Journal of Colloid and Interface Science 363 (2011) 585–594

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Journal of Colloid and Interface Science

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Positive cooperative mechanistic binding of proteins at low concentrations: Acomparison of poly (sodium N-undecanoyl sulfate) and sodium dodecyl sulfate

Susmita Das a, Monica R. Sylvain a, Vivian E. Fernand a, Jack N. Losso b, Bilal El-Zahab a, Isiah M. Warner a,⇑a Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USAb Department of Food Science, Louisiana State University, Baton Rouge, LA, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 May 2011Accepted 14 July 2011Available online 27 July 2011

Keywords:Intrinsic fluorescencePoly-SUSSDSMolecular micelleProtein–surfactant interactionCooperative bindingSDS–PAGE

0021-9797/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.jcis.2011.07.044

⇑ Corresponding author. Fax: +1 225 578 3971.E-mail address: iwarner@lsu.edu (I.M. Warner).

The interactions of the negatively charged achiral molecular micelle, poly (sodium N-undecanoyl sulfate)(poly-SUS), with four different proteins using intrinsic and extrinsic fluorescence spectroscopic probes,are studied. A comparison of poly-SUS with the conventional surfactant, sodium dodecyl sulfate (SDS),and the monomeric species, SUS, is also reported. In this work, we observed that poly-SUS preferentiallybinds to acidic proteins, exhibiting positive cooperativity at concentrations less than 1 mM for all pro-teins studied. Moreover, it appears that the hydrophobic microdomain formed through polymerizationof the terminal vinyl group of the monomer, SUS, is largely responsible for the superior binding capacityof poly-SUS. From these results, we conclude that the interactions of poly-SUS with the acidic proteins arepredominantly hydrophobic and postulate that poly-SUS would produce superior interactions relative toSDS at low concentrations in polyacrylamide gel electrophoresis (PAGE). As predicted, use of poly-SUSallowed separation of the His-tagged tumor suppressor protein, p53, at sample buffer concentrationsas low as 0.08% w/v (2.9 mM), which is 24 times lower than required for SDS in the standard reducingPAGE protocol. This work highlights the use of poly-SUS as an effective surfactant in 1D biochemicalanalysis.

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1. Introduction

Studies of protein–surfactant interactions are important innumerous aspects such as biochemical, industrial (food and cos-metic), pharmaceutical and in the development of analytical tech-niques for protein separation and detection [1–3]. Understandingsuch interactions definitely gives us better insight into proteinstructure and function [1–8]. Interactions of different classes ofsurfactants, such as cationic, anionic, zwitterionic, and neutral aswell as surfactants with variable alkyl chain lengths and geminisurfactants, have been studied using various model proteins [9–12]. Among the various surfactants, characterization of proteininteraction with sodium dodecyl sulfate (SDS) continues to be along-standing area of ambiguity [2,3,7]. SDS–PAGE (polyacryl-amide gel electrophoresis) is an established technique for proteinseparations [4–8]. Though this technique has been used for a widevariety of protein separations, it exhibits weak resolution for sep-arating hydrophobic proteins or complex protein mixtures [13].In some cases, a mixture of cationic and anionic surfactants is usedto overcome this problem and zwitterionic surfactants are also

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sometimes considered a better alternative [13,14]. Protein separa-tions are primarily based on differences in binding affinities of thesurfactant to the various proteins in a mixture.

In evaluating the literature on recent investigations of protein–surfactant interactions, it is evident that such studies have almostalways been restricted to single or double chain monomericspecies [9,10,15]. However, to the best of our knowledge interac-tions of proteins with polymerized surfactants (herein termedmolecular micelles) have not been reported. Molecular micellesgarner attention because of their unique physicochemical proper-ties in comparison to those of single and double chain surfactants.Therefore, we believe this issue is of practical interest and rele-vance to using molecular micelles as separation reagents in 1Dand 2D gel electrophoresis for hydrophobic proteins and as probesto further our understanding of their solubilization [16]. In thestudies reported here, the interactions of four proteins in solutionwith a molecular micelle, poly (sodium N-undecanoyl sulfate)(poly-SUS), its monomeric species, SUS, and the conventional sur-factant, SDS, have been examined using fluorescence spectroscopyand circular dichroism.

Poly-SUS is an amphipathic molecule with an achiral hydro-philic head group and covalently bound hydrophobic tails(Fig. 1).

+Na -O3SO

n

+Na -O3SO

60Co Irradiation

(a) (b) (c)+Na -O3SO

Fig. 1. Structures of the (a) monomer, sodium undecylenic sulfate (SUS), (b)molecular micelle, poly (sodium N-undecanoyl sulfate) (poly-SUS), and (c) theconventional surfactant, SDS.

586 S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594

In general, poly-SUS is formed by polymerizing the double bondat concentrations five times higher than the critical micelle con-centration (CMC) using c-irradiation. This concentration ensuresthat a spontaneous self-assembled phase exists. Subsequently,the dynamic equilibrium between the surfactant monomer andthe micelle is largely eliminated after irradiation/polymerization.Therefore, molecular micelles do not have a CMC, and their overallstability is not compromised when interacting with proteins. Theirradiation process imparts a unique morphology to the molecularmicelle through formation of a covalently bound highly hydropho-bic micro-domain. In this study, such molecular micellar hydro-phobicity has been confirmed by use of the hydrophobic probe8-anilino-1-napthalenesulfonic acid (ANS), and it is found to bein stark contrast to conventional and second generation surfac-tants. Thus, it is expected that poly-SUS should have access togreater numbers of sites on proteins that are improbable as a resultof the dynamic assembly and disassembly of a conventional mi-celle. As a result, we hypothesize that protein binding would beachieved at much lower concentrations using a molecular micellethan is achieved with a conventional surfactant such as SDS. More-over, our group has demonstrated that interactions with molecularmicelles provide superior separation schemes relative to conven-tional micelles due to their improved interactions [17–19]. Forexample, we have demonstrated that by systematically changingthe concentration of poly-SUS in the running buffer, resolution ofsixteen polycyclic aromatic hydrocarbons with relatively high effi-ciency and small k0 values are achievable [17].

