physicochemical and conformational studies of papain/sodium dodecyl sulfate system in aqueous medium

Colloids and Surfaces A: Physicochem. Eng. Aspects 264 (2005) 6–16

Physicochemical and conformational studies of papain/sodiumdodecyl sulfate system in aqueous medium

Soumen GhoshCentre for Surface Science, Department of Chemistry, Jadavpur University, Calcutta 700032, West Bengal, India

Received 19 May 2004; received in revised form 31 December 2004; accepted 14 February 2005Available online 6 July 2005

Abstract

Interaction between a globular protein, papain and the anionic surfactant, sodium dodecyl sulfate (SDS) has been studied in aqueous mediumin detail using conductometric, tensiometric, calorimetric, fluorimetric, viscometric, circular dichroism techniques. The physicochemicalproperties, e.g. critical micellar concentration (CMC), counterion binding, free energies, enthalpies and entropy of micellization, interfacialadsorption, micellar aggregation number and micellar polarity of SDS have been determined in presence of papain. The results show thatthe CMC values of SDS increase with the increasing concentration of papain. The energetics of micellization of papain–SDS system ise e of proteina lts show theh©

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ndothermic and the interaction of SDS with papain is an entropy controlled process. Such physicochemical studies in presencre rare. Also, the conformational behavior of papain in aqueous solution has been investigated in the presence of SDS. The resuigh �-helical content and unfolded structure of papain in the presence of SDS due to strong electrostatic repulsion.2005 Elsevier B.V. All rights reserved.

eywords: Globular protein; Surfactant; Physicochemical; Conformational interaction

. Introduction

Interactions between surfactants and globular proteinsave been extensively studied[1–17] because of the variouspplications in the fields of industrial, biological, pharma-eutical and cosmetic systems. There are technical applica-ions for drug delivery and in several biochemical separationethods. Recently, proteolytic enzymes are widely used inetergent industry to remove any proteinaceous stain (e.g.tains of blood, egg, meat-soup, etc.) by hydrolysis of pro-eins into amino acids or other compounds. Physicochemicaltudies on the interaction between surfactant and globularrotein are limited[17]. In most of these studies, it has beenonsidered that an amphiphile binds to a single protein andorms a complex[6]. This binding of surfactant molecule torotein can lead to unfold and disrupt the native structuref most globular proteins[1,4]. Generally, ionic surfactantsind to proteins and the most widely used anionic surfactant,DS unfolds and denatures proteins more than do cationic

E-mail address:[email protected].

surfactants[1,7–9,12]. Relatively, studies of the interactioof proteins with cationic and nonionic surfactants are lim[18].

The globular protein, papain (EC 3.4.22.2) is a tenzyme from the latex and unripe fruit ofCarica papaya(tropical melon or papaw). The cysteine protease, papaunusually stable to high temperatures and to high contration of denaturing agents, such as, 8 M urea or orgsolvents like 70% EtOH. Papain is a carbohydratebasic, single chain protein. Papain has molecular weig23,350 Da and consists of 212 amino acid residues (menine absent; IP 8.75) with four disulfide bridges and catically important cysteine (position 25) and histidine resid(position 158)[19]. Under acidic conditions, papain exiin a molten globule state and its unfolding was found tonon-cooperative. Drenth et al.[20] reported that papain hfive stretches of�-helices at residues 24–41, 49–57, 67–117–128 and 137–143 and the molecule contains a displeated sheet structure of about 30 residues. The moleca rotational ellipsoid, divided by a cleft, and contains adominance of antiparallel�-structures. For activity, papa

927-7757/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfa.2005.02.032

S. Ghosh / Colloids and Surfaces A: Physicochem. Eng. Aspects 264 (2005) 6–16 7

requires a free SH group at pH 5–5.5. SH blocking agents,like iodoacetic acid are powerful papain inhibitors whereasSH compounds, like mercaptoethanol are potent activators.Papain has a broad specificity and it catalyses the cleavageof the wide variety of peptide bonds indicating a fairly lowspecificity for peptide bond cleavage[19]. The active site ofpapain can be divided into seven “subsites”, each accommo-dating one amino acid residue of the substrate. Four of thesesubsites are on one side of the catalytic site, three on the other:

H2N R1 R2 R3 R4 R5 R6 R7 COOH

↑cleavage

Papain is used medically with fetal as well as postnatal brainregions to provide maximal dissociation and viability of neu-rons and for the treatment of necrotic tissue and eczema[19]. It is used in protein chemistry for cleaving proteins intolarge peptides[19]. Papain is also used in detergents and hasrecently been patented[21].

