Characterization of a partially folded intermediate of papain induced by fluorinated alcohols at low...

Characterization of PartiallyFolded Intermediates ofPapain in Presence ofCationic, Anionic, and NonionicDetergents at Low pH

Aabgeena Naeem

Sadaf Fatima

Rizwan Hasan KhanInterdisciplinary

Biotechnology Unit,Aligarh Muslim University,

Aligarh-202002, India

Received 22 September 2005;revised 17 March 2006;accepted 23 March 2006

Published online 5 April 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20520

Abstract: A systematic investigation of the effects of detergents [Sodium dodecyl sulphate (SDS),hexa decyltrimethyl ammonium bromide (CTAB) and Tween-20] on the structure of acid-unfoldedpapain (EC.3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence,and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits a substantial amount of sec-ondary structure and is relatively less denatured compared with 6 M GdnHCl (guanidine hydro-chloride) but loses the persistent tertiary contacts of the native state. Addition of detergents causedan induction of a-helical structure as evident from the increase in the mean residue ellipticity valueat 208 and 222 nm. Near-UV CD spectra also showed the regain of native-like spectral features inthe presence of 8 mM SDS and 3.5 mM CTAB. Induction of structure in acid-unfolded papain wasgreater in the presence SDS followed by CTAB and Tween-20. Intrinsic tryptophan fluorescencestudies indicate the change in the environment of tryptophan residues upon addition of detergents toacid-unfolded papain. Addition of 8 mM SDS resulted in the loss of ANS binding sites exhibited by adecrease in ANS fluorescence intensity, suggesting the burial of hydrophobic patches. MaximumANS binding was obtained in the presence of 0.1 mM Tween-20 followed by CTAB, indicating a com-pact ‘‘molten-globule’’–like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in the presence of detergents showed the partial recovery of enzymatic activity. Theseresults suggest that papain at low pH and in the presence of SDS exists in a partially folded state char-acterized by native-like secondary structure and tertiary folds. While in the presence of Tween, acid-unfolded papain exists as a compact intermediate with molten-globule–like characteristics, viz.enhanced hydrophobic surface area and retention of secondary structure. While in the presence ofCTAB it exists as a compact intermediate with regain of native-like secondary and partial tertiarystructure as well as high ANS binding with the partially recovered enzymatic activity, i.e., a moltenglobule state with tertiary folds. # 2006 Wiley Periodicals, Inc. Biopolymers 83: 1–10, 2006

This article was originally published online as an accepted preprint. The ‘‘Published Online’’ datecorresponds to the preprint version. You can request a copy of the preprint by emailing theBiopolymers editorial office at [email protected]

Keywords: anion-induced state; circular dichroism; molten globule; papain; partially foldedintermediate state

INTRODUCTION

To understand the factors governing the formation of

three-dimensional protein structures, it is important

to elucidate the hierarchy of interactions that stabilize

the native and molten globule states. A molten globule

exists as an intermediate between native and denatured

states, is defined as a compact conformation with a

Correspondence to: R. H. Khan; e-mail: [email protected] or [email protected]

Biopolymers, Vol. 83, 1–10 (2006)

# 2006 Wiley Periodicals, Inc.

1

comparable amount of native-like secondary structure

but a high enhancement in the intramolecular motion,

i.e., a largely disordered tertiary structure.1 A large

barrier to crack the protein-folding code is the high

cooperativity of folding reactions at equilibrium. A

solvent perturbation study is a good approach to evalu-

ate the stabilizing force of a protein structure. The

interesting cosolvents in this context can be either

polyols and sugars or alcohol and surfactants since

they have potential protecting effects on various types

of protein denaturation.2,3

One of the main objectives in the solution of the

‘‘protein-folding problem’’ is to characterize these par-

tially folded stable intermediate states.4,5 Several stud-

ies have shown that the amount of secondary structure

and the compactness of the intermediate state formed

in the folding pathway of protein are not necessarily

close to those of the native state, but vary greatly

depending on the protein species.6,7 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 experimental