Thus, the present work was undertaken to study the mechanismof interaction of four proteins, namely Bovine Serum albumin(BSA), ovalbumin (OVA), a-chymotrypsinogen A (aCHY) anda-lactalbumin (aLAC), with the achiral molecular micelle, poly-SUS and compare these results to interactions with the monomerSUS and the more commonly used conventional surfactant SDS.The focus of this study was to provide a basis for the developmentof a new analytical tool for use in biochemistry and biotechnology.Our results suggest that poly-SUS indeed exhibits stronger interac-tions with the proteins under consideration at much lower concen-trations, as compared to the monomeric species SUS and SDS. Thetype of interactions as interpreted using Scatchard analysis is dis-tinctly different, which explains the stronger interactions andhigher binding constants of poly-SUS as compared to SDS andSUS. Hence, these observations suggest that due to its unique prop-erties, poly-SUS can serve as a more favorable alternative to SDS inapplications such as gel electrophoresis, protein extraction, andsolubilization.

2. Materials and methods

2.1. Materials

Serum albumin (bovine, 66 kDa, BSA, 98%), ovalbumin (egg,45 kDa, OVA, 98%), a-chymotrypsinogen A (bovine pancreas,26 kDa, aCHY), a-lactalbumin (bovine milk, 14.2 kDa, aLAC, 85%),p53 (43 kDa) and 8-anilino-1-napthalenesulfonic acid (ANS) wereobtained at the highest purity available from Sigma–Aldrich (St.Louis, MO, USA) and used as received. Tris/Glycine buffer was usedfor all studies since this is the buffer traditionally used in 1D gelelectrophoresis. Ultrapure water (18.2 MX) was obtained usingan Elga PURELAB Ultra water purifier (Lowell, MA, USA). SDS(>98%), Trishydroxymethylaminomethane, and Glycine were ob-tained from Invitrogen Corporation (Carlsbad, CA, USA) and thePrecision Plus Protein All Blue Standard marker was obtained fromBio-Rad Laboratories (Hercules, CA). Polyethyleneglycol (PEG,M.W. 200–1000) were obtained from Polysciences Inc. (Warring-ton, PA). All chemicals were used as received without furtherpurification.

2.2. Methods

2.2.1. Synthesis of the molecular micelle, poly-SUSPoly-SUS was synthesized according to a procedure previously

reported by Warner et al. [17] and Bergstrom [20]. The criticalmicelle concentrations (CMC) of monomeric SUS [�25 mM (aq)]and SDS [�8 mM (aq)] were determined by use of surface tensionmeasurements at room temperature with a KSV Sigma 703 digitaltensiometer (Fig. S1). Polymerization of SUS was achieved at a con-centration of five times the CMC under c-irradiation using a 60Cosource. ESI-MS experiments suggest the presence of differentmolecular weights viz. 295.115, 567.214, 381.315, 567.214,839.309, 1111.405, 1385.5055, 1657.6 corresponding to differentspecies ranging from monomer to hexamer + Na. However, averagemolecular weight of the polymer as determined by use of viscositymeasurement suggests a molecular weight of 534 which is close toa dimer. Viscosity measurements were performed using Aton Paarautomated micro viscometer based on falling sphere methodwhich is PC controlled by use of Visolab Filmware software (AtonPar, Austria). PEGs of different known molecular weights between200 and 1000 were used as standards. The intrinsic viscosity foreach polymer–solvent system was determined from the intersec-tion of a Huggins plot (gred vs. c) and Kraemer plot lngrel/c vs. c,where gred is the reduced viscosity and grel is the relative viscosity.Then Mark-Howink equation [g] = KMa was used to obtain K and a,the Mark–Howink constants, using the standard. Using K, a and theintrinsic viscosity ([g]) of the unknown polymer the molecularweight poly-SUS was estimated. Monomeric SUS and polymericSUS (poly-SUS) were characterized by use of 1H NMR (Fig. S2, sup-porting material) in deuterium oxide (D2O) on a Bruker–250 MHzinstrument. Complete polymerization was confirmed by observingthe disappearance of the NMR chemical shift signals (5.0–6.0 ppm)associated with the terminal vinyl group. All poly-SUS solutionsare reported using the equivalent monomer concentration, namelycalculations were based on the molecular weight of the individualsurfactant unit (i.e., SUS, 272 g/mol).

2.2.2. InstrumentationFluorescence spectra were recorded at 25 �C using a SPEX

Fluorolog-3 spectrofluorimeter (Jobin Yvon, Edison, NJ) equippedwith a 450-W xenon lamp and R928P photomultiplier tube(PMT) emission detector. A quartz cuvette with an optical path-length of 1 cm was used and bandwidths for both the excitationand emission monochromators were set at 3 nm unless otherwise

S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594 587

stated. Excitation was performed at 295 nm (Trp) and 364 nm(ANS), while emission spectra were respectively measured in theranges of 335–360 nm and 460–525 nm. Fluorescence spectrareported herein were obtained from proteins at concentrations of1 mg/mL in 25 mM Tris/192 mM Glycine, pH 8.4 unless otherwiseindicated. Circular dichroism (CD) data were obtained using anAVIV Model 62DS (AVIV Associates, Lakewood, N.J.) spectropho-tometer at 25 �C fitted with a 1 mm pathlength quartz cell. TheCD spectra of native protein samples in 25 mM Tris/192 mM Gly-cine, pH 8.4, were acquired at concentrations that produced opti-mal CD signal. The Tris/Glycine buffer was filtered through a0.45 lm nylon filter prior to sample preparation. All CD scans wereconducted in triplicate in the far UV (200–240 nm) and near UV(240–320 nm) regions of the spectrum, respectively, and averagespectra were recorded. All CD spectra were also corrected for back-ground intensity of the buffer. The CD response is reported as ellip-ticity and displayed in units of millidegree (mdeg).

