Formation of Complexes between the ConjugatedPolyelectrolyte Poly{[9,9-bis(6′-N,N,N-

trimethylammonium)hexyl]fluorene-phenylene} Bromide(HTMA-PFP) and Human Serum Albumin

Maria Jose Martınez-Tome, Rocıo Esquembre, Ricardo Mallavia, and C. Reyes Mateo*

Instituto de Biologıa Molecular y Celular, Universidad Miguel Hernandez de Elche,03202 Elche (Alicante), Spain

Received February 1, 2010; Revised Manuscript Received April 14, 2010

The interaction between the conjugated polyelectrolyte poly{[9,9-bis(6′-N,N,N-trimethylammonium)hexyl]fluorene-phenylene} bromide (HTMA-PFP) and human serum albumin (HSA) has been investigated from changes observedin both the spectroscopic properties of HTMA-PFP and the intrinsic fluorescence of HSA. Absorption andfluorescence spectra of HTMA-PFP suggest that HTMA-PFP and HSA form polymer-protein complexes due toelectrostatic interactions between the cationic side chains of HTMA-PFP and the negatively charged surface ofthe protein. Interaction between both macromolecules induces an increase in the fluorescence signal of HTMA-PFP, which suggests that hydrophobic forces also contribute to the polymer-protein complex stabilization. Inaddition, this interaction causes a decrease in the HSA fluorescence, partially due to static quenching and energytransfer between both macromolecules. Effects of HTMA-PFP on the thermal stability and protein conformationwere explored from CD experiments. Results indicate that as polymer is added it binds to HSA and initiatesunfolding. This unfolding process induces HTMA-PFP chains to become more extended, disrupting backboneinteractions and increasing polymer fluorescence intensity.

Introduction

Fluorescent conjugated polyelectrolytes (CPEs) constitute aninteresting class of materials with a wide range of properties.They are π-conjugated polymers, their rigid backbone consistingof an alternation of simple and double bonds with water-solubleside chains containing cationic or anionic groups. Electrondelocalization facilitates rapid intra- and interchain excitonmigration, conferring collective optical responses and amplifiedsignals when comparing with conventional fluorophores.1,2 Inaddition, these new materials can undergo spontaneous self-assembly through reversible, electrostatic, and/or hydrophobicinteractions with some other species, generally of oppositecharge, resulting in supramolecular structures with interestingoptical and material properties.3 Given these properties, CPEshave received great attention during the last years because oftheir potential applications in areas such as chemical andbiological sensing.2-8

The use of CPEs as biomolecule sensors is based on changessuffered by the polymer upon interaction with the biomolecules.This interaction can cause different conformational transitionsof the polyelectrolyte backbone, aggregation or separation ofthe polyelectrolyte chains, and energy or electron transferprocesses between polymer and biomolecule, which yieldimportant changes in the polymer intrinsic fluorescence.9,10

Several authors have reported the existence of nonspecificinteractions between CPEs and biomolecules such as proteinsthat perturb the fluorescence of the polyelectrolytes.10-12 Theseinteractions generate a distinct fluorescence response pattern fora given protein and therefore have opened opportunities for thedevelopment of new biosensors.2,13,14 However, the existenceof such interactions becomes a double-edge sword because many

of these sensors are designed with the aim of being used inliving cells or human biological fluids that contain a complexmixture of proteins and other biomolecules. For instance, humanserum contains more than 20 000 proteins with an overall proteincontent >1 mM. Interactions between such proteins and thepolyelectrolyte should result in a nonspecific background thatcould be very high and obscure any fluorescence specificsignal.12 Therefore, it should be of great interest to know thefluorescence response of CPEs to human serum proteins to betterdefine the real-world sensing applications of this class ofpolymers.

Human serum albumin (HSA) is the most abundant andprinciple extracellular protein, forming ∼60% of the mass ofhuman plasma proteins, with a typical concentration of 5 g/100mL in the bloodstream.15 It is a globular protein composed ofa single polypeptide of 585 amino acids with three R-helicaldomains I-III, each containing two subdomains A and B (Figure1). Its isoelectric point is 4.9;16 therefore, the protein displaysa negative net charge at neutral pH. However, the chargedistribution is not homogeneous through the three domains;domains I and II more acidic than domain III.17 The proteincontains multiple binding sites that allow its interaction withmany organic and inorganic compounds and make this proteinan important regulator of intercellular fluxes.17 Its 3D structure,determined through X-ray crystallographic measurements, in-dicates that the principal regions of ligand binding sites in HSAare located in hydrophobic cavities in subdomains IIA and IIIAand the sole tryptophan residue (Trp-214) of the protein islocated in subdomain IIA.18,19 The molecular interactionsbetween HSA and many compounds have been successfullyinvestigated, including drugs,18,20 surfactants,21,22 lipids andliposomes,23,24 and macromolecules such as dendrimers,25

polyoxometalates,26 or polethyleneglycol.27 However, to our* Corresponding author. Fax: +34 966 658 758. E-mail: rmateo@umh.es.

Biomacromolecules 2010, 11, 1494–15011494

10.1021/bm100123t 2010 American Chemical SocietyPublished on Web 04/27/2010

knowledge there has been no study exploring the nature of theinteractions of CPEs with HSA.

