Báo cáo Y học: Characterization of a partially folded intermediate of stem bromelain at low pH ppt

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

bind the ¯uorescent probe as well as consider able retention
of secondary structure. All the above data taken together
suggest the existence of a partially folded intermediate state
under low pH conditions.
Keywords: acid denaturation; circular dichroism; partially
folded interme diate; s tem b romelain.
The molecular mechanism of the spontaneous folding of
proteins from a random polypeptide chain to the well
ordered native conformation is still unknown. Results of
kinetic refolding experiments in vitro as well as theoretical
considerations suggest that folding of large proteins is a
sequential hierarchical process [1]. Various proteins have
been observed to e xist in stable conformations that are
neither f ully folded nor unfolded and are said t o be in the
Ômolten globuleÕ state [2]. These partially folded in termedi-
ates can be made to accumulate in equilibrium by mild
concentrations of chemical denaturants, low pH, covalent
trapping or by protein engineering [3]. It is now generally
accepted that protein folding involves a discrete pathway
with interme diate states between native and denatured states
[4]. A number of globular proteins are known to s how the
equilibrium unfolding transition that does not obey the two-
state rule but exhibits a compact intermediate that has an
appreciable amount of secondary structure [5±8]. Acid-
induced unfolding of proteins is often incomplete and the
acid-unfolded proteins assume conformations that are
different from the fully unfolded ones observed in the
presence of 6
M
GdnHCl or 9

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

proposed that bromelain f orms may have t he same folding
pattern shown by other members of the papain family as the
spectral characteristics displayed by stem bromelain are
similar to those observed in case of papain and proteinase W
namely, a bilobal structure with predominantly a and
Correspondence to R. Hasan Khan, Interdisciplinary Biotechnology
Unit, Aligarh Muslim U n iversity, Aligarh 202002, India.
Fax: + 9 1 571 701081, Tel.: + 91 571 701718,
E-mail:
Abbreviations: ANS, 1-anilino 8-naphthalene sulfonic acid.
Enzymes: s tem bromelain (EC 3.4.22.32).
(Received 25 June 2001, revised 17 October 2001, accepted 19 October
2001)
Eur. J. Biochem. 269, 47±52 (2002) Ó FEBS 2002
antiparallel b sheet domains [20,21]. Stem bromelain
belongs to the a + b protein class as other cysteine
proteinases do a nd the h ighly identical amino-acid sequenc-
es of papain [22], actinidin [23], proteinase W [24,25]
chymopapain [26,27] and stem bromelain [28] indicate that
the polypeptide chains of these proteins share a common
folding pattern. This has been con®rmed for the ®rst three
proteinases by d etailed X-ray diffraction studies [21,29,30].
In the present communication, we demonstrate the presence
of a partially folded intermediate at pH 2.0 having disor-
dered side chain interactions but with considerable second -
ary structure and relatively more exposed hydrophobic
surface as seen by ¯uorescence, CD and ANS binding.
MATERIALS AND METHODS
Materials
Bromelain (EC 3.4.22.32) lot no. B4882 and 1-anilino

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

distilled water was prepared and c oncentration determined
using an extinction coef®cient of e
M
 5000
M
)1
Ácm
)1
at
350 n m [34]. The molar ratio o f protein to ANS was 1 : 50 .
Acid denaturation
Acid-induced unfolding of stem bromelain was carried ou t
in 10 m
M
solutions of the following buffers: glycine/HCl
(pH 0 .8±2.2), sodium acetate (pH 2.5±6.0), sodium phos-
phate (pH 7.0±8.0) and glycine/NaOH (pH 9.0±10.0). pH
measurements were carried out on an Elico digital pH
meter (model LI 610) with a least count of 0.01 pH unit.
Stem bromelain (12.6±37.8 l
M
) was incubated with the
buffers of desired pH at 4 °C a nd allowed t o equilibrate for
4 h before taking the s pectrophotometric m easurements. In
order to assess the reversibility of acid induce d unfolding,
stem bromelain a t pH 2.0 was extensively dialyzed against
10 m
M
sodium pho sphate buffer, pH 7.0. This dialyzed
preparation was compared to stem bromelain at pH 7.0 and

