Colloids and Surfaces B: Biointerfaces 41 (2005) 209–216

Conformational study of papain in the presence of sodiumdodecyl sulfate in aqueous medium

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

Received 31 May 2004; accepted 9 December 2004Available online 25 January 2005

Abstract

The interactions between a globular protein, papain and the anionic surfactant, sodium dodecyl sulfate (SDS) have been investigated inaqueous medium using fluorimetric, circular dichroism, Fourier transform infra-red, UV–vis spectrophotometric, dynamic light scattering,and nuclear magnetic resonance techniques. The conformational change of papain in aqueous solution has been studied in the presence ofSDS. The results show the high�-helical content and unfolded structure of papain in the presence of SDS due to strong electrostatic repulsionl©

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eading to a “necklace and bead model” in protein–surfactant complexes.2004 Elsevier B.V. All rights reserved.

eywords: Conformational study; Protein–surfactant interaction; Papain; SDS

. Introduction

The study of surfactant–protein interaction creates muchnterest for many physicochemical as well as conformationalhenomena. Such interaction has been widely studied forany years because of its applications in industry, chemical,iological, pharmaceutical and cosmetic laboratories[1–15].ow-a-days, proteolytic enzymes (peptide-bond cleaving en-ymes) are extensively used in soap and detergent industriesor their very effective role to remove any stain, particularlytains of blood, egg-yolk, meat-soup, etc. In most of the in-eractions between proteins and surfactants, the “surfactantinding” to a single protein is considered, which can un-

old and sometimes denature the globular protein[3,5]. Twoeneral cases may appear for denatured proteins[16]: mix-

ures of anionic surfactants with proteins below and abovehe isoelectric point (IEP). Below the IEP, the protein isonsidered as a “cationic biopolymer” where the interac-ions with anionic surfactants are dominated by precipita-ion phenomena. Above the IEP, the interactions can formtable, fully solubilized complexes, which can change the

topology and conformation of the protein molecule in stion. The most widely used anionic surfactant, SDS unfand denatures proteins more than that of cationic surfac[5,7–8,17].

The water-soluble globular protein, papain (EC 3.4.2is a thiol enzyme obtained from the latex and unripe fruCarica papaya(tropical melon or papaw). Papain is a carhydrate free, basic, single chain protein. Papain has moleweight of 23,350 Da and consists of 212 amino acid resi(methionine absent; IP 8.75) with four disulfide bridgescatalytically important cysteine (position 25) and histidresidues (position 158)[18]. Papain is used medically ffetal as well as postnatal brain regions to provide maxdissociation and viability of the neurons and for the treatmof necrotic tissue and eczema.

Usually, various modes of association are obsefor surfactant–protein interactions due to dipole–dipion–dipole or ion–ion forces. Six different types ofsociations are discussed by Nagarajan et al.[19] onpolymer–surfactant interactions involving either individsurfactant molecules or surfactant clusters along with

E-mail address:gsoumen70@hotmail.com.

chains of polymers. The protein–SDS complex has beenproposed by different models, such as rod shaped model

927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved.

oi:10.1016/j.colsurfb.2004.12.004

210 S. Ghosh / Colloids and Surfaces B: Biointerfaces 41 (2005) 209–216

[2], necklace model[14,15], deformable prolate ellipsoidmodel [4], etc. Among these, the ‘necklace model’ seemsto have the strongest support. Originally, results from freeboundary electrophoresis proposed that an unfolded proteinbinds SDS in the form of micelle-like clusters[12]. Theproposal of necklace model is based on the results from freeboundary electrophoresis[20], SANS [21], quasi-elasticlight scattering [22], NMR [23], ESR [24], viscometry[14,25], circular dichroism[14,15] and fluorescence tech-nique[24,26], where the partially or fully unfolded proteinwraps around the micelle-like aggregates. This type ofmodel is also observed between surfactants with polymers[20,27]and polyelectrolytes[28].

The physicochemical properties have already been stud-ied [15] by tensiometry, conductometry, calorimetry, etc. forthe interaction between SDS and papain. In this paper, thework has been done to study the conformation of papain inpresence of SDS using a number of techniques like fluorime-try, circular dichroism, Fourier transform infra-red, UV–visabsorption and dynamic light scattering spectrophotometry,and nuclear magnetic resonance spectrometry. This investi-gation helps to determine the shape of the protein–surfactantaggregates.