2.2.3. Determination of binding parameters of SDS, SUS, and polySUSto protein

A spectrophotometric titration procedure was used to deter-mine the characteristic binding parameters of poly-SUS, SUS, andSDS interacting with the four proteins employed in this study.The proteins (1 mg/mL) were allowed to equilibrate for 30 minwith a range of concentrations of poly-SUS, SUS, and SDS (0–20 mM) in 25 mM Tris/192 mM Glycine, pH 8.4 at 25 �C. The bind-ing isotherms, stoichiometries, and dissociation constants weredetermined employing Scatchard Analysis [21,22]. In biologicalsystems where a ligand, L, binds to a receptor (macromolecule),Scatchard analysis [21,22] is typically used to determine the re-gions of binding in the isotherm, the binding constant for each re-gion, and the number of ligand binding sites. The variousparameters characteristic of such analyses were determined as de-scribed below:

Fraction of surfactant bound;a ¼ ðI � I0Þ=ðIm � I0Þ ð1Þ

The concentration of the bound surfactant Sb

¼ a½Total surfactant� ð2Þwhere I0 is the fluorescence intensity of the protein in the absenceof poly-SUS (or SUS or SDS), I is the fluorescence intensity when theprotein and poly-SUS (or SUS, SDS) are in equilibrium, and Im is thefluorescence intensity when the protein is completely saturatedwith poly-SUS (or SUS, SDS). The concentration (in M) of freepoly-SUS ([free poly-SUS]), was determined by 1-[bound poly-SUS]. The parameter, m, is defined as a[Total surfactant]/[Total pro-tein] and the concentration of free surfactant (c) was obtained from[Total surfactant](1 � a). Each linear portion of a Scatchard plot (m/cvs. m) was given a linear fit and the equilibrium binding constant (K)and number of binding sites (n) for a particular concentrationregion were obtained from the slope and intercept respectively.

2.2.4. Gel electrophoresis2.2.4.1. Instrumentation. A Bio-Rad Laboratories Mini-PROTEAN 3Electrophoresis Module was used for PAGE separations (Hercules,CA, USA). A constant voltage of 200 V was applied for each separa-tion by a 1000 V Bio-Rad power supply. During staining anddestaining, gels were placed in plastic containers and set on a rock-er (Midwest Scientific, St. Louis, MO, USA). Typical staining anddestaining times for SDS–PAGE were used. The protein bands wereanalyzed for each gel using a Kodak Gel Logic 200 Image Analyzer(Rochester, NY, USA).

2.2.4.2. Preparation of sample and running buffers. Standard 10�running buffer (RB) stock solution contained 25 mM Tris and192 mM Glycine (pH 8.4). The RB solution was prepared by

measuring an appropriate amount of poly-SUS (or SDS) into a vol-umetric flask, dissolving it with 50 mL of electrode buffer stocksolution, and diluting it to a final volume of 500 mL with ultrapurewater (18.2 MX). Desired pH values of the electrode buffer wereachieved by the drop-wise addition of either 1 M NaOH or 1 MHCl. All poly-SUS solutions were prepared by using the equivalentmonomer concentration, namely calculations were based on themolecular weight of the individual surfactant unit (i.e. SUS,272 g/mol). Concentrations of 0.0125%, 0.025%, 0.0375%, and0.053% w/v poly-SUS were used in the running buffer for the opti-mization and validation separations. The sample buffer (SB) wasprepared in 1.7 mL eppendorf tubes by combining appropriateamounts of ultrapure water, 50 mM Tris HCl, pH 6.8, glycerol, bro-mophenol blue, and 0.078%, 0.156%, 0.313%, 0.625%, 1.25%, 2.0%, or2.51% w/v poly-SUS (or SDS). Model proteins of equal concentra-tion were added in the SB at a protein:SB ratio of 2:1. The reducingagent, b-mercaptoethanol, was added at 5% v/v of the SB.

2.2.4.3. Electrophoretic separation. Running buffer totaling 325 mLwas loaded into the upper and lower chambers of the Mini-PRO-TEAN 3 module. Each sample was heated at 95 �C for 5 min on adry bath incubator from Fisher Scientific (Pittsburgh, PA, USA)unless noted otherwise. Dry bath incubator temperatures wereadjusted during optimization of the poly-SUS separation protocol.Twenty microliters (20 lL) of sample was loaded into each wellof the 4–20% Tris HCl gradient mini gels. When SDS was in thesample, a wide range SDS marker (6.5–205 kDa) from Sigma–Aldrich (St. Louis, MO, USA) was used. The migration time was lessthan 35 min for all separations. After each separation, gels wererinsed with ultrapure water (18.2 MX), stained with approxi-mately 25 mL of Colloidal Blue Stain, and placed on a rocker. Gelswere destained with ultrapure water (18.2 MX) until a clear back-ground was visible.

3. Results and discussion

3.1. Intrinsic fluorescence spectroscopy

Intrinsic Trp fluorescence of the protein was used to determinethe relative binding properties of the three ligands, poly-SUS, SUS,and SDS, to the four proteins. The intrinsic fluorescence of a pro-tein, contributed by the aromatic amino acids of Trp, Tyr, andPhe, is often used to study the mechanism of ligand binding to pep-tides and proteins [23].

All four proteins have intrinsic Trp residues in the native state,which are either buried in hydrophobic pockets or located towardthe outer surfaces, as indicated in Table 1. Trp excitation at 295 nmwas used to minimize excitation of tyrosine residues and subse-quent heterotransfer to Trp [24]. In the absence of poly-SUS, SUS,or SDS, the intrinsic Trp fluorescence of BSA, OVA, aCHY, and aLACdisplayed typical emission maxima (i.e., kmax) of 352 nm, 346 nm,338 nm, and 346 nm, respectively (see Fig. S3). Increasing the con-centrations of poly-SUS, SUS, or SDS in the presence of the four pro-teins resulted in various fluorescence emission responses andemission maxima shifts (Fig. 2, S4 and S5). The variability in emis-sion responses was expected due to heterogeneity in the number ofTrp residues and their locations in each native protein. For exam-ple, BSA and aCHY have two (1 solvent accessible, 1 buried) andeight (6 solvent accessible, 2 buried) Trp residues, respectively.With increasing concentration of poly-SUS, the Trp emission ofaCHY was gradually quenched which was accompanied by a redshift until a point of saturation was reached, where further in-creases in [poly-SUS] concentration did not result in additionalquenching or shifting of emission maxima (Fig. 2A–C) For BSA,with increasing poly-SUS (or SUS or SDS) concentration, quenching

Table 1Physical properties of proteins studied.

