Characterization of a partially folded intermediate of stem
bromelain at low pH
Soghra Khatun Haq, Sheeba Rasheedi and Rizwan Hasan Khan
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, India
Equilibrium studies on the acid included denaturation of
stem bromelain (EC 3.4.22.32) were performed by CD
spectroscopy, ¯uorescence emission spectroscopy and
binding of the hydrophobic dye, 1-anilino 8-naphthalene
sulfonic acid (ANS). At pH 2 .0, stem bromelain lacks a well
de®ned tertiary structure as seen by ¯uorescence and near-
UV CD spectra. F ar-UV CD spectra show retention of some
native like s econdary structure at p H 2.0. T he mean residue
ellipticities at 208 nm plotted against pH showed a transition
around pH 4.5 with loss of s econdary structure leading to
the formation of an acid-unfolded state. With further
decrease in pH, this unfolded state regains most of its sec-
ondary structure. At pH 2.0, stem bromelain exists as a
partially folded intermediate c ontaining about 42.2% of th e
native state s econdary structure E nhanced binding of ANS
was observed i n this s tate compared to the n ative folded state
at neutral pH or completely unfolded state in the presence of
6
M
GdnHCl indicating the exposure of hydrophobic regions
on the protein molecule. Acrylamide quenching of the
intrinsic tryptophan residues in the protein molecule showed
that at pH 2.0 the protein is in an unfolded conformation
with more tryptophan residues exposed to the solvent as
compared to the native conformation at neutral pH. Inter-
estingly, stem bromelain at pH 0.8 exhibits some charac-
teristics o f a molten globule, such as an enhanced ability to

M
urea [9±11]. Such stable
conformational states located b etween the n ative and
unfolded states have been found for several proteins [12].
Several studies have shown that the compactness and the
amount of secondary structure of the intermediate states
formed in the folding pathway of proteins are not neces-
sarily close to those of the native state, but vary greatly
depending on the protein species [1,13]. This suggests the
presence of various intermediate states, from one close to
the fully unfolded state to one close to the native state
depending upon the protein and the experimental condi-
tions [14].
The characteristic fe atures of a Ômolten-globuleÕ are: (a) i t
is less compact than the native state; (b) i t i s m ore c ompact
than the un folded state; (c) it contains extensive secondary
stricture; and ( d) it has loose t ertiary contacts without tight
side-chain packing. Recently, increasing evidence supports
the idea that the molten globule may possess well-de®ned
tertiary contacts [15±18]. Proteins in the molten g lobule s tate
contain high level of secondary structure, as well as a
rudimentary, native like tertiary topology. Thus, the struc-
tural similarity between the molten globule and native
proteins may h ave a s igni®cant bearing in understanding the
protein-folding problem [19].
While a detailed s tudy on the denaturation a nd refolding
aspects of p apain, a thiol protease has b een made by s everal
workers; no studies on the acid denaturation of stem
bromelain, a protelytic cysteinyl protease from Ananas
comosus has been made till date. A rroyo-Reyna et al. have

8-naphthalene sulfonic acid (ANS) were purchased from
Sigma Chemical Co., USA. Guanidine hydrochloride
(GdnHCl) was obtained from Qualigens, India. Acrylamide
and urea were purchased from Sisco Research Laboratories,
India. All other reagents were of analytical grade.
Autolysis inhibition
To avoid complications due to autocatalysis, enzyme
samples were irreversibly inactivated by the method of
Sharpira & Arnon [31] with certain modi®cations. Reduc-
tion was carried o ut i n 0 .32
M
2-mercaptoethanol for 4 h a t
room temperature, followed by addition of solid iodoace-
tamide to give a ®nal concentration of 0.043
M
.After
stirring for 30 min at 4 °C, the solutions were dialyzed
overnight a gainst 10 m
M
sodium phosphate buffer, pH 7.0.
This inactive derivative was used throughout the present
study.
Spectrophotometric measurements
The protein concentration was determined on a Hitachi
U-1500 Spectrophotometer using an extinction coef®cient
e
1%
1cmY280nm
 20.1 [32]. The molecular mass of the protein
was taken as 23 800 [33]. A stock solution of ANS in

the partially folded state at pH 2.0 using ¯uo rescence and
CD.
Fluorescence measurements
Fluorescence measurements were carried out on a Shimadzu
Spectro¯uorometer (model RF-540) equipped with a data
recorder DR-3 and on a Hitachi Spectro¯urometer (model
F-2000). The concentrat ion of stem bromelain used was in
the range 13.9±14.5 l
M
. For the intrinsic tryptophan
¯uorescence, the excitation wavelength was set at 280 nm
and the emission spectra recorded in the range of 300±
400 n m with 5- and 10-nm slit widths for excitation and
emission, respectively. Binding of ANS to stem bromelain at
various pH values was studied by exciting the dye at 380 nm
and the emission spectra wer e recorded from 400 to 600 nm
with 10-nm slit width for excitation and emission.
CD measurements
CD measurements were carried out on a Jasco J-720
Spectropolarimeter equipped with a microcomputer and
precalibrated with (+)-10-camphorsulfonic acid. All the
CD measurements were carried out at 30 °C and each
spectrum was recorded as an average of two scans. The
near-UV spectra were recorded in the wavelength region of
250±300 nm with a p rotein concentration of 0.9 mgÁmL
)1
in a 10-mm pathlength cuvette. The far-UV C D s tudies were
made in the wavelength region of 200±250 nm with a
concentration of 0.3 mgÁmL
)1

