Appl Biochem Biotechnol
DOI 10.1007/s12010-013-0163-9
Evidence for a Molten Globule State in Cicer α-Galactosidase
Induced by pH, Temperature, and Guanidine Hydrochloride
Neelesh Singh & Reetesh Kumar & M. V. Jagannadham &
Arvind M. Kayastha
Received: 31 December 2012 / Accepted: 19 February 2013
# Springer Science+Business Media New York 2013
Abstract Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the
present study, we have investigated the temperature, pH, and guanidine hydrochloride
(GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the
presence of both α and β structures in Cicer α-galactosidase and showed that its secondary
structure belongs to α+β class of proteins with 31 % α-helicity. For Cicer α-galactosidase
the emission maximum was found to be 345 nm which suggests that tryptophan residues are
less exposed to solvent. However, at pH2.0, protein showed blue-shift. This state of protein
lacked activity but it retained significant secondary structure. Enhanced binding of ANS at
pH2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded
state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in
the fluorescence intensity. The enzyme maintained its native structure and full activity up to
40 °C; however, above this temperature, denaturation was observed.
Keywords α-Galactosidase . Fluorescence . Circular dichroism . Molten globule . Unfolding .
Guanidine hydrochloride
Abbreviations
GuHCl Guanidine hydrochloride
CD
Circular dichroism
MG
Molten globule
RFOs
Raffinose family oligosaccharides
pNPGal p-Nitrophenylα-D-galactopyranoside
ANS
8-Anilino-1-napthalenesulfonic acid
N. Singh : A. M. Kayastha (*)
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221005, India
e-mail: [email protected]
R. Kumar : M. V. Jagannadham
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India
Appl Biochem Biotechnol
Introduction
Protein folding studies have attracted noteworthy attention of both industrial and biotechnological researchers in search of finding ways to increase protein/enzyme stability. The
molecular mechanism of spontaneous folding of proteins from random polypeptide chain to
the well-stabilized native form is still unknown [1]. In their biologically active state proteins
are characterized by well-defined three dimensional structures. The three dimensional
structure of proteins are maintained under specific environmental conditions. Outside their
particular environmental condition, proteins have the tendency to undergo denatured or
unfolded state. Thus, the study of the factors (such as pH, temperature, ionic strength, and
solvent composition) creating suitable environmental conditions for proteins is essential for
describing the forces responsible for conformational stability of proteins. The simplest
method for such studies involves the monitoring of conformational changes due to
perturbation of protein molecule by various agents such as temperature, pH, and
GuHCl. CD and fluorescence spectroscopy are two powerful techniques in structural
biology and have been used here for understanding the intricacies of protein folding
mechanism.
At present, it is a well-accepted view and this has been also proved experimentally that
there is existence of stable “molten globule” (MG) conformations between native and
denatured states [2, 3]. A MG-state conformation is characterized by folding intermediate
with significant secondary structure, no tertiary structure, and exposed hydrophobic patches
[1]. Thus, the understanding structural similarity and differences between MG and native
proteins may be possibly useful to sort out protein folding problem.
α-Galactosidase [α-D-galactoside galactohydrolase, E.C. 3.2.1.22] is universally distributed in plants, microorganisms and animals and catalyze the hydrolysis of terminal galactose
[4]. Raffinose family oligosaccharides (RFOs) are α-galactosyl derivatives of sucrose (e.g.
raffinose and stachyose). Since the humans and other monogastric animals lack the
α-galactosidase necessary for the breakdown of stachyose and raffinose [5], these oligosaccharides accumulated in the lower intestine and undergo fermentation carried out by the
anaerobic microorganisms causing flatulence [6]. Thus, the reduction in the RFOs is highly
desirable so that soy based food products can be exploited maximally for serving human
population. This could be a boon for poor people to fulfil their protein requirement and a best
substitute of milk for lactose intolerant population. α-Galactosidase is used in sugar and
paper industries [7, 8]. α-Galactosidase is used for Fabry disease treatment [9] and also
possesses efficiency of converting ‘B’ blood group to ‘O’ (universal) blood group [10, 11].