2

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w rime-t tureo asc hor-s ingt onso erem them ettet darys ered of2 ture.F reaset was5

2.1.2. FluorimetryThe fluorescence emission spectra of native papain and of

papain–SDS mixtures were measured using a F-3010 Fluo-rescence spectrophotometer, Hitachi (Japan) with a slit widthof 1 cm. The excitation wavelength was 292 nm with the emis-sion range of 300–400 nm. All spectra were measured thricein a constant temperature water bath and the mean valueswere used for data processing.

2.1.3. Fourier transform infra-red spectroscopySolutions used for Fourier transform infra-red (FTIR)

measurements were prepared by mixing of papain and SDSsolutions in D2O at different concentrations in equal propor-tion. Infra-red spectra were measured with a Nicolet, Impact410 Fourier transform infra-red spectrophotometer (USA)at 298 K. Samples of a soluble or aggregated protein wereplaced between two CaF2 discs (32 mm diameter) separatedby a 25�m thick spacer. The CaF2 discs containing the pro-tein sample were packed into a thermally insulated cell holderassembly placed on a mount inside the sample compartmentof the FT-IR instrument, whose temperature was maintainedby circulating water from a constant temperature water bath.For each spectrum, 64 interferograms were averaged andcollected at absorbance mode with Happ–Genzel apodiza-tion function and Fourier transformed to give a resolutiono −1 −1 rD tion( -t ssiblen uriers idtha -h geo ered

2using

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. Experimental section

.1. Materials and methods

The globular protein, papain was crystalline powderoduct of Merck, Germany. It was dissolved in double

illed water and the pH was unadjusted. The anionic suant, sodium dodecyl sulfate (SDS) was a product of SigSA. In all preparations, the used double distilled waterpecific conductance of 3�S cm−1 at 303 K. All measureents were done at constant temperature in a wateraintained at 298± 0.01 K.

.1.1. Circular dichroism spectroscopyFar- and near-UV circular dichroism (CD) measurem

ere performed on a Jasco, J-600 recording spectropolaer (Japan) attached with a chiller to control the temperaf Xe-lamp and electronic circuit. The instrument walibrated with an aqueous solution of d-10-campulphonic acid. Solutions for CD were prepared by mixhe papain solution in water with different concentratif SDS in equal proportion. Far-UV CD spectra weasured between 200 and 250 nm wavelength withixture of papain and SDS in a 1 mm path length cuv

o study the conformational changes in the secontructure of the protein. Near-UV CD measurements wone with a 10 mm path length cuvette in the region50–320 nm to study the changes in the tertiary strucive scans of each spectrum were signal averaged to inc

he signal-to-noise (S/N) ratio and the scan speed0 nm/min.

f 4 cm from 4000 to 400 cm region. The spectrum fo2O was subtracted from the spectrum of the protein solu

or the mixture of protein–SDS) in D2O, the resultant proein difference spectra were smoothed to remove the pooise, and the baseline correction was made. Then Foelf-deconvolution (FSD) was carried out using a bandwt half height of the peak of 20 cm−1 and a resolution enancement factor (K value) of 2.5, over the frequency ranf 1700–1600 cm−1. Such computational procedures wone employing the Omnic software.

.1.4. UV–vis spectrophotometrySpectrophotometric measurements were performed

Shimadzu 160A UV–vis spectrophotometer (Japan)ng temperature-controlling arrangement; 1 cm quartzettes were used for sample holding. Initially, the absorbeading was taken at 280 nm with a sample containingliquots of 0.01 gm dl−1 papain solution and the referenell containing 2 ml water; then subsequent reading wasfter each addition of SDS solution to both cells at 298 K

.1.5. Dynamic light scattering spectrophotometryThe particle size of microwater droplets of prote

urfactant complex was determined using a dynamiccattering (DLS) instrument of Otsuka electronics, Jahe used light source was 632 nm Ne laser, which acted oolution of protein–surfactant mixture. The solution of papr papain–SDS mixtures (1:1, v/v) in aqueous medium (

ipore water) at different concentrations of SDS was takecylindrical quartz cell. The DLS measurements have

aken at the angle of 90◦, the scattered light from the so

S. Ghosh / Colloids and Surfaces B: Biointerfaces 41 (2005) 209–216 211

tion was detected by the photomultiplier tube, and the photocurrent was suitably amplified. The intensity data were pro-cessed in a computer. Essentially, the instrument measuresthe diffusion coefficient (DDLS) of the dispersed dropletsand determines the hydrodynamic diameter (dh) through theStokes–Einstein relation

dh = kT

3πηDDLS

wherek is the Boltzmann constant,T is the absolute temper-ature, andη is the viscosity of the solvent.

2.1.6. Nuclear magnetic resonance spectroscopyThe experiments of nuclear magnetic resonance (NMR)

were made on a Bruker DRX 500 MHz spectrometer, USAand water suppression was achieved by using WATERGATEpulse sequence. The diffusion measurement was carried outwith stimulated echo based pulse sequence [90◦-gradientpulse-90◦-delay for diffusion-90◦-gradient pulse acquisi-tion]. Solutions for NMR were prepared by mixing thepapain and SDS solutions in aqueous medium at differentconcentrations of SDS in equal proportion. Typical delaysfor diffusion for pure protein and protein–SDS mixtureswere set at 200 and 100 ms, respectively. The strength of thegradient pulse was varied and ln [intensity] versusG2 wasp

3

es ina spe-c tion( tes ata ation( AtC boveC MCv minedb etry,c al-ua MCvT DSi n intt plex[

3

ut toe ringu ovidei s of

Fig. 1. Ellipticity vs. wavelength curves (far-UV CD) of papain–SDS mix-tures at 298 K. Spectra A and B represent 0.1 M SDS and 0.1 gm dl−1 papain,respectively. Spectra C–F represent 0.001, 0.003, 0.005, and 0.1 M SDS, re-spectively, in the mixtures of equal volume of SDS and 0.1 gm dl−1 of papainsolutions.

secondary structure and changes in the tertiary structure ofpapain. The CD spectra of proteins are generally measured intwo wavelength regions: far-UV and near-UV.Fig. 1 showsthe far-UV CD spectra of native papain and papain–SDS mix-tures at 200–250 nm wavelength, where the concentration ofSDS increases keeping the concentration of papain constant.The spectrum (B) of pure papain differs from the spectra ofpapain–SDS complexes. In the presence of SDS, the spec-tra are different near 210 and 220 nm. The spectra (specially,E and F) of complexes show a hump at 220–222 nm at thehigh concentrations of SDS, indicating the less random coilwith the increase in�-helix [6] of the papain molecule towhich SDS binds. These spectra also show a minimum at<210 nm compared with pure papain, indicating the partialunfolded structure of papain in the presence of SDS. Suchtype of observation is reported in my earlier publication[14].The percentages of�-helix, �-structure, and random coils ofpapain molecule in SDS micelle have been calculated on thebasis of the experimental curves using a software program,k2d.zip.[29], where the input CD spectra file must contain 41CD values ranging from 200 to 240 nm. These are presentedin Table 1. It shows that at low concentration of [SDS], thehelicity of papain is low and increases with the increasingvalue of SDS. Actually,�-helicity cannot be exposed in thepresence of low concentration of SDS due to irregular confor-

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lotted. The temperature was set at 300 K.

. Results and discussion

In aqueous medium, SDS molecules form aggregatregular pattern known as micelle, after reaching a

ific concentration called the critical micelle concentraCMC). In papain–SDS systems, SDS usually aggregaconcentration known as critical aggregation concentr

CAC) substantially lower than the normal CMC of SDS.AC, protein bound micelles are formed where at and aMC, free micelle formation is possible. The CAC and Calues of SDS in the presence of papain have been detery different techniques such as tensiometry, conductomalorimetry, etc.[15]. The results show that the CAC ves of SDS in papain solution (around 1× 10−3 mol dm−3)re almost of the order of magnitude lower than the Calues of SDS in that solution (around 9× 10−3 mol dm−3).hese also show that both CAC and CMC values of S

ncrease with increase in the concentration of papaihe mixture, indicating the adsorption of DS− of SDS onhe surface of the papain molecule forming a com15].

