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: rizwanhkhan@hotmail.com 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 . Characterization of a partially folded intermediate of stem bromelain at low pH Soghra Khatun Haq, Sheeba Rasheedi and Rizwan Hasan Khan Interdisciplinary. 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