Báo cáo Y học: Artocarpus hirsuta lectin Differential modes of chemical and thermal denaturation potx

Artocarpus hirsuta
lectin
Differential modes of chemical and thermal denaturation
Sushama M. Gaikwad, Madhura M. Gurjar* and M. Islam Khan
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India
Unfolding, inactivation and dissociation o f the lectin from
Artocarpus hirsuta seeds were studied by chemical (guanidine
hydrochloride, GdnHCl) and thermal denaturation. Con-
formational transitions were monitored by intrinsic fluor-
escence and circular dichroism. The gradual red shift in t he
emission maxima of the native p rotein from 33 5 to 356 nm,
change in the e llipticity at 218 nm and simultaneous decrease
in the sugar binding activity were observed with increasing
concentration o f G dnHCl in the pH range between 4.0 and
9.0. The unfolding and inactivation by GdnHCl were par-
tially reversible. Gel filtration of the lectin in presence o f
1–6
M
GdnHCl showed that the protein dissociates rever-
sibly into partially unfolded dimer and then irreversibly into
unfolded inactive monomer. Thermal denaturation was
irreversible. The l ectin loses activity rapidly above 45 °C.
The exposure of h ydrophobic patches, distorted secondary
structure and formation o f insoluble aggregates of the
thermally inactivated protein probably leads to the irre-
versible d enaturation.
Keywords: Artocarpus lectin; denaturation; intrinsic fluores-
cence; unfolding; aggregation.
Proteins that bind carbohydrates specifically and reversibly
are termed as lectins. They occur ubiquitously in nature and
have diverse role in plants, animals and microbes. T he

recognition of c arbohydrate m oieties b y l ectins h as import-
ant applications in a number of biological processes such a s
cell–cell i nteractions, signal transduction, and cell growth
and differentiation [1]. a-Galac toside specific lectin present
in the s eeds of Artocarpus hirsuta [2–4], is a homotetrameric
protein with molecular mass of 60 000 Da and high
specificity f or methyl a-
D
-galactopyranoside (Me a-gal).
The folding pathways of oligomeric proteins involve both
intramolecular and intermolecular interactions. The dena-
turation of pea and peanut lectins, both oligomeric proteins,
has b een studied in great detail [ 5–7]. In t his paper we show
the progressive unfolding and inactivation of the lectin in
presence of GdnHCl and heat, and refolding and reactiva-
tion under renaturing conditions.
MATERIALS AND METHODS
Materials
GdnHCl was a product o f Sigma Chemical Co. A ll other
chemicals were of t he highest purity available. The l ectin
from A. hirsuta was purified as described previously [2].
Stocks of 7
M
GdnHCl were freshly prepared in appropriate
buffers and filtered through 0.45-lm filter. Buffers used
were acetate f or pH 4.0, citrate/phosphate for pH 5.0 and
6.0, phosphate for p H 7.0 and Tris/HCl for pH 8.0 and 9.0
(all 100 m
M
).

Fluorescence studies
Protein samples (1.5 l
M
) were equilibrated for 4 h at the
desired denaturant concentration at 30 °C in the pH range
of 4.0–9.0. Unfolding as a function of GdnHCl concentr a-
tion was monitored by intrinsic tryptophan fluorescence
emission in a 1-cm quartz cell in the 300–400 nm region,
when excited at 280 nm, in a Perkin-Elmer LS 50B
spectrofluorimeter with attached circulating water bath.
Excitation and emission band passes of 5 n m were u sed.
Activity of the sample was measured at the same time.
Unfolding as a function of temperature w as carried out
by incubating the protein samples (1.5 l
M
) in duplicates at
the temperature from 30–70 °C for 15 min. One of the
duplicates was u sed to r ecord the spectra and activity a t the
respective temperature. The other sample was brought to
35 °C, centrifuged to remove any particulate matter, spectra
were recorded and activity was estimated.
Circular dichroism studies
Far UV CD (210–250 nm) spectra of the protein samples
(15 l
M
) t reated with different concentrations GdnHCl in
respective buffers of pH 4.0–9.0 and incubated for 14 h were
recorded in a 1-mm path length cell, on a Jasco J715
spectropolarimeter connected to a circulating water bath.
For thermal denaturation studies, the protein sample was