Generally, surfactant–protein interactions involve variousmodes of association caused by dipole–dipole, ion–dipoleor ion–ion forces. Nagarajan and Kalpakci[22] discussedsix possible types of associations on polymer–surfactantinteractions involving either individual surfactant moleculeso ers.T pro-p ”mRq[ es m-p them r tot sur-f

e thei r oft rime-t ntp rstandt tes.

2

2

DS)u wasa er-m d thep uct ofF aterm

Double distilled water of specific conductance, 3�S cm−1

at 303 K was used in all preparations. All measurements weredone at constant temperature in a water bath maintained at298± 0.01 K.

2.2. Conductometry

The conductance values were measured with a Systron-ics 304 conductivity meter (India) using a cell of cell con-stant 1.0 cm−1. The conductance values were accurate within±0.5%. The concentrated surfactant solution was progres-sively added with the help of a Hamilton microsyringe toprotein solution taken in a small beaker and mixed thor-oughly at constant temperature and then the conductancevalues were measured. The detailed procedure is describedelsewhere[17,31–33].

2.3. Tensiometry

The air/solution surface tensions at various concentra-tions were measured with a du Nouy tensiometer (Kruss,Germany) using a platinum ring by the ring detachmenttechnique. The tensiometer was calibrated against water andthe measured surface tension values were corrected usingthe technique of Harkins and Jordan[34]. With the help ofa Hamilton microsyringe, the concentrated surfactant solu-t keni sur-f within± rlier[

2

s ofc ctant( lu-t py ofm wasm tionc ea-s iths umh else-w

2

lubi-l a F-3 ) witha gthso . ThecA aturew ssing.

r surfactant clusters along with the chains of polymhe protein–SDS complex has been described by theosal of three models: a rod like particle[3], a “necklaceodel[17,23]and a deformable prolate ellipsoid model[5].esults from NMR[10], free boundary electrophoresis[22],uasi-elastic light scattering[24], SANS [25], viscometry

26], circular dichroism[17], ESR [27], and fluorescenctudy[27,28] agree with the “necklace model” of the colex where the partially unfolded protein wraps aroundicelle-like aggregates. This necklace model is simila

he structures reported for complexes formed betweenactants with polymers[23,29]and polyelectrolytes[30].

The present work has been performed to investigatnteraction between SDS and papain using a numbeechniques such as conductometry, tensiometry, calory, fluorimetry, viscometry, circular dichroism. The differehysicochemical processes have been applied to unde

he nature and insight of the protein–surfactant aggrega

. Experimental

.1. Materials and methods

The anionic amphiphile sodium dodecyl sulfate (Ssed in this study was a product of Sigma, USA. Papainliophilized, crystalline powder, a product of Merck, Gany. Papain was dissolved in double distilled water anH was unadjusted. The dye safranine T (ST) was a prodluka, Switzerland, crystallized twice from an ethanol–wixture.

ion was progressively added to the protein solution tan the glass vessel and thoroughly mixed and thenace tension was measured. The results are accurate0.1 mN m−1. The detailed explanation can be found ea

17,31–33].

.4. Calorimetry

The calorimetric titration experiment consisted of serieonsecutive additions of concentrated solution of surfaconcentration� CMC) from a burette to the protein soion taken in a calorimeter sample vessel and the enthalicellization of surfactant in aqueous protein solutioneasured with the help of a Tronac 458 Isoperibol titra

alorimeter (USA). The instrument was calibrated by muring the heat of neutralization of hydrochloric acid wodium hydroxide. The calorimeter can measure a minimeat change of 4.2 J. The detailed procedure is reportedhere[17].

.5. Fluorimetry

The fluorescence emission spectra of safranine T soized in the investigated systems were obtained using010 Fluorescence spectrophotometer, Hitachi (Japanslit width of 1 cm. The excitation and emission wavelenf the probe dye ST were 520 and 587 nm, respectivelyoncentration of ST was maintained at the order of 10−5 M.ll spectra were measured thrice in a constant temperater bath and the mean values were used for data proce

8 S. Ghosh / Colloids and Surfaces A: Physicochem. Eng. Aspects 264 (2005) 6–16

2.6. Viscometry

Viscosity measurements of solutions were performedusing a Cannon–Fenske capillary viscometer suspended ina temperature-regulated water bath, at 298 K with the accu-racy of±0.2 K. The flow time for water is 123 s.