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

in a 1-mm pathlength
cuvette.
GdnHCl induced denaturation
Denaturation of stem bromelain a t pH 2.0 in the presence
of guanidine hydrochloride was studied by far-UV CD.
Increasing amounts o f 7.2
M
GdnHCl were added t o a ®xed
concentration (21 l
M
) of p rotein and a llowed to e quilibrate
before taking CD measurements at 222 nm. Mean residue
ellipticity (MRE) values were calculated a ccording t o Chen
et al . [35] and plotted against denaturant concentration.
Fraction of protein denatured ( f
D
) was calculated according
to Tayyab et al.[36].
Acrylamide quenching
Quenching of intrinsic tryptophan ¯uorescence was per-
formed on a Hitachi Spectro¯uorometer (model F-2000)
using a stock solution of 5
M
acrylamide. To a ®xed amount
(17.2 l
M
) of protein, increasing amounts of acrylamide
(0.1±1.0
M
) were added and the samples incubated for

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

environment. A similar blue-shifted ¯uoresence has been
reported earlier for glucose isomerase [37], bovine growth
hormone [38] and interferon-c [39]. The addition of 2
M
urea
to the protein at pH 2.0 further decreases the ¯uorescence
intensity a pparently without altering the m icroenvironment
of the aromatic ¯uoropho re. The completely unfolded s tate
of bromelain in the presence of 6
M
GdnHCl shows a red
shift of 4 nm with a concomitant decrease in the ¯uores-
cence intensity. Th ese observations suggest that the protein
at pH 2.0 is present in a conformational state that is
different from the native state at pH 7.0 as well as
completely unfolded state in the presence of 6
M
GdnHCl.
Figure 3 shows the near UV CD spectra of the native
state of the protein, the denatured state of t he protein a nd of
the acid-induced state at pH 2.0. As s een in the ®gure, the
spectrum of stem bromelain at pH 2.0 differs from that at
pH 7.0 a nd resembles the denatured state of the p rotein in
presence of 6
M
GdnHCl. This suggests t hat the protein at
pH 2.0 has most of its tertiary contacts disrupted. However,
the presence of loose tertiary i nteractions in the absence of
tight side chain packing cannot be ruled out.
The changes in the secondary structure of stem b romelain

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

non-native, i ntermediate c onformations of globular proteins
[40]. T his property o f ANS was also u sed t o study th e a cid-
unfolding of stem bromelain (Fig. 6). The ANS ¯uorescence
intensity increases constantly with decrease in pH and is
maximum at pH 0.8. As shown in Fig. 7, stem bromelain at
pH 2.0 shows a marked increase in ANS ¯uorescence
intensity as compared to the native protein at pH 7.0 or
unfolded in the presence of 6
M
GdnHCl. These observa-
tions suggest the presence of a large number of solvent-
accessible nonpolar clusters in the protein molecule at
pH 2.0 as w ell as p H 0.8 as the ANS dye b inds to
hydrophobic surfaces on the protein with greater af®nity.
Denaturation of stem bromelain at pH 2.0 in the presence
of varying amounts of GdnHCl was also investigated by far-
UV CD. As seen in Fig. 8, GdnHCl further induces the
unfolding of the residual secondary structure detected in
stem bromelain at pH 2. 0. E arlier s tudies on the GdnHCl-
induced unfolding of the molten slobule state of
a-lactalbumin also showed a sigmoidal transition curve
[41,42].
The Stern±Volmer plot and the modi®ed Stern±Volmer
plot for quenching of intrinsic protein ¯uorescence by
acrylamide at pH 7.0 and 2.0 are depicted in Fig. 9. The
quenching constants ( K
SV
values) calculated for pH 7.0 and
2.0 w ere 5 .88 and 9.36
M