conditions. There has been considerable interest in

whether a new type of intermediate state, which may

be located between the native state and the molten

globule state or the molten globule state and the

unfolded state, exists.4,8 Characterization of the transi-

tion state of proteins, which is believed to be located

between the molten globule state and the native state,

is an important topic in protein folding.7,9–11 A number

of equilibrium intermediates have been detected and

characterized. The high cooperativity and complexity

of the protein-folding process makes the characteriza-

tion of conformational transitions and equilibrium in-

termediate states hard to achieve; different methods

are employed for the characterization of folding inter-

mediates and for understanding the different factors

leading to the formation and stabilization of these

intermediates. There is evidence that supports the idea

that the molten globule may also possess well-defined

tertiary contacts.12–15

Surfactants have a polar head and a nonpolar tail;

the latter causes a hydrophobic effect, which arises pri-

marily from the strong attractive forces between water

molecules themselves. The forces involved between

protein and surfactants are both electrostatic and hydro-

phobic. Surfactants are unique among protein denatur-

ants; even as low as 1 mM SDS, for instance, can often

alter protein conformation. The addition of surfactant

can either increase or decrease the helical conforma-

tion. This is unlike the unfolding of most proteins in 5–

6 M guanidine hydrochloride or 8–10 M urea. Deter-

gent extraction of integral proteins from the membranes

often results in disruption of protein subunits or even

converts � forms into helices.16,17

Papain, one of the sulfhydryl proteases from the la-

tex of Carica papaya, is well studied because of the

molecular structure and many industrial applications.

The interesting feature of this protein is its molecular

structure, which is divided essentially in the form of

two distinct lobes separated by a cleft. The structural

stability of each lobe is at least partly due to the hydro-

phobic core to which many side chains contribute. It is

suggested that the folding of each lobe is independent

of the other and this feature of the papain may indicate

its binuclear nature. The folding of the polypeptide

chain is seen to be different in the two lobes.18,19 Here

we report the different equilibrium intermediates of pa-

pain in the presence of different detergents. The

approach taken was to obtain different intermediates

starting from a common point, i.e., the acid-unfolded

state to detergent-induced states.

MATERIALS AND METHODS

Materials

Papain (EC.3.4.22.2) from the dried latex of papaya was

purchased from Sigma (St. Louis, MO, USA) and the pu-

rity was checked by SDS–PAGE. Sodium dodecyl sulphate

(SDS), hexa decyltrimethyl ammonium bromide (CTAB),

and Tween (Sigma) were used without any further purifica-

tion. All other chemicals used in this study were of analyti-

cal grade.

Methods

All of the measurements were carried out at room tempera-

ture. Typically, papain stock solution (5 mg/mL) was pre-

pared in and dialyzed against 20 mM Gly HCl buffer,

pH 2.0. The concentration of native papain in 20 mM phos-

phate buffer was determined from the extinction coefficient

of 25 M–1cm–1 at 280 nm, pH 7. For studying the effect of

detergents on acidic pH, the unfolded protein was incu-

bated at room temperature for 30 min to carry out spectro-

scopic measurements.

Circular Dichroism Measurements. Circular dichroism

(CD) was measured with a JASCO J 715 spectropolarim-

eter (Tokyo, Japan) calibrated with ammonium D-10

camphorsulfonate. Cell path lengths of 0.1 and 1 cm were

used for scanning between 250–200 and 300–250 nm,

respectively. Protein concentrations of the samples were

typically 20 �M for far-UV CD and 50 �M for near-UV

CD studies. The results were expressed as the mean residue

ellipticity (MRE in deg cm2/dmol), which is defined as

MRE ¼ � obs ðmdegÞ=ð10� n� Cp� 1Þ

where � obs is the observed ellipticity in degrees, Cp is the

molar fraction, and l is the length of the light path in cm.