2.7. Circular dichroism spectroscopy

Circular dichroism (CD) measurements were made toexamine the possibility of conformational changes occurringupon binding of anionic surfactant, SDS to papain. Solutionsfor CD were prepared by mixing the papain and SDS solu-tions in water at different concentrations in equal proportion.Far and near-UV CD measurements were done on a Jasco,J-600 recording spectropolarimeter (Japan) attached with achiller to control the temperature of Xe lamp and electroniccircuit. The instrument was calibrated with an aqueous solu-tion ofd-10-camphor-sulfonic acid. Far-UV CD spectra weremeasured between 200 and 250 nm wavelength with the mix-ture of papain and SDS in a 1 mm path length cuvette to studythe conformational changes in the secondary structure of theprotein. Near-UV CD measurements were done with a 10 mmpath length cuvette in the region of 250–320 nm to study thechanges in the tertiary structure. Each spectrum representsthe average of five scans and the scan speed was 50 nm/min.

3

3

elf-a gularp cen-t s ofs ainw inF hefi con-c thec ndm os-s peso rp ft pro-t n theC ndC sing[

ora showni heb plotd will

Fig. 1. The plots of specific conductance (κ) vs. concentration of SDS inthe presence of different [papain] at 298 K. Effect of papain concentration(g dl−1): (I) 0.001; (II) 0.005; (III) 0.008; (IV) 0.01.

result in a decrease inγ value as SDS adsorbs at the air-waterinterface and after CMC,γ is almost constant. Similar inter-actions between protein and surfactant are also studied earlier[17,37].

From the calorimetric titration experiment, the heat ofdilution of SDS in aqueous papain solution at a constantconcentration is recorded and typical thermograms are exem-plified in Fig. 3. The break points correspond to the CMCvalues[17]. The thermograms are highly endothermic innature and give the enthalpy of micellization. Such calori-metric study on protein–surfactant interaction is rare[17,38].

The CMC values of pure SDS and papain–SDS mixtures atdifferent concentrations of papain obtained from tensiomet-ric, conductometric and calorimetric methods are presentedin Table 1. The results fromTable 1show that the CMC val-ues of SDS increase with increasing concentration of papain

Table 1Evaluation of critical micellar concentration (CMC) of SDSa andpapain–SDS mixtures in aqueous medium by different methods at 298 K

[Papain] (%, w/v) CMC (× 103 mol dm−3)

Tensiometry Conductometry Calorimetry

0 7.94 7.94 7.370.001 8.41 8.80 (1.0) 7.510.005 8.91 9.00 (1.1) 8.600.008 10.00 9.20 (1.2) 9.400

T

. Results and discussion

.1. Critical micelle concentration (CMC)

In aqueous medium, pure amphiphilic molecules sssemble to form a special type of aggregates in a reattern known as micelle after reaching a specific con

ration, called the critical micelle concentration. The plotpecific conductance (κ) at constant concentration of papith varying [SDS] (i.e.κ versus [SDS]) are presentedig. 1 which shows two distinct breaks in each plot. Trst break point corresponds to the critical aggregationentration (CAC) while the second one corresponds toritical micelle concentration (CMC). At CAC, protein bouicelles are formed where free micelle formation is p

ible at and above CMC. Conductometrically, such tyf behavior are observed in trypsin–SDS[17] and otheolymer–surfactant systems[14,35,36]. In both systems o

rypsin–SDS and papain–SDS, the CAC values of SDS inein solution are almost an order of magnitude lower thaMC values of SDS in that protein solution. Both CAC aMC values of SDS are observed to increase with increa

papain] (Table 1).The surface tension (γ) versus log[SDS] behaviors f

queous papain at several concentrations have beenn Fig. 2. The upper portion of each plot is curved. Treak point between two straight lines for a particularenotes CMC. Addition of SDS to a papain solution

.010 10.59 9.40 (1.3) 10.00

he values in parentheses indicate CAC.a The CMC values of SDS solution were taken from Ref.[17].


Fig. 2. Surface tension (γ) vs. log (SDS) profile of the papain–SDS mixturesat 298 K. The curves I, II, III, and IV represent 0.001, 0.005, 0.008, and0.01 g dl−1 papain, respectively, in the mixture.

indicating strong interaction between SDS and papain. Here,DS− of SDS gets adsorbed on the surface of papain moleculeand amphiphile self-assembles resulting in the formation offree micelles at the higher concentration than that of pureSDS solution[17,39]when the concentration of free surfac-tant monomers in the aqueous solution reaches the CMC.Such method dependent CMC values have been reported inthe literature[17,40]. These CMC values are plotted as afunction of [papain] inFig. 4 which presents a comparativeprofile of those by different methods. Again, the experimen-tal ratio CAC/CMC obtained by conductometric method is

F int MC.S tration(

Fig. 4. CMC of SDS as a function of [papain]. CMC is evaluated followingtensiometric (�), conductometric (�) and calorimetric (�) techniques.

plotted as a function of [papain] inFig. 5which indicates theincreasing probability of formation of complex with increas-ing concentration of papain.