In this work, we present a spectroscopy study of theinteraction in aqueous solution of HSA with poly{[9,9-bis(6′-N,N,N-trimethylammonium)hexyl]fluorene-phenylene} bromide(HTMA-PFP, Figure 1), a cationic CPE that has been previouslydemonstrated to interact with surfactants and DNA.28-32 Theinteraction was characterized from spectroscopic changesobserved in both macromolecules. Effects of polymer on thethermal stability and conformation of the protein were alsoexplored from CD experiments to get more insight into thenature of this interaction.

Experimental Section

Materials. HSA was purchased from Sigma-Aldrich Chemical(Milwauke, WI). HSA was dissolved in deionized doubly distilled water.The cationic CPE HTMA-PFP (Mj ) 8340 g/mol, repeat unit molecularweight, 694.71 g/mol; n′ ) 12 based on polyfluorene calibration) wasobtained and characterized in our laboratory, as was previouslydescribed.33,34 In brief, a low-molecular-weight batch of the neutralpolymer, poly[9,9-bis(6′-bromohexyl)fluorene phenylene], was synthe-sized by Suzuki coupling reaction with Pd(II) as catalyst and treatedwith gas-phase trimethylamine to obtain the corresponding cationicpolyelectrolyte. Other chemicals were of analytical or spectroscopicreagent grade. The pH of all aqueous solutions was controlled, andvariations were always lower than (0.15, even at the maximalconcentrations of HSA and HTMA-PFP used in this study.

Preparation of HTMA-PFP Solutions. When CPEs are dissolvedin water, they self-assemble into aggregates through the interactionbetween the charged side chains and the hydrophobic backbone. Thedegree of aggregation depends on the charge density in the CPEs,however, the addition of organic cosolvents as dimethyl sulfoxide(DMSO), methanol, acetonitrile, or dioxane break up these aggregatesfacilitating their solubilization. In this work, we have used DMSO ascosolvent of HTMA-PFP, with the exception of the CD experiments,in which dioxane was selected because of the strong absorption ofDMSO in the far UV region. In all cases, the proportion of the cosolventin the water mixture was always lower than 1% (v/v). For absorptionand fluorescence experiments, stock solutions of HTMA-PFP (3.65 ×10-4 M, in repeat units) were prepared in DMSO. Aliquots of this stocksolution were dissolved in deionized doubly distilled water, obtaininga clear and transparent solution, which suggests the nonexistence ofaggregates.

Absorption and Steady-State Fluorescence Measurements. Ab-sorption measurements were carried out at room temperature using aShimadzu spectrophotometer (UV-1603, Tokyo, Japan). Fluorescencemeasurements were performed in a PTI-QuantaMaster spectrofluorom-eter interfaced with a Peltier cell. Excitation wavelengths at 280 and380 nm for HSA and HTMA-PFP, respectively, were utilized. Thermalscans were collected from 25 to 88 °C with a heating rate of 10 °C/min. Spectra were taken 10 sec after the temperature equilibrium wasreached. The experimental samples were placed in 10 × 10 mm pathlength quartz cuvettes. Background intensities were always checkedand subtracted from the sample when it was necessary.

Quenching Experiments. Fluorescence emission of HSA wasstudied in the absence and presence of different concentrations ofHTMA-PFP. Analysis was applied to the fluorescence quenching data,according to the standard Stern-Volmer equation (eq S1 in theSupporting Information) to obtain the Stern-Volmer constant, KSV. Inthis work, samples were excited at 280 nm to minimize polymerabsorption, and the emission intensity was measured as the area of theemission spectrum, calculated from 340 to 380 nm, to avoid the tyrosineemission. The significance of KSV depends on the nature of thequenching process: it may represent the association constant for complexformation or the rate of dynamic quenching (KSV ) kqτ0), where kq isthe bimolecular rate constant of the quenching process, and τ0 is thelifetime of the biomolecule. Dynamic or static quenching can bedistinguish, among others, by their differing dependence on temperatureand by the different alterations induced in the fluorescence lifetime orin the absorption spectrum of the fluorophore.35

Energy Transfer Experiments. For energy transfer experiments,the CPE HTMA-PFP was used as an acceptor of the HSA tryptophanexcitation. Data analysis was carried out using the Forster equations,as is described in the Supporting Information (eqs S2-S4). As forquenching experiments, samples were excited at 280 nm to minimizepolymer absorption, and the emission intensity was measured as thearea of the emission spectrum, calculated from 340 to 380 nm, to avoidthe tyrosine emission. Artifacts, such as the effect of the acceptor(HTMA-PFP) absorption at the donor (tryptophan) excitation wave-length (280 nm), were corrected according to eq S5 in the SupportingInformation.

Circular Dichroism Experiments. CD measurements of HSA andits polymer complexes were carried out with a Jasco spectropolarimeter,model J-815 (JASCO, Easton, MD), interfaced with a PTC-423S/15Peltier-type cell holder for temperature control. Spectra were collectedat 25 °C with a scan speed of 50 nm per min, response time of 4 s, anda bandwidth of 1 nm. For each spectrum, four scans were accumulatedand averaged to improve the signal-to-noise ratio. Spectra were recordedfrom 260 to 197 nm using 0.1 cm quartz cells. HSA concentration waskept constant (3.0 µM) while each polymer concentration was varied(1.5, 9, 12 µM). