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

Eur. J. Biochem. 269, 47–52 (2002) (cid:211) FEBS 2002

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

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 of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable 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 intermediate; stem bromelain.

Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near- UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary 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 containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state

conformational states located between the native 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 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 exist in stable conformations that are neither fully folded nor unfolded and are said to be in the (cid:212)molten globule(cid:213) state [2]. These partially folded intermedi- 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 intermediate states between native and denatured states [4]. A number of globular proteins are known to show 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

The characteristic features of a (cid:212)molten-globule(cid:213) are: (a) it is less compact than the native state; (b) it is more compact than the unfolded state; (c) it contains extensive secondary stricture; and (d) it has loose tertiary contacts without tight side-chain packing. Recently, increasing evidence supports the idea that the molten globule may possess well-defined tertiary contacts [15–18]. Proteins in the molten globule state 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 have a significant bearing in understanding the protein-folding problem [19].

While a detailed study on the denaturation and refolding aspects of papain, a thiol protease has been made by several workers; no studies on the acid denaturation of stem bromelain, a protelytic cysteinyl protease from Ananas comosus has been made till date. Arroyo-Reyna et al. have proposed that bromelain forms may have the 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 University, Aligarh 202002, India. Fax: + 91 571 701081, Tel.: + 91 571 701718, E-mail: rizwanhkhan@hotmail.com Abbreviations: ANS, 1-anilino 8-naphthalene sulfonic acid. Enzymes: stem bromelain (EC 3.4.22.32). (Received 25 June 2001, revised 17 October 2001, accepted 19 October 2001)

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the partially folded state at pH 2.0 using fluorescence and CD.

Fluorescence measurements

antiparallel b sheet domains [20,21]. Stem bromelain belongs to the a + b protein class as other cysteine proteinases do and the highly 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 confirmed for the first three proteinases by detailed 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 fluorescence, CD and ANS binding.

M A T E R I A L S A N D M E T H O D S

Fluorescence measurements were carried out on a Shimadzu Spectrofluorometer (model RF-540) equipped with a data recorder DR-3 and on a Hitachi Spectroflurometer (model F-2000). The concentration of stem bromelain used was in the range 13.9–14.5 lM. For the intrinsic tryptophan fluorescence, the excitation wavelength was set at 280 nm and the emission spectra recorded in the range of 300– 400 nm 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 were recorded from 400 to 600 nm with 10-nm slit width for excitation and emission.

Materials

CD measurements

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

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 (cid:176)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 protein concentration of 0.9 mg(cid:1)mL)1 in a 10-mm pathlength cuvette. The far-UV CD studies were made in the wavelength region of 200–250 nm with a concentration of 0.3 mg(cid:1)mL)1 in a 1-mm pathlength cuvette.

GdnHCl induced denaturation

To avoid complications due to autocatalysis, enzyme samples were irreversibly inactivated by the method of Sharpira & Arnon [31] with certain modifications. Reduc- tion was carried out in 0.32 M 2-mercaptoethanol for 4 h at room temperature, followed by addition of solid iodoace- tamide to give a final concentration of 0.043 M. After stirring for 30 min at 4 (cid:176)C, the solutions were dialyzed overnight against 10 mM sodium phosphate buffer, pH 7.0. This inactive derivative was used throughout the present study.

Spectrophotometric measurements

Denaturation of stem bromelain at pH 2.0 in the presence of guanidine hydrochloride was studied by far-UV CD. Increasing amounts of 7.2 M GdnHCl were added to a fixed concentration (21 lM) of protein and allowed to equilibrate before taking CD measurements at 222 nm. Mean residue ellipticity (MRE) values were calculated according to Chen et al. [35] and plotted against denaturant concentration. Fraction of protein denatured ( fD) was calculated according to Tayyab et al. [36].

Acrylamide quenching

The protein concentration was determined on a Hitachi U-1500 Spectrophotometer using an extinction coefficient e1% 1cm;280nm (cid:136) 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 concentration determined )1(cid:1)cm)1 at using an extinction coefficient of eM (cid:136) 5000 M 350 nm [34]. The molar ratio of protein to ANS was 1 : 50.

Acid denaturation

Quenching of intrinsic tryptophan fluorescence was per- formed on a Hitachi Spectrofluorometer (model F-2000) using a stock solution of 5 M acrylamide. To a fixed amount (17.2 lM) of protein, increasing amounts of acrylamide (0.1–1.0 M) were added and the samples incubated for 30 min prior to taking the fluorescence measurements. For the intrinsic tryptophan fluorescence spectra, the protein samples were excited at 295 nm and emission spectra recorded between 250 and 550 nm and the data obtained were analyzed according to the Stern–Volmer equation [37].