The �-helical content of papain was calculated from the

2 Naeem, Fatima, and Khan

Biopolymers DOI 10.1002/bip

MRE value at 222 nm using the following equation as

described by Chen et al.20:

% �� helix ¼ MRE222 � 2340=30300� 100

Fluorescence Measurements. Fluorescence spectra were

recorded with a Shimadzu RF 540 spectrofluorophotometer

(Japan) in a 10-mm path-length quartz cell. Samples con-

taining different concentrations of detergents were equili-

brated at room temperature for 30 min before recording for

tryptophan fluorescence measurements. The excitation

wavelength was 280 nm and the emission was recorded

from 300 to 400 nm. The final protein concentration was

10 �M for all experiments.

ANS-Fluorescence Measurements. 8-Anilino-1-naphtha-

lene-sulfonic acid (ANS) binding was measured by fluores-

cence emission with excitation at 380 nm and emission

was recorded from 400 to 600 nm. Typically, ANS concen-

tration was 100 molar excess of protein concentration and

protein concentration was in the vicinity of 10 �M.

Percent Residual Activity. In the presence of detergents the

% residual activity was calculated relative to native papain as

100% from the hydrolysis of casein at pH 7.0 and 378C.

Caseinolytic Activity Measurement. Proteolytic activity

measurements were made using 2% casein as substrate and a

stock enzyme concentration of 0.1 mg/mL. Casein (0.25 mL)

was treated with enzyme solution (0.3 mL) in 0.1 M sodium

phosphate buffer, pH 7.0 at 378C for 15 min, and the reaction

was stopped by the addition of 5% TCA (0.25 mL). The pre-

cipitated material, from each reaction mixture, was removed

by centrifugation and the TCA soluble fraction was assayed

by the method of Lowry et al.21 An appropriate blank solu-

tion was also prepared and absorbance values at 700 nm were

measured with reference to the blank. The absorbance values

obtained could be correlated with the amount of product

formed. The results are expressed in terms of % residual ac-

tivity compared with native.

Data Analysis

The folding curves for the D$ N transition were normalized to

the apparent fraction folded, fN, using the following equation22:

fN ¼ ðY � YDÞ=ðYN � YDÞwhere Y is the observed variable parameter and YN and YDare the values of the variable characteristics of the folded

and unfolded conformations. The difference in free energy

between the folded and the unfolded states, DG was calcu-

lated by the following equations:

�G ¼ �RT lnK ¼ �RT ln ½ð1� fDÞ=fD�¼ �RT ln ½fN=1� fN�:

fN ¼ 1� fD or fN þ fD ¼ 1

where fN is the fraction folded, fD is the fraction unfolded, Kis the equilibrium constant, R is the gas constant (1.987 cal/

deg/mol), and T is the absolute temperature (298 K).

RESULTS AND DISCUSSION

CD Measurements

The effect of detergents on acid-unfolded papain was

studied by far UV and near UV circular dichroism.

Figure 1A shows the far-UV CD spectra in 200–250 nm

range of the native and acid-unfolded papain at pH 2.0

FIGURE 1 (A) Far-UV CD spectra of papain as a function

of SDS in 20 mM glycine–HCl buffer, pH 2.0. The concentra-

tions of SDS for different spectra from curves 1 to 6 are 0, 2,

4, 6, 7, and 8 mM. Curve 7 was obtained in 20 mM sodium

phosphate buffer, pH 7.0. The protein concentration was

20 �M and the pathlength was 0.1 cm. (B) Effect of increasing

concentrations of detergents (mM), i.e., SDS (l), CTAB (^),

and Tween-20 (~) on papain at pH 2 as followed by ellipticity

measurements at 208 nm.

Characterization of Papain Intermediates 3


both in the absence and presence of various concen-

trations of SDS (0–8 mM). Curve 1 represents the

far-UV CD spectra of acid-unfolded papain, whereas

the far-UV CD spectrum of native papain is repre-

sented by curve 7. The spectra of acid-unfolded pro-

tein obtained in the presence of different concentra-

tions (2–8 mM) of SDS are shown in curves 2 and 6.

