3506 DOI: 10.1021/la903118c Langmuir 2010, 26(5), 35063513Published on Web 12/15/2009

pubs.acs.org/Langmuir

2009 American Chemical Society

Structure and Activity of Lysozyme on Binding to ZnO Nanoparticles

Soumyananda Chakraborti, Tanaya Chatterjee, Prachi Joshi, Asim Poddar,

Bhabatarak Bhattacharyya, Surinder P. Singh, Vinay Gupta, ) and Pinak Chakrabarti*,

Department of Biochemistry, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India,National Physical Laboratory, New Delhi 110012, India, Department of Engineering Sciences and Materials,University of Puerto Rico, Mayaguez, Puerto Rico 00681-9000, and )Department of Physics and Astrophysics,

University of Delhi, New Delhi 110007, India

Received August 21, 2009. Revised Manuscript Received November 23, 2009

The interaction between ZnO nanoparticles (NPs) and lysozyme has been studied using calorimetric as well asspectrophotometric techniques, and interpreted in terms of the three-dimensional structure. The circular dichroismspectroscopic data show an increase in R-helical content on interaction with ZnO NPs. Glutaraldehyde cross-linkingstudies indicate that the monomeric form occurs to a greater extent than the dimer when lysozyme is conjugated withZnONPs. The enthalpy-driven binding between lysozyme andZnOpossibly involves the region encompassing the activesite in the molecule, which is also the site for the dimer formation in a homologous structure. The enzyme retains highfraction of its native structure with negligible effect on its activity upon attachment to NPs. Compared to the freeprotein, lysozyme-ZnO conjugates are more stable in the presence of chaotropic agents (guanidine hydrochloride andurea) and also at elevated temperatures. The possible site of binding of NP to lysozyme has been proposed to explainthese observations. The stability and the retention of a higher level of activity in the presence of the denaturing agent ofthe NP-conjugated protein may find useful applications in biotechnology ranging from diagnostic to drug delivery.

Introduction

Adsorption of proteins on inorganic surfaces may lead tostructural and functional changes1,2 that are dependent on boththe nature of the adsorbed proteins and the physicochemicalproperties of the inorganic surfaces.3,4 Protein surface recognitionoffers a powerful tool in understanding protein-protein interac-tion,5 which is a key aspect of many complex cellular functions.6

Nanoparticles (NPs), because of their small size, have distinctproperties compared to the bulk form of the same material, thusoffering many new developments in the fields of biosensors,biomedicine, and bionanotechnology. The adsorption of proteinon NPs and its consequence on the structure and function arestrongly dependent on the size and shape of the NPs.7

Chicken egg white lysozyme (molecular weight (MW) = 14.6kDa) is a small globular protein, consisting of 129 amino acidresidues with four disulfide bonds. The importance of lysozymerelies on its extensive use as a model system to understand theunderlying principles of protein structure, function, dynamics,and folding through theoretical and experimental studies.8,9 Highnatural abundance is also one of the reasons for choosing

lysozyme as a model protein for studying protein-NP interac-tion. Another important aspect of lysozyme is its ability to carrydrug.10 According to the X-ray crystal structure, lysozymepossesses a relatively rigid structure.11 It contains six tryptophan(Trp) residues. Three of them are located in the substrate bindingsite, two are located in the core hydrophobic region, and one isseparated from all other residues. Trp62 and Trp108 are the mostdominant fluorophores.12

NPs have been widely explored for a wide range of biotechno-logical applications from sensing to drug delivery. In the past fewyears, there has been a great deal of work to identify theinteraction of lysozyme with silica and single-walled carbonnanotubes of varying shape and size.13,14 It is reported thatlysozyme retains a considerable amount of native-like secondaryand tertiary structure when adsorbed on small hydrophilic silicaNPs as compared to that on larger NPs. The fact that NPs withgreater surface area cause higher degrees of perturbation ofstructure and function of the protein has also been shown bystudying the effect of the increasing size of silica NPs on thethermodynamic stability and enzymatic activity of RNase A.15

Despite all these studies, little is known about the fundamentalrole of NPs in governing protein structure and function, and theregion on the protein surface where NPs bind still remainsnebulous. Zinc oxide (ZnO), with wide band gap (3.3 eV) andhigh excitonic binding energy (60 MeV), is a potential nanoma-terial for biomedical application because of its biocompatibility

*Corresponding author. E-mail: pinak@boseinst.ernet.in.(1) Larsericsdotter, H.; Oscarsson, S.; Buijs, J. J. Colloid Interface Sci. 2001, 237,

98103.(2) Billsten, P.; Carlsson, U.; Jonsson, B. H.; Olofsson, G.; Hook, F.; Elwing, H.

