more reactivity than the native disulfide bonds of TAP,
Hir and RNase A, respectively).
High reactivity of any disulfide bond in a protein maybe
due to two major factors. First is the electrostatic effect
exerted by any positively charged groups located near the
disulfide bond, which increases the effective concentration
of the thiolate anion of the reducing agent, thus enhancing
the apparent reactivity. This was observed in the disulfide
bond of thioredoxin from Escherichia coli where the reac-
tivity was 102–103 times that of normal disulfide owing to
the presence of a positively charged group of lysine residue
nearby [31]. By altering the electrostatic environment, it was
possible to achieve a 106-fold enhancement of the rate
constant of disulfide intercharge reactions in peptides [32].
The second important factor, which induces high reactivity
of disulfide bonds, is the geometry of the disulfide bond
induced by the native tertiary fold of the protein. When the
disulfide bond is constrained and forced to have strained
geometry, it exhibits enhanced reactivity to the reducing
agents. This has been demonstrated in small disulfide con-
taining ring compounds [33] and in short peptides contain-
ing disulfide loops [34]. The torsion energy (Etorsion) of the
disulfide bond is a very good measure of the geometrical
strain imposed on the disulfide bond.
To check the electrostatic contribution for the reactivity
of the disulfide bonds, the first-order reaction of the reduc-
tion was measured with 10 mM DTT at various ionic
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291 289
strengths decided by the presence of increasing concen-
trations of KCI (Fig. 6). The rate constant (kr) was found to
increase at low ionic strengths of 0.05 and 0.1. At ionic
strengths 0.2 and 0.4, the rate constants were comparatively
less and decreased further with increasing ionic strength
(0.6 and 0.8) to reach the normal value 0.2 min� 1, which
was comparable to the rate constant in the absence of KCI.
The initial increase of the rate constant might be due to the
electrostatic screening of the long-range repulsion of the
thiolate anion of DTT and the inhibitor, which had a net
negative charge at pH 8.0. No decrease in the reduction
rate was observed by the presence of KCI, which ruled out
the possibility of the electrostatic effect being responsible
for high reactivity of disulfides. Etorsion values of the
individual disulfide bonds may be an important factor in
deciding the reactivity. However, the lack of high resolu-
tion X-ray structure of the horsegram BBI prevents verify-
ing this.
When the fully unfolded inhibitor was allowed to fold
under optimum conditions, the fully reduced and denatured
inhibitor completes folding within 4 h (Fig. 7A). With a
very high content of free cysteines, the possibility of the
formation of the nonnative intramolecular disulfide bonds is
very high. The presence of the redox combination of the
glutathiones allows the breakage and reformation of the
nonnative disulfide bonds by the ‘disulfide–thiol’ exchange
reactions. A recovery of 40% activity in the absence of
glutathiones indicated that 60% of the total folding inhibitor
is trapped in the wrong conformations owing to the for-
mation of wrong combinations of disulfide bonds. There
was a significant decrease in the yield of the fully folded
active form when folding was performed in the presence of
denaturants. The formation of the noncovalent interactions
resulting in the secondary structural elements enhanced the
possibility of the correct disulfide bonds by bringing the
right combinations of cysteines in the vicinity of each other.
In most of the globular proteins, the ‘hydrophobic effect’
where the nonpolar residues tend to sequester into a ‘core’
to largely avoid the contact of the external water molecules
drives folding. In the absence of the ‘hydrophobic core’, the
formation of other noncovalent interactions and the secon-
dary structural elements may be an important supplementing
force apart from the disulfide formation, which decide the
efficiency of folding of horsegram BBI. It can be inferred
that the formation of the right disulfide bonds and the
secondary structural elements complement each other and
decide the folding efficiency of horsegram BBI. The recov-
ery of 30–40% of inhibitory activity within 4 h of folding in
the presence of denaturants and up to 80% recovery after 24
h of folding in the presence of 8 M urea indicates the
intrinsic stability of the protein to the action of denaturants.
The recovery of 30–40% of the activity when folding was
performed in adverse conditions like absence of gluta-
thiones, presence of denaturants and increased protein con-
centrations indicate that 30–40% of the total inhibitor
refolding has a high folding propensity.