)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

pH 7.0 (ÐÁÐ), acid-induced state at pH 2.0 (Ð) and 6
M
GdnHCl
denatured state (± ±).
50 S. Khatun Haq et al. (Eur. J. Biochem. 269) Ó FEBS 2002
protein reportedly unfolds gradually beyond pH 10.0 and is
extensively denatured above pH 12.0.
Goto et al. [ 44] have proposed that acid denaturation of
proteins leads t o unfolding of the protein molecule due to
intramolecular charge repulsion. However, proteins exhibit
differential behaviour upon acid denaturation [10]. Our stu-
dies on the acid-induced unfolding of stem b romelain reveal
that stem bromelain exhibits unfolding behaviour charac-
teristic of Type I prote ins as classi®ed by Fink et al. [45].
Results of spectroscopic studies on the reversibility of the
partially folded state at pH 2.0 (data not shown) lead us to
believe that the acid induced unfolding of stem bromelain is
irreversible.
Fluorescence and CD data support the involvement of a n
intermediate state at p H 2.0. This state retains considerable
secondary structure and is c haracterized by its hydrophobic
dye-binding capacity that is lower t han that of t he possible
molten globule state at pH 0.8 but greater than that of the
native state. Acrylamide quenching data clearly show that
stem bromelain at p H 2.0 is in an u nfolded state a s
compared to the protein at neutral pH. The properties of the
pH 2.0 s tate proteins are intermediate between those i n t he
native state and molten globule state and justify its
occurrence on the native (N) ® molten globule (MG)
pathway, therefore w e h ave t ermed th is the p artially f olded

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.
REFERENCES
1. Kuwajima, K. (1989) The molten globule state as a clue for
understanding the foldin g and cooperativity of glo bular-protein
structure. Pr oteins 6, 87± 103.
2. Ohgushi, M. & Wada, A. (1983) ÔMolten-globule stateÕ:acompact
form of globular pro teins with mobile-sid e-chain. FEBS Lett. 164 ,
21±24.
3. Sanz, J.M. & G imenez-Gallego, G. (1997) A partly f olded state of
acidic ®broblast growth fact or at lo w pH. Eur. J. Biochem. 240,
328±335.
4. Kim, P.S. & Baldwin, R .L. ( 1990) Intermediates in the folding
reactions of small proteins. Ann u. Rev. Biochem. 59, 631±660.
5. Kuwajima, K. (1992) Protein folding in vitro. Curr. Opin. Bio-
technol. 3, 462± 467.
6. Ptitsyn, O.B. (1987) Protein folding: hypotheses and experiments.
J. Pro t . Chem. 6, 273 ±293.
7. Ptitsyn, O.B. (1992) Protein Folding (Creighton, T.E., eds),

pp. 2 43±300. W.H. Fre eman, New York.
8. Barrick, D. & Baldwin, R.L. (199 3) Three-state analysis of sperm
whale a pomyoglobin f olding. Bioc hemistry 32, 379 0±3796.
9. Matthews, C.R. (1993) Pathways of protein folding. Annu. Rev.
Biochem. 62, 653±683.
10. Tanford, C . (1968) Prot ein denaturation. Adv. Protein C hem. 23 ,
121±282.
11. Dill, K.A. & Shortle, D. (1991) Denatured states of proteins.
Annu.Rev.Biochem.60, 7 95±825.
12. Nishii,I.,Kataoka,M.&Goto,Y.(1995)Thermodynamicsta-
bility o f the m olten globule states o f apomyoglobin. J. Mol. Biol.
250, 223 ±238.
13. Privalov, P.L. (1996) Intermediate states in protein folding. J. Mol.
Biol. 250, 707± 725.
14. Khan, F., Khan, R.H . & Muzammil, S. (2000) A lcohol-ind uced
versus a nion induced s tates of a-chymotrypsinogen A at low p H.
Biochem. Biophys. Acta. 1481, 229±236.
15. Kay, M.S. & Baldwin, R.L. (1996) Packing interactions in the
apomyglobin folding intermediate. Nat. Struct. Biol. 3, 4 39±445.
16. Song, J., Bai, P., L uo, L. & Peng, Z.Y. (1998) C ontribution of
individual resid ues to formation of t he native-like tertiary topol-
ogy in the alpha-lactalbumin molten globule. J. Mol. Biol. 280,
167±174.
Fig. 9. Stern±Volmer plot (A) a nd modi®ed S tern±Volmer plot (B) of acrylamide quenching. Native stem bromelain at pH 7.0 (s) and ac id-induce d
state at p H 2.0 ( d).
Ó FEBS 2002 Partially folded intermediate of stem bromelain (Eur. J. Biochem. 269)51
17. Wu, L.C. & Kim, P.S. ( 1998) A speci®c hydrophobic core in the
alpha-lactalbun i n molten g lobule. J. Mol. Biol. 280, 175±182.
18. Shortle, D. & Ackerman, M .S. (2001) Persistence of native-like
topology in a denatured p rotein in 8