Protein Accession # MW (kDa) Trpa % a-Helixb % b-Sheetb Theoretical pIc Theoretical charge at pH 8.4c

a-Lactalbumin (aLAC) P00711 14.7 4 (2/2) 43 11 5.0 �12a-Chymotrypsinogen A (aCHY) P00766 26 8 (6/2) 14 32 8.2 �2Ovalbumin (OVA) P01012 45 3 (1/2) 32 32 5.3 �16Albumin, bovine serum (BSA) P02768 66 2 (1/1) 70 – 5.9 �37

a Tryptophan (Trp) residues that are solvent accessible (sa) and buried (b) are indicated as (sa/b).b The secondary structure data was obtained from http://www.pdb.org.c The theoretical values were obtained from http://www.scripps.edu/~cdputnam/protcalc.html.

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Fig. 2. Fluorescence wavelength maxima shifts of TrpaCHY and TrpBSA in the presence of increasing monomeric concentration (0–20 mM) of SDS (open circles), poly-SUS (opendiamonds), and SUS (open triangles) in association with aCHY (38 lM, A–C) and BSA (15 lM, D–F), respectively, determined by steady state fluorescence (kex = 295 nm, 25 �C).The lines have been included to guide the eye.

588 S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594

was observed in the Trp emission while an initial blue shift was fol-lowed by a continual red shift in emission wavelength maxima.Similarly, saturation of BSA with poly-SUS (or SUS or SDS) coin-cided with no further Trp quenching or red shifting in the emissionwavelength maxima (Fig. 2D–F). The red shift and quenching (datanot shown) in the Trp emission maxima of BSA and aCHY withincreasing concentration of the ligand are attributed to changesin the native conformation of the proteins [10,25]. Such a confor-mational change induced by the binding of ligand to the proteinsuggests leads to exposure of Trp residues to a relatively hydro-philic microdomain [23]. Similar changes in the intrinsic Trp

fluorescence for the other two proteins, OVA and aLAC (S4 andS5). In the presence of all three surfactants, both OVA and aLACTrp exhibited a red shift, which is indicative of exposure to a morehydrophilic environment. The shifts were followed to understandthe binding-associated conformational changes in the presence ofthese surfactants.

3.2. Binding studies and Scatchard analysis

Analyses of Scatchard plots reveals the type of binding, particu-larly when multi-site ligand binding is suspected [26]. Generally,

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Fig. 3. Fraction bound of poly-SUS (solid diamonds), SUS (open triangles), and SDS (open circles) to BSA (15 lM) with increasing monomeric surfactant concentration (0–20 mM).

Table 2Cooperative binding type for poly-SUS, SUS and SDS in the low concentration region(<1.0 mM).

Surfactant BSA OVA aCHY aLAC

pSUS Region1 sp, + sp, + sp, + sp, +Region2 + � � �Region3 � + � �Region4 n.sp n.sp n.sp n.sp

SUS Region1 sp, + sp sp sp,�Region2 � � + �Region3 n.sp n.sp � �Region4 n.sp n.sp n.sp �

SDS Region1 sp sp,� sp, + sp, �Region2 � � � �Region3 + � + n.spRegion4 n.sp n.sp n.sp �

Sp = specific binding, + = positive cooperative binding, � = negative cooperativebinding, n.sp = non specific binding.

S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594 589

the binding isotherm displays four characteristic regions withincreasing surfactant concentration: (1) specific binding to highenergy sites on the protein, which are believed to be electrostatic,(2) noncooperative association, (3) cooperative binding as evi-denced by a marked increase in binding and where protein unfold-ing is believed to occur, and (4) saturation in which no furtherbinding occurs and micelles co-exist with the saturated protein[8,27]. Moreover, distinct differences in binding, namely positiveor negative cooperative binding of the ligand L (or surfactant) tothe target, are obtainable [28].

With regard to the binding studies reported here, it was ob-served that binding of poly-SUS to BSA occurred in a significantlylower concentration regime as compared to SUS and SDS. Interest-ingly, for BSA at concentrations between 0 and 0.8 mM, the slope ofthe fraction bound curve (see inset of Fig. 3) for poly-SUS is ob-served to be 8.4 (�23) and 16.6 (�24) times that for SDS and SUS,respectively, suggesting that poly-SUS binding to BSA is highlycooperative. Thus, a surfactant with one more methylene group(SDS) binds BSA twice as fast as a surfactant with one less carbon(SUS), while overall binding is exponentially greater when the ter-minal double bond of the monomer is polymerized (poly-SUS). Atconcentrations as low as 0.8 mM, the fraction of poly-SUS boundto BSA reached 90% while it required five times more SDS andtwelve times more SUS to reach the same bound fraction (Fig. 3).

A similar trend was observed for aCHY, where the slope of thefraction bound curve at low concentrations was greater for poly-SUS ([poly-SUS] < 1.0 mM) than SDS or SUS. However, the corre-sponding slope for aLAC was similar for poly-SUS and SDS andthe slope for OVA was greater for SDS than for poly-SUS (datanot shown). It is evident from Figs. 2 and 3 that the binding ofpoly-SUS to BSA and aCHY are significantly better as comparedto SDS and SUS. These results suggest that the saturation bindingpoints were attained at much lower concentrations (�1 mM) withpoly-SUS, while saturation for the other two surfactants was at-tained at remarkably higher concentration (�4.7 mM for SDS and�10 mM for SUS) which corresponds to their CMC in this medium(determined experimentally by tensiometry) [29]. Furthermore,we believe that the better binding performance of poly-SUS inthe presence of BSA and aCHY as compared to the other two surfac-tants is due to the absence of any transition from a monomeric spe-cies to a low-aggregated state followed by a full micellar state tosaturate the proteins [10]. Similar binding behavior was observedwith aLAC. However, with OVA, the binding was relatively weakerfor SDS as compared to poly-SUS. This is attributed to the signifi-cantly lower surface hydrophobicity of OVA [30]. This observation

again suggests that the binding of poly-SUS with proteins are as-sisted primarily through hydrophobic interactions.