The folding of the two subdomains was cooperative. The
simultaneous recovery of both TIA and CTIA indicate the
cooperative folding of both the subdomains. Mutational
analysis of the disulfide in the tryptic inhibitory domains
of soybean BBI [13] indicated that the mutation of certain
disulfide bonds created local disturbances, which in turn
disrupted the ionic interactions between the subdomains
decreased the folding ability. The drastic decrease of folding
of the horsegram BBI in the presence of denaturants may
also be due to the disruption of the formation of these long-
range ionic interactions between the two subdomains. RP-
HPLC analyses of the ‘acid-trapped’ folding intermediates
of horsegram BBI showed the presence of 10 fractions (a–j)
where fraction ‘f’ comprises the major fraction (Fig. 7B). A
high heterogeneity of the folding intermediates of PCI [35]
and Hir [36], both of which are proteins with three disulfide,
has been reported. The folding intermediates of horsegram
BBI that has seven disulfide bonds will also be exception-
ally heterogeneous.
The folding rate was remarkably enhanced in the pres-
ence of PDI, which catalyzed the oxidation and isomer-
ization of the disulfide bonds in proteins [37–39]. There was
a 3-fold enhancement of rate in the presence of PDI in the
ratio of 1:20 (PDI/inhibitor). The presence of PDI enhanced
the efficiency of folding of the misfolded protein with
nonnative disulfide bonds by catalyzing the breaking of
wrong disulfide bonds and their shuffling to form the native
disulfide bonds, thus significantly increasing the folding
rate. This clearly indicates the shuffling or isomerization is
the rate-limiting factor in the folding of horsegram BBI. The
presence of PPI in high molar concentration (Fig. 8A)
marginally enhanced the folding rate. This shows that the
cis– trans isomerization across the prolyl imidic peptide
bond may be resulting in the slow folding of the horsegram
BBI but is not rate limiting. The catalytic efficiency of PPI
depends on may factors like affinity to the substrates and the
accessibility of the prolyl residues. As the folding of horse-
gram BBI is disulfide-coupled, the formation of the disulfide
bonds induces local rigidity and stability, which restricts the
easy accessibility of the prolyl residues to PPI. Similar
phenomenon was observed in the case of PPI catalyzed
refolding of RNAse T1 where the refolding catalysis was
more efficient when the disulfides were reduced and carbox-
ymethylated, which prevented the formation of disulfides
when allowed to refold and this strongly decreases the
stability of the intermediates. This was possible in the case
of RNAse T1 as the protein could attain the folded form
even when disulfide formation was prevented. A similar
folding of fully reduced and carboxymethylated horsegram
BBI would not be possible as the characteristic tertiary fold
attained and stabilized exclusively by the disulfide bonds
would be prevented. A decrease in the catalytic efficiency of
PPI was also observed when refolding of RNAse T1 was
done in the presence of NaC1, which stabilized the folding
intermediates and hence decreased the accessibility of pro-
lines [40]. A marginal enhancement of rate of folding of
R. Rajesh Singh, A.G. Appu Rao / Biochimica et Biophysica Acta 1597 (2002) 280–291290
horsegram BBI was observed (from 2.9- to 3.4-fold) when
both PDI and PPI were present at optimum concentrations
during folding. These two enzymes have been shown to
enhance the refolding rate of RNAse T1 when they were
present individually and in combination. A 6.5 fold increase
of the folding rate of RNAse T1 was observed when they
were used in combination in comparison to 3-fold enhance-
ment in the presence of PDI and 2.5-fold enhancement of
rate with PPI, respectively [41]. A synergy of the two
enzymes of this proportion was not observed in the case
of horsegram BBI.
In summary, we have, for the first time, conducted a
detailed study of the reductive unfolding and oxidative
refolding of a protease inhibitor belonging to the Bow-
man–Birk class. These protein share unique features like
the lack of a ‘hydrophobic core’ and presence of seven
conserved disulfide bonds. The native disulfide bonds were
found to be ‘hyperreactive’, which may be attributed to their
high torsional energies. In the absence of the ‘hydrophobic
effect’, which is a very important factor in folding of many
globular proteins, the formation of the noncovalent inter-
actions and the secondary structural elements were impor-
tant supplementary factors in maintaining the folding
efficiency. Significant enhancement of the folding rate by
PDI indicated that the rate-limiting factor was the formation
and isomerization of the disulfide bonds during folding.
Marginal improvement of the folding rate by PPI indicated
that the cis– trans isomerization of the prolyl-imidic bond
may be involved in the slow folding of the inhibitor but was
not rate limiting.
Acknowledgements
We thank Dr. V. Prakash, Director, Central Food Tech-
nological Research Institute for keen interest in this work R.
Rajesh Singh is the recipient of Senior Research Fellowship
from Council for Scientific and Industrial Research, New
Delhi, India.
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