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Process Biochemistry 46 (2011) 458464

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Comparison of activity and conformational changes of cin during denaturation


by urea and guanidine hydrochloride
K.B. Devaraj, Parigi Ramesh Kumar, V. Prakash
Department of Protein Chemistry and Technology, Central Food Technological Research Institute (A Constituent Laboratory of CSIR), Mysore 570020, India

a r t i c l e

i n f o

Article history:
Received 17 July 2010
Received in revised form 6 September 2010
Accepted 21 September 2010
Keywords:
Cysteine protease
Ficin
Guanidine hydrochloride
Protein denaturation
Stability
Urea
Unfolding

a b s t r a c t
The activity and conformational changes of cin (EC 3.4.22.3), a cysteine protease from Ficus carica
have been investigated during the denaturation by urea and guanidine hydrochloride (GuHCl). The
denaturation of cin was followed by activity measurements, uorescence and circular dichroism (CD)
spectroscopic studies. The enzyme activity decreased signicantly at low concentration of both urea
and GuHCl before unfolding of the enzyme molecule. The enzyme molecule was resistant for unfolding by urea under neutral conditions even at higher concentrations. However, the protein is susceptible
to unfolding by urea at lower pH and transition follows a cooperative two-state rule with increasing
concentration of urea. On the other hand, cin molecule loses its complete structure in presence of 4 M
GuHCl under neutral conditions. The GuHCl-induced unfolding occurs in a simple two-state cooperative
process. These results indicate the differential structural stability and fragility of active site of the enzyme
towards denaturation by urea and GuHCl.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
The stability of proteins in solutions is a major concern of biologists and pharmacologists. Although many mechanisms are in
vogue, the protein folding from a denatured state to a biologically
active form is not very well understood. The denatured state of
proteins is equally important as much as the native state in determining the stability and folding pathway of proteins. Therefore
more in depth knowledge of protein denaturation and unfolding/refolding process are central to understanding the protein
stability. Further, studying of the denaturation process of proteins
provides a basis for the protein designing and helps to establish
a molecular description of protein folding. It is well known that
urea and GuHCl are the most frequently used classical denaturants
to study the protein stability and folding pathways. In comparison
to either acid or thermal unfolding, chemical agents such as urea
and GuHCl are more effective in disturbing the non-covalent interactions. The extent of unfolding is generally greater than that of
any other means of denaturation [13]. Despite their widespread
use, the mode of action of these agents on protein conformation is not clearly known. They may exert their effect directly,

Abbreviations: ANS, 8-anilino-1-naphthalene-sulfonic acid; CD, circular dichrosism; GuHCl, guanidine hydrochloride; Fu , fraction unfolded; Ksv , SternVolmer
constant.
Corresponding author. Tel.: +91 821 2517760; fax: +91 821 2516308.
E-mail address: prakash@cftri.com (V. Prakash).
1359-5113/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.09.016

by binding to the protein molecule, or indirectly, by altering the


solvent environment [4,5]. It has been shown in several studies that the inactivation of many enzymes occurs before protein
molecule undergoes signicant conformational changes which can
be detected during the process of denaturation by urea, GuHCl and
temperature [68].
Ficin (EC 3.4.22.3) is one of the commercially important plant
cysteine protease isolated from the latex of Ficus species. The
available information indicates that cin apparently shares many
common properties with papain with respect to substrate specicity, esterase activity, transpeptidase reactions, and activation by
reducing agents [9]. Very few references are available on the structural aspects of cin till date as compared to papain [10,11] as well
as other related cysteine proteases [1215]. In our initial studies,
we have reported the purication and the properties of cin from
F. carica. It is a single polypeptide chain with a molecular mass
of 23.1 kDa. The enzyme was active at neutral pH and complete
inactivation of the enzyme occurs below pH 3.0 [16]. The characterized enzymatic properties of this cin shared many similarities
with many other plant cysteine proteases. The results of our earlier studies demonstrated that the pH-induced denaturation of cin
leads to a partially unfolded state at acidic pH. The partial unfolded
structure of cin at low pH showed the characteristics of molten
globule like intermediate state as studied by various biophysical
techniques [17]. In the present study we primarily focus on the
effect of chemical denaturants such as urea and GuHCl on the structure and stability aspects of cin. These studies help to understand
the mechanism of denaturation and stability of cin.

