Professional Documents
Culture Documents
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
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
459
2.1. Materials
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)
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.
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].
460
100
450
d
375
80
b
a
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
Wavelength (nm)
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.
1.5
300
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
e
d
c
Fo /F
g
-1.5
Wavelength (nm)
Wavelength (nm)
0.0
-7.5
50
b
a
1
0.00
0.08
0.16
0.24
0.32
0.40
C
120
A N S i ntensi t y at 4 7 8 nm ( A U )
461
100
80
60
40
20
0
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
A
g
375
e
d
c
b
a
300
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
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
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].
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
B
1.0
0.6
6
4
0.4
G (kcal/mol)
Fraction unfolded
0.8
0.2
2
0
-2
0.0
-4
463
Table 1
Urea and GuHCl-induced unfolding parameters of cin.
Condition
Method
1. Urea at pH 3.0, 25 C
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
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.
References
[1] Tanford C. Protein denaturation. Adv Protein Chem 1968;23:121282.
[2] Matthews CR. Pathways of protein folding. Annu Rev Biochem 1993;62:65383.
[3] Zou Q, Habermann-Rottinghaus SM, Murphy KP. Urea effects on protein stability: hydrogen bonding and the hydrophobic effect. Protein: Struct Funct Genet
1998;31:10715.
[4] Schellman JA. Fifty years of solvent denaturation. Biophys Chem
2002;96:91101.
[5] Vanzi F, Madan B, Sharp K. Effect of the protein denaturants urea and guanidinium on water structure: a structural and thermodynamic study. J Am Chem
Soc 1998;120:1074853.
[6] Ma YZ, Tsou CL. Comparison of the activity and conformational changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride. Biochem
J 1991;277:20711.
[7] Tsou CL. Conformational exibility of enzyme active sites. Science
1993;262:3801.
[8] Rai S, Dwivedi UN, Goyal N. Leishmania donovani trypanothione reductase: role
of urea and guanidine hydrochloride in modulation of functional and structural
properties. Biochim Biophys Acta 2009;1794:147484.
[9] Liener IE, Friedenson B. Ficin. Methods Enzymol 1970;19:26173.
[10] Shapira E, Arnon R. Cleavage of one specic disulde bond in papain. J Biol
Chem 1969;244:102632.
[11] Edwin F, Jagannadham MV. Sequential unfolding of papain in molten globule
state. Biochem Biophys Res Commun 1998;252:65460.
[12] Haq SK, Rasheedi S, Khan RH. Characterization of a partially folded intermediate
state of stem bromelain at low pH. Eur J Biochem 2002;269:4752.
[13] Ahmad B, Shamim TA, Haq SK, Khan RH. Identication and characterization
of functional intermediates of stem bromelain during urea and guanidine
hydrochloride unfolding. J Biochem 2007;141:2519.
[14] Ahmad B, Khan RH. Studies on the acid unfolded and molten globule states of
catalytically active stem bromelain: a comparison with catalytically inactive
form. J Biochem 2006;140:5018.
[15] Dubey VK, Jagannadham MV. Differences in the unfolding of procerain
induced by pH, guanidine hydrochloride, urea and temperature. Biochemistry
2003;42:1228797.
[16] Devaraj KB, Ramesh Kumar P, Prakash V. Purication, characterization and
solvent-induces thermal stabilization of cin from Ficus carica. J Agric Food
Chem 2008;56:141723.