All of the spectra were characterized by the presence

of minima at 208 and 222 nm, the characteristic fea-

tures of helical secondary structure. As can be seen

from the figure, papain in acidic conditions retains

some amount of secondary structure. Addition of SDS

on acid-unfolded papain up to 8.0 mM led to an in-

crease in helical content as evidenced by an increase in

negative MRE value at 208 and 222 nm, hence ap-

proaches the native value. The 12-carbon hydrophobic

chain of SDS creates a nonpolar environment that de-

creases the polarity of the solvent at pH 2 causing an

increase in hydrophobicity and also interacting with

the hydrophobic cluster of the protein. The addition

of sulphate anion to papain leads to electrostatic inter-

actions with the positively charged centers on the pro-

tein (binding of SO42– anion), so as to effectively shield

the repulsive forces, which favor unfolding, and con-

sequently the intrinsic hydrophobic interactions of

protein in an aqueous environment manifest them-

selves.11 This results in protein folding, for the protein

to adopt a compact structure with increased hydropho-

bic surface area. Further increase in the SDS concen-

tration beyond that which maximum ellipticity was

observed leads to the transition of the SDS monomer

to the formation of micelles. The critical micellar con-

centration of SDS in water is reported to be 8.3 mM,

whereas it further increases with increase in dielectric

constant of the solvent.23 Hiramatsu and Yang16 have

reported a similar study on cytochrome c.SDS induces the anion transition in acid-unfolded

papain. The helical contents calculated from the MRE

value at 222 nm by the method of Chen et al.20 for the

native state at pH 7.0, the acid-unfolded state at pH 2.0,

and the acid-unfolded state at pH 2.0 in the presence of

8 mM SDS were 29, 16.7, and 28%, respectively.

The effect of detergents, i.e., cationic (CTAB),

anionic (SDS), and nonionic (Tween-20) on the acid-

unfolded state of papain at pH 2.0 was studied by

measuring MRE at 208 nm (Figure 1B). All three

detergents used were found to induce helical struc-

ture on acid-unfolded papain. However, SDS was

found to be more effective than CTAB or Tween 20,

with maximum ellipticity being observed at 8 mM.

Further increases in detergent concentration led to

protein precipitation. Detergents form micelles at a

particular concentration called the critical micelle

concentration. It has been observed that, at concen-

trations beyond their ‘‘CMC’’ values, detergents in-

duce precipitation in proteins. Detergent-induced tran-

sition of acid-unfolded papain as observed in this study

can be explained on the basis of addition of deter-

gent, known to stabilize the helical structure of a pro-

tein. A summary of respective maximum MREs at

208 and 222 nm of detergents, their Cm values (the

concentration of detergent at which half of the struc-

ture is induced), and their �-helix is given in Table I.

Figure 2A shows the far UV CD spectra in the

200–250 nm range of native papain (curve 6), acid-

unfolded papain, and in the presence of various

detergents at their respective maximum concentrations.

As can be seen from the figure, papain in acidic condi-

tions (curve 2) retains some amount of secondary

structure compared with 6M GdnHCl (curve 1). Maxi-

mum induction of structure at 208 nm is seen in 8 mMSDS (curve 5) followed by 3.5 mM CTAB (curve 4)

and 0.1 mM Tween-20 (curve 3). Hence SDS is found

to be most effective in inducing secondary structure

almost approaching native (curve 6), among the above-

mentioned detergents.

Table I Comparison of Different Spectral Properties of Acid-Unfolded Papain and in the

Presence of 8 mM SDS, 3.5 mM CTAB, and 0.01 M Tween

Variable

Acid-Unfolded

Papain SDS CTAB Tween

MRE at 208 nm �7,400 �12,100 �11,200 �10,100

MRE at 222 nm �7,100 �11,000 �11,000 �9,900

MRE at 292 nm — 146 122 54

Cm value (mM) — 3.5 0.07 0.08

% Helixa 16.7 28 28 25

Intrinsic fluorescence at 337 nm 31 52 68 33

lmax 333 337 337 338

ANS fluorescence at 480 nm 58 37 172 260

a Calculated by the method of Chen et al.20



Figure 2B shows near-UV CD spectra in the 300–

250 nm range of native papain (curve 5), acid-

unfolded papain (curve 1), and acid-unfolded papain

in the presence of 0.1 mM Tween-20 (curve 2),

3.5 mM CTAB (curve 3), and 8 mM SDS (curve 4).