K.B. Devaraj et al. / Process Biochemistry 46 (2011) 458464

459

2. Materials and methods

2.6. Fluorescence quenching experiments

2.1. Materials

Acrylamide quenching studies of the intrinsic uorescence were performed


by adding aliquots from the stock solution of the quencher (5 M acrylamide) into
a cuvette containing protein solution. The intrinsic uorescence was excited at
295 nm, and the emission was recorded between 300 and 400 nm. Final uorescence intensities were corrected for dilution effects. The decrease in uorescence
intensity at max was analyzed by the SternVolmer equation [21]

Ficin (F. carica) Lot #031K76652, 8-anilino-1-naphthalene-sulfonic acid (ANS),


urea, guanidine hydrochloride (GuHCl) and acrylamide were purchased from
SigmaAldrich Chemical Co., St. Louis, Mo, USA. Sodium acetate, acetic acid,
hydrochloric acid, monosodium and disodium salts of phosphate, glycine, and
hydrochloric acid were purchased from E-Merck, Mumbai, India. All the above chemicals used were of analytical grade. Quartz triple distilled water was used in all
experiments.

Ficin was puried from the commercial crude preparation (Sigma) according
to the method described in our earlier report [16]. The homogeneity of the puried
cin was evaluated by SDS-PAGE to conrm the purity. Concentration of the enzyme
was determined by measuring absorbance at 280 nm using an absorption coefcient
of E1% 280 = 20.9 on Shimadzu spectrophotometer [16] or alternatively by Lowrys
method [18].
2.3. Equilibrium denaturation experiments
Denaturation of cin was induced by incubating or dialyzing the enzyme with
various concentrations of denaturants till the equilibrium was attained. Refolding
was performed using the cin that had been completely denatured by 8 M urea
or 6 M GuHCl. The denatured cin was either diluted with appropriate concentration of urea or GuHCl and the mixture was incubated till the reaction reached to the
equilibrium or alternatively the denatured protein was dialyzed against desired concentration of GuHCl/urea with several changes. The extent of unfolding and refolding
of cin was measured either by changes in the emission maximum or ellipticity values at 222 nm by uorescence or circular dichroic measurements, respectively. The
data are expressed in terms of the fraction unfolded (Fu ) calculated from the standard
equation [19,20]:
Fobs Fn
Fu Fn

(1)

where Fobs is the observed value of the signal at a given denaturant concentration
and Fn and Fu are the values of native and unfolded protein, respectively. From these
measurements, values of GNU for a two-state process were determined using the
relation:
GNU = RT ln

Fobs Fn
Fu Fn

(2)

If a standard two-state model is assumed, the GuHCl and urea transitions are
tted to the equation
GNU = G(H2 O) m[D]

(4)

where Fo and F are the uorescence intensities at an appropriate wavelength


in the absence and presence of quencher (acrylamide), respectively; Ksv is the
SternVolmer constant and [Q] is the concentration of the quencher.