α-Galactosidase can also be used for improving the gelling properties of galactomannans,
used as a food thickeners [12]. In addition to the above, use of α-galactosidase for
transglycosylation and reverse hydrolytic reactions is well-reported [13].
Although physiological importance and structure details are well-established, little is
known about the stability and folding mechanism of α-galactosidase. Recently,
α-galactosidase has been isolated from white chickpea seeds (Cicer arietinum) and
purified to 340-fold with final specific activity of 61 U/mg [11]. Further Cicer α-galactosidase
was immobilized on different matrices [14]. We have used α-galactosidase from white chickpea
seeds as a model system to understand folding of α-galactosidase in general and Cicer
α-galactosidase in specific. In the present communication, the effect of pH, temperature, and
GuHCl on the structure of Cicer α-galactosidase was investigated by fluorescence spectroscopy
and CD. We also demonstrated the existence of partially folded intermediate state
(MG) at pH2.0 by CD, fluorescence, and 8-anilino-1-napthalenesulfonic acid (ANS)
binding studies.
Appl Biochem Biotechnol
Materials and Methods
Plant Material and Chemicals
White chickpea seeds (Pusa 1053) were purchased from local market and used in the present
study. All the chemicals for buffers and other reagents were of analytical grade or
electrophoresis grade. Unless otherwise specified, all the chemicals were purchased
from Sigma (St. Louis, MO, USA). Milli Q water (Millipore, USA) with a resistance
of higher than 18 MΩcm was used throughout the experiments.
Enzyme Activity Assay
The activity of Cicer α-galactosidase was routinely assayed as described by
Malhotra and Dey [15] using p-nitrophenylα-D-galactopyranoside (pNPGal) as substrate with certain modifications. Aliquots of 0.1 ml of pNPGal (2 mM) and 0.5 ml
of 50 mM acetate buffer (pH 4.5) were pre-incubated at 37 °C temperature for 2 min
before adding 0.1 ml of suitably diluted enzyme to start the reaction. The reaction
was stopped after 10 min by adding 3 ml of 0.1 M Na2CO3 and the released
p-nitrophenol was determined spectrophotometrically at 410 nm. One unit (U) of
α-galactosidase activity was defined as the amount of enzyme liberating 1.0 μmol of
p-nitrophenol/min under the assay conditions.
Protein Assay
Protein was estimated as described by Bradford [16] with the Bradford reagent, calibrated
with bovine serum albumin.
Enzyme Preparation
α-Galactosidase was purified from white chickpea seeds using the method of Singh and
Kayastha [11].
Fluorescence Spectroscopy
Fluorescence measurements were carried out on a Perkin-Elmer LS-50 B spectrofluorimeter.
All the measurements were performed at 25 °C on a thermostatically controlled cell
holder attached to Julabo F 25 water bath. Protein concentration for all the fluorescence
measurements were 0.1 mg/ml. Excitation wavelength of 290 was used while emission spectra
were recorded from 300 to 400 nm with 5- and 10-nm slit widths for excitation and emission,
respectively.
Circular Dichroism
CD measurements in the far-UV (200–260 nm) and near-UV (260–320 nm) region were
recorded on a JASCO J-500 A spectropolarimeter equipped with a 500 N data processor and
was calibrated with ammonium (+)-10-camphorsulfonate. All the measurements were
performed at 25 °C on a thermostatically controlled cell holder attached to Julabo F 25
water bath. A cell of path lengths 1 and 10 mm were used for far-UV and near-UV,
respectively. Each spectrum was the average of three scans. Protein concentrations used
Appl Biochem Biotechnol
for far-UV and near-UV were 0.2 and 1 mg/ml, respectively. After subtracting the appropriate blanks, mean residues ellipticities were calculated using formula:
½θ ¼ θobs MRW=10cl
where, θobs is observed ellipticity in degrees, MRW is mean residue weight, c is concentration of protein (gram per cubic meter) and l is path length in centimeters [17]. A mean
residue molecular weight 110 was used. Sensitivities of 1 and 2 m°/cm (millidegree per
centimeter) were used for far-UV and near-UV measurements, respectively. Cell cuvette
thickness and protein concentration were chosen in such a way that the maximum hightension voltage at the photomultiplier does not exceed 800 V at the shortest wavelength
(200 nm). Percentages of the different secondary structures (α helix, β sheets) of the protein
were estimated using the K2D2 program [18].