incubated at various temperatures for 10 min and the
spectra were recorded. The spectra were collected with
response time of 4 s, and scan speed of 100 n mÆs
)1
.Each
data point was an average of three accumulations.
Correspondence to S. M. Gaikwad, Division of Biochemical Sciences,
National Chemical Laboratory, Pune, 411008, India.
Fax: + 9 1 20 5884032, Tel.: + 91 20 5893034,
E-mail:
Abbreviations: GdnHCl, guanidine hydrochloride; Me a-gal, methyl
a-
D
-galactopyranoside; ANS, 1-anilino-8-naphthalene sulfonate.
*Present address: University of Medic ine and D e ntistry New J ersey,
Robert Wood J ohn son Medical School, D ivision of Biochemistry
(Research t ower), Piscataway, Ne w Jersey 0 8854, USA.
(Received 21 December 2 001, acc epted 14 Ja nuary 2002)
Eur. J. Biochem. 269, 1413–1417 (2002) Ó FEBS 2002
Hydrophobic dye binding studies
8-Anilino-1-naphthalene sulfonate (ANS) emission spectra
were recorded in the range of 400–500 nm with excitation at
375 n m using slit widths of 5 nm. The changes in the ANS
fluorescence induced by the binding to the lectin were
followed by r ecording the spectra at constant concentration
of protein (1 l
M
)andANS(50l
M
), in different concentra-

tions of GdnHCl (1–5
M
).
Determination of the lectin activity
Sugar binding activity of the samples was measured by the
enhancement in the intrinsic flu orescence of the prote in at
335 n m after addition of the nonfluorescent ligand Me a-gal
(4 m
M
) at saturating c oncentration. Increase in the fl uores-
cence of the native lectin after adding Me a-gal was taken as
100% activity [3].
Light scattering studies
Rayleigh light scattering experiments w ere carried out with
the s pectrofluorimeter t o f ollow p rotein aggregation during
GdnHCl and thermal denaturation. Both excitation and
emission wavelengths were set at 400 nm and the time
dependent change in scattering intensity was followed.
Renaturation studies
Two-hundred microliters aliquot was removed from the
samples treated with different c oncentrations of GdnHCl
(3.0–5.0
M
)for4hat30°C a t p H 7 .0 and d iluted 1 0 t imes
with 100 m
M
buffer of pH 7.0. After 30 min, the fluores-
cence spectra and activity of the original (treated w ith
GdnHCl) a s well as diluted samples were recorded. Protein
sample without GdnHCl tre ated under identical conditions

was taken as control.
The renaturation of thermally denatured protein was
followed b y c ooling t he heate d samples to 3 5 °C, re moving
any p articulate matter by centrifuging, and t hen recording
the fluorescence spectra and the activity.
Gel filtration studies
Lectin samples (10 l
M
in 100 lL) were incubated for 14 h
with GdnHCl (1.0–6.0
M
) a nd injected onto Protein Pak
300SW HPLC column (7.8 · 300 mm) connected to a
Waters HPLC syste m preequilibrated a nd eluted with
different concentrations of GdnHCl (1.0–6.0
M
)in
100 m
M
buffer of pH 7 .0 at a flow rate of 0.5 mLÆmin
)1
.
Elution was monitored by absorbance at 280 nm. The
standard molecular mass markers run in the presence of
buffer were, BSA (66 kDa), o valbumin (45 kDa), carbonic
anhydrase (29 kDa) and cytochrome c ( 14.5 k Da).
RESULTS AND DISCUSSION
Unfolding studies
The fluorescence emission spectrum of the native lectin
showed a maximum a t 335 nm which characterizes no n-

polar environment o f t he tryptoph an residues. On dena-
turation of the lectin with increasing concentration of
GdnHC l (1–5
M
) a lthough fluorescence intensity at 335 nm
does not change much, the fluorescence at 356 n m
increases significantly thus changing the emission maxi-
mum from 335 to 356 nm (Fig. 1A) indicating that due to
unfolding, most of the tryptophans in the protein are
getting exposed to th e solvent. The ratio of fluorescence
intensities at 3 35 and 356 F(335/356) (Fig. 1B) decreases
from 1.35 to 0.82. Similar trend of denaturation with
GdnHCl is observed in the pH range of 5.0–8.0, while it is
more drastic at pH 4.0 and 9.0. The concentration of
GdnHCl required for 50% unfolding of the protein
between the pH range 6.0–8.0 is higher (3.2
M
)thanat
pH 4.0 and pH 9.0 (2.2
M
).
The l ectin showed a typical far UV CD spectrum
observed for proteins with high proportion of b sheet
content, with minimum at 218 nm [2]. The relative
percentage of structural elements calculated using CDPro
software package for analyzing protein CD spectra was
a helix 2%, b sheet 44%, turns 2 3% and random coil 30%
for native protein. The CD spectra of the G dnHCl treated
protein when analysed using the above programs did not
show any significant change in the different structural