2.2. Purication of cin

Fu =

Fo
= 1 + Ksv [Q ]
F

(3)

where G(H2 O) and GNU are the free energy of the folding in water and at a denaturation concentration D, respectively. m is the slop of the GNU vs [denaturant]
plot, and D is the denaturant concentration [20].
2.4. Ficin assay
Enzyme activity of cin was quantied by measuring its ability to cleave an
amide bond in small-molecular-weight synthetic substrate benzoyl-d,l-arginine pnitroanilide hydrochloride (BAPNA) as described in our earlier studies using 0.05 M
sodium phosphate buffer containing 5 mM cysteine hydrochloride [16]. The extent
of hydrolysis was determined by measuring the product (p-nitroaniline) formed
at 410 nm ( = 8800 for p-nitroaniline). All the enzyme activity was measured in
both the presence of different concentrations of urea or GuHCl in 0.05 M sodium
phosphate buffer containing 5 mM cysteine hydrochloride at 55 C [16]. The enzyme
activity in the absence of the denaturants served as the control, and the percentage
residual activity was calculated on the basis of its original activity.
2.5. Fluorescence measurements
Fluorescence measurements of cin under different conditions were carried
out on Shimadzu (Model RF 5000) spectrouorimeter. All the measurements were
made at 25 C using appropriate blanks for baseline correction of uorescence intensity. The intrinsic uorescence was recorded in the wavelength raging from 300 to
400 nm and the excitation of protein solution was set at 280 nm. The slit width for
both excitation and emission was set at 5 nm. For binding studies of ANS, cin samples at different conditions were incubated with 100-fold molar excess of ANS for
30 min at 25 C in dark. The uorescence of ANS was excited at 380 nm and emission
was collected between 400 and 600 nm. In order to give correction for the unbound
ANS uorescence emission intensities, assays were performed with ANS and buffer
only.

2.7. Circular dichroic measurements


Circular dichroic (CD) spectral measurements of cin under different conditions were carried out using an automatic recording Jasco J-810 spectropolarimeter
(Japan Spectroscopic Co., Tokyo, Japan) tted with a xenon lamp and calibrated with
+d-10-camphor sulfonic acid. All the measurements were performed at 25 C on a
thermostatically controlled cell holder attached to a water bath. Far-UV CD measurements were recorded in the range of 190260 nm using protein concentration
of 10 M with 1 mm path length cell. The scan speed was 10 nm/min using a bandwidth of 1 nm and the spectra were taken as an average of three scans. The results
were expressed as the mean residual ellipticity []MRW obtained from the relation
[] =

100 obs
lc

(5)

where  obs is the observed ellipticity in degrees. The []MRW was calculated using
a value of 115 for mean residue mass of the protease, c is the concentration in g/l
and l is the length of the light path in cm. The values obtained were normalized
by subtracting the base line recorded for the buffer under similar conditions. The
analysis of the data for the secondary structure elements was done according to
computer program of Yang et al. [22].

3. Results and discussion


3.1. Effect of urea on the conformation of cin under neutral
condition
The effect of urea on structural and functional properties of cin
was studied by equilibrating the enzyme at neutral conditions (pH
7.0) in the presence of different concentrations of urea. The enzyme
was equilibrated for 36 h before taking all spectral measurements.
Changes in the uorescence emission spectra of cin after equilibration in presence of increasing concentrations of urea are shown
in the Fig. 1A. As seen from the spectra, a slight increment in the
uorescence intensity was observed with increasing concentration
of urea without any shift in the emission maximum. This increase
in the intensity is probably due to the loss of specic interaction
of tryptophan residues with vicinal quenching groups, which consequently resulted in the increment in the uorescence intensity
[23]. These results suggest that cin molecule under neutral conditions resist to unfold even in the presence of high concentration
of urea. The stability or conformational rigidity of cin at neutral
conditions was further evaluated by far-UV CD spectral studies.
Far-UV CD spectra of cin in presence of different concentrations
of urea are shown in Fig. 1B. The spectra of cin in presence of different concentrations of urea remained unaffected except for marginal
changes in the ellipticity values at 222 nm. The mean residual ellipticity value at 222 nm for native enzyme is 5150 deg cm2 dmol1
and in presence of 8 M urea is 4504 deg cm2 dmol1 . These results
clearly indicate the structural stability of cin molecule towards
urea denaturation. The structural stability of cin, isolated from
Ficus glabrata towards urea denaturation has been reported earlier
by Englund et al. [24]. Similar to the results indicated here stem
bromelain and procerain, a cysteine proteases from Calotropis procera also showed unusual structural stability against urea in neutral
conditions [15,25]. -Amylase from malted sorghum (Sorghum
bicolor) and lectin from Erythrina indica are also stable towards
denaturation by urea under neutral conditions [26,27].