As can be seen from the figure, native papain is char-

acterized by the presence of three prominent peaks at

292, 280, and 269 nm. At pH 2.0, the protein com-

pletely lost its tertiary structure with the disappear-

ance of all of the prominent peaks. In the presence of

8 mM SDS, all three peaks reappear but the MRE

value does not approach the native value. Whereas in

the case of 3.5 mM CTAB, the only peak reappear-

ance was at 292 nm. In the presence of 0.1 mMTween protein there was a peak at around 272 nm in

comparison to acid-unfolded papain, in which the

spectrum was almost zero. Thus, acid-unfolded pa-

pain in the presence of 8 mM SDS and 3.5 mMCTAB retains its partial tertiary structure.

Intrinsic Fluorescence

The intrinsic fluorescence maximum (lmax) is an

excellent parameter to monitor the polarity of the

tryptophan environment in the protein and is sensi-

tive to the protein conformation.24 Contribution of

detergents alone to the emission spectra was taken

into account and we report here the subtracted fluo-

rescence spectra. To rule out aggregation of protein

and hence light scattering, the samples were read at

350 nm and the negligible absorbance was observed.

Intrinsic tryptophan fluorescence studies were per-

formed to monitor the modification of the microen-

vironment of tryptophan in the presence of deter-

gents (SDS, Tween-20, and CTAB). The emission

maximum of the native protein was obtained at 337 nm

(curve 2) and a red shift of 13 nm was observed in

the completely unfolded state in 6 M Gdn HCl (curve

1) as all the five tryptophans are exposed to the sol-

vent. A decrease in the fluorescence intensity was

observed at pH 2 (curve 6) with a blue shift of

17 nm, i.e., lmax at 333 nm with a 30% decrease in

fluorescence signal relative to 6 M Gdn HCl. This ob-

servation suggests that the papain molecule at pH 2.0

acquires a conformation different from native and

denatured states. This blue-shift fluorescence of pa-

pain at pH 2.0 can be attributed to the conformational

changes in the vicinity of surface-exposed trypto-

phans, presumably because of internalization in a

more hydrophobic environment. A similar blue-

shifted fluorescence has been reported earlier for

glucose isomerase,25 bovine growth hormone,26 and

interferon-�.27 An alternative possibility is that sur-

face-exposed tryptophans are quenched differently

from the internal tryptophans at low pH without inter-

nalizing them, leading to a greater proportion of the

remaining signal coming from the more buried tryp-

tophans that are located in the interior of the native

protein. Upon addition of 8 mM SDS, the internalized

trytophans in the acid-unfolded state (curve 6) get

partially exposed from a nonpolar to a polar environ-

ment as indicated by a red shift of 4 nm and an

increase in fluorescence intensity (curve 3). This indi-

cates that tryptophan residues of the acid-unfolded

FIGURE 2 (A) Far-UV CD spectra of acid-unfolded pa-

pain (curve 2) in the presence of detergents: 8 mM SDS

(curve 5), 3.5 mM CTAB (curve 4), and 0.1 mM Tween-20

(curve 3) at their respective maximium concentrations in

20 mM glycine–HCl buffer, pH 2.0. Curve 1 represents Gdn–

HCl–denatured papain. Curve 6 represents papain at pH 7.

The protein concentration was 20 �M and the pathlength was

0.1 cm. (B) Near-UV CD spectra of native papain (curve 5),

acid-unfolded papain at 8 mM SDS (curve 4), 3.5 mM CTAB

(curve 3), and 0.1 mM Tween-20 (curve 2). Curve 1 repre-

sents papain at pH 2. The protein concentration was 50 �Mand the pathlength was 1 cm.



state in the presence of SDS acquire a state that

resembles more closely native protein. Similar spectra

were obtained in 3.5 mM CTAB (curve 4) and

0.1 mM Tween (curve 5) but the effect was less pro-

nounced in CTAB and the least in Tween. This result

is consistent with our CD results, in which the sulfate

ions present in the electronegative series were found

to be effective in inducing secondary and partial terti-

ary structure. The electronegative series given by

Goto and collaborators28–31 is as follows:

FeðCNÞ3�6 >SO2�4 >CCl3COO�>SCN�>ClO�4

>I�>NO�3 >CF3COO

� >Br� >Cl�

Figure 3B depicts the fluorescence intensity at 337 nm

as a function of detergent concentration (mM). On

increasing the concentration of detergents there was

increase in fluorescence intensity. More enhancement

was found in case of SDS followed by CTAB and

Tween-20.