elements compared to the native protein, while there was
visible difference in the respective CD spectra. This was
probably due to the incompatibility o f the data with the
programmes used. Because GdnHCl was interfering
the CD s pectra below 2 10 nm, d ata in the ran ge of
210–250 nm could be collected. The negative ellipticity of
the protein at 218 nm increases in 1–2
M
GdnHCl and
then decreases at h igher c oncentration ( Fig. 1C). The
change in the structure at 1–2
M
GdnHCl is concomitant
with loss of activity and therefore cannot be a stable
Fig. 1. GdnHCl-induced unfolding of A. hirsuta lectin at 30 °C. Protein
(1.5 l
M
) at the required GdnHCl concentration was incubated for 4 h
and the fluorescence emission spectra were re co rded between 300 and
400 nm with the excitation wavelength of 280 nm (A), shift in the
emission max (B), ratio F(335/356) (C), mean residue ellipticity at
218 nm in far UV region and (D) activity of the GdnHCl treated
protein. The s ym bols used for all the figure s a re p H 4.0 (.), pH 5.0
(d), pH 6.0 ( m), pH 7.0 (,), pH 8.0 (s)andpH9.0(n).
1414 S. M. Gaikwad et al. (Eur. J. Biochem. 269) Ó FEBS 2002
conformation. When Rayleigh light scattering studies of
the s amples were carried out, the sample incubated at 1
M
GdnHCl at 30 °C showed lower light scattering intensity
than the native p rotein. At 2.0

M
GdnHCl, there was
lower light scattering than that with 1.0
M
and at s till
higher concentrations of GdnHCl, there was n o light
scattering at all (data not shown). Thus, the increase in
the negative ellipticity at 218 n m at low concentrations
of GdnHCl could be due to the solubilization of the
aggregates in the protein. There is substantial loss in
the secondary structure of t he lectin as indicated b y the
decrease in the n egative ellipticity at 218 nm with
increasing concentration of GdnHCl. Similar trend was
observed for denaturation between p H 5.0–8.0, while the
rate of unfolding was faster at pH 4 .0 and 9.0.
The inactivation of the lectin was proportional to the
concentration of GdnHCl (Fig. 1D). The maximum
enhancement in the intrinsic fluorescence of the lectin due
to the binding of sugar, Me a-gal, taken as measure of
100% activity of the lectin [3] determined at different pH
was diffe rent. T he percentage decrease in the enhancement,
i.e. activity w ith increasing concentration o f GdnHCl,
however, was equivalent in the pH range of 4.0–9.0 a nd
the loss in the activity of the lectin is concomitant with the
unfolding of the protein. A t 3
M
GdnHCl, more than 5 0%
activity was lost with 60% decrease in the ratio (F335/356)
and 25–35% shift in the emission maximum.
Refolding of the protein

Renaturation or re folding of t he protein w as measured as
the extent of reappearance of the original spectra (F 335/
356) and recovery of the sugar binding activity. After
dilution of the r eaction mixture containing lectin and
GdnHCl (10 t imes), partial reactivation o f the lectin was
observed. The lectin treated with 3 , 4, and 5
M
GdnHCl
had 45, 13 and 7% activity, which increased to 75, 37,
and 23%, respectively ( Table 1 ) on renaturation. Re fold-
ing of the protein w as indicated by s ubstantial increase i n
the F(335/356) ratio. GdnHCl probably unfolds the
protein in such a way that substantial interactions are
reformed after removal of the denaturant, leading to the
significant reformation of the structure and regaining of
activity.
Gel filtration studies
The native protein gets dissociated first into dimer (M
r
30 000) and then into monomer (M
r
14 000) with increasing
concentration of G dnHCl (Fig. 2). At 3–4
M
GdnHC l, a
single peak at 10.4 min appears that seems arise f rom
the totally denatured monomer. Complete dissociation of
the tetramer does not take place even at 6
M
GdnHCl. The