460

K.B. Devaraj et al. / Process Biochemistry 46 (2011) 458464

100

450
d

375

80

b
a

Percent residual activity

F lu o r e s c e n c e e m is s io n in te n s ity (A U )

300

225

150

320

340

360

380

20

400

Concentration of urea (M)

Wavelength (nm)

[ ] MRW x 10 -3 deg.cm 2 dmol -1

40

75
300

60

Fig. 2. Activity prole of cin as a function of urea at pH 7.0. Activity of cin was
carried out in presence of various concentrations urea using BAPNA as substrate:
unfolding () and refolding ().

1.5
0.0
-1.5
-3.0
-4.5
d
c

-6.0
b
a

-7.5
200

210

220

230

240

250

260

Wavelength (nm)
Fig. 1. (A) Intrinsic uorescence emission spectra and (B) far-UV CD spectra of cin
in presence of different concentrations of urea at neutral condition at pH 7.0. The
enzyme solutions in presence of different concentrations of urea are equilibrated for
36 h before taking all spectral measurements. (a) Control in pH 7.0 sodium phosphate
buffer, (b) in 4 (c) in 6 M and (d) in 8 M urea.

3.2. Effect of urea on the enzyme activity of cin


Enzyme activity measurements in presence of different concentrations of denaturants are also excellent probe to monitor the small
changes induced by the denaturants. The effect of urea on the activity of cin at neutral conditions is shown in Fig. 2. The enzyme
loses 50% of its activity at 2 M urea and is completely inactivated
by 6 M urea. In order to check the reversibility of the activity, the
denatured protein sample diluted to desired concentrations of urea.
The reversibility studies showed that the inactivation of enzyme
was reversible. A recovery of about nearly 90% of the activity was
observed after complete removal of urea. The inactivation of the
enzyme at low concentrations of urea could be due to the changes
in micro environment of the active site. Several earlier studies have
also shown that the inactivation of enzymes occurs before significant conformational changes of the molecule as a whole during
denaturation by urea or GuHCl [68,28]. These results suggest the
fragility of the active site of the cin molecule. The catalytic activity
is lost before enzyme undergoes major conformational changes.
3.3. Effect of urea on the structure and stability of cin under
acidic conditions
3.3.1. Fluorescence spectral studies
Since cin is resistant to unfolding by urea under neutral pH, the
unfolding of the enzyme was carried out at lower pH. The enzyme

was susceptible to urea unfolding at pH 3.0 and the changes were


reversible. The maximum incubation time sufcient for achieving
equilibrium was standardized and the changes occurred within a
maximum time of 3 h with no further changes up to 12 h. The modications of microenvironment of aromatic residues of cin due to
denaturation by urea were studied by monitoring the changes in
the uorescence spectra as a function of urea concentration. The
changes in uorescence emission spectra and emission maximum
of cin in presence of increasing concentrations of urea are shown
in Fig. 3A. For native cin, the emission maximum is at 347 nm
as studied under neutral pH and the emission maximum shifts
to 345 nm at pH 3.0, suggesting the aromatic residues are in the
hydrophobic core of the protein at acidic condition. A red shift in
the uorescence emission maximum, as well as increase in uorescence intensity was observed by urea induced denaturation of
protein. At 4.5 M urea, the maximum red shift of 8 nm was observed
in the emission maximum. The emission maximum remains unaffected further even though the concentration of urea increased up
to 8.0 M.
3.3.2. Fluorescence quenching
The uorescence quenching experiments allow us to assess the
relative solvent exposure of uorophores [21]. The exposure of
the protein interior and the red shift in the uorescence emission
maximum of cin upon urea denaturation at pH 3.0 was further
evaluated by acrylamide quenching of tryptophan uorescence.
Fig. 3B represents the SternVolmer plot for analyzing the acrylamide uorescence quenching data of cin in presence of different
concentrations of urea. It is evident from the plot that the uorescence quenching was more effective by acrylamide as urea
concentration increased. The maximum quenching was observed
at 4.5 M. The results of SternVolmer plot thus indicate that the
aromatic amino acids of cin are more exposed to solvent during unfolding process induced by urea. These results are consistent
with the intrinsic uorescence emission results. The red shift in
the uorescence emission maximum and high quenching of tryptophan uorescence are indicative of the unfolding of protein
molecule.
3.3.3. ANS binding
The exposure of any hydrophobic regions, which are buried
inside the enzyme, on urea unfolding was monitored by ANS binding studies. The level of ANS binding to the protein in presence of
different concentrations of urea is shown in Fig. 3C. From the gure,