Examination of the Scatchard plots for binding of poly-SUS, SDS,and SUS clearly suggests that the binding mechanism of thesethree surfactants to the various proteins studied is significantly dif-ferent from each other. Different characteristics of the Scatchardplots in different concentration region suggest that the binding ofthese surfactants to the four different proteins follows separatemechanisms in various concentration regions (Table 2).

The analysis (Figs. 4 and 5, Tables 2 and 3) revealed that poly-SUS followed a highly cooperative binding mechanism especiallyin the low concentration regions, wherein it was either unboundor fully bound over small changes in concentration spending littletime in partially bound intermediate states [31]. The observed co-operativity may also be attributed to raveling and unraveling capa-bilities of the molecular micelle depending on the environment(Scheme S1).

This was presumably due to polymerization of the terminaldouble bond. According to these studies, the binding mechanismof poly-SUS to BSA is in direct opposition to what has been ob-served for the conventional anionic surfactant SDS. The concavedownwards nature of the Scatchard plot with very high bindingconstant suggested positive cooperative binding of poly-SUS inthe low concentration regime (0.2 < [poly-SUS] < 0.8 mM). Thebinding constant was found to be two orders of magnitude greaterthan that observed for SDS in the same concentration range. Thus,

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Fig. 5. Scatchard plots of OVA with (left) SDS and (right) poly-SUS. The inset expands the low concentration regions of the corresponding plots.

Table 3Scatchard analysis data for the interaction of the four proteins with the surfactants.

Protein Poly-SUS SUS SDS

Concentration range (mM) K (M�1) n Concentration range (mM) K (M�1) n Concentration range (mM) K (M�1) n

BSA 0.2–0.8 1.30 � 104 0.4 0.2–0.8 3.65 � 102 227.7 0.2–0.8 6.80 � 102 67.31.0–2.7 4.00 � 104 23.7 1.0–4.4 5.90 � 101 9950 1.0–3.4 2.40 � 103 0.633.4–4.7 8.00 � 102 1997.0 4.7–8.0 2.73 � 102 74.9 4.1–5.4 4.50 � 104 5.35.0–12.0 2.80 � 103 21.9 10.0–16.0 1.19 � 103 481.1 6.0–16.0 3.71 � 103 81.7

OVA 0.2–1.0 1.42 � 103 0.4 0.7–1.0 4.01 � 102 8.5 0.2–0.5 2.36 � 103 0.11.7–2.7 1.07 � 103 2.2 1.7–3.4 2.60 � 102 17.4 0.7–1.0 1.64 � 103 0.13.4–8.0 2.85 � 103 28.5 4.1–5.1 4.24 � 102 29.3 1.7–2.7 8.58 � 104 0.510.0–18.0 8.14 � 103 43.7 5.4–7.0 1.28 � 103 76.1 3.4–4.4 2.65 � 104 3.97

5.1–12.0 6.63 � 104 3.09

aCHY 0.2–0.5 2.31 � 103 1.3 0.2–0.5 2.01 � 103 0.7 0.2–0.5 2.20 � 103 0.20.7–0.1 2.93 � 103 0.3 0.7–1.0 1.16 � 103 2.9 0.7–1.0 1.07 � 103 3.21.7–3.4 5.38 � 102 173.9 1.7–3.4 7.27 � 102 11.2 1.7–3.4 9.61 � 102 1.04.1–6.0 8.45 � 102 41.6 4.1–5.1 6.35 � 102 28.6 4.1–6.0 2.57 � 103 20.68.0–12.0 1.75 � 103 43.6 5.4–8.0 5.74 � 102 120.3

aLAC 0.5–1.0 2.24 � 103 0.7 0.2–1.0 1.24 � 100 0.31 0.2–0.5 2.44 � 102 1.71.7–4.4 6.81 � 102 148.7 1.7–3.4 8.43 � 100 13.4 0.7–2.2 4.80 � 101 7.24.7–8.0 1.35 � 103 53.3 4.1–5.1 3.08 � 101 36.4 2.7–4.1 1.06 � 100 2.710.0–16.0 2.15 � 103 113.4 5.4–8.0 1.58 � 101 95.0 5.4–8.0 2.30 � 103 0.2

590 S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594

below 2.7 mM the binding of poly-SUS to one site on BSA increasesthe poly-SUS binding affinity to subsequent sites of the protein.The negative cooperative binding of SDS to BSA in the similarlow concentration region (0.2 < [SDS] < 0.8 mM) was evident fromthe concave upwards nature of the Scatchard plot. Typically ob-served in this low concentration region, negative cooperative bind-ing occurs when the surfactant monomer binds to a site on the

protein with no allosteric effects present to facilitate additional li-gand binding [32]. Thus, SDS binding is highly specific in the lowconcentration regime (0.2 < [SDS] < 0.8 mM). Above 2.7 mM, poly-SUS exhibited a negative cooperative binding suggesting that inthis concentration region, BSA was already saturated with poly-SUS and thus binding at one site lowers the binding affinity atadjacent sites. However, at higher concentrations (3.4–5.4 mM),

S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594 591

positive cooperative binding was observed for SDS followed by alinear region at higher concentrations characteristic of non-specificbinding [21]. The highest binding constant for SDS was calculatedto be 4.5 � 104 M�1 in region III (4.1–5.4 mM) where the onset ofSDS micellization occurs. The binding of SDS agreed with theisotherm observed by Takeda et al. (1981) who used conductomet-ric and chromatographic methods to study the binding events forSDS associating with BSA [33]. The high n values observed in cer-tain regions especially for poly-SUS binding to proteins suggestssubstantial unfolding of the protein due to the already boundsurfactants leading to exposure of additional binding sites. How-ever relatively low binding constants in these regions may beattributed to steric factors and other thermodynamic factors asso-ciated with the binding [34].