protein components corresponding to the peaks 1, 2, 3 and 0
were analysed separately for sugar binding activity. Peak 1
was found to be the f olded and active fraction of the total
population o f the lectin molecules t reated with GdnHCl.
Peak 2 is p artially unfolded form of t he lectin with traces of
activity. Peak 3 is unfolded, inactive monomer. Peak 0 is the
totally denatu red m onomer similar t o t hat observed in c ase
of peanut lectin [6]. The dissociation of the native protein in
presence of GdnHCl into dimer is reversible, that into
monomer is irreversible as observed by rechromatography
of the individual peaks on gel filtration column under
renaturing conditions (data not shown). Based on the
dissociation pattern, the following scheme can be written:
T () D () M () M*
where T is tetramer, D is dimer, M is monomer and M* is
totally denatured monomer.
The monomer seems to be unstable and the conforma-
tional stability of the oligomer seems to be contributed
wholly by the q uaternary i nteractions. I n case of p eanut
lectin, folded m onomer is obtained after dissociation of the
protein [7] and the molten globule-like state of the monomer
was d etected during its unfolding [6], both of which retain
the sugar binding activity.
Thermal denaturation
The A. hirsuta lectin loses sugar binding activity and starts
precipitating above 45 °C. The fluorescence emission spec-
trum broadens, but the emission maxima does not shift
from 335 to 356 even at 70 °C where almost total
inactivation of the lectin takes place. T he decrease in the
ratio F(335/356) observed for thermally denatured protein,

from 1.36 (native) to 1.03 (70 °C, 15 min) (Table 1)
was less than that observed with GdnHCl denaturation
(at pH 7.0), 1.35 (native) to 0.82 (5
M
GdnHCl) ( Fig. 1B).
Table 1. Effect of treatment GdnHCl and thermal denaturation and renaturation on A. hirsuta lectin. The samples treated w ith GdnHCl were diluted
10 times with 100 m
M
phosphate buffer, pH 7.0, the spectra were recorded and activity was estimated as described in Materials and methods. The
lectin samples i ncubated at respective temperatures were cooled to 35 °C, spectra were recorded and activity was estimated.
Treatment
Activity (%) F 335/356
On denaturation On Renaturation On denaturation On Renaturation
Lectin + GdnHCl (0
M
) 100 100 1.35 1.35
Lectin + GdnHCl (3
M
) 45 75 1.15 1.26
Lectin + GdnHCl (4
M
) 13 37 0.86 1.2
Lectin + GdnHCl (5
M
) 7 23 0.82 1.16
Lectin Þ 35 °C,15 min 100 100 1.36 1.36
Lectin Þ 45 °C,15 min 75 70 1.26 1.18
Lectin Þ 50 °C,15 min 53 35 1.23 1.1
Lectin Þ 60 °C,15 min 13 5 1.09 0.98
Lectin Þ 70 °C,15 min 7 0 1.03 0.95

Ó FEBS 2002 Denaturation studies of Artocarpus hirsuta lectin (Eur. J. Biochem. 269) 1415
On thermal denaturation, the p rotein forms insoluble
aggregates before total unfolding and loses its sugar
binding activity. When the temperature of the samples
incubatedfrom45to70°C was brought down slowly to
35 °C, the activity was not restored and no refolding was
observed a s there is decrease in the F(335/356) ratio
(Table 1) indicating that the thermal denaturation is
irreversible. Because the protein s tarts a ggregating o n
thermal denaturation, Rayleigh light scattering studies were
carried out. The protein shows higher light scattering
intensity a t 45 °C (Fig. 3A) which goes on increasing with
further i ncrease i n the temperature.
ANS binding studies
Binding of ANS to the proteins occurs upon the exposure of
hydrophobic clusters during the unfolding process. ANS
does not bind to the native or t he denatured states of t he
A. hirsuta lectin but binds at the intermediate stage (at 2
M
GdnHCl), showing increase in t he fluorescence intensity,
indicating temporary exposure of the hydrophobic patches
of the protein during unfolding (Fig. 3B). The p ossibility of
the occurrence of the molten globule during unfolding of
A. hirsuta lectin as observed in t he peanut lectin [6], was
ruled out because a significant amount of th e tertiary and
secondary structure was intact. T he ANS binding to the
protein samples exposed to 50, 60, and 70 °C was more than
those incubated a t 30 a nd 40 °C (Fig. 3C) i ndicating the
exposure of hydrophobic patches are due to therm al
denaturation. The tendency of the protein to aggregate