M
urea. Science 293, 487±489.
19. Bai, P., Song, J., L uo, L. & Peng, Z.Y. (2001) A model of dynamic
side-chain±side-chain interactions in the alpha-lactalbumin molten
globule. Protein S ci. 10, 53±62.
20. Arroyo-Reyna, A ., Hernandez-Arana, A. & Arreguin-Espinosa,
R. (1994) Circular dichroism of stem b romelain a third spectral
class w ithin the fam ily of cysteine proteinases. Biochem. J. 300,
107±110.
21. Kamphuis, I.G., Kalk, K.H., Swarte, M.B.A. & Drenth, J. (1984)
Structure of p apain re®ned at 1 .5 A
Ê
resolution. J. M ol. Biol. 179,
233±257.
22. Cohen, L.W., Coghlan, V.M. & D ihel, L.C. (1 986) Cl oning and
sequencing o f papain-encoding cDNA. Gene 48, 21±227.
23. Carne, A . & Moore, C.H. ( 1978) The amino ac id sequence of the
tryptic peptides f rom actinidin, a pro teolytic enzyme from the fruit
of Actinidia c hinensis. Biochem. J. 173, 73± 83.
24. Dubois, T., Kleinschmidt, T., Schnek, A.G., Looze, Y. &
Braunitzer, G. (1988) The thiol proteinases from the latex of
Carica papaya L. III. The primary structure of p roteinase omega.
Biol. C hem. Hoppe-Seyler 369 , 741±754.
25. Topham, C.M., Salih, E., Frazao, C., Kowlessur, D., Overington,
J.P.,Thomas,M.,Brocklehurst,S.M.,Patel,S.M.,Thomas,E.W.
& Brocklehurst, K. ( 1991) Structure±function relationships in the
cysteine proteinases actinidin, papain and papaya proteinase
omega. Three dimensional structure of papaya proteinase om ega
deduced by kno wledge-based m odellin g and ac tive-centre c har-
acteristics determined by two-hydronic-state reactivity probe

kinetics a n d kinetics of c atalysis. Biochem. J. 280, 79±92.
26. Jacquet, A., Kleinschmidt, T., Schnek, A.G., Loozer, Y. &
Braunitzer, G. (1989) The thiol proteinases from the latex of
Carica papaya L. III. The primary structure of chymopapain. Biol.
Chem. H oppe-Seyler 370, 425±434.
27. Watson, D.C., Yaguchi, M. & Lynn, K.R. (1990) The amino acid
sequence of chymopapain from Carica papaya. Biochem. J. 266,
75±81.
28. Ritonja, A., Rowan, A.D., Buttle, D.J., Rawlings, N.D., Turk, V.
& Barett, A.J. (1989) Stem bromelain: amino acid sequence and
implications for weak binding of cystatin. FEBS Lett. 247, 419±424.
29. Baker, E.N . ( 1980) Structure of actinidin af ter re®nement at 1.7 A
Ê
resolution. J. Mol. Biol. 141, 4 41±484.
30. Pickersgill, R.W., Sumner, I.G. & Goodenough, P.W. (1990) Eur.
J. Bioc hem. 190, 443±449.
31. Sharpira, E. & Arnon, R . (1969) Cleavage of one speci®c disul®de
bondinpapain.J. Biol. Chem. 24 4, 4989±4994.
32. Arroyo-Reyna, A. & Hernandez-Arana, A. (1995 ) T he therm al
denaturaton of stem bromelain is consistent with an irreversible
two-state model Biochem. Biophys. Acta. 1248, 123±128.
33. Vanhoof, G . & Cooreman, W. (1997) Bromelain. Pharmaceutical
Enzymes (Lauwers, A. & Scharpe, S., eds), Marcel Dekker Inc.,
New Y ork.
34. Khurana, R. & Udgaonkar, J.B. (1994) Equilibrium unfolding
studies of barstar: evidence for an alternative conformation which
resembles a molten globule. Biochemistry 33, 106±115.
35. Chen, Y .H., Yang, J.T. & Martinez, H.M. (1972) Determination
of the s econd ary struc ture of proteins by circu lar d ichro ism a nd
optical rota tory dispersion. Bioc hemistry 11, 4120±4131.