.1. Circular dichroism

Circular dichroism (CD) measurements are carried oxamine the probability of conformational changes occurpon binding of SDS to papain. Such measurements pr

nformation of the fractional content of different element

able 1valuated percentages of�-helix, �-structure, and random coil of papaa

n the presence of different concentrations of SDS from CD curves

SDS] (mol dm−3) �-Helix (%) �-Structure (%) Random coil (%

79 0 21.001 8 46 46.003 11 33 56.005 26 20 53.100 28 30 42

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

212 S. Ghosh / Colloids and Surfaces B: Biointerfaces 41 (2005) 209–216

mation and also weak interaction between SDS and papain.But at the high concentration of SDS,�-helicity is exposed innature due to the strong electrostatic repulsion, while the per-centages of�-structure and random coil decreases, i.e. partialunfolding of the protein occurs and the papain–SDS aggre-gate is formed as a result of spreading of negative charge onthe polypeptide chains causing the denaturation of protein[14]. The unfolding process could be discussed on the basisof the initial electrostatic attraction between the positivelycharged amino acid residues of the protein and the nega-tively charged dodecyl sulfate (DS−) molecules[30]. Afterthe neutralization of all the positive charges by the negativecharges of the surfactants, repulsion between the negativelycharged head groups of SDS causes the unfolding. With addi-tion of excess SDS, its hydrophobic groups can penetrate theglobular structure of the protein to interact with its hydropho-bic backbone causing repulsion and ultimately the completeunfolding of the protein. According to Turro et al.[24], thedenaturation is due to SDS-induced unfolding of the papain.This unfolding occurs in the co-operative binding region.

The near-UV CD spectrum contributes from aromaticamino acid residues and disulfide bonds.Fig. 2 shows thenear-UV CD spectra of papain–SDS mixtures, where the con-centration of papain remains constant and [SDS] increases.The CD intensity in near-UV region of disulfide of cysteiner : thed dt f2 atef tureo ce ofS state[

F ix-t ,r SDS,rp

3.2. Fluorescence spectroscopy

Fluorescence is an excellent probe to investigate confor-mational changes of proteins, and such measurements areused to find information about the size of the surfactant aggre-gates formed along the chain of the protein. The aggregationnumber of SDS in the presence of papain has been deter-mined by the steady state fluorescence measurements usinga probe, safranine T. The results show that the aggregationof papain–SDS micelles takes place at higher concentrationof SDS than the CMC of pure micelles and the aggrega-tion number of SDS is lower in the presence of papain thanpure SDS micelle. The aggregation number of 0.1 M SDS inaqueous medium is 72 and that value decreases from 70 to63 with increasing concentration of papain (from 0.001 to0.01%). This suggests that the polypeptide chain of proteinmay wrap around several aggregates anchoring its hydropho-bic portions in the micellar phase and thereby disturbing onlythe outer parts of the micelles. Again, fluorescence is usuallydominated by the contribution of the tryptophan residues be-cause both their absorbance at the wavelength of excitationand their quantum yield of emission are considerably greaterthan the respective values of other amino acids. The changesin protein conformation, such as unfolding, very often leadto large changes in the fluorescence emission. In protein thatc andc lding[ ecu-lt tra oft s ares ofe ds –SDSm res-e

F S mix-t nt0 SDSac

esidues may be evaluated by the following three factorsihedral angle of the disulfide, the CS S bond angle, an

he interaction with the protein matrix[31]. In the range o57.6–292.2 nm, the positive molar ellipticity may origin

rom the interaction of the disulfides. The tertiary strucf the native papain (curve B) is changed in the presenDS and almost completely destroyed in the unfolded

32] in SDS micelle.

ig. 2. Ellipticity vs. wavelength curves (near-UV CD) of papain–SDS mures at 298 K. Spectra A and B denote 0.1 M SDS and 0.1 gm dl−1 papainespectively. Spectra C–F represent 0.001, 0.003, 0.004, and 0.1 Mespectively, in the mixtures of equal volume of SDS and 0.1 gm dl−1 ofapain solutions.

ontains tryptophan residues, both shifts in wavelengthhanges in intensity are generally observed upon unfo33]. As papain has five tryptophan residues in its molar structure, excitation was carried out at 292 nm[32] withhe emission range of 300–400 nm. The emission speche native papain (curve I) and of papain–SDS mixturehown inFig. 3. This figure shows the maximum intensitymission spectrum of the native protein at∼345 nm, the rehift and the decreased intensity of the spectra of papainixtures. This indicates the unfolding of papain in the pnce of SDS.

ig. 3. Fluorescence emission spectra of native papain and papain–SDures. Spectrum I represents 0.01 gm dl−1 papain and spectra II–IV represe.5, 20 and 100 mM SDS, respectively, in mixtures of equal volume ofnd 0.01 gm dl−1 papain solutions. Spectra were recorded in 1 cm× 1 cmells in a Hitachi F-3010 fluorimeter at 298 K.

S. Ghosh / Colloids and Surfaces B: Biointerfaces 41 (2005) 209–216 213

Fig. 4. Deconvoluted infra-red spectra in the amide I region of SDS, papain,and papain–SDS mixtures in D2O. Spectra A and B represent 1 mM SDS and3 gm dl−1 papain, respectively. Spectra C and D represent 0.1 and 20 mMSDS, respectively, in mixtures of equal volume of SDS and 6 gm dl−1 ofpapain solutions.

3.3. FT-IR spectroscopy

Fourier transform IR spectroscopy is one of the mostcommonly used spectroscopic techniques for determiningthe secondary structure of proteins in solution[34–36],and is used as an independent technique to confirm the CDmeasurements.Fig. 4 shows the infra-red spectra in theamide I region of pure papain and mixtures of papain–SDSdissolved in D2O. The presence of a number of amide I bandfrequencies resulting from theC O stretching vibration ofthe protein backbone has been correlated with the presence of�-helix,�-sheet,�-turn and random coil structures. The FSDspectra of theFig. 4 show nine components for each curve(B–D). These components are assigned inTable 2showing�-sheet,�-turn,�-helix and random coiled conformations atdifferent positions of amide I component band of papain andpapain–SDS mixtures in D2O. The appearance of a band at1616 cm−1 has been explained as due to the strong hydro-gen bonding in intermolecular�-sheet formation[36]. Aspectrum of pure papain inFig. 4(curve B) is different fromthe spectra of the mixtures of papain–SDS systems. In thepresence of very low concentration (0.0001 M) of SDS, thespectrum (curve C) of the mixture deviates highly from thatof papain indicating the change in the conformation of the

Fig. 5. Variation of absorbance at 280 nm vs. [SDS] on interaction with0.01 gm dl−1 of papain at 298 K.

protein. At the high concentration (0.02 M) of SDS, the bandposition of the spectrum D has higher absorbance comparedto the spectrum C, probably due to the conversion of certainpercentages of random coil into�-helix, which is highlyexposed in nature due to strong electrostatic repulsion indicat-ing the partial unfolding of protein supported by CD spectra(Table 1).

3.4. Absorbance spectrophotometry

Absorbance spectroscopic technique in UV region canbe used to study binding and folding–unfolding of the pro-tein initiated by surfactant.Fig. 5shows the absorbance val-ues of papain–SDS mixture at 280 nm as a function of theconcentration of SDS. The plot exhibits an initial sharp in-crease of absorbance at the low concentration of SDS (up to0.00255 mol dm−3) followed by an exponential decrease ofthat value. The initial sharp rise has been attributed to thebinding of anionic SDS to high affinity sites like cationichistidine exposed on the surface of papain[5]. The markedchange of absorbance value of the mixture at concentrationsbelow the CMC of SDS (0.008 mol dm−3) corresponds to thechange of the conformation of the protein, i.e. the protein un-folding [37]. In fact, DS− of SDS binding to cationic sitesof papain increases the absorption. In contrast, the unfoldingd

TD nd pap

M (cm−1)

111 in11111

able 2econvoluted amide I band frequencies and assignments of papain a

ean frequencies of curve B (cm−1) Mean frequencies of curve C

616.67 1614.34626.27 1622.88632.98 1639.19644.05 1649.29653.17 1658.58663.28 1667.65678.02 1684.23689.90 1695.62

ecreases the absorption[37].

ain–SDS mixtures in D2O at 298 K

Mean frequencies of curve D (cm−1) Assignment

1616.93 �-Sheet1626.50 �-Sheet1634.20 �-Sheet and extended cha1644.37 Un-ordered1653.44 �-Helix1664.57 �-Turn1678.78 �-Turn1690.44 �-Sheet

214 S. Ghosh / Colloids and Surfaces B: Biointerfaces 41 (2005) 209–216

Fig. 6. A profile of hydrodynamic diameter (dh) of papain–SDS mixturesvs. [SDS] obtained by DLS method at 298 K. The concentration of papainsolution in aqueous medium is 0.1 gm dl−1.

3.5. Dynamic light scattering spectrophotometry

The DLS measurements provide information on the hy-drodynamic diameter (dh), diffusion coefficient (DDLS) andpolydispersity index (PDI) of the solution of papain–SDScomplex. These measurements (dust free) give the indirectinformation on the conformation of papain in the complex.Fig. 6shows that the hydrodynamic diameter of 0.1 gm dl−1

pure papain in aqueous medium is 4.2 nm and increases withincreasing concentration of SDS, indicating an unfolded andextended papain molecule[37,38]. Here, the diameter of thefully denatured protein is around 14 nm. Actually, the largeamounts of adsorbed ionic surfactants (SDS) can be expectedboth to break the inter chain hydrophobic bonding and pro-vide an electrostatic repulsion favoring an extended structure.The interaction is mostly between SDS micelles and polypep-tide chain of papain, resulting in an expansion of the chainstabilized through the necklace and bead model (unfoldedprotein with SDS micelle-like clusters bound to it)[39].Fig. 7shows the exponential decrease in translational diffusion co-efficient (DDLS) as a function of the SDS concentration. ThePDI values obtained are all greater than 0.1 (requirement formonodispersity) and fall in the range of 0.46–0.60, indicatingthat the droplets in the complexes are fairly polydisperse andthe particle size distribution is heterogeneous.

3

anti ulesasa howna ,fa ase

Fig. 7. Diffusion coefficient vs. [SDS] plots whereDDLS andDNMR representdiffusion coefficient values obtained by DLS and NMR methods respectively.

of the values ofDNMR. The lower diffusion coefficient in-dicates an extended and unfolded structure of papain in thepapain–SDS complex. From this figure, it is also observedthatDNMR values are slightly higher thanDDLS values. Thisis probably due to not only a change in conformation of thecomplexes, but also some contribution from complex aggre-gation for the heterogeneity of the structure observed and alsodue to sensitivity of the instruments.

The surfactant, SDS is used for the interaction with globu-lar proteins because it induces these proteins to expand fromtheir native structures[6]. Hence it is said that the confor-mational properties of the polypeptide chain of papain are

F fu-sa equalv

.6. Nuclear magnetic resonance

Translational diffusion coefficient can provide importnformation about the hydrodynamic radius of the molecnd thus indicates the degree of compactness in protein.Fig. 8hows the plot of ln [intensity] versusG2 for pure papainnd papain–SDS mixtures. The slopes of the curves sre 0.00315, 0.00157 and 0.00136 cm2/Gauss2, respectively

rom which translational diffusion coefficient values (DNMR)re calculated.Fig. 7 exemplifies the exponential decre

ig. 8. ln [intensity] vs.G2 profile for measurements of translational difion coefficients of papain–SDS mixtures at 300 K. I, 2 gm dl−1 papain, IInd III represent 0.01, and 0.1 M SDS, respectively, in the mixtures ofolume of SDS and 2 gm dl−1 papain solutions.