As papain consists of more basic amino acids, sul-

phate ions, being negatively charged, interacted with

the positive charge on the protein more effectively

than cationic (CTAB) or nonionic (Tween 20) deter-

gents. As the concentration of SDS (0–8 mM) is

increased, it acts as source of sulphate anion, which

binds positively charged groups of the protein result-

ing in screening of the repulsive effect, thus over-

coming charge–charge repulsion, causing a compact

structure. Alternatively, the electrostatic effects of

the formation of ionic couples at the protein surface

due to specific sulfate anion on the conformation of

acid-unfolded papain can be partially interpreted

within the framework of Debye–Huckel theory with

simple electrostatic consideration. The sulphate

anion exerts an ‘‘ionic atmosphere’’ effect around the

protein. The counterion presence around the charge

groups will weaken the repulsion, thus permitting the

other forces favoring folding to become relatively

strengthened and resulting in folding of the protein.

Thus, SDS is found to be effective in inducing sec-

ondary structure (as observed by far-UV CD) and

partial tertiary structure (as observed by near-UV CD

and tryptophan fluorescence).

ANS Fluorescence

The intermediate state induced by detergents is fur-

ther confirmed by extrinsic fluorescence studies. Since

ANS is known to bind to hydrophobic patches on pro-

FIGURE 3 (A) Tryptophan fluorescence emission spectra

of papain in the native state in 20 mM sodium phosphate

buffer, pH 7.0 (curve 2); acid-unfolded state in 20 mM gly-

cine–HCl buffer, pH 2.0 (curve 6); in the presence of 8 mMSDS (curve 3) at pH 2.0; in the presence of 3.5 mM CTAB

(curve 4) at pH 2.0; and in the presence of 0.1 mM Tween-20

(curve 5) at pH 2.0; curve 1 represents 6 M Gdn–HCl–dena-

tured state. The protein concentration was 10 �M. (B) Effect

of increasing concentrations of detergents (mM), i.e., SDS

(l) CTAB (^), and Tween-20 (~) on papain at pH 2 as fol-

lowed by fluorescence intensity at 337 nm.



tein, binding of ANS to hydrophobic regions of protein

has been widely used to study the folding intermedi-

ates formed during protein folding.17,23,32–35 Maximum

ANS binding occurs in the presence of 0.1 mM Tween

(curve 1), followed by 3.5 mM CTAB (curve 2) (Fig-

ure 4). As can be seen in Figure 4A, the acid-unfolded

state of papain (curve 3) opened more hydrophobic

regions of the protein, making them available for ANS

binding. Addition of 8 mM SDS to this state caused a

significant decrease in ANS binding (curve 4), thus

suggesting ordering of the hydrophobic regions of the

protein, which produced a conformational change

close to the tertiary structure of the protein (curve 5).

Thus, from ANS binding, we conclude that, in the

presence of SDS, the spectrum of acid-unfolded papain

has a greater resemblance to the native than the

unfolded state at pH 2.0. Contrary to SDS, Tween

showed higher ANS binding followed by CTAB, thus

indicating in the presence of Tween, a molten-glob-

ule–like state is formed with enhanced exposure of

hydrophobic surface area.35 Titration of the papain so-

lution at pH 2 by detergents was monitored by ANS

fluorescence at 480 nm (Figure 4B). As can be seen

from the figure, ANS fluorescence gradually was

quenched in the presence of SDS and was enhanced in

the presence of Tween.