R E S U L T S A N D D I S C U S S I O N

Acid-induced unfolding of stem bromelain was carried out in 10 mM 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 lM) was incubated with the buffers of desired pH at 4 (cid:176)C and allowed to equilibrate for 4 h before taking the spectrophotometric measurements. In order to assess the reversibility of acid induced unfolding, stem bromelain at pH 2.0 was extensively dialyzed against 10 mM sodium phosphate buffer, pH 7.0. This dialyzed preparation was compared to stem bromelain at pH 7.0 and

The acid denaturation of stem bromelain was studied over a pH range of 0.8–10.0. Stem bromelain contains five tryptophan residues [28] and extensive sequence homology with papain suggests that three tryptophans are buried in

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Partially folded intermediate of stem bromelain (Eur. J. Biochem. 269) 49

hydrophobic core whereas two of them are located near the surface of the molecule. As the intrinsic fluorophore tryptophan is highly sensitive to the polarity of its surrounding environment, the pH dependent changes in the conformation of stem bromelain were followed using fluorescence spectroscopy. As seen from Fig. 1, with the lowering of pH, the relative fluorescence 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.

cence intensity. These 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 the protein and of the acid-induced state at pH 2.0. As seen in the figure, the spectrum of stem bromelain at pH 2.0 differs from that at pH 7.0 and resembles the denatured state of the protein in presence of 6 M GdnHCl. This suggests that the protein at pH 2.0 has most of its tertiary contacts disrupted. However, the presence of loose tertiary interactions in the absence of tight side chain packing cannot be ruled out.

The changes in the secondary structure of stem bromelain as a function of pH were also followed by far-UV CD by measuring mean residue ellipticity values at 208 nm (Fig. 4). A cooperative transition from the native to the unfolded state occurs in the vicinity of pH 4.5 reflecting loss of secondary structure. However, at pH 2.0, stem bromelain retains some secondary structural features (Fig. 5). On further lowering of pH; stem bromelain regains a significant 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.

The emission spectrum of stem bromelain at pH 7.0 (Fig. 2) shows a maximum at 347 nm that suggests that some of the tryptophan residues of the protein are relatively more exposed to solvent. However at pH 2.0 there is a decrease in the fluorescence emission intensity with a slight blue shift ((cid:25) 3–4 nm). This blue-shifted fluorescence of stem bromelain at pH 2.0 can be attributed to the conforma- tional changes in the vicinity of the surface exposed tryptophans; in this case internalization in a hydrophobic environment. A similar blue-shifted fluoresence 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 fluorescence intensity apparently without altering the microenvironment of the aromatic fluorophore. The completely unfolded state of bromelain in the presence of 6 M GdnHCl shows a red shift of 4 nm with a concomitant decrease in the fluores-

Fig. 3. Near UV-CD spectra of stem bromelain. Native protein at pH 7.0 (—(cid:1)—), acid-induced state at pH 2.0 (—) and 6 M GdnHCl denatured state (– –).

Fig. 1. E(cid:128)ect of pH on the emission fluoresence intensity of stem bromelain. Ten millimolar solutions of glycine/citrate/phosphate buf- fers were used in the pH range 0.8–10.0.

Fig. 4. E(cid:128)ect of pH on the mean residue ellipticity (MRE) of stem bromelain. Ellipticity was monitored at 208 nm by far UV CD. Fig. 2. Spectroscopic characterization of stem bromelain: fluoresence emission spectra of stem bromelain at pH 7.0 (1), pH 7.0 + 6 M GdnHCl (2), pH 2.0 (3) and pH 2.0 + 2 M urea (4). Excitation and emission wavelengths were 280 nm and 345 nm, respectively.

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Fig. 5. Far UV-CD spectra of stem bromelain. Native protein at pH 7.0 (—(cid:1)—), acid-induced state at pH 2.0 (—) and 6 M GdnHCl denatured state (– –).

the molten slobule

unfolding of the residual secondary structure detected in stem bromelain at pH 2. 0. Earlier studies on the GdnHCl- induced unfolding of state of a-lactalbumin also showed a sigmoidal transition curve [41,42].

Changes in ANS fluoresence are frequently used to detect non-native, intermediate conformations of globular proteins [40]. This property of ANS was also used to study the acid- unfolding of stem bromelain (Fig. 6). The ANS fluorescence 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 fluorescence 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 well as pH 0.8 as the ANS dye binds to hydrophobic surfaces on the protein with greater affinity.

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

The Stern–Volmer plot and the modified Stern–Volmer plot for quenching of intrinsic protein fluorescence by acrylamide at pH 7.0 and 2.0 are depicted in Fig. 9. The quenching constants (KSV values) calculated for pH 7.0 and )1, respectively. The Stern–Volmer 2.0 were 5.88 and 9.36 M plot indicates that the aromatic amino-acids in the protein at pH 2.0 are more exposed to the solvent as compared to the native folded conformation at pH 7.0; therefore tryptophan fluorescence is quenched more in case of the former.

Earlier studies on the effect of alkaline media on stem bromelain have reported no comformational change in the protein from pH 7.0–10.0 as no significant change in physical parameters is detected in this pH region [43]. The

Fig. 7. Interaction of ANS with various forms of stem bromelain. Native protein at pH 7.0 (1); 6 M GdnHCl-denatured state (2); acid-induced state at pH 2.0 (3); acid-induced state in the presence of 2 M urea (4).

Fig. 6. E(cid:128)ect of pH on the ANS fluorescence intensity of stem brome- lain. (kex (cid:136) 380 nm). 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 were added to a fixed amount of protein (21 lM). Inset shows fraction denatured ( fD) against denaturant concentration.

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Partially folded intermediate of stem bromelain (Eur. J. Biochem. 269) 51

Fig. 9. Stern–Volmer plot (A) and modified Stern–Volmer plot (B) of acrylamide quenching. Native stem bromelain at pH 7.0 (s) and acid-induced state at pH 2.0 (d).

R E F E R E N C E S

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