3.2. Interfacial activity

Amphiphile adsorbs at the air/protein solution interfaceand decreases surface tension of protein solution. The inter-facial adsorption per unit area of surface at various concen-trations of amphiphile can be calculated with the help of theGibbs adsorption equation. The detailed explanation of Gibbsadsorption equation has already been reported in my earlierwork [32,33]and also by Chattoraj and Birdi[41].

Based on the surface tension results, the total maximumsurface excess,Γ tot

max at CMC can be evaluated according tothe modified Gibbs adsorption equation[41]:

Γ totmax = 1/2.303RT lim

CDS−→CDS− (at CMC)dπ/d logCDS− , (1)

F duc-t

ig. 3. The profile of temperature (millivolt) vs. time of titration of SDShe presence of different [papain] at 298 K. The break point denotes C

and E represent the start and end of a run. Effect of papain conceng dl−1): (I) 0.001; (II) 0.005; (III) 0.008; (IV) 0.01.

ig. 5. The plot of experimental CAC/CMC ratio obtained from the conometric method as a function of [papain] at 298 K.


Table 2Surface chemical behaviors of SDSa and papain–SDS mixtures at 298 K

[Papain] (%, w/v) πCMC (× 103 J m−2) Γ totmax (× 106 mol m−2) Atot

min (nm2) f

0 44.0 0.77 2.16 0.280.001 29.5 1.53 1.08 0.450.005 28.6 1.72 0.96 0.420.008 31.0 1.74 0.95 0.410.010 22.8 1.81 0.92 0.39

a The values of SDS were taken from Ref.[17].

and the total area minimum value (Atotmin) at CMC is calculated

by the equation:

Atotmin = 1018/NΓ tot

max. (2)

Here,π represents the surface pressure obtained from surfacetension of water minus surface tension of surfactant solution,C is the surfactant concentration in mol dm−3 andN is Avo-gadro’s number. The slope of the linear plot ofπ versus logCcorresponds to the value of (dπ/d logC) (plots not shown tosave space). The evaluated values ofπCMC, Γ tot

max, andAtotmin

are shown inTable 2. For pure SDS, the value ofΓ totmax is

minimum and this value increases with increasing [papain].Consequently,Atot

min decreases indicating the increase of inter-action among the adsorbed papain molecule in the layers ofthe micelles.

3.3. Thermodynamics of micellization and interfacialadsorption

The standard free energy of micellization per mole ofmonomer unit ( G◦

m) of ionic surfactant was evaluated fromthe equation:

G◦m = (1 + f )RT ln CMC, (3)

wheref is the fraction of the counter-ions bound to the micelle.H (w io oft cificcm isf Thevf ofi trendo n of

papain. Such evaluation off in protein–surfactant system isvery rare[17].

The standard free energy of interfacial adsorption ( G◦ad)

at the air/saturated monolayer interface has been performedby the relation[42]:

G◦ad = G◦

ad − πCMC/Γ totmax, (4)

whereπCMC is the surface pressure at the CMC.The standard enthalpy of micellization ( H◦

m) can be eval-uated by calorimetric method[38,43]. The standard entropyof micellization ( S◦

m) has been evaluated by applying theGibbs–Helmholtz equation:

G◦m = H◦

m − T S◦m. (5)

The values of G◦m, H◦

m, S◦m, and G◦

ad are presentedin Table 3. The minimum value of G◦

m is obtained for pureSDS and it slightly increases in papain–SDS mixture. Thevalue of G◦

ad of pure SDS is the maximum and decreasesin the mixture. The heat of micellization ( H◦

m) is very low(negative) in pure SDS solution and increases with increasein concentration of papain in solution. The values of H◦

mand S◦

m, both are positive in the mixture of papain–SDS.Such positive values are also reported in trypsin–SDS system[17]. Comparing two systems, it is observed that H◦

m forthe system of papain–SDS is much higher (approximatelyt Thev n-c ye valueo ro-c ,t her-m ayi ind-i f the