30 min p rior to taking the ¯uorescence m easurements. For
the intrinsic tryptophan ¯uorescence spectra, the protein
samples were excited at 295 n m and emission spectra
recorded between 250 and 550 n m and the data obtained
were analyzed according t o the Stern±Volmer equation [37].
RESULTS AND DISCUSSION
The acid denaturation of stem bromelain was studied over a
pH range of 0.8±10.0. Stem bromelain contains ®ve
tryptophan residues [ 28] and extensive sequence homology
with papain suggests that three tryptophans are buried in
48 S. Khatun Haq et al. (Eur. J. Biochem. 269) Ó FEBS 2002
hydrophobic core w hereas two o f them a re located n ear the
surface of the molecule. As the intrinsic ¯uorophore
tryptophan is highly sensitive to the polarity of its
surrounding environment, the pH dependent changes in
the conformation of stem bromelain were followed using
¯uorescence spectroscopy. As seen from Fig. 1, with the
lowering of pH, the relative ¯uorescence of stem bromelain
gradually decreases to pH 2.0 and becomes more or less
constant, indicative of the presence of a non-native stable
intermediate at low pH.
The emission spectrum of stem bromelain at pH 7.0
(Fig. 2) shows a maximum at 347 nm that suggests that
some of the t ryptophan residues of the protein are relatively
more exposed to solvent. However at pH 2.0 there is a
decrease in the ¯uorescence emission intensity with a slight
blue shift (% 3±4 nm). This blue-shifted ¯uorescence of stem
bromelain at pH 2.0 can be attributed to the conforma-
tional changes in the vicinity o f t he surface exposed
tryptophans; in this case internalization in a hydrophobic

as a function of pH were also followed by far-UV CD by
measuring mean residue ellipticity values a t 208 nm (Fig. 4 ).
A cooperative transition from the native to the unfolded
state occurs in the vicinity of pH 4.5 re¯ecting loss of
secondary structure. Howe ver, at pH 2.0, stem bromelain
retains some secondary structural features (Fig. 5). On
further lowering of pH; stem bromelain regains a signi®cant
amount (42.2%) of the lost secondary structure due to
effective shielding of repulsive forces by the anions but the
tertiary structural loss as seen by near-UV CD is not
regained.
Fig. 1. Eect of pH on the emission ¯uoresence intensity of stem
bromelain. Ten millimolar solutions of glycine/citrate/phosphate buf-
fers wer e used in the pH r ange 0.8±10.0.
Fig. 2. Spectroscopic characterization of stem bromelain: ¯uoresence
emission spectra of stem bromelain at pH 7.0 (1), pH 7.0 + 6
M
GdnHCl (2), p H 2 .0 (3) and pH 2 .0 + 2
M
urea (4). Excitation and
emission wave lengths were 280 nm and 345 nm, respectively.
Fig. 3. Near UV-CD spectra of stem bromelain. Native protein at
pH 7.0 (ÐÁÐ), acid-induced state at pH 2.0 (Ð) and 6
M
GdnHCl
denatured state (± ±).
Fig. 4. Eect of pH on the mean residue ellipticity (MRE) of stem
bromelain . Ellipticity w as monitored a t 208 nm by far UV CD.
Ó FEBS 2002 Partially folded intermediate of stem bromelain (Eur. J. Biochem. 269)49
Changes in ANS ¯uoresence are frequently used to detect

)1
, r espectively. The Stern±Volmer
plot indicates that the aromatic amino-acids in the protein at
pH 2.0 are more exposed to t he solvent as compared to t he
native folded conformation at pH 7.0; therefore tryptophan
¯uorescence is quenched more in case of the former.
Earlier studies on the e ffect of alkaline media on stem
bromelain have reported no comformational change in the
protein f rom pH 7.0±10.0 as n o s igni®cant change i n
physical parameters is detected in this pH region [43]. The
Fig. 6. Eect o f pH o n t he ANS ¯uorescence intensity of s tem b rome-
lain. (kex  38 0 nm).
Fig. 7. Interaction of ANS with various forms of stem bromelain. Native
protein a t pH 7.0 (1); 6
M
GdnHCl-denatured state (2); a cid-induc ed
state a t pH 2.0 ( 3); acid-induced state in t he presence of 2
M
urea (4).
Fig. 8. GdnHCl induced transition of stem bromelain at pH 2.0 as
monitored by far-UV CD changes at 222 nm . Increasing amounts of
7.2
M
GdnHCl we re ad ded to a ®xed amount of protein (21 l
M
). Inset
shows fraction d en atured ( f
D
) a gainst denaturant c oncentration.
Fig. 5. Far UV-CD spectra of stem bromelain. Native protein at

state. A similar intermediate state on the N ® MG
pathway, termed the premolten globule state, has been
localized at pH 5.0 for the apo-a-lactalbumin by Lala &
Kaul [46] and between pH 3.7 and 4.0 for Ca
2+
-saturated
bovine a-lactalbumin by Gussakovsky & Haas [47].
ACKNOWLEDGEMENT
Facilities provided by the Aligarh Muslim University are gratefully
acknowledged Financial a ssistance in the form of research fellowship to
S. K. H. by Council of Scienti®c and Industrial Research and
studentship to S . R . by D epartment o f B iotechnology, G ovt of I ndia
is gratefully ackno wledged.
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