K.B. Devaraj et al. / Process Biochemistry 46 (2011) 458464

1.5
300

[]MRW x 10-3 deg.cm2 dmol -1

Fl uor e s cenc e i nt en s i t y ( A U )

A
f
e
d
c
b
a

250
200
150
100

300

320

340

360

380

400

-3.0

-4.5

b
a

-6.0

200

210

220

230

240

250

260

Fig. 4. Effect of different concentrations of urea on far-UV CD spectra of cin at


acidic condition (pH 3.0). (a) Native enzyme at pH 7.0; (b) at pH 3.0 in absence of
urea and in presence of (c) 2 M, (d) 2.5 M, (e) 3 M, (f) 4 M and (g) 4.5 M urea.

e
d
c

Fo /F

g
-1.5

Wavelength (nm)

Wavelength (nm)

0.0

-7.5

50

by the strong ANS binding in the region of 0.52.0 M urea under


acidic condition.

b
a

1
0.00

0.08

0.16

0.24

0.32

0.40

Concentration of acrylamide (M)

C
120

A N S i ntensi t y at 4 7 8 nm ( A U )

461

100
80
60

3.3.4. Far-UV CD spectral studies


Far-UV CD studies on urea-induced unfolding of cin were carried out to investigate the effect of urea on the secondary structure
of the protein molecule (Fig. 4) The spectrum of the cin at pH 3.0
is similar to that of cin at neutral pH except some loss in the ellipticity values. At pH 3.0, the enzyme retains about 70% of secondary
structures as compared to native state at pH 7.0, which is calculated
based on the ellipticity values at 222 nm. As seen from the gure,
the loss of secondary structure occurs as the concentration of urea
is increased. The maximum loss of secondary structures is observed
at 4.5 M urea. The decrease of the ellipticity values at 222 nm with
increase in urea concentration reects the disruption of secondary
structures by urea. A marked decrease in ellipticity between 2 and
4.5 M concentration of urea indicated that the majority of secondary structures are disrupted in the transition region. There was
no further major change in the ellipticity observed as the concentration of urea was increased up to 8 M. These results indicate that the
maximum loss of secondary structure occurs at 4.5 M urea under
acidic condition.
3.4. Effect of GuHCl on the structure and stability of cin

40
20
0

Concentration of urea (M)


Fig. 3. (A) Intrinsic uorescence emission spectra and (B) SternVolmer plot for the
acrylamide uorescence quenching of cin in presence of various concentrations
of urea at pH 3.0. (a) Control in Gly/HCl pH 3.0 buffer, (b) in 1.5 M (c) in 2.5 M, (d)
in 3 M, (e) in 4 M and (f) in 4.5 M urea. (C) Effect of urea-induce unfolding (at pH
3.0) on ANS uorescence. Protein to ANS concentration was in the ratio 1:100 and
the spectra were recorded in the region of 400600 nm after exciting at 380 nm at
25 C.