TT K

[ (kJ mol

00000

ere, f = 1− degree of dissociation of ionic surfactantα)hereαcan be evaluated conductometrically from the rat

he postmicellar and premicellar slopes of the plot of speonductance vs. concentration of the solution[31–33]. Thisethod of determination off (although not very accurate)

requently used for its simplicity and easy adoptability.alue of f has been presented inTable 2and f is minimumor pure SDS solution. In presence of papain, the valuefs higher than that of pure SDS although a decreasingf f values is observed with the increasing concentratio

able 3hermodynamic behaviors of SDS and papain–SDS mixtures at 298

Papain] (%, w/v) − G◦m (kJ mol−1) H◦

m

15.45 −3.80a

.001 17.24 1.91

.005 16.64 3.92

.008 16.25 5.64

.010 15.86 6.54a The values of SDS were taken from Ref.[17].

wo to four times) than that for trypsin–SDS system.alues of H◦

m and S◦m are maximum for the highest co

entration of papain (0.01 g dl−1). This interaction is mainlntropy controlled process. Heat is released (negativef H◦

m) for the formation of pure SDS micelle and the pess of micellization is exothermic in nature[32]. Essentiallyhe binding of SDS micelle to the chain of papain is atal and initial binding at low concentration of papain m

nvolve SDS monomers. In aqueous media, micellar bng with complete shielding involves some aggregate o

−1) S◦m (J mol−1 K−1) − G◦

ad (kJ mol−1)

39.09 72.5964.26 36.5268.99 33.2773.46 34.0775.17 28.46


SDS molecules along the length of the polypeptide chain ofpapain. This process absorbs heat and positive value of H◦

m(Table 3) denotes the endothermic process in nature[17,36].At higher binding, endothermic contributions are importantin the overall process due to unfolding of the protein[1].

3.4. Aggregation number and dielectric constant

Fluorescence measurements are used to gain informationabout the size of the surfactant aggregates formed on proteinas well as to investigate the micropolarity at the interface andin the micellar core in solution[16].

The aggregation number of papain–SDS complex has beendetermined by the steady state fluorescence measurementsusing the equation[44]:

1/(1 − FR) = (Kc/n)([S]/FR) − Kc[DT], (6)

whereFR = (F − F0)/(Fmax− F0). Here,F0 andF are the flu-orescence intensities without and with 0.1 M SDS in papainsolution, respectively, andFmax is the maximum value ofFwith SDS in papain solution;S, DT, andKc represent surfac-tant (SDS), total dye (ST), and dye-micelle binding constantin papain solution, respectively. The aggregation number,ndepends on concentration of surfactant[45–47].

The graphical presentations of the experimental results ofpbf ctricc atedb ectrai wn

F Sm and0

Table 4Aggregation numbera, dielectric constanta, binding constant (Kc)b and vis-cosity B-coefficient for SDS and papain–SDS mixtures at 298 K

[Papain](%, w/v)

Aggregationnumber,n

Dielectricconstant,D

Kc × 10−5 −B (dm3 mol−1)

0 72.0 29 1.27 –0.001 70.0 26 1.23 0.440.005 67.7 24 1.22 0.580.008 64.8 22 1.22 0.950.010 63.4 20 1.23 1.60

a Then andD values for pure amphiphiles were taken from Ref.[17].b From Eq.(6), Kc was obtained using fluorescence data.

dielectric constant. The detailed procedure and data analysisare reported in literature[31–33,44]. The values ofn, ε andKc are presented inTable 4. Both the values ofnandε of SDSdecrease with increase in concentration of papain whileKcremains invariant. Similar results are obtained in the system oftrypsin–SDS complex[17]. As aggregation starts, the probepasses from aqueous medium into more hydrophobic envi-ronment and there may be a relation with the distribution ofsurfactants between aggregates and the free and molecularlyadsorbed states and aggregation number decreases. The lowervalue ofn also suggests that the polypeptide chain of proteinmay wrap around several aggregates anchoring its hydropho-bic portions in the micellar phase and thereby perturbing onlythe outer parts of the micelles. The fluorescence intensity val-ues of papain solution at various concentrations in absence ofSDS are almost same. This signifies that there is no interac-tion between papain and safranine T. But an interaction occursbetween papain and safranine T at the micellar interface. Thereducing value of dielectric constant of the micelle may bedue to the increase of the size of micelles as a result of addi-tion of polymer inducing a decrease in the area per head groupwith a simultaneous decrease in the interface polarity. Thisindicates a less open and less hydrated palisade layer of theaggregated surfactant phase as compared to regular micelles[17]. The plot ofnversusD of papain–SDS mixtures inFig. 7