By comparison of the cooperativity profiles for all of theproteins studied (Table 2), it is apparent that positive cooperativitydominated for poly-SUS in the low concentration region (0 < [poly-SUS] < 1.0 mM). This observation suggests maximum molecularmicelle complexation with each protein at lower concentrations.The positive cooperative binding to all four proteins (Fig. 5 andS5) in this concentration range suggests that poly-SUS binding tothe proteins studied is independent of protein size and charge.We attribute this phenomenon to the specificity (absence of mi-celle assembly and disassembly due to the covalently bound micel-lar structure), flexibility (ability to adopt different conformations),and hydrophobicity (large hydrophobic microdomain) that existsin poly-SUS, giving it access to more binding sites and thus promo-tion of positive allosteric modulation [32]. Also, the observed posi-tive cooperativity at such low concentrations may be attributableto the greater number of anionic head groups on the polymerizedmolecular micelle as compared to its monomeric species. Summa-rizing the results from the intrinsic fluorescence data, poly-SUSbinding to the proteins studied is (1) more cooperative than SDSand SUS, (2) occurs at much lower concentrations (<1.0 mM) thanSDS (>4.1 mM) and SUS (>10 mM) for two of the proteins, and (3)occurs through a primarily hydrophobic interaction under theexperimental conditions explored.

For the number of binding sites, values of 24 and 5 were foundon BSA for poly-SUS and SDS, respectively. Although the number ofSDS binding sites is in agreement with previous reports [34,35], itis significantly lower than the number of binding sites found forpoly-SUS. The large number of poly-SUS binding sites on BSA offersan explanation for the efficiency with which poly-SUS wasobserved to interact with BSA (at concentrations as low as0.8 mM). In addition, the amount of poly-SUS bound to BSA ascompared to SDS at concentrations <1.0 mM may be explainedby the large number of sites made available for binding by poly-SUS-induced conformational changes to BSA (see Section 3.4).

0

2

4

6

8

10

12

ExtrinsicProbe

IntrinsicProbe

ExtrinsicProbe

poly-SUS

Satu

ratio

n C

once

ntra

tion

(mM

)

BSAOVAaCHYaLAC

Fig. 6. Comparison of saturation concentrations (mM) for poly-SUS, SUS, and SDS assointrinsic and extrinsic fluorescence spectroscopy. Saturation was not attained for SUS/B

3.3. Extrinsic fluorescence spectroscopy

In addition to intrinsic fluorescence spectroscopy, the extrinsicprobe, 8-anilino-1-naphthalene-sulfonate (ANS), has been used tomonitor surfactant saturation concentrations and surfactant-in-duced unfolding of the four proteins [34]. ANS is a well knownhydrophobicity probe whose fluorescence is extremely weak inhydrophilic environment and increases abruptly as it migrates toa hydrophobic environment such as the hydrophobic patches with-in proteins. These changes in fluorescence of the external probe areused as a tool to understand the surfactant binding to the proteins.

In this study, fluorescence data were collected for excitationwavelengths at 295 nm and 364 nm representing Trp and ANS,respectively. ANS is both hydrophobic, i.e. containing a naphtha-lene moiety, and hydrophilic, i.e. possessing an anionic sulfonatehead group. An increase in ANS emission accompanied with blueshift was observed in the presence of each protein due to hydro-phobic interaction and subsequent energy transfer from Trp. ANSis known to bind to the hydrophobic sites of proteins and exhibitenhanced fluorescence emission due to Forster Resonance EnergyTransfer (FRET) from Trp residues of proteins [34]. The extent ofFRET depends on the proximity of ANS to the Trp residues. Thegreatest intensity increase resulted from the interaction with BSA(see Fig. S6). This was attributed to efficient FRET from Trp residuesin BSA to bound ANS suggesting that these two molecules were inclose proximity (<10 nm). However, little energy transfer was ob-served from TrpaCHY to ANS which suggested the inaccessibilityof the ANS to the Trp.

As poly-SUS (or SDS or SUS) was titrated into the cuvette con-taining 1 mg/mL of protein and 20 lM ANS and allowed to equili-brate, we envisaged ANS amphipathic contacts would be replacedby poly-SUS (or SDS or SUS) amphipathic contacts on the proteins.This explanation was complementary to the observation that theANS emission dropped on addition of polySUS (or SDS or SUS) asit was replaced by polySUS (or SDS or SUS) from the hydrophobicpatches of the protein due to stronger hydrophobic interactionsof the later. In addition, the ANS emission maximum red shiftedindicating ANS was moving to a more hydrophilic environment.After addition of a certain amount of poly-SUS (or SDS or SUS),ANS fluorescence was again found to increase accompanied witha blue shift. When the protein–surfactant interaction was com-plete, we suppose that ANS would be found either in the hydropho-bic core of the molecular micelle (poly-SUS), in the bulk aqueousenvironment due to its hydrophilicity, or sterically situated be-tween the two environments. An increase in the ANS emissionafter the saturation point (concentration of surfactant for whichthe protein binding sites are saturated) suggested that ANS onceagain migrates to a relatively hydrophobic environment. This

IntrinsicProbe

ExtrinsicProbe

IntrinsicProbe

SUS SDS

ciating with the proteins, BSA, OVA, aCHY, and aLAC (1 mg/mL), determined fromSA using the extrinsic probe; therefore, no concentration is indicated.

592 S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594

signature in ANS fluorescence emission is used to obtain the satu-ration binding concentrations of a surfactant to a protein. The con-centration of poly-SUS (or SDS or SUS) coinciding with completeANS displacement was believed to be the saturation concentrationof poly-SUS (or SDS or SUS), interacting with the protein since ex-cess poly-SUS (or SDS or SUS) was then available for hydrophobicinteraction with ANS. The saturation concentrations determinedby extrinsic fluorescence spectroscopy (Fig. 6) were consistentwith the values obtained from intrinsic Trp fluorescence of the pro-teins studied.