Langmuir 1999, 15, 63956399.(3) Hobora, D.; Imabayashi, S.-I.; Kakiuchi, T. Nano Lett. 2002, 2, 10211025.(4) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 81688173.(5) Janin, J.; Bahadur, R. P.; Chakrabarti, P.Q.Rev. Biophys. 2008, 41, 133180.(6) Degterev, A.; Lugovskoy, A.; Cardone, M.; Mulley, B.; Wagner, G.;

Mitchison, T.; Yuan, J. Y. Nat. Cell Biol. 2001, 3, 173182.(7) Shang,W.; Nuffer, J. H.;Muniz-Papandrea, V. A.; Colon,W.; Siegel, R.W.;

Dordick, J. S. Small 2009, 5, 470476.(8) Buck, M.; Schwalbe, H.; Dobson, C. M. Biochemistry 1995, 34, 13219

13232.(9) Ghosh, A.; Brinda, K. V.; Vishveshwara, S. Biophys. J. 2007, 92, 25232535.(10) Zhang, Z.; Zheng, Q.; Han, J.; Gao, J.; Liu, J.; Gong, T.; Gu, Z.; Huang, Y.;

Sun, X.; He, Q. Biomaterial 2009, 30, 13721381.

(11) Blake, C. C. F.; Koenig, D. F.; Mair, G. A.; North, A. C. T.; Phillips, D. C.;Sarma, V. R. Nature 1965, 206, 757761.(12) Imoto, T.; Foster, L. S.; Ruoley, J. A.; Tanaka, F. Proc. Natl. Acad. Sci.U.

S.A. 1972, 69, 11511155.(13) Asuri, P.; Bale, S. S.; Pangule, R. C.; Shah, D. A.; Kane, R. S.; Dordick, J.

S. Langmuir 2007, 23, 1231812321.(14) Vertegal, A. A.; Siegel, R.W.; Dordick, J. S.Langmuir 2004, 20, 68006807.(15) Shang, W.; Nuffer, J. H.; Dordick, J. S.; Siegel, R. W. Nano Lett. 2007, 7,

19911995.

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and high stability.16-18 It has been reported that ZnO nanowiresget solubilized in biofluids after a survival time of a few hours,which may find applications in drug delivery.19

In the present paper, we show that lysozyme, when bound toZnO NPs of 7 nm diameter at pH 7.4, takes on a more regularstructure in comparison to its free form. Isothermal calorimetry(ITC) has been used to quantify the interaction.When conjugatedto NP, lysozyme undergoes a lesser degree of unfolding inducedby guanidine hydrochloride (GdnHCl) and urea, and as a result,some residual activity is retained in the presence of denaturingagent. A molten globule intermediate can be detected in the urea-induced unfolding of the protein in the presence of NPs. ZnOpossibly binds around the active site of lysozyme and prevents thedimerization of the molecule.

Experimental Section

Materials. Chicken egg white lysozyme and Micrococcuslysodeikticus cells were purchased from Sigma (St. Louis, MO)as salt-free, dry powders and were used without further purifica-tion. GdnHCl, urea, glutaraldehyde, and all other chemicals werepurchased from Merck, India, and used as received. All otherreagents were of analytical grade, and double-distilled water wasused throughout the experiment.

NPPreparation. ZnOquantumdots (QDs) were prepared bywet chemical route,20 using zinc nitrate hexahydrate (Zn(NO3)2 36H2O) as a precursor. Ten millimolars of the compound wassonicated in water to get a clear solution. Twenty millimolarNaOHwas also sonicated inwater and added dropwise to the zincnitrate solution with stirring. The solution was allowed to stir for4-5 h. The precipitate was centrifuged, washed three to fourtimes, and collected after drying at 70 C.Sample Preparation.Abuffer solution, consisting of 0.1mM

sodium phosphate at pH 7.4, was used in all the experiments.Protein solution (concentration 10 M) was exhaustively dia-lyzed; using membrane (Spectra biotech membrane; molecularweight cutoff (MWCO) = 3500, Spectrum Laboratories, Comp-ton, CA) against buffer solution at 4 C. For studying NP-lyso-zyme interaction a fixed amount of the NP solution was added tothe protein solution, mixed by vortexing, and incubated at roomtemperature for overnight. Longer incubation time did not alterthe spectroscopic results. For unfolding studies, urea solutionwasprepared immediately before use. Commercially availableGdnHCl powder was used for preparing 10MGdnHCl solution.Different amounts of the stock solution of urea orGdnHCLwereused to obtain samples with 0-8 M concentration of urea and0-6MGdnHCl, butmaintaining the sameprotein concentration.A fixed amount (0.01 M of dithiotheritol (DTT) was used forreducing the disulfide bonds.

Circular Dichroism (CD) Spectropolarimetry. We mea-sure the far-UV CD spectra to evaluate the structural change oflysozyme induced by the addition of ZnO NP. The CD spectrawere obtained using a JASCO-810 spectropolarimeter equippedwith a thermostatically controlled cell holder. Protein concentra-tion was 10 M for all the experiments. The far UV region wasscanned between 200 and 260 nm with an average of three scansand also a bandwidth of 5 nm at 25, 70, and 80 C, respectively.The final spectra were obtained by subtracting the buffercontribution from the original protein spectra. The CD resultswere expressed in terms of mean residual ellipticity (MRE) in

deg 3 cm23 dmol

-1 according to the following equation:

MRE fobserved CD in m degg=Cpnl 1Cp is themolar concentration of protein, n is the number of aminoacid residues (129), and l is the path length (0.1 cm). Deconvolu-tion of the far-UV CD spectra to determine percentage composi-tion of the different secondary structural elements was done withCDNN (http://bioinformatik.biochemtech.uni-halle.de/cd-spec/cdnn).

Fourier Transform Infrared (FT-IR) Spectroscopy. Tenmilligrams of lyophilized lysozyme powder was added to asolution containing 0.1 mg of ZnONPs and made (using SPEEDVAC, Savant, Inc.) into a dry powder (protein/NP ratio of 100:1).Protein FT-IR spectra were recorded on a Perkin-Elmer spectro-meter equipped with a DTGS KBr detector and a KBr beamsplitter. All the spectra were taken via the absorbance mode withconstant nitrogen purging. Spectra were obtained at 4 cm-1

resolution with 50 scans. Spectra of background were collectedand subtracted from the original protein spectra. If not specifi-cally mentioned, all the spectra were collected in the range of1400-1800 cm-1.Fluorescence Spectroscopy. Fluorescence spectroscopy was

used tomonitor the tertiary structural change in lysozyme inducedby ZnO NP. All measurements were carried out using a HitachiF3000 spectrofluorimeter with 10 Mprotein concentration. Theslits were 5 nm for excitation and emission scans. Fluorescencewas measured by excitation at 295 nm and emission at 310-430 nm. Unfolding of lysozyme was monitored by noting thechanges of Fluorescence max as a function of GdnHCl concen-tration. The signals were fitted to an equation describing a two-state model of unfolding.21 8-Anilinonaphthalene-1-sulfonic acid(ANS), a hydrophobic fluorescence dye, is popularly used tomonitor the exposure and/or disruption of hydrophobic patchesof protein during its unfolding/folding process.22 We also usedANS fluorescence to study ANS binding; the excitation wave-length was set at 340 nm, and the emission spectra were recordedin the range of 440-600 nm.For fluorescence quenchingmeasurements,ZnONPwasadded

to the protein from a 500 M stock solution. The fluorescenceintensities were determined at the max and inner filter correction,and data analysis was done using the Stern-Volmer equation:23

FO=FC 1KSV NP 1Kq0NP 2Fo and Fc denote the steady-state fluorescence intensities in the

absence and in the presence of a quencher (ZnONP), respectively;KSV is the Stern-Volmer quenching constant, and [NP] is theconcentration of quencher. Kq is the bimolecular quenchingconstant, and 0 is the lifetime of fluorophore. Equation 2 wasused for determining KSV.

ITC. ITC measurement was performed on a VP-ITC calori-meter (Microcal Inc., Northampton, MA). Lysozyme was dia-lyzed extensively against 0.1M sodium phosphate buffer, and theligand (ZnO NPs) was dissolved in the last dialysate. A typicaltitration involved 12 injections of the NPs (20 L aliquot perinjection from a 400 M stock solution) at 5 min intervals intothe sample cell (volume 1.4359 mL) containing lysozyme(concentration, 35M).The titration cellwas stirred continuouslyat 310 rpm. The heat of the ligand dilution in the buffer alone wassubtracted from the titration data for each experiment. The datawere analyzed todetermine the binding stoichiometry (N), affinityconstant (Ka), and other thermodynamic parameters

24 of the

(16) (a) Singh, S. P.; Arya, S. K.; Pandey, P.; Malhotra, B. D.; Saha, S.;Sreenivas, K.; Gupta, V. Appl. Phys. Lett. 2007, 91, 063901.(17) Wu, Y. L.; Lim, C. S.; Fu, S.; Tok, A. I. K.; Lau, H. M.; Boey, F. C.; Zeng,

X. T. Nanotechnology 2007, 18, 215604.(18) Kumar, A. S.; Chen, M. S. Anal. Lett. 2008, 41, 141158.(19) Guo, D.; Wu, C.; Jiang, H.; Li, Q.; Wang, X.; Chen, B. J. Photochem.

Photobiol. B 2008, 93, 119126.(20) Joshi, P.; Chakraborti, S.; Chakrbarti, P.; Haranath, D.; Shanker, V.;

Ansari, Z. A.; Singh, S. P.; Gupta, V. J. Nanosci. Nanotechnol. 2009, 9, 64276433.

(21) Pace, C. N. Methods Enzymol. 1986, 13, 266.(22) Semisotonov, G. V.; Rodionova, N. A.; Kutysheno, V. P.; Elbert, B.;

Blank, J.; Pitiqyn, O. B. FEBS Lett. 1987, 224, 913.(23) Lakowicz, J. R. Principle of Fluorescence Spectroscopy, 3rd ed.; Springer:

New York, 2006; 278-285.(24) Goobes, G.; Goobes, R.; Shaw, J. W.; Gibson, M. J.; Long, R. J.;

Raghunathan, V.; Schueler-Furman, O.; Popham, M. J.; Baker, D.; Campbell,T. C.; Stayton, S. P.; Drobny, G. P. Magn. Reson. Chem. 2007, 45, S32S47.

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Article Chakraborti et al.

reaction usingOrigin software. Calorimetric titration of lysozymewith ZnO NPs was carried out at 25 C. The reported thermo-dynamic quantities were the average of two parallel experiments.

Glutaraldehyde Cross-Linking. A glutaraldehyde cross-linking experiment was carried out to monitor the oligomericstatus of lysozyme in the presence of ZnONPs. Ten micromolarsof protein was treated with 0.1 and 0.2% glutaraldehyde andincubated at room temperature for different time period. Thereactionwas then terminatedby the additionof 1MTris-HCl (pH8.0) and 1X sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS-PAGE) gel loading buffer. After being boiled in awater bath,25 samples were loaded on 10% tris-glycine SDS-PAGE along with a molecular weight marker from Bio-Rad.The bands were subjected to densitometry analysis (using theMOLECULARANALYST software (Bio-Rad, USA) for deter-mining the dimer/monomer ratio.

Lytic Activity of Lysozyme. The rate of lysis ofMicrococcuslysodeikticus (M. luteus) by lysozymewasmeasured as reported.26

The lytic activity was monitored turbidometrically at 450 nm atpH 7 and 30 C. To a 1 mL suspension ofM. luteus in 0.1 mM ofsodium phosphate buffer, 50 L lysozyme solution was added.Change in the turbidity at 450 nmwas recordedperminute using aShimadzu UV-2401 spectrophotometer with a thermostaticallycontrolled cell holder. One unit is equal to a decrease in turbidityof 0.001 per minute at 450 nm at pH 7.0 and 30 C under thespecified conditions. The formulas used to obtain the activity aregiven below.

Units=mg A450=minute 1000=mg enzyme in reaction mixture 3

MgP=mL A280 0:39 4Determination of Binding Stoichiometry between Lyso-

zyme and ZnO NPs by UV Spectroscopy. In this experiment,10 Mof lysozyme in phosphate buffer was equilibrated for 4 h at37 Cwith varying protein/NP ratios, that ranged from 5:1 to 1:4.After exposure, this suspension was centrifuged at 8000 rpm, andthe protein concentration in the supernatantwas determined fromUV absorbance at 280 nm using a Shimadzu UV-2401 spectro-photometer. The difference between the initial and final concen-tration of the protein, i.e., the amount of adsorbed lysozyme, wasnormalized to the milligram of protein adsorbed per unit area ofZnO NP and plotted against the mole fraction of the NP. Thestoichiometry of the protein-NP complex was determined by themolar-ratio method using a Jobs plot. The breakpoint in the plotcorresponds to themole fraction of theNP in its protein complex,giving the binding stoichiometry.27

Structural Analysis. The three-dimensional coordinates ofhen egg white lysozyme (code: 2 VB1, determined at a resolutionof 0.65 A)28 were obtained from the Protein Data Bank (PDB).29

Asa representativeofdimeric formof themolecule,weusedTapesjaponica lysozyme (code: 2DQA, resolution 1.6 A).30 The struc-tures were superimposed using the DALI server,31 and theresidues in the equivalent positions were used to make a sequencealignment (Figure S1, Supporting Information). The residues

forming the dimeric interface in 2DQA were identified usingPROFACE32 and were mapped into 2 VB1, thus identifying theputative interface for the hen eggwhite lysozyme thatmay exist insolution. The surface potential of the molecule was calculatedusing GRASP.33 Pockets and cavities in lysozyme were identifiedusing the CASTp (Computed Atlas of Surface Topography ofproteins) server34 located at http://cast.engr.uic.edu with thedefault probe radius of 1.4 A. PyMol35 was used to makemolecular diagrams.

Results

Properties of ZnO NPs. For all the experiments, colloidalZnO NPs were used. ZnO NPs are spherical in shape, asconfirmed by transmission electron microscopy (TEM) measure-ments, with size ranging from 4 to 7 nm.20 The isoelectric point,pI, of ZnO has been reported to be9.5.18However, as theremaybe some differences depending on the method of synthesis, andthere could be some residual nitrate anions adsorbed on surface ofour ZnONPs,20 we also determined the zeta potential at differentpH values (Figure S2), and the isoelectric point (9) was found tobe quite close. Thus the ZnO NPs are slightly positively chargedunder the experimental conditions. Dynamic light scattering datashowed that the NPs have a natural tendency to form aggregatesin solution (data not shown). To prevent aggregation, prolongedsonication was done to achieve a monodisperse solution of ZnONPs.Secondary Structure of Lysozyme in the Presence ofNPs.

Far-UV CD analysis provides information regarding the changein secondary structures, at pH 7.4 with the addition of ZnO NPs(Figure 1). The bands at 208 and 222 nm, characteristics of anR-helical structure, become more negative, indicating an increasein the helical content of lysozyme at the expense of the coil region(Table S1, Supporting Information) with the addition of NPs.ThusNPs induce the protein to acquire amore regular conforma-tion. Even when lysozyme is unfolded in the presence of NPs, thehelical content is more as compared to the unfolding of the freeformof the protein by urea orGdnHCl (Table S2). Also, whenwecompare the free andNP-conjugated forms of lysozyme, the latterseems to have a higher helical content when the temperature isincreased (Figure S3).The decrease in the random coil content of lysozyme induced

by NP conjugation is also revealed using FT-IR spectroscopy(Figure 2). Among the different bands of protein, the amide I in

Figure 1. Far-UV CD spectra of lysozyme (10 M in 0.1 Msodiumphosphate buffer) in the absence andpresence ofZnONPs.

(25) Wang, Y.; Guo, C. H. J. Biol. Chem. 2003, 278, 32103219.(26) Shugar, D. Biochim. Biophys. Acta 1952, 8, 302309.(27) Ghosh, K. S.;Maiti, T. K.;Mandal, A.; Dasgupta, S. FEBS Lett. 2006, 580,

47034708.(28) Wang, J.; Dauter, M.; Alkire, R.; Joachimiak, A.; Dauter, M Acta

Crystallogr. D 2007, 63, 12541268.(29) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;

Weissig, H.; Shindyalov, N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235242.(30) Goto, T.; Abe, Y.; Kakuta, Y.; Takeshita, K.; Imoto, T.; Ueda, T. J. Biol.

Chem. 2006, 282, 2745927467.(31) Holm, L.; Kaariainen, S.; Rosenstrom, P.; Schenkel, A. Bioinformatics

2008, 24, 27802781.(32) Saha, R. P.; Bahadur, R. P.; Pal, A.; Mandal, S.; Chakrabarti, P. BMC

Struct. Biol. 2006, 6, 11.

(33) Nicholls, A.; Sharp, K. A.; Honig, B. Proteins: Struct., Funct., Genet. 1991,11, 281296.(34) Binkowski, T. A.; Naghibzadeh, S.; Liang, J. Nucleic Acids Res. 2003, 31,

33523355.(35) DeLano, W. L. The PyMOLMolecular Graphics System; Delano Scientific:

Palo Alto, CA, 2002; http://www.pymol.org.

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the region 1600-1700 cm-1 (mainly CdO stretch) has a relation-ship with the secondary structure of protein, whereas the absor-bance intensity of the amide II band in the region 1500-1600 cm-1 (C-N stretch coupled with N-H bending mode)has been reported to be proportional to the amount of the proteinabsorbed on a surface.36 A shift in the amide II band from 1530 to1533.5 cm-1 with a loss of intensity indicates absorption oflysozyme on the NP surface. Different regions of the amide Iband are contributed by different secondary structural elements:1620-1645 cm-1 by -sheet, 1645-1652 cm-1 by random coil,1652-1662 cm-1 by R-helix, and 1662-1690 cm-1 by turns.37 Asmall peak around 1649 cm-1, observed for free lysozyme,disappears completelywhen it is bound to theZnONP, indicatinga loss in the nonregular structural region in the protein. Further ashift from 1657.6 to 1656.8 cm-1 is suggestive of small alterationsin the helical structure of lysozyme in the presence of the ZnONP.Unfolding of Lysozyme in the Presence of Urea and

GdnHCl. The effect of NPs on the unfolding of lysozyme wasstudied using Trp fluorescence. Eight molar urea and 6 MGdnHCl were used as denaturing agents. The effect of NP ismore on the urea-treated sample (Figure 3); while the proteinalone has a max at 340 nm in the folded form, which moves to349 nm in the presence of 8 M urea, the shift is only to 346 nmwhen NP is present. Thus the protein is not completely unfolded,and is possibly trapped in a molten globule-like intermediate dueto the presenceofNPs (elaboratedon in the next section).No suchintermediate is apparent when the unfolding is caused byGdnHCl. Moreover, there is no significant difference in theunfolding transitionmonitored by CD and fluorescence spectros-copy (Figure S4). As such, the unfolding induced by GdnHCl(Figure 4) was fitted with a two state model (N T U) and theparameters are presented in Table 1. On the basis of the unfoldingfree energy (4GNU), ZnO NPs stabilize the folded form oflysozyme by 0.3 kcal/mol. Also the unfolding transition midpointis shifted by 0.2 M to higher GdnHCl concentration.ANS Binding Studies. As the data given in the previous

section suggested that the presence of an intermediate whenlysozyme is unfolded in the presence of NP, we characterized itusing the fluorescence spectra of ANS-lysozyme complex in the440-600 nmwavelength range (Figure 5). At 5M urea, there is asubstantial enhancement of the fluorescence intensity, likely to becaused by exposure of hydrophobic residues. Although there is areduction in the ANS fluorescence intensity when the urea

concentration is increased to 8 M, it is still substantial. Theunfolding of free lysozyme does not cause any change in ANS

Figure 2. FT-IR spectra of lysozyme in 0.1 M sodium phosphatebuffer (pH 7.4) and treated with ZnO NPs.

Figure 3. Fluorescence spectra (ex = 295) of lysozyme in thepresence and absence of ZnO NPs on being treated with (a) 6 MGdnHCl and (b) 8 M urea.

Figure 4. Shift inmax of the fluorescence spectrum(ex=295nm)of free and NP-treated lysozyme as a function of GdnHCl con-centration. Data were fitted with a two-state model, and theparameters obtained are shown in Table 1.

Table 1. GdnHCl-Induced Unfolding of Lysozyme: Two-StateAnalysis Using Fluorescence Dataa

max-monitored data lysozyme NP-conjugated lysozyme

SN 340 340.0SU 351 350.04GNU (kcal/mol) 5.86 ( 0.25 6.16 ( 0.15mNU (kcal/mol/M) 1.35 ( 0.06 1.36 ( 0.09

aBased on data shown in Figure 4.

(36) Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Biochemistry 1993, 32,389394.(37) Speare, J. O.; Rush, T. S., III. Biopolymer 2003, 72, 193204.

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fluorescence intensity (data not shown). ANS does not normallybind to the native or fully unfolded protein, both the forms beingdevoid of sufficiently organized, exposed hydrophobic patchesthat can constitute a binding site for the dye.38 The binding ofANS indicates the existence of a molten globule-like intermediatewhen the unfolding of lysozyme by urea is carried out in thepresence of ZnO NPs.Quenching of Trp Fluorescence by ZnO NP. To study the

proximity of the NP binding sites on lysozyme to the location ofTrp residues in the protein structure, we analyzed the change inintrinsic fluorescence spectra with increasing NP concentrations(0-26 M). Figure 6 indicates a steady reduction in the fluores-cence signal from lysozyme.Mechanisms of fluorescence quench-ing are usually based on dynamic or static processes. However, ithas been reported that ZnO NPs form a ground-state complexwith Trp,39 and it is quite likely that the quenching of the intrinsicfluorescence of lysozyme results from a complex formationbetween the protein and the NP. The analysis of the Stern-Volmer plot provides a value of 2 104M-1 as theKSV constant.Thermodynamic Data on Lysozyme-NP Interaction.We

used ITC to investigate protein-NP interaction. The raw data ofthe binding of the ZnO NPs to lysozyme at 25 C is shown at thetop of Figure 7, while at the bottom is shown a plot of the heatflow per mole of the titrant (NP) versus the molar ratio (NP:lysozyme) at each injection, after subtraction of the backgroundtitration. The addition of ZnONPs exhibits an exothermic ligandbinding event, the various parameters for which are shown inTable 2. The values of G and Ka indicate moderate bindingbetween the two components.Although there is some reduction inentropy (and the CD data do indicate a slight increase in helicalcontent at the expense of nonregular structure), this gets ade-quately compensated by the enthalpy, and overall the bindingreaction is enthalpically driven. Although the data have beenpresented for 7 nm particles, very similar results are observed forsmaller (4 nm) particles also (data not shown).According to the fitted parameters for the ITC measurements

(Table 2) two protein molecules interact with one ZnO NP.However, as the calorimetric results do not always coincide withthe biding isotherm data,24 a plot (Figure S5) showing theadsorption isotherm for lysozyme onto the NP surface has alsobeen made using the protocol discribed in the Experimental

Section. This indicates a binding ratio of 1:1, i.e., a lesser surfacecoverage of NPs by lysozyme molecules as compared to the ITCdata.

Figure 5. Fluorescence emission spectra (ex = 340) of ANSbound to NP-conjugated lysozyme (curve 1), and on being treatedwith 8M (curve 2), and 5M (curve 3) urea. The 2.5Murea-treatedsample shows same fluorescence intensity as curve 1.

Figure 6. Quenching of Trp fluorescence of lysozyme in the pre-sence of varying concentrations of ZnO NPs. The correspondingStern-Volmer plot is shownbelow; the equationof the fitted line isFo/Fc = 1 0.0201 [NP] (R2 = 0.988).

Figure 7. ITC data from the titration of 35 M lysozyme in thepresence of 0.4 mM ZnO NPs. Heat flow versus time duringthe injection of ZnO NPs at 25 C and heat evolved per moleof added NPs (corrected for the heat of ZnO NP dilution) againstthe molar ratio (NP to lysozyme) for each injection, shown at thetop and bottom, respectively. The data were fitted to a standardmodel.

(38) Mukherjee, D.; Saha, R. P.; Chakrabarti, P. Biochim. Biophys. Acta 2009,1794, 11341141.(39) Mondal, G.; Bhattacharya, S.; Ganguly, T. Chem. Phys. Lett. 2009, 472,

128133.

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Glutaraldehyde Cross-Linking. Lysozyme from hen egg hasa tendency to form a weak dimer.40 Glutaraldehyde cross-linkingexperiments carried out at physiological pH confirmed theexistence of both monomer and dimer forms of lysozyme(Figure 8), but in the presence of NP the relative content of thedimeric formwas reduced (Table 3). The dimer-to-monomer ratiois 0.11when lysozyme is incubatedwith 0.1%glutaraldehyde for 1min (lane 1), which increases to 0.19 with 0.2% glutaraldehydeand 3 min incubation (lane 4). In the presence of NPs, thecorresponding ratios are 0.07 (lane 6) and 0.1 (lane 9), respec-tively. This may possibly indicate that the association betweentwo lysozyme chains is hindered by the direct binding of NPs atthe same region that is involved in the dimer formation, or that theinterference is caused indirectly by the perturbation broughtabout in the structure by NP binding.Lysozyme Activity. To test whether adsorption of lysozyme

to a ZnO NP has any role in the enzymatic activity, we examinedthe activity of the protein adsorbed on the ZnO NP surfacerelative to that of the free protein (Figure 9). Even when thelysozyme/NP ratio is 1:500, the enzyme retains about 90% of itsactivity. We also tested the activity under denaturing conditions.While only 8%of the activity is retainedwhen lysozyme is treated

with 8 M urea, it is 14% when the protein is in the conjugatedform. Incubated with 6 M GdnHCl, there is drastic reduction inthe activity, but still the NP treated protein showed 3% higheractivity over the untreated protein. These results indicate thatZnONP stabilizes the integrity of the active site in the presence ofchaotropic agents.

Discussion

Effect of ZnONP on the Secondary Structure Content ofLysozyme. There is an approximately 4% increase of the helicalcontent (at the expense of random coil structures) of lysozyme inthe presence of NPs, as can be seen from the CD and IR data(Figures 1 and 2, and Table S1). Interestingly, ZnONPs have alsobeen reported to bring about a very similar change in theR-helicalcontent of glucose oxidase.41 Lysozyme as well as horseradishperoxidase and subtilisin Carlsberg, when covalently attached tosingle-walled carbon nanotubes, were found to retain a highfraction of their native structure and activity, and were morestable in the presence of GdnHCl and at elevated temperaturerelative to the free enzyme.13 Bovine serum albumin whenconjugated to gold NPs underwent substantial conformationalchanges, i.e., a decrease in helical structures and an increase in -sheet structure, becoming more flexible.42 On the other hand,chymotrypsinwasdenatured completely by functionalizedmixed-monolayer protected gold clusters43 and single-walled carbonnanotubes.44 Similarly, nano-TiO2 induced transition of R-helixinto -sheet, resulting in a substantial inactivation of lysozyme.45

It is likely that the hydrophobic/hydrophilic nature of the NP, itssize, and surface curvature, the charge distribution on the protein,and so forth would have consequences on the site of binding onthe protein surface and how the binding of NP affects thestructure of the protein.14,15 Although the existing data on thedetails of NP-protein interactions are rather meager, the discus-sion below provides some insight into the possible binding site ofZnO NPs on lysozyme.

Table 2. Thermodynamics Parameters Involved in the Binding be-tween Lysozyme and ZnO NP, Derived from ITC Measurements

parameter value (standard deviation)

N (NP: protein stoichiometry) 0.54 ( 0.01Ka (binding constant, M

-1) 1.03 106 ( 0.2H (binding enthalpy, kcal/mol) -10.3 ( 0.3S (entropy change, cal/mol.K) -6.38G (free energy change, kcal/mol) -8.37

Figure 8. Glutaraldehyde cross-linking of lysozyme. SDS-PAGEof glutaraldehyde cross-linked samples of lysozyme, untreated(lanes 1-4) and treated with ZnO NPs (lanes 6-9). Lane 5 showsthe protein marker. Concentration and incubation period ofglutaraldehyde for various samples are as follows. Lane 1: 0.1%,1 min; 2: 0.2%, 1 min; 3: 0.1%, 3 min; 4: 0.2%, 3 min; 6: 0.1%,1 min; 7: 0.2%, 1 min; 8: 0.1%, 3 min; 9: 0.2%, 3 min.

Table 3. Dimer-to-Monomer Ratio of Lysozyme in the Presence ofDifferent Concentrations of Glutaraldehyde at pH 7.4

sample

glutaraldehydeconcentration

(%)

time ofincubation

(min)

ratioa

(dimer/monomer)

lysozyme 0.1 1 0.11lysozyme 0.2 3 0.19lysozymeNP 0.1 1 0.07lysozymeNP 0.2 3 0.1

aFrom densitometry analysis of the bands in Figure 8.

Figure 9. The relative activity (%) (with respect to the free en-zyme) of lysozymewith varying concentration ofNPs (the first twobars) and of unfolded lysozyme (with 8 M urea, middle two bars,and 6MGdnHCl, the last two bars) treated with fixed concentra-tion of NPs.

(40) Onuma, K.; Inaka, K. J. Crystal Growth. 2008, 310, 11741181.

(41) Ren, X.; Chen, D.; Meng, X.; Fangqiong, T.; Hou, X.; Han, D.; Zhang, L.J. Colloid Interface Sci. 2009, 334, 183187.(42) Shang, L.; Wang, Y.; Jiang, J.; Dong, S. Langmuir 2007, 23, 27142721.(43) Fischer, N. O.; McIntosh, C. M.; Simard, J. M.; Rotello, V. M. Proc. Natl.

Acad. Sci.U.S.A. 2002, 99, 50185023.(44) Karajanagi, S. S.; Vertegel, A. A.; Kane, R. S.; Dordick, J. S. Langmuir

2004, 20, 1159411599.(45) Xu, Z.; Liu, X. W.; Ma Y. S.; Gao, H. W. Environ. Sci. Pollut. Res. [Online

early access]. DOI: 10.1007/s11356-009-0153-1. Published on the web: April 2009.

3512 DOI: 10.1021/la903118c Langmuir 2010, 26(5), 35063513

Article Chakraborti et al.

Putative Binding Site of ZnO NPs on Lysozyme and ItsConsequence. The catalytic residues, Glu35 and Asp52, lie in acleft that is contiguous to the largest pocket (with a volume of84 A3 and molecular surface area of 73 A2) harboring the bindingsite (Figure 10a). This largest depression on the protein surfacewould allow a close approach by NPs providing the maximumcontact surface area. The interaction would also be facilitated bythe electrostatic charge distribution (Figure 10b), a negativepotential at the active site; at pH below 9.5, the surface of ZnONPs becomes positively charged by absorption of surroundingH.18 As the entropy contribution to binding is not large(Table 2), there may not be much change in the conformationand water structure around the active site induced by the binding.Because of this, binding the integrity of the active site would bepreserved, even at higher concentrations of urea, explaining the

residual catalytic activity (Figure 9). Moreover, as the bindingconstant is not very large (Table 2), NPs can be replaced by thesubstrate, and the enzyme can still function at the 90% level in thepresence of NPs.The active site of lysozyme contains two Trp residues that are

important for substrate binding (Figure 10a). Our conjecture ofthis is that the NP binding site is also supported by the fluores-cence quenching data (Figure 6), as this positionwouldbringZnOparticle in close proximity to the two Trp residues. From theequilibrium adsorption isotherm (Figure S5), one protein mole-cule interacts with one NP. For a proper perspective of thegeometry of binding, the distance between the opposite tips ofthe pocket in Figure 10a is21 A (= 0.21 nm). Interestingly, theincrease in the helical content of lysozyme brought about by thebinding of NP at the active site has also been observed during thebinding of a drug molecule, menadione at the same site.46

ZnONPBinding and theOligometric State of Lysozyme.A dimeric form of lysozyme is also known to exist in solution.40

However, as there is no known crystal structure of hen egg whitelysozyme in this oligomeric form, we used the dimeric structure ofTapes japonica30 to model the possible interface region of theputative dimer. Although the C-terminal region of the moleculesfrom the two organisms differ considerably (Figure S1), thestructures are similar enough to enable us to transfer informationon dimerization from one molecule to the other. Comparison ofpanels b and c in Figure 10b shows that there is considerableoverlap between the active site and the interface regions, indicat-ing thatNPs bound to the former would prevent the formation ofthe dimer, as has been indicated by cross-linking studies (Figure 8and Table 3).Urea-Induced Unfolding of Lysozyme in the Presence of

ZnO NPs. The Trp fluorescence did not indicate a completeunfolding of lysozyme by 8Murea (Figure 3b). The existence of amolten globule intermediate in the unfolding pathway is indicatedby the binding and consequent enhancement of ANS fluores-cence, which persisted even at 8 M concentration of urea(Figure 5).Molten globule-like structures have also been reportedduring the unfolding of lysozyme adsorbedon silicaNPs,47 as alsofor free lysozyme under various denaturing conditions.48,49 Theexposure of hydrophobic patches with consequent ANS-bindinghas also been observed during the unfolding of -lactoglobulinadsorbed on silica NP surfaces.50

Conclusions

In conclusion, using lysozyme as a model protein, we haveshown that NPs are capable of disrupting protein-proteinassociation. ZnO NPs bind to the largest cleft on the proteinsurface, thereby helping it to retain the secondary structures to agreater degree and exhibit enzymatic activity even under denatur-ing conditions. There have been promising applications of ZnO-based nanomaterials in biosensors even at elevated tempera-tures.16-18 The stabilizing influence of ZnO NPs on lysozymeand the mode of interaction elucidated in this article would beuseful for the fruitful application of NPs in biotechnology.

Acknowledgment. We thank Dr. K. Chattopadhyay for hishelp in zeta potential measurement. T.C. and P.C. are supported

Figure 10. Cartoon and surface representations of the structure ofhen egg white lysozyme showing the largest pocket on the surface.(a) The active site pocket with the catalytic residues (Glu35 andAsp52) in red and the other residues in the binding site (Gln57,Ile58, Asn59, Trp63, Ile98, Ala107, Trp108) in light orange.(b) Surface potential of the molecule with the scale shown ontop. (c) The putative dimeric interface of the molecule is shown inred. The orientations of the molecules in b and c are roughly thesame and approximately 90 rotated about a horizontal axisrelative to that in a.

(46) Banerjee, S.; Choudhury, D. S.; Dasgupta, S.; Basu, S. J. Lumin. 2008, 128,437444.(47) Wu, X.; Narsimhan, G. Biochim. Biophys. Acta 2008, 1784, 16941701.(48) Hameed, M.; Ahmed, B.; Fazili, K. M.; Andrabi, K.; Khan, R. H. J.

Biochem. 2007, 141, 573583.(49) Bhattacharjya, S.; Balaram, P. Protein Sci. 1997, 6, 10651073.(50) Wu, X.; Narsimhan, G. Langmuir 2008, 24, 48884893.

DOI: 10.1021/la903118c 3513Langmuir 2010, 26(5), 35063513

Chakraborti et al. Article

by grants from the Department of Science and Technology. S.P.acknowledges the funding from IFN-EPSCoR.

Supporting Information Available: Two tables (Tables S1and S2) describing data on the secondary structural contentof lysozyme (at different temperatures and in the presence ofdenaturants) based on CD spectra, and five figures (Figures

S1-S5) showing temperature-dependent CD spectra, over-lay of unfolding of lysozyme monitored by CD and fluores-cence spectroscopy, the sequence alignment of two homo-logues of lysozyme, the adsorption pattern of lysozyme onthe NP surface, and measurement of zeta potential of ZnONP at different pH values. This material is available free ofcharge via the Internet at http://pubs.acs.org.