it can be observed that there was a minimal ANS binding to cin in


absence of urea at pH 3.0. ANS binding to the enzyme is maximal
at 0.52.0 M urea concentration, which is suggestive of exposure of
hydrophobic patches in this region of urea-induced unfolding pathway. The minimal binding of ANS was observed above the 2.0 M
urea concentration. These results suggest the formation of a molten
globule like state in the unfolding pathway of cin is substantiated

In majority of studies, urea or GuHCl is used as chemical denaturing agents to induce the protein unfolding. However, GuHCl
has been found to be more potent denaturant than urea [2,29].
GuHCl, a salt and also a bi-functional reagent have its strong
denaturing effect associated with the guanidinium ion. Thus, the
GuHCl-induced modulation of protein stability through mechanisms is related to both its denaturant and ionic properties [30].
The above results show that cin molecule under neutral pH conditions is resistant to denaturation by urea. In order to further
characterize, the effect of GuHCl on the structure, function and stability of cin under neutral conditions was determined. The enzyme
was incubated with desired concentrations of GuHCl under neutral
condition for 12 h to attain the equilibration before all the measurements.
3.4.1. Effect of GuHCl on uorescence spectra of cin
The uorescence emission spectral properties such as position,
shape and intensity are mainly determined by the polarity and specic interactions of uorophores. These unique properties are very
sensitive to any small changes in uorophores environment. The

462

K.B. Devaraj et al. / Process Biochemistry 46 (2011) 458464

A
g

375

e
d

c
b
a

300

[ ]MRW x 10-3 deg.cm2 dmol-1

F l uo re s ce nc e emi s s i on i nt e ns i t y ( A U )

225

150

75

1.5
0.0
f

-1.5

e
d

-3.0

c
b

-4.5
a
-6.0
-7.5

300

320

340

360

380

200

400

210

220

230

240

250

Wavelength (nm)

Wavelength (nm)

100

Percent residual activity

80
6
f

F0 /F

e
c

b
a

3
2

60

40

20

0
1
0
0.00

0.08

0.16

0.24

0.32

0.40

Concentration of GuHCl (M)

Concentration of acrylamide (M)


Fig. 5. (A) Intrinsic uorescence emission spectra and (B) SternVolmer plot for the
acrylamide uorescence quenching of cin in presence of various concentrations of
GuHCl at pH 7.0. (a) In absence (only in buffer) and in presence of (b) 2 M, (c) 2.5 M,
(d) 3 M, (e) 3.5 M, (f) 4 M and (g) 6 M GuHCl.

changes in the uorescence emission spectra of cin after denaturation with different concentrations of GuHCl solutions are shown
in Fig. 5A. The spectrum of cin remains unaffected by GuHCl up
to 1.8 M concentration. With the increasing concentration above
1.8 M of GuHCl, the uorescence emission intensity increased and
the emission maximum has a red shift. The red shift in the wavelength maximum indicates the more exposure of uorophores of
the protein to the solvent environment, which is the characteristic of unfolding. The increase in uorescence intensity may be the
result of an increased distance between uorophores and specic
quenching groups of the protein [23,31]. Fluorescence quenching titrations were performed with sequentially added aliquots
of acrylamide solutions to cin samples equilibrated at different
concentrations of GuHCl solutions. SternVolmer plot for the tryptophans uorescence quenching of cin by acrylamide at various
concentrations of GuHCl is shown in Fig. 5B. As seen from the plot,
the solvent accessibility of tryptophan residues was increased with
increase in the concentration of GuHCl. These results are consistent
with the data obtained from the intrinsic uorescence experiments.
The results obtained in this study are comparable to the effect of
urea and GuHCl on total cin fractions. GuHCl markedly affects the
cin conformation than urea as reected from red shift in uorescence maxima [32].