For example, the saturation concentration of poly-SUS, SUS, andSDS for interaction with aLAC was observed to be 1.1 mM, 2.9 mM,and 4.1 mM, respectively using extrinsic fluorescence which isclose to the values obtained using intrinsic fluorescence (1.0 mM,2.9 mM, and 4.4 mM, for poly-SUS, SUS, and SDS respectively).Thus, it was confirmed that poly-SUS binds to aLAC at concentra-tions at least four times less than SDS. At pH 8.4, all of the proteinsshould have a net negative charge (Table 1). Therefore, the interac-tion of ANS with the four acidic proteins was expected to be pri-marily hydrophobic, although electrostatic contacts (e.g., withlysine, arginine, or histidine) are possible, but to a lesser extent.When taken in aggregate, analyses of the results using the extrinsicreporter molecule indicate binding of poly-SUS to the proteinsstudied is primarily hydrophobic.

Summarizing the results thus far, we conclude that the interac-tion between the acidic proteins and poly-SUS, SDS, and SUS wouldbe primarily hydrophobic due to the hydrophobicity of the core ofpoly-SUS (or hydrophobicity of the aliphatic chain in the conven-tional micelle). Another noticeable observation from these studieswas a greater increase in ANS emission in the presence of poly-SUSover SDS, which suggests that poly-SUS is significantly morehydrophobic than SDS (Fig. S7).

3.4. Effect of poly-SUS on protein denaturation: circular dichroismstudies

To evaluate the effect of poly-SUS on protein conformation ascompared to the conventional surfactant SDS, circular dichroism

-35.0

-30.0

-25.0

-20.0

200 205 210 215 220 225 230

Wavelength (nm)

CD

Sig

nal (

mde

g)

Native BSA

0.156% w/v poly-SUS

(A)

-35.0

-30.0

-25.0

-20.0

200 205 210 215 220 225 230

Wavelength (nm)

CD

Sig

nal (

mde

g)

Native BSA

0.156% w/v SDS

(C)

(

(

Fig. 7. Changes in secondary structure of BSA in the native (A) and (C) and reduced (B) anThe buffer was 25 mM Tris/192 mM Glycine at pH 8.4 and 25 �C. A 1 mm pathlength qu

measurements were used to monitor changes in the secondarystructure. Far UV CD spectroscopy is the characteristic region ofthe electromagnetic spectrum where secondary structure transi-tions in proteins are gauged. In this region, the peptide backboneis the chromophore; a protein that is primarily a-helical in struc-ture exhibits two negative bands at 208 nm and 222 nm. Altera-tions in the far UV spectra for aLAC in the absence and presenceof poly-SUS and SDS are shown in Fig. S8. A greater predominantshift in the minimum at 208 nm is observed for poly-SUS relativeto SDS in the presence of aLAC at a concentration of �4 mM (or0.12%w/v). No appreciable differences in CD spectra were observedfor poly-SUS and SDS at �2.9 mM (or 0.08% w/v). In principle, theCD spectrum of a protein is the sum of percentages of all possiblesecondary structural motifs (i.e., a-helix, b-sheet, b-turn, and ran-dom coil). Thus, as the 208 nm minimum (�h208nm) increases withincreasing concentration of poly-SUS, aLAC begins to unfold. Theshift in the CD signal for the 208 nm minimum at �4 mM (or0.12%w/v) suggests a transition in aLAC conformation toward ran-dom coil in the presence of poly-SUS [36]. Additional evidence forthe poly-SUS-induced secondary structural change in aLAC was ob-served upon addition of greater amounts of poly-SUS, resulting indramatic shifts in the minimum at 208 nm relative to SDS (datanot shown). In addition, when binding to BSA in either the nativeor reduced states, poly-SUS perturbs the secondary structure caus-ing significant unfolding (Fig. 7A–D) as compared to SDS.

To further probe the concentration effect of poly-SUS on thesecondary structure change in BSA, we have monitored the changein ellipticity at 208 nm. In the concentration range from 0 to25 mM the ellipticity at 208 nm (�h208nm) for poly-SUS increaseddramatically compared to SDS (see Fig. S9) suggesting decreaseda-helical content and significant unfolding of BSA. Though an in-crease in ellipticity was observed at 222 nm, the trend was notas pronounced as at 208 nm. Over the concentration range studied,SDS did not appreciably change the conformation of BSA. Fromthese data, one can deduce that poly-SUS overcomes the lowdielectric constant of the hydrophobic interior [37] to induce majorconformational changes. This exponentially increases the capacityof poly-SUS to bind to the interior sites complementing its ability

-35.0

-30.0

-25.0

-20.0

200 205 210 215 220 225 230

Wavelength (nm)

CD

Sig

nal (

mde

g)

Reduced BSA

0.156% w/v poly-SUS

B)

-35.0

-30.0

-25.0

-20.0

200 205 210 215 220 225 230

Wavelength (nm)

CD

Sig

nal (

mde

g)

Reduced BSA

0.156% w/v SDS

D)

d (D) states in the presence of 0.156% w/v poly-SUS and 0.156% w/v SDS as indicated.artz cuvette was used.

1 2 3 1 2 3 4

kDa

250 –

150 – 100 –

75 –

50 –37 –

25 –20 –15 –10 –

– 66 kDa

– 45 kDa

(A) SDS (B) poly-SUS

Fig. 8. PAGE of p53 on a 4–20% gradient gel using (A) the conventional surfactant,SDS, and (B) the molecular micelle, poly-SUS. (A) SDS: lane 1, precision plusmolecular weight marker (MWM, 10–250 kDa); lane 2, 2% w/v SDS; lane 3, 0.08% w/v SDS. (B) poly-SUS: lane 1, 2% w/v poly-SUS; lane 2, 0.08% w/v poly-SUS; lane 3,BSA standard and 2% w/v poly-SUS; lane 4, OVA standard and 2% w/v poly-SUS.Lanes 3 and 4 were used as markers to identify the approximate molecular weightof p53 in the poly-SUS separation.

