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

269, 47-52 2002 © FEBS 2002 Characterization of a partially folded intermediate of stem bromelain at low pH Soghra Khatun Haq, Sheeba Rasheedi and Rizwan Hasan Khan Interdisciplinary

Eur J Biochem 269, 47-52 (2002) © 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

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

at neutral pH or completely unfolded state in the presence of 6M 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

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

‘molten globule’ 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 mM GdnHCl or 9 m urea [9-11] Such stable

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)

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 characteristic features of a ‘molten-globule’ 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 Q namely, a bilobal structure with predominantly œ and

antiparallel B sheet domains [20,21] Stem bromelain

belongs to the « + § protein class as other cysteine

proteinases do and the highly identical amino-acid sequenc-

es of papain [22], actinidin [23], proteinase Q [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

MATERIALS AND METHODS

Materials

Bromelain (EC 3.4.22.32) lot no B4882 and _ I-amlino

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 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 °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

The protein concentration was determined on a Hitachi

U-1500 Spectrophotometer using an extinction coefficient

E14 080nm — 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

using an extinction coefficient of ex, = 5000 m'-cm™ at

350 nm [34] The molar ratio of protein to ANS was | : 50

Acid denaturation

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 uM) was incubated with the

buffers of desired pH at 4 °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 partially folded state at pH 2.0 using fluorescence and

CD

Fluorescence measurements

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 um 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

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 protein concentration of 0.9 mg-mL™ ina 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-mL7! in a l-mm pathlength cuvette

GdnHCl induced denaturation 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 GdnHC] were added to a fixed concentration (21 um) 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 (/p) was calculated according

to Tayyab et al [36]

Acrylamide quenching 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 um) of protein, increasing amounts of acrylamide (0.1-1.0 mM) 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]

RESULTS AND DISCUSSION

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

© FEBS 2002

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

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 (& 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-y [39] The addition of 2 mM 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 Mm GdnHCl shows a red

shift of 4 nm with a concomitant decrease in the fluores-

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Fig 1 Effect 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

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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

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

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

Fig 3 Near UV-CD spectra of stem bromelain Native protein at

pH 7.0 (——), acid-induced state at pH 2.0 (—) and 6 m GdnHCl denatured state (— -)

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Fig 4 Effect of pH on the mean residue ellipticity (MRE) of stem bromelain Ellipticity was monitored at 208 nm by far UV CD.

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Wavelength (nm)

Fig 5 Far UV-CD spectra of stem bromelain Native protein at

pH 7.0 (——), acid-induced state at pH 2.0 (—) and 6 m GdnHCl

denatured state (— —)

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

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Fluorescence

Intensity

pH Fig 6 Effect of pH on the ANS fluorescence intensity of stem brome-

lain (Aex = 380 nm)

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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)

unfolding of the residual secondary structure detected in stem bromelain at pH 2 0 Earlier studies 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 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 (Ksy values) calculated for pH 7.0 and 2.0 were 5.88 and 9.36 m _, respectively The Stern—Volmer 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 1s detected 1n this pH region [43] The

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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 um) Inset shows fraction denatured (fp) against denaturant concentration.

© FEBS 2002

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

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Fig 9 Stern-Volmer plot (A) and modified Stern-Volmer plot (B) of acrylamide quenching Native stem bromelain at pH 7.0 (©} and acid-induced state at pH 2.0 (@)

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 to 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 bromelain reveal

that stem bromelain exhibits unfolding behaviour charac-

teristic of Type I proteins as classified by Fink ef 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 an

intermediate state at pH 2.0 This state retains considerable

secondary structure and is characterized by its hydrophobic

dye-binding capacity that is lower than that of the 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 pH 2.0 is in an unfolded state as

compared to the protein at neutral pH The properties of the

pH 2.0 state proteins are intermediate between those in the

native state and molten globule state and justify its

occurrence on the native (N) > molten globule (MG)

pathway, therefore we have termed this the partially folded

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” ” -saturated

bovine o-lactalbumin by Gussakovsky & Haas [47]

ACKNOWLEDGEMENT

Facilities provided by the Aligarh Muslim University are gratefully

acknowledged Financial assistance in the form of research fellowship to

S.K H by Council of Scientific and Industrial Research and

studentship to S R by Department of Biotechnology, Govt of India

is gratefully acknowledged

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