increases a s the hydrophobic patches get exposed due to
thermal denaturation. The CD s pectra of the protein
exposed at 45–70 °C for 10 min s hows progressive loss in
the secondary structure (Fig. 3D).
There s eem to be two different modes o f d enaturation o f
the A. hirsuta lectin with GdnHCl and heat. The former
unfolds and inactivates the protein, allowing it to fold back
and reactivate to certain extent after removal of the r eagent.
Thermal denaturation l eads to unfolding and simultaneous
formation of insoluble aggregates and is therefore irrevers-
ible. Different modes of folding and unfolding observed
under different conditions could be due to the unusual
Fig. 2. Gel filtration of A. hirsuta lectin in presence of GdnHCl in
100 m
M
potassium phosphate buffer (pH 7.0). Molarity of GdnHCl is
indicated o n t he figure. M
r
values of the standards used were as f o l-
lows, 1, BSA 6 6 kDa, 2 , ov albumin, 45 kDa, 3, carbonic anhydrase,
29 kDa and 4, cytochrome c, 14. 5 k Da.
Fig. 3. Rayleigh light scattering (A) and ANS fluorescence (B,C) studies
of A. hirsuta lectin. (A) The l ectin (1.5 l
M
) was incubated at d iff erent
temperatures for 10 min an d the light scattering was m onitored by
setting kex ¼ kem ¼ 400nm1,50m
M
bufferofpH7.0,2,30°C, 3,
35 °C, 4, 40 °C and 5, 45 °C. (B) C hange in A NS fluorescence in the

presence of A. hirsuta lectin an d GdnHCl. T he fluorescence emission
spectra of the lectin (2.0 l
M
) in the presence of ANS (50 l
M
).
(kex, 375 nm). Numbers on the curves indicate the molarity of
GdnHCl. (C) Change in ANS fl uorescence in the p resence of t he
A. hirsuta lectin a t various temperatures. T he spectra were t aken as
described in (B) prote in sam ples treated a t, 1, 30 °C, 2, 40 °C, 3, 50 °C,
4, 60 °C, and 5, 70 °C. (D) N ear UV CD s pectra of A. hirsuta lectin
(15 l
M
), lectin exposed at 1, 35 °C , 2, 45 °C, 3, 50 °C, 4, 55 °C, 5,
60 °C, 6, 65 °C and 7, 70 °C f or 15 min.
1416 S. M. Gaikwad et al. (Eur. J. Biochem. 269) Ó FEBS 2002
folding and association of subunits of the lectin as compared
to other plant lectins [5,6].
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Opin. Struct. Biol. 1, 741–749.
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binding lectin from Artocarpus hirsuta: c haracterization of the
sugar specificity and binding site. Biochim. Biophys. Acta 1381,
256–264.
3. Gaikwad, S.M., Gurjar, M.M. & Khan, M.I. (1998) Fluorimetric
studies on saccharide binding to the b asic lectin fr om Artocarpus
hirsuta Biochem. Mol . Biol. I nt. 46,1–9.
4. Rao, K.N., Gurjar, M.M., Gaikwad, S.M., Khan, M.I. &
Suresh, C .G. (1999) Crystallization and preliminary X-ray studies

of the basic lectin from the seeds of Artocarpus hirsuta. Acta
Crystallo. D 55, 120 4–1205.
5. Ahmed, N ., Srinivas, V .R., Reddy, G.B. & Surolia, A. (1998)
Thermodynamic characterization of the confor mational stability
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16772.
6. Reddy, G .B., Srinivas, V.R., Ahmed, N . & Surolia, A. (1999)
Molten globule like state of peanut l ectin monomer retains its
carbohydrate specificity. J. Biol. Chem. 274, 4500–4503.
7. Reddy, G.B., Bharadwaj, S. & Surolia, A. (1999) Thermal stability
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