S. Ghosh / Colloids and Surfaces B: Biointerfaces 41 (2005) 209–216 215

changed in the presence of SDS. The polypeptide chain islong and flexible with aggregates of SDS forming along thechain. Since this polypeptide chain is unfolded, there is aprobability that in the denatured region, some hydropho-bic portions of the polypeptide chain of papain tends to getpartially buried into the micelle, extending the structure ofthe complex and behaves like a nucleus of micellization tostabilize micelle-like clusters of small aggregation numbers(smaller than in protein-free SDS aqueous solutions). Thus,the chain of the protein must wrap around the polar shellof the micellar cluster of SDS supporting the “necklace andbead” model of the complex[14,15,24,26].

4. Conclusions

The papain–SDS system was studied by different methodsto understand the nature of structure of papain–SDS complex.

Earlier [15], it is observed that the interaction of SDSwith the papain is endothermic in nature (∆H

◦max of SDS is

positive, 1–6 kJ mol−1 in the presence of papain), which isconfirmed from calorimetric study. SDS forms the clusterin the presence of papain due to binding of DS− ion on thepapain molecule, and the micellization process of SDS ishindered to some extent (confirmed from tensiometric andc opy-c e ina risont freem thei -coilda

atet dw ofp r-UVC in isg n them res-c easedi e ofS mix-t dc entbi old-i allys ng ofS rkedc con-c g oft iam-e ationo thec sing

values of diffusion coefficients of papain–SDS mixtures withincreasing concentration of SDS indicating the unfolded andextended structure of papain in the complex. The PDI valuesshow that the complexes in the solution are fairly polydis-perse.

Ultimately, the polypeptide chain of papain is long andflexible with micellar aggregates of SDS adsorbed on thesurface along the chain. Papain belongs to random-coil and�-helix, and has an unfolded structure in the co-operative bind-ing region in presence of SDS supported by spectroscopictechniques. The increase in viscosity supports an expansionof a protein coil in the cluster, and the calorimetric resultsindicate the endothermic nature of the process, which entailsthe protein unfolding and causes the denaturation of protein.Hence, there is a probability that in the denatured region,the hydrophobic portions of the polypeptide chain of papainstabilize micellar clusters of small aggregation numbers andwrap around those clusters of SDS. This idea supports the“necklace and bead” model of the papain–SDS aggregate.

Acknowledgments

I acknowledge Jadavpur University for giving me a mi-nor project (unassigned MRP grant) to complete this work.I io-p d in-s f. S.R ionsa runM gan-m

R

try 6

nd

5)

man-teins,

76)

em-

ins,

em.

[ 17

[ 1980)

[ 16

onductometric studies). This interaction is also entrontrolled process. From fluorimetric study, decreasggregation number of papain–SDS system in compa

o SDS indicates that micellar clusters are smaller thanicelles. The increase in viscosity of the solution with

ncrease in [SDS] indicates the expansion of proteinue to electrostatic repulsion among the attached DS− ionslong the polypeptide chain of the protein.

The far-UV CD spectra of papain–SDS complex indiche less random coil and higher�-helical content compareith the structure at low [SDS] and unfolded structureapain due to strong electrostatic repulsion. The neaD spectra show that the tertiary structure of native paparadually destroyed in the presence of increasing [SDS] iixture, indicating the unfolded state of protein. The fluo

ence spectra of the complex show the red shift and decrntensity indicating the unfolding of papain in the presencDS. The infra-red spectra of papain and papain–SDS

ures in D2O show�-sheet,�-turn,�-helix and random coileonformations at different positions of amide I componand. These spectra show the exposure of�-helix of papain

n the presence of high [SDS], indicating the partial unfng of the protein. UV-spectrophotometric spectrum initihows the sharp rise of absorbance, indicating the bindiDS along with the polypeptide chain of papain. The mahange of absorbance value of the mixture at the lowerentration than the CMC of SDS indicates the unfoldinhe protein. DLS studies reveal that the hydrodynamic dter of the complex increases with increasing concentrf SDS, indicating an unfolded structure of papain inomplex. Both DLS and NMR results show the decrea

thank heartily Prof. Asok Banerjee, Department of Bhysics, Bose Institute, Calcutta, who always helped anpired me to pursue the whole work. I also thank Prooy and Dr. K.P. Das of Bose Institute for useful discussbout NMR, CD and FT-IR spectra. I acknowledge Baazumdar for performing the NMR experiments and Jaoy Guin for helping with the CD experiments.

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