Percent Residual Activity of Papain in thePresence of Detergents

Figure 5 depicts the % residual activity versus deter-

gent concentration (mM). Percent residual activity

was calculated by taking the activity of native en-

zyme as 100%. Proteolytic activity measurements

were made using 2% casein as substrate. Adequate

FIGURE 4 (A) ANS fluorescence emission spectra of

papain in 20 mM sodium phosphate buffer, pH 7 (curve 5);

acid-nfolded state in 20 mM glycine–HCl buffer, at pH 2.0

(curve 3); pH 2 containing 8 mM SDS (curve 4); pH 2 con-

taining 3.5 mM CTAB (curve2); and pH 2 containing

0.1 mM Tween-20 (curve 1). The protein concentration

was 10 �M and the ANS concentration was 100 molar

excess the protein concentration. (B) Effect of increasing

concentrations of detergents (mM), i.e., SDS (l), CTAB

(^), and Tween-20 (~) on papain at pH 2 as followed by

ANS fluorescence at 480 nm.

FIGURE 5 Percent residual activity of papain at pH 2 in

the presence of detergents (mM): SDS (l), CTAB (^),

and Tween-20 (~). The activity of the native enzyme was

taken as 100% at pH 7 (n).



controls of substrate casein in the presence of deter-

gents were taken to rule out its chaperone activity

and changes induced by casein on acid-unfolded pa-

pain or being itself denatured by detergents. The pre-

cipitated material was removed and the TCA soluble

fraction was assayed by the method of Lowry et al.21

At pH 2, in the absence of detergents, the activity of

papain was reduced to 35%. While in the presence of

detergents, there was partial recovery of enzymatic

activity of acid-unfolded papain. The % recovery in

enzyme activity, calculated in the presence of 8 mMSDS, 3.5 mM CTAB, and 0.1 mM Tween-20, was

43, 41, and 39%, respectively. No distinct difference

was observed in the activity on acid-unfolded papain

among the detergents. This result is consistent with

near- and far-UV CD spectra, as acid-unfolded papain

in the presence of 8 mM SDS and 3.5 mM regains the

tertiary structure and almost native-like secondary

structure (Figure 2). The gain of signal in the near-UV

CD region with low activity may be attributed to the

initial occurrence of nonnative structures and subse-

quent reorganization toward the native one.

Thermodynamic Parameter Derivedfrom the Detergent-DependentTransition Curve

The fraction of protein-folded at pH 2 in the pres-

ence of detergents is shown in Figure 6. fN of papain

at pH 2 in the presence of detergents was calculated

by taking papain at pH 7 as the native value. In the

presence of SDS and CTAB at pH 2, no intermediate

state could be observed, as the curves were sharp,

suggesting a single-step two-state transition. No dis-

tinct transition could be seen in the Tween-20 curve

(fluorescence intensity at 337 nm). However, the tran-

sition curve of the ellipticity change at 208 nm shows a

single-step, two-state transition. Nonoverlap of the two

transitions suggests a noncooperative transition. The

calculated free energy of folding, DG, in the presence

of detergents is shown in Table II.36

CONCLUSION

Detergents have gained an important status in protein

chemistry and are applied in the biosciences and in

many (bio)technological processes owing to the fact

that detergents mimic the native, hydrophobic envi-

ronment of the phospholipid bilayer in vivo. Keeping

in view the wide applicability of detergents in mem-

brane protein solubilization, electrophoresis, etc., it

becomes imperative to develop a better understand-

ing of the effects of detergents on proteins. Acid-

unfolded papain in the presence of different deter-

gents attains different conformations, making avail-

able different types of information for the usage of

protein purification. The primary role of detergents

in protein purification is to affect solubilization. The

nondenaturing property is the most difficult issue to

address in the selection of a detergent. A precise def-

inition of denaturation is usually given in terms of

effects on secondary and tertiary structure. It is there-

fore essential to recognize the detergents that will ac-

complish the task of maintaining solubility while not

significantly compromising the native structure and

consequently the biological function of the protein.