Fa

apain–SDS system are described inFig. 6following Eq.(6)y drawing 1/(1− FR) against [S]/FR to evaluaten andKc

rom the slope and the intercept respectively. The dieleonstant (ε) of the papain–SDS clusters has been evaluy comparing the Stokes shifts in the fluorescence sp

n the micelle–protein media with that in solvents of kno

ig. 6. The plots of (1− FR)−1 vs. [S]/FR of pure SDS and papain–SDixtures at 298 K: (I) SDS; (II)–(V) represent 0.001, 0.005, 0.008,.01 g dl−1 papain, respectively.

ig. 7. Dielectric constant (ε) vs. aggregation number (n) plot of pure SDSnd mixed systems of papain–SDS followingTable 4.


produces a straight line which is an important physicochem-ical behavior of protein–surfactant complex. The probe andsurfactant molecules adsorb in active sites on the protein andtransferred into the micelle-like aggregates[28].

3.5. Viscosity

The conformational and rheological behavior of proteinin aqueous solution in the presence of an ionic surfactant isgoverned by the viscosity of the solution.Fig. 8traces the rel-ative viscosity of surfactant-doped papain solution (ηr) versus[SDS], where the ratio of the viscosity of the surfactant-dopedprotein solution to that of the surfactant solution is called therelative viscosity of the solution. A characteristic break ineach plot occurs at [SDS] = 0.015 mol dm−3 and then the rel-ative viscosity curve rises at a different rate than the initialone with increasing [SDS]. Initially viscosity increases due tothe charging up of the protein molecule by binding of surfac-tants[1]. The subsequent increase of viscosity at high [SDS]is possibly due to the cross linking of the several aggregatesby free surfactant micelles in the solution[1]. The relativeviscosity of solution is also higher at higher concentration ofpapain. The reduced viscosity,ηred, is defined as

ηred = ηsp/C = (ηr − 1)/C, (7)

w -t sity,η tanta dt dlT ositya wnf uced

F at20

Fig. 9. The profile ofηred vs. [papain] of papain–SDS mixtures at differentconcentrations of SDS at 298 K. Effect of SDS concentration (M): (I) 0; (II)0.003; (III) 0.008; (IV) 0.015; (V) 0.05; (VI) 0.1.

viscosity becomes gradually more marked as the surfactantconcentration increases indicating enhanced aggregates in adilute papain solution and expansion of protein coil due toionic repulsion effect among the attached DS− ions along thepolypeptide chain[17]. The extrapolations of the reducedviscosity curves to zero protein concentration indicate theintrinsic viscosity values (η), where

[η] = limC→0

(ηsp/C). (8)

A plot of [η] versus [SDS] is drawn inFig. 10which showsthat intrinsic viscosity of the solution is high at high concen-tration of SDS. This type of nature indicates an expansionof protein coil in the cluster which is a result of enhancedelectrostatic repulsion[17]. In this case, the system can beconsidered to consist of a necklace shaped structure[17,48]made of polypeptide chains of papain loaded with boundmicelles.

Viscosity B-coefficient has been derived from Jones–Doleequation [49] for dilute solution (s≤ 0.1 M) using the

F K.

hereηsp indicates the specific viscosity andC is the concenration of protein solution in g/100 ml. The reduced viscored is plotted as a function of [papain] relative to surfact different [SDS] inFig. 9. All the plots are nonlinear, an

he characteristic break appears at [papain] = 0.005 g−1.he figure demonstrates an upsweep in reduced visct low protein concentrations, an effect that is well kno

or polymers and polyelectrolytes. The upsweep in red

ig. 8. Variation ofηr as a function of [SDS] of papain–SDS mixtures98 K. Effect of papain concentration (g dl−1): (I) 0.001; (II) 0.005; (III).008; (IV) 0.01.
ig. 10. Intrinsic viscosity (η) vs. [SDS] of papain–SDS mixtures at 298


Table 5Voluminosity, intrinsic viscosity, and Simha shape factor of papain andpapain–SDS mixtures at 298 K

[SDS] (mol dm−3) VE η (dl g−1) ν

0 2.78 9.4 3.380.003 4.33 14.1 3.260.008 5.79 19.0 3.280.015 8.37 27.3 3.260.050 10.18 33.3 3.270.100 11.50 38.2 3.32

following equation:

ηred = 1 + A√

s + Bs, (9)

where s is the concentration of SDS solution. The plotsof [(ηred − 1)/

√s] versus

√s were found to be linear. The

slope (B) of plot yields viscosity B-coefficient and the valuesare presented inTable 4. Originally, the values of viscosityB-coefficients were introduced as an empirical form depen-dent upon solute–solvent interactions and on the relativesize and shape of solute and solvent molecules. ViscosityB-coefficient values of aqueous papain–SDS mixtures arenegative indicating the unfolding structure of papain in pres-ence of SDS.