ANS Binding
The extent of exposure of hydrophobic surfaces in the enzyme was measured by its ability to
bind with fluorescent dye ANS [19]. ANS stock solution was prepared in methanol and dye
concentration was determined by using extinction coefficient Є5,000 M−1 cm−1 at 350 nm
[20]. The protein sample was incubated with a 100-fold molar excess of ANS for 45 min in
dark at room temperature. ANS binding was recorded by fluorescence emission spectra with
excitation at 380 nm and emission was recorded from 400 to 600 nm. Slit widths for
excitation and emission were 5- and 10-nm, respectively.
Acid Denaturation
Acid-induced unfolding of Cicer α-galactosidase was carried out in 50 mM of the following
buffers: KCl/HCl (pH1.0–1.5), glycine/HCl (pH2.0–3.5), sodium acetate (pH4.0–5.5), and
sodium phosphate (pH6.0–7.0). pH measurements were carried out on an Cyberscan 510
digital pH meter with a least count of 0.01 pH unit. Cicer α-galactosidase was dialyzed
against respective buffer at 4 °C. The final pH and concentration of the protein in each
sample were measured again.
GuHCl-Induced Denaturation
GuHCl-induced denaturation of enzyme at a given pH was studied by incubating the enzyme
sample with varying concentration of denaturant for 24 h at 4 °C to attain equilibrium. Data
are expressed in terms of the fraction unfolded (Fu) given by following equations:
Fu ¼ ðFobs Fn Þ=ðFu Fn Þ
where, Fobs is the observed value of the signal at any denaturant concentration. Fn and Fu are
the values of native and unfolded protein, respectively.
Thermal Unfolding
For temperature-induced unfolding, the samples were measured at several different temperatures (20–80 °C). Enzyme samples were incubated at desired temperature for 10 min,
before taking any measurements.
Appl Biochem Biotechnol
Results and Discussion
The CD spectra of Cicer α-galactosidase, in both near- and far-UV wavelength regions, are
shown in Fig. 1. Near-UV CD spectra are appropriate for assessing the tertiary structure of
proteins. The spectra in the region 260–320 nm arise from the aromatic amino acids. In the
aromatic region, the CD spectrum of Cicer α-galactosidase exhibits positive peak which
a
pH 7.0
pH 2.0
6 M GuHCl
100
80
2
-1
Molar ellipticity (deg cm dmol )
120
60
40
20
0
-20
-40
260
270
280
290
300
310
320
Wavelength (nm)
-3
2
-1
Molar ellipticity x 10 (deg cm dmol )
b
0
pH 7.0
pH 2.0
6 M GuHCl
-2
-4
-6
-8
200
210
220
230
240
250
260
Wavelength (nm)
Fig. 1 a Near-UV and b far-UV spectra of Cicer α-galactosidase at pH7.0, 2.0, and 6 M GuHCl
Appl Biochem Biotechnol
suggests that many of the aromatic amino acids (Phe, Tyr, and Try) side chains are ordered in
the native structure. However, the obtained near-UV CD spectrum at pH2.0 and 6 M GuHCl
is featureless, indicating that the aromatic side chains are disordered during protein
unfolding. The far-UV CD spectrum is an important technique for examining the secondary
structure of proteins in solution. Cicer α-galactosidase showed two well-resolved negative
peaks at 208 nm and around 220–222 nm. Strong negative ellipticities at 208, 215, and
222 nm for Cicer α-galactosidase, at pH7.0 (native state), is composed of α-helix and βsheet regions. At pH7.0, Cicer α-galactosidase showed α helix 31 % and β sheet 15.4 %.