Fig. 6. (A) Effect of different concentrations of GuHCl on far-UV CD spectra of cin at


pH 7.0. (a) In absence of GuHCl (only in buffer) and in presence of (b) 2 M, (c) 2.2 M, (d)
2.5 M, (e) 3 M and (f) 4 M GuHCl. (B) Activity prole of cin as a function of GuHCl at
pH 7.0. Activity of cin was carried out in presence of various concentrations GuHCl
using BAPNA as substrate: unfolding () and refolding ().

3.4.2. Effect of GuHCl on secondary structural properties of cin


The effect of GuHCl on the structural changes of cin was further
characterized by far-UV CD spectral measurements. The secondary
structure of cin is unaffected till 1.8 M concentration of GuHCl.
The loss of secondary structure of cin by increasing concentrations
of GuHCl is shown in Fig. 6A. It can be seen from the spectra and
ellipticity values, the loss in the secondary structure of the protein
was observed with increase in the concentration of GuHCl. Most
of the secondary structure was disturbed in the region between 2
and 4 M GuHCl. Further increase in concentration of GuHCl above
4 M did not contribute to the any major changes in the spectra as
indicated by the complete loss of structure which occurs at 4 M
GuHCl concentration.
3.5. Effect of GuHCl on the activity of cin
The effect of various concentrations of GuHCl on the activity of
cin is shown in Fig. 6B. Ficin loses its half of the activity at around
1 M GuHCl, and the complete inactivation occurs at 3 M concentration. These results indicate that inactivation of cin occurred
very rapidly before major conformational changes in the enzyme
molecule. It is therefore suggested that perhaps the active site of
cin is situated in a limited and relatively fragile regions whose
conformational integrity is more sensitive to denaturation than
the molecule as a whole. A slight perturbation to geometry and

K.B. Devaraj et al. / Process Biochemistry 46 (2011) 458464

A
1.0

Concentration of urea(M)

0.6

G (kcal/mol)

Fraction unfolded

0.8

0.4

0.2

2
1
0
-1
-2

0.0
0

Concentration of urea (M)

B
1.0

Concentration of GuHCl (M)

0.6

6
4

0.4

G (kcal/mol)

Fraction unfolded

0.8

0.2

2
0
-2

0.0

-4

Concentration of GuHCl (M)


Fig. 7. (A) The urea-induced equilibrium unfolding transition curves of cin at pH
3.0 and (B) GuHCl at pH 7.0 as measured by changes in the CD ellipticities at 222 nm
() and uorescence emission maximum (). Insets: plot of G vs [denaturants]
for determining the stability of cin. The G was calculated according to standard
equation [19,20].

dynamics of the active site responsible for catalysis destroys


enzyme activity before the molecule as a whole [68,33].

463

mid point of 2.4 0.1 M under neutral conditions. The refolding


was studied by dilution of the protein sample which had been
completely equilibrium unfolded in 8 M urea or 6 M GuHCl. The
transition curves for the process of refolding were close to that of
unfolding curves. Thus, urea and GuHCl-induced unfolding of cin
can be described by a simple two-state model. It is generally recognized that protein denaturation is a highly cooperative process,
for which small globular proteins may be approximated by a twostate model and no signicant intermediates are present during the
transitions between native denatured states [29,3436]. Several
results have suggested that these chemical denaturants unfold proteins by interacting with polypeptide bonds and forming hydrogen
bonds with the backbone of protein molecule [24,37]. The denaturants tend to increase the solubility of the proteins by preferential
interactions and exposing more hydrophobic surfaces which are
buried inside the native molecules. In a more generalized mechanism the extent of binding show that two urea molecules bind to a
single peptide bond and additionally one urea molecule to each of
the to aromatic amino acids [3840].
The analysis of solvent denaturation curves using reagents can
provide a measure of conformational stability of proteins. The equilibrium constants obtained from the fraction of native protein in
the transition region of normalized curve were used to calculate the
free energy change (GNU ). The use of liner extrapolation method,
in which GNU is linearly related to the denaturant concentration
(Fig. 7, insets), is the simple method of estimating the protein stability [19,20]. The transition midpoints of urea and GuHCl-induced
unfolding of cin obtained by far-UV CD and uorescence wavelength maximum and their respective thermodynamic parameters
are summarized in Table 1. The conformational stability G(H2 O)
of cin determined under acidic condition using urea unfolding was
3.1 kcal/mol, which is signicantly lower than the stability determined using GuHCl under neutral conditions (6.1 kcal/mol). This
phenomenon may be due to an increased level of protonation of
the acidic groups, which disrupts the salt bridges at this lower pH.
Hence, these results suggest that the salt bridges are important in
determining the stability of this protein molecule and the thermodynamic evaluations of many reversible transitions from native
to unfolded state have demonstrated that the native structure of
proteins is highly sensitive to solution conditions and very fragile
[29,34,36].
4. Conclusions