The relative viscosity data at different concentrations ofprotein are used to calculate voluminosity (VE) of proteinsolutions at a given temperature[50]. VE is a measure ofvolume of solvated polymer molecules. The value ofVE isevaluated by plottingY against concentration of protein,C(g dl−1) where

Y = (η0.5r − 1)/C(1.35η0.5

r − 0.1). (10)

The extrapolation of the straight line obtained to zero proteinconcentration (C= 0) yields the intercept,VE. The values arelisted in Table 5. The shape factor,ν was calculated fromSimha equation[51]:

[

T pe ofm orcv orma-t

3

on-t ngesi ts ofp ons:f aina ares ant.T a ofp apain

Fig. 11. Far-UV CD spectra of papain–SDS mixtures at 298 K. Spectra 1and 2 represent 0.1 M SDS and 0.1 g dl−1 papain, respectively. Spectra 3–10represent 0.003, 0.008, 0.01, 0.02, 0.03, 0.05, 0.07 and 0.1 g dl−1 papain,respectively, in mixtures of equal volume of 0.1 M SDS and papain solutions.

near 210 and 220 nm. The spectra (specially, 8–10) of com-plexes show a hump at 220–222 nm at the high concentrationsof papain indicating the less random coil with the increasein �-helix [6] of the papain molecule to which SDS binds.These spectra also show a minimum at <210 nm indicatingthe partial unfolded structure of papain in the presence ofSDS. Similar observations using SDS are seen in my previ-ous paper[17]. Fig. 12shows the CD spectra of SDS–papainmixtures where the concentration of SDS increases keep-ing the concentration of papain constant. The percentagesof �-helix, �-structure, and random coils of papain in SDSmicelle have been evaluated on the basis of the experimen-tally obtained curves using a software program, k2d.zip[54].These are presented inTable 6which shows that papain is high

F 1 and2 ep-r ctively,i

η] = νVE. (11)

he Simha shape factor gives an idea about the shaacromolecules in solution[52]. The values of shape fact

alculated for various systems are presented inTable 5. Allalues are around 3.30, suggesting near-spherical confions of the macromolecules in solution[53].

.6. Circular dichroism

The CD spectrum gives information of the fractional cent of different elements of secondary structure and chan the tertiary structure of the protein. CD measuremenroteins are usually performed in two wavelength regi

ar UV and near UV. The far-UV CD spectra of native papnd papain–SDS mixtures at 200–250 nm wavelengthhown inFig. 11where the concentration of SDS is consthe spectrum (2) of pure papain differs from the spectrapain–SDS complexes. SDS changes the spectrum of p

ig. 12. Far-UV CD spectra of papain-SDS mixtures at 298 K. Spectrarepresent 0.1 M SDS and 0.1 g dl−1 papain, respectively. Spectra 3–9 r

esent 0.001, 0.002, 0.003, 0.004, 0.005, 0.05 and 0.1 M SDS, respen mixtures of equal volume of SDS and 0.1 g dl−1 papain solutions.


Table 6Estimated percentages of�-helix, �-structure, and random coil of papaina

in the presence of different concentrations of SDS from the curves ofFig. 12

Curve [SDS](mol dm−3)

�-Helix (%) �-Structure (%) Randomcoil (%)

2 0 79 0 213 0.001 8 46 466 0.004 18 26 569 0.100 28 30 42

a The concentration of papain is 0.1 g dl−1.

�-helical content enzyme and this helicity is low in presenceof low concentration of SDS, while�-structure and randomcoil of the enzyme are high. But at high concentration of SDS,�-helicity is exposed in nature and the papain–SDS clusteris formed. The strong electrostatic repulsion of those clus-ters entails the protein unfolding and causes the denaturationof protein[17]. The denaturation is considered to be due tothe SDS-induced unfolding of the papain. This unfolding isbelieved to occur in the co-operative binding region[27].

The near-UV CD spectrum reflects the contributions fromaromatic amino acid residues and disulfide bonds.Fig. 13shows the near-UV CD spectra of papain–SDS mixtureswhere the concentration of SDS remains constant. The CDintensity in near-UV region of disulfide of cysteine residuesmay be determined by the following three factors: the dihe-dral angle of the disulfide, the CS S bond angle, and theinteraction with the protein matrix[55]. The positive molarellipticity between 257.6 and 292.2 nm may originate fromthe interaction of the disulfides. Tertiary structure of nativepapain (curve 2) is completely changed in presence of SDS.Actually, tertiary structure of protein is absent in the unfoldedstate [56] in SDS micelle. Same nature is also observedin Fig. 14where [papain] is constant and [SDS] increases.