S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594 593

to bind the solvent accessible hydrophobic regions. This is alsocomplementary to the Scatchard analysis data where we find thatthe binding constant of poly-SUS with aLAC is orders of magnitudehigher than SUS and SDS, suggesting stronger binding of poly-SUSat significantly lower concentration as compared to the conven-tional surfactants.

In summary, the present work emphasizes the critical interac-tions that contribute to the binding mechanism involving a molec-ular micelle and four proteins with the aim of contributing to thegrowing need for new and enhanced analytical tools for biochem-istry and biotechnology. By use of intrinsic and extrinsic fluores-cence spectroscopy, we have determined that the molecularmicelle, poly-SUS, exhibits a positive cooperative binding mecha-nism for all proteins studied and binds two of the proteins at lowerconcentrations than SUS and SDS. Further, our circular dichroismresults show that poly-SUS disrupts the secondary structure ofBSA and aLAC at low concentrations, which is in stark contrast toSDS.

3.5. Comparison of poly-SUS and SDS in polyacrylamide gelelectrophoresis

The fluorescence and CD studies clearly suggest the distinctivebinding property of poly-SUS with the proteins studied, irrespec-tive of their size. We initially postulated that poly-SUS would per-form superior to SDS at low concentrations in polyacrylamide gelelectrophoresis due to greater hydrophobic interactions and great-er binding constants observed from our spectroscopic data. Whenstudied in 1D-gels, poly-SUS indeed produced separations at signif-icantly lower concentration as compared to SDS. To verify this un-ique behavior of poly-SUS, a His-tagged protein, p53, containinglong regions of unordered structure [38] was selected as the ana-lyte of interest. This protein was selected because it is a major tar-get for anticancer therapy due to mutations that alter its ability tocontribute to tumor suppression [39,40]. Facile detection of poly-SUS, SUS, and SDS binding efficiency to p53 was evaluated in the

porous gel medium. Representative PAGE-SDS and PAGE-poly-SUS separations are shown in Fig. 8. Migration of p53 throughthe gel was influenced by the ligand binding efficiency as judgedby the location of the band in the gel. In a gradient gel, whichwas used here, the stacking region of the gel contains large poresthat are capable of accommodating large aggregates whereas theresolving region of the gel contains progressively smaller poresas the gel is traversed. It was observed (Fig. 8A) that at a loadingof 0.08% w/v (2.9 mM) SDS in the sample buffer a trail of p53was essentially smeared down the lane presumably due to a lackof binding with SDS.

We propose that the water molecules that solvate the hydro-philic residues on the protein surface began interacting with thehydrophilic head groups of the SDS monomers and micelles, caus-ing a reduction in protein–protein repulsive forces and subsequentaggregation or precipitation of p53. The SUS monomer separationexhibited bands that were diffuse (see Fig. S10). Examination ofthis gel suggests lower protein binding with the addition of one de-gree of unsaturation in the molecule, which is consistent with theintrinsic fluorescence results. From the work by Sprague et al. [41],we know that the presence of unsaturation originating from theterminal double bond in SUS increases the CMC by a factor oftwo over its saturated counterpart. Thus, micellar species withwidely differing aggregation numbers interacting with the proteinmay contribute to the diffuse bands and streaking. Conversely,poly-SUS binding to p53 is highly efficient down to concentrationsin the sample buffer as low as 0.08% w/v (2.9 mM) as observed inFig. 8B. Thus, it was concluded from these data, as predicted by re-sults from our fluorescence spectroscopy studies, that poly-SUS hasrobust association with water soluble proteins like p53 that isattainable at concentrations as high as 2% w/v and as low as0.08% w/v. This robustness is attributed to polymerization ofpoly-SUS, having conformational flexibility, and having highlyhydrophobic microdomains providing it with access to sites onthe protein that are not attainable with a conventional micelle. Col-lectively, these PAGE separations are consistent with our hypothe-sis that poly-SUS at low concentrations would exhibit greaterbinding efficiency and separation effectiveness in PAGE as com-pared with SDS.

4. Conclusions

Detailed examination of the data reported herein show thatpoly-SUS is a robust surfactant that is facile, flexible, and hydro-phobic with high affinity for globular proteins over an appreciablylow concentration range. The salient positive cooperative bindingmechanisms of the four proteins with poly-SUS as compared tothe mostly negative co-operative binding mode with SDS and theirpoly-SUS binding affinities at low concentration are striking out-comes of the present study. These properties were understoodthrough coupling of data from intrinsic and extrinsic fluorescencespectroscopy, circular dichroism, and polyacrylamide gel electro-phoresis, thereby revealing potential applications of molecularmicelles in protein separations over the conventional surfactants[9–12,42–44]. Extrapolating from our data, we propose molecularmicelles as solubilizing agents for hydrophobic proteins. Theextraordinary hydrophobicity and other structural features suchas specificity achieved through the molecular micelle formationof the monomeric surfactant molecules as compared to the dy-namic micellar self-assembly makes them highly competent candi-dates in proteomics. Moreover, results from the data presentedhere suggest that water soluble, hydrophobic, amino acid-basedmolecular micelles may exhibit greater recognition and higherbinding affinity of proteins, which may facilitate enhanced solubi-lization, separation, and identification in proteomics studies.

594 S. Das et al. / Journal of Colloid and Interface Science 363 (2011) 585–594

Preliminary gel electrophoresis studies also reveal better perfor-mance of poly-SUS as compared to SDS at low concentrations. Fur-ther studies are ongoing in our laboratory to exploit the applicationof this and other molecular micelles in 1D and 2D gels as a signif-icantly better alternative to existing surfactants [9–12,42–44].

Acknowledgments

I.M. Warner acknowledges support from the grant through theNational Institutes of Health (Grant # 1R21RR024 431-01) and thePhilip W. West Endowment at Louisiana State University. M.R.S.acknowledges a National Science Foundation Graduate ResearchFellowship and a UNCF/Merck Graduate Science DissertationFellowship.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jcis.2011.07.044.

1HNMR of SUS, polySUS, Fluorescence data analysis, CD dataanalysis, gel electrophoresis with SUS are provided in supportingmaterial.

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