3.6. Urea and GuHCl-induced equilibrium unfolding prole of


cin
The equilibrium unfolding transitions of cin at pH 3.0 as
a function of urea (Fig. 7A) and GuHCl at pH 7.0 (Fig. 7B) are
monitored by changes in the uorescence emission maximum
and ellipticity values at 222 nm by uorescence spectroscopy and
circular dichroic measurements, respectively. The data obtained
were normalized and analyzed according to the standard equation [19,20]. It can be seen from the plots that both urea and
GuHCl unfolding transitions of cin are cooperative and coincidental as indicated by sigmoidal curves. In urea, the maximum
changes occurred between 2 and 4.5 M with a mid point of transition of 2.5 0.1 M under acidic conditions. In case of GuHCl, the
maximum changes occurred between 2 and 3.5 M with a transition

In summary, the differential effect of urea and GuHCl on the


structural, functional-stability properties of cin provide a comparative analysis of the mechanism of changes of enzymatic activity
and structural stability in these denaturants. Several experimental
properties that reect different aspects of the structural integrity
of cin were monitored using different spectroscopic parameters through the course of equilibration denaturation. The results
obtained in this study show that active site of cin is situated in a
limited region of the enzyme molecule that is more fragile to denature that the protein as a whole. The unfolding/refolding results
indicate that a high rigid nature of the protein molecule towards
the unfolding by urea, even at 8 M concentration under neutral
conditions, as studied by uorescence and CD spectroscopic measurements. However, cin molecule was susceptible to unfolding

Table 1
Urea and GuHCl-induced unfolding parameters of cin.
Condition

Method

Transition mid point (Cm ) (M)

1. Urea at pH 3.0, 25 C

(a) Fluorescence emission maxima


(b) Far UV-circular dichroism
(a) Fluorescence emission maxima
(b) Far UV-circular dichroism

2.5
2.5
2.4
2.4

2. GuHCl at pH 7.0, 25 C

0.1
0.1
0.1
0.1

G(H2 O) (kcal/mol)
3.1
3.2
6.0
6.1

0.2
0.2
0.3
0.4

mUN (kcal/mol/M)
1.22
1.28
1.30
1.35

0.1
0.1
0.1
0.1

464

K.B. Devaraj et al. / Process Biochemistry 46 (2011) 458464

by urea under acidic condition and by GuHCl at neutral condition. The equilibrium transition curves as determined by various
methods, for both urea (at pH 3.0) and GuHCl (at pH 7.0) induced
denaturation were coincidental, suggesting the unfolding of the
protein is a simple two-state transition (native unfolded). The
measurements of conformational stability of the protein indicate
that salt bridges play a major role in the structural stability of
cin. The results presented here have demonstrated the differential
structural stability processes of cin towards denaturation effect by
urea and GuHCl.
Acknowledgement
Senior Research Fellowship to Mr. K.B. Devaraj from Council of
Scientic and Industrial Research, Government of India, New Delhi,
India is gratefully acknowledged.
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