The crystallographic models of rice [21], Trichoderma reesi [22] and Human [23] αgalactosidases showed that these proteins consist of N-terminal (β/α)8-barrel (catalytic
domain) and a C-terminal β-sheet domain. At pH2.0, Cicer α-galactosidase still showed
presence of secondary structure with a significantly low ellipticity, than native state. In the
presence of 6 M GuHCl, enzyme showed complete loss of secondary structure.
Fluorescence Spectra
Figure 2, showed intrinsic fluorescence spectra of Cicer α-galactosidase at pH7.0 and 2.0,
respectively and at 6 M GuHCl. The fluorescence spectrum of native Cicer α-galactosidase
at pH7.0 showed maximum emission intensity at 345 nm, indicating tryptophan residues of
the protein were relatively less exposed to solvent. However, at pH2.0 there was a decrease
in the fluorescence emission intensity with a slight of blue-shift (≈5 nm). The blue-shift at
pH2.0 indicates exposure of tryptophan to hydrophobic environment after pH denaturation.
A similar type of blue-shifted fluorescence has been reported earlier for glucose isomerase
[24] and stem bromelain [1]. The unfolded state of Cicer α-galactosidase with 6 M GuHCl
suffered a red-shift from 345 to 360 nm along with a decrease in fluorescence intensity.
This indicates complete exposure of tryptophan to solvent. These shifts are a result of
environmental changes in the vicinity of the surface exposed tryptophan residues. Overall
180
Fluorescence intensity (a.u.)
160
pH 7.0
pH 2.0
6 M GuHCl
140
120
100
80
60
40
20
0
300
320
340
360
380
400
420
440
Wavelength (nm)
Fig. 2 Intrinsic fluorescence spectra of Cicer α-galactosidase at pH7.0, 2.0, and 6 M GuHCl
Appl Biochem Biotechnol
data indicates that the protein at pH2.0 is at an intermediate state distinct from native and
denatured state. With 6 M GuHCl, similar observations for α-amylase have been reported
earlier [25]. Furthermore, these observations indicate that the Cicer α-galactosidase at pH2.0
is present in a conformational state that is different from the native state as well as
completely unfolded state.
Acid-Induced Unfolding
At extreme pH, the main factor responsible for protein unfolding is the repulsion
between charged groups on the protein molecule. The changes in the secondary
structure of Cicer α-galactosidase as a function of pH were followed by far-UV CD
by measuring ellipticity values at 222 nm (Fig. 3). Cooperative transition is obtained
during the unfolding of Cicer α-galactosidase due to pH effect from native to
unfolded state. In order to understand the changes in the secondary structure of Cicer
α-galactosidase as a function of pH, far-UV CD spectra were measured over wide pH
range (1.0–7.0).
Acidic environment induced a significant reduction of the regular conformations,
suggesting that Cicer α-galactosidase failed to preserve its secondary structure at very low
pH. At pH2.0, the enzyme showed complete loss of activity due to loss of native structure in
addition to protonation of the active site nucleophile. However, it retained some secondary
structure, which again indicates that it is an intermediate state between the native and
completely unfolded state.
ANS is a fluorescent dye that has the affinity for hydrophobic regions of proteins. This
hydrophobic affinity is helpful in the identification of a stable intermediate termed as molten
globule [26]. The loose tertiary interactions present in the MG enable the dye to bind with
-1
Molar ellipticity x 10 (deg cm dmol )
0
-3
2
-1
-2
-3
-4
-5
-6
0
1
2
3
4
5
6
7
pH
Fig. 3 Effect of pH on the ellipticity of Cicer α-galactosidase. Ellipticity was monitored at 222 nm
by far-UV CD
Appl Biochem Biotechnol
the exposed hydrophobic patches [26]. The increased ANS fluorescence of Cicer α-galactosidase at pH2.0 indicates significant unfolding and exposure of hydrophobic regions
(Fig. 4).