F tra 1a –8d y,i

Fig. 14. Near-UV CD spectra of papain–SDS mixtures at 298 K. Spectra1 and 2 denote 0.1 M SDS and 0.1 g dl−1 papain, respectively. Spectra 3–9denote 0.001, 0.002, 0.003, 0.004, 0.01, 0.05, and 0.1 M SDS, respectively,in mixtures of equal volume of SDS and 0.1 g dl−1 papain solutions.

The tertiary structure of papain is gradually destroyed withincreasing mole fraction of SDS in the mixture indicating theunfolded state of protein.

4. Conclusions

The papain–SDS system was studied by different meth-ods to understand the physicochemical properties of SDS andnature of structure of papain due to protein–surfactant interac-tion. The experimental values obtained by different methodspropose the structure of the complex due to the interaction ofpapain and SDS.

The addition of papain to SDS micelle changes the bulk aswell as interfacial properties of the solution. The CMC val-ues of SDS increase with increasing concentration of papaindue to adsorption of DS− on the papain molecule result-ing the formation of free micelle when the concentration offree surfactant monomers in the aqueous solution reaches theCMC. Increase in total maximum surface excess,Γ tot

max andcorresponding decrease in surface area,Atot

min indicates theinteraction among the adsorbed papain molecule in the lay-ers of the micelles. The values of the fraction of counter ionbinding,f of papain–SDS solution are higher than that of pureSDS micelle. The energetics of micellization of pure SDS ise ◦ ft tivelyh isa SDSs con-s thatm ty ofp ellei in coilda

ig. 13. Near-UV CD spectra of papain-SDS mixtures at 298 K. Specnd 2 denote 0.1 M SDS and 0.1 g dl−1 papain, respectively. Spectra 3enote 0.003, 0.008, 0.01, 0.02, 0.03, and 0.05 g dl−1 papain, respectivel

n mixtures of equal volume of 0.1 M SDS and papain solutions.

xothermic in nature and Hm is very low whereas that ohe papain–SDS cluster is endothermic and has comparaigh values of H◦

m. The interaction of SDS with papainn entropy-controlled process. The addition of papain toolution decreases aggregation number and dielectrictant, i.e. polarity of the micellar interface. This signifiesicellar clusters are smaller than free micelles. Viscosirotein-decorated micelle is higher than that of pure mic

ndicating enhanced aggregates and expansion of proteue to electrostatic repulsion among the attached DS− ionslong the polypeptide chain of protein.


The far-UV CD spectra of papain–SDS complex indicatethe less random coil and high�-helical content and unfoldedstructure of papain due to strong electrostatic repulsion. Thenear-UV CD spectra show that the tertiary structure of nativepapain is gradually destroyed in the presence of increas-ing [SDS] in the mixture indicating the unfolded state ofprotein.

Ultimately, the surfactant, SDS is used for the interactionwith globular proteins because it induces these proteins toexpand from their native structures[6]. Hence, it is said thatSDS changes the conformational properties of the polypep-tide chain of papain. The polypeptide chain is long and flexi-ble with micellar aggregates of SDS adsorbed on the surfacealong the chain. Papain belongs to random-coil and�-helixand has an unfolded structure in the co-operative bindingregion in presence of SDS supported by spectroscopic tech-niques. The increase in viscosity supports an expansion of aprotein coil in the cluster, and the calorimetric results indicatethe endothermic nature of the process which entails the pro-tein unfolding and causes the denaturation of protein. Suchstudy needs further investigations in future.

Acknowledgments

ern-m orka ject( ner-j whoa rk. Ia us-s n forh

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ins,

[ 209.[ 987)

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[ ang-

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I acknowledge the Department of Biotechnology, Govent of India for the partial financial assistance to this wnd also Jadavpur University for giving me a minor prounassigned MRP grant). I thank heartily Prof. Asok Baee, Department of Biophysics, Bose Institute, Calcuttalways helped and inspired me to pursue the whole wolso thank Dr. K.P. Das of Bose Institute for useful discions about CD spectra. I acknowledge Jaganmoy Guielping with the CD experiments.

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physicochemical and conformational studies of papain/sodium dodecyl sulfate system in aqueous medium

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