Earlier scattering and hydrodynamic data were mainly

used to probe the structural changes in protein–deter-

gent complexes. However, more recently, spectro-

scopic approaches have gained more attention. Oneda

FIGURE 6 Fraction folded versus detergent: fraction

folded at acid-unfolded papain in the presence of SDS (l,

*), CTAB (^, ^), and Tween (~, ~) by the change in

MRE at 208 nm and fluorescence intensity at 337 nm.

Filled symbols represent the changes monitored by MRE

208 nm and open symbols represent changes monitored by

fluorescence intensity at 337 nm.

Table II DG Values for the Detergent-Induced

Transition of Acid-Denatured Papain Studied by MRE

Measurements at 208 nm and Fluorescence Intensity

Measurements at 337 nm

Probe

DGH2O

(kcal/mol)

SDS

DGH2O

(kcal/mol)

CTAB

DGH2O

(kcal/mol)

Tween-20

MRE208 �1.39 �1.85 �1.82

Fluorescence337 �1.23 �0.42 �0.014



and Inouye37 have used Brij-35 to refold and recover

the recombinant human matrix metalloproteinase 7

(matrilysin) from inclusion bodies expressed by Esche-richia coli. Cardamone et al.38 have used CTAC (cetyl

tri methyl ammonium chloride), a cationic detergent

for spectroscopic and equilibrium binding analysis

using a recombinant porcine growth hormone. SDS at

a low surfactant/protein ratio acts as a structure-stabi-

lizing additive on BSA. A similar study shows that in-

teraction of HSA with surfactant becomes stronger

with increasing alkyl chain length.39 SDS is used to

solubilzed interferon and interleukin 2 (IL-2) from in-

clusion bodies. In our study, all three detergents were

able to induce �-helix in acid-unfolded papain, as

evident from the induction of MRE values at 208 and

222 nm. The anion-induced state at low pH in the pres-

ence of SDS showed a diminished ANS binding com-

pared with acid-unfolded papain or that of the Tween-

induced state. Moreover, in presence of SDS there was

gain of signal in the near-UV (aromatic) region of the

CD spectrum, suggesting an ordering of the tertiary

structure. Also, in the presence of SDS, acid-unfolded

papain recovers partial activity followed by CTAB and

Tween-20. While in the case of Tween, an intermedi-

ate state with native-like secondary structure, loss of

tertiary structure and high ANS binding was obtained.

This state can be characterized as a molten-globule–

like state. In the case of CTAB, the intermediate state

obtained can also be characterized as molten globule,

due to high ANS binding and a native-like secondary

structure that also retains native-like tertiary contacts.

These species may hence be described as compact

molten-globule–like intermediates, as similar proper-

ties have been observed for the molten globule states

of a number of proteins. Molten globules are partially

structured a protein-folding intermediate that adopt a

native-like overall backbone topology in the absence

of extensive detectable tertiary interactions. It is im-

portant to determine the extent of the specific tertiary

structure present in molten globules and to understand

the role of specific side-chain packing in stabilizing

and specifying the molten globule structure.40 Moreover,

the A-state of equine ferricytochrome c has native-likecompactness, native-like helix content, native helix–

helix interactions, and one native heme ligand.41–43

Tertiary A state has been reported for �-lactalbumin,14

RNase H,44 myoglobin,45 and ubiquitin.46 A summary

of observations on acid-unfolded papain in presence of

detergents at pH 2 is presented in Table I. The effect of

detergents on proteins depends on the nature of both

the detergent and the protein component; in addition,

the environmental conditions are of importance. In our

studies, SDS and CTAB proved to be the better deter-

gents, as the information obtained from experiments

shows that protein regains secondary as well as tertiary

structures. Nevertheless, there are still a lot of promis-

ing possibilities of protein–detergent interaction that

need to be explored for solubilization of inclusion

bodies and membrane protein purification.

The authors are very thankful for the facilities obtained at

AMU Aligarh. The authors are also thankful to DST–FIST

for financial support. A. N. is the recipient of CSIR-SRF

and S.F. is the recipient of CSIR-JRF.

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Reviewing Editor: Yuji Kobayashi



Characterization of a partially folded intermediate of papain induced by fluorinated alcohols at low...

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