The data from intrinsic and extrinsic (ANS) fluorescence studies together with
near-UV CD indicates that the protein at pH 2.0 has highly exposed hydrophobic
patches; tryptophan in hydrophobic environment with significant secondary structure. The state of the protein at this pH seems to be different from native and
denatured state of the protein and suggests existence of a folding intermediate
state. Overall, the properties of the intermediate state indicate it to be a molten
globule-like state.
GuHCl-Induced Denaturation
GuHCl-induced changes in Cicer α-galactosidase under different pH conditions were
followed by CD, fluorescence, and activity measurements (Fig. 5). The transition curves
obtained due to the unfolding of Cicer α-galactosidase with increasing GuHCl concentration
is cooperative with two state transitions. This suggests that there is no other intermediate
state between native (pH7.0) and denatured state as well as molten globule (pH2.0) and
denatured state. The activity loss of enzyme was in good correlation with the disturbances in
secondary structure of enzyme.
Thermal Unfolding
The effect of temperature on secondary structure of Cicer α-galactosidase was monitored by changes in the mean residue ellipticity at 222 nm. At higher temperature the
interactions in the protein molecule are disrupted and thus cause denaturation.
ANS Fluorescence intensity (a.u.)
250
pH 7.0
pH 2.0
6 M GuHCl
200
150
100
50
0
400
450
500
550
600
Wavelength (nm)
Fig. 4 Interaction of ANS with Cicer α-galactosidase. Native enzyme at pH7.0, 6 M GuHCl (denatured state)
and acid-induced state at pH2.0
Appl Biochem Biotechnol
a
1.0
Far-UV CD (222 nm)
Fluorescence
Activity
Fraction unfolded
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
6
GuHCl (M)
b
1.0
Far-UV CD (222 nm)
Fluorescence
Fraction unfolded
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
6
GuHCl (M)
Fig. 5 The GuHCl-induced equilibrium unfolding transition curve of Cicer α-galactosidase a at pH7.0 and b
at pH2.0
Temperature-induced unfolding for Cicer α-galactosidase is completely irreversible.
The irreversible thermal denaturation of many proteins generally attributed to alterations such as aggregation or chemical alteration of residues that hindered the protein
to refold to native state. The enzyme maintains its native structure and full activity up
Appl Biochem Biotechnol
1.0
Far-UV CD (222 nm)
Fluorescence
Fraction unfolded
0.8
0.6
0.4
0.2
0.0
0
20
40
60
80
o
Temperature ( C)
Fig. 6 Unfolding transitions of Cicer α-galactosidase induced by temperature
to approximately 40 °C and beyond this temperature the gradual loss in ellipticity
obeyed a cooperative transition (Fig. 6).
Fig. 7 Proposed unfolding pathway of Cicer α-galactosidase
Appl Biochem Biotechnol
Conclusion
The study of intermediates of folding pathway is essential to understand the protein folding
mechanism. In the present study Cicer α-galactosidase, which was isolated and purified
recently, was investigated systematically with respect to its stability against pH, GuHCl, and
temperature-induced denaturation (Fig. 7). Fluorescence and CD data supported the existence of MG state. The MG state retained considerable secondary structure and is characterized by ANS (hydrophobic dye) binding where ANS fluorescence intensity is greater than
native enzyme or unfolded enzyme. Furthermore the idea of stability mechanism studied in
this case may be helpful in further development of enzymes/recombinant protein with
improved stability for biotechnological applications.
Acknowledgments One of us (N.S.) would like to thank the University Grants Commission (UGC), New
Delhi, India, for financial support in the form of junior and senior research fellowships.
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