36. Tayyab, S., Siddiqui, M.U. & Ahmad, N. (1995) Experimental
determination of the free energy of unfolding of proteins. Biochem.
Ed. 3, 162±164.
37. Pawar, S.A. & D eshpande, V.V. (2000) Characterization of acid-
induced u nfo lding in terme diates o f glucose/xylose i somerse. Eur.
J. Bioc hem. 267, 6331±6338.
38. Holzman, T.E., Do ugherty, J.J., Brems, D.N . & Ma cKenz ie, N.E.
(1990) pH-induced conformational states of bovine growth hor-
mone. Biochemistry 29 , 1255±1261.
39. Nandi, P .K. (1998) Evidence of molten globule like state(s) of
interferon gamma i n acid ic and so dium perc hlorate solutio ns. Int.
J. Biol. M ac romo l. 22, 23±31.
40. Semisotnov, G .V., Rodionova, N.A., Razgulyaev, O.I., Uversky,
V.N., Gripas, A.F. & Gilmanshin, R.I. (1991) Study of the Ômolten
globuleÕ intermediate state in protein f olding by a hydrophobic
¯uorescent probe. Bio polymers 31, 119±128.
41. Kuwajima, K., Nitta, K., Yoneyama, M . & Suga i, S. (1976) Three-
state denaturation of a-lactalbumin by guanidine hydrochloride.
J. Mol. Biol. 106, 3 59±373.
42. Ikeguchi, M., Kuwajima, K ., Mitani, M . & Sugai, S. (1986) Evi-
dence for identity between the equilibrium unfolding intermediate
and a transien t f olding inte rm ediate: a comparative study of the
folding reactions of a-lactalbumin and lysozyme. Biochemistry 25,
6965±6972.
43. Murachi, T. & Yamazaki, M. (1970) Changes in conformation
and e nzymatic a ctivity of stem brome lain i n alkaline media. Bio-
chemistry 9, 1935 ±1938.
44. Goto, Y., Takahashi, N. & Fink, A.L. (1990) Mechanism of Acid-
induced folding of proteins. Biochemistry 29, 3480±3488.
45. Fink, A .L., Calciano, L.J., Goto, Y., Kurotsu, T. & Palleros, D.R.

(1994) Classi®cation of acid denaturation of proteins: inter-
medates and u nfo lded states. Biochemistry 33 , 12504±12511.
46. Lala, A .K. & Kaul, P. ( 1992) Increased exposure of hydrophobic
surfaceinmoltenglobulestateofa-lactalbumin: ¯uorescence and
hydrophobic pho tolabellin g st udies . J. Biol. Chem . 267, 19914±
19918.
47. Gussakovsky, E .E. & Haas, E. (1995) Two s teps in the transition
between the native and acid states o f bovine a-lactalbumin
detected by circular polarization of luminescence: evidence for a
pre-molten globule state. Pro tein Sci. 4, 2319±2326.
52 S. Khatun Haq et al. (Eur. J. Biochem. 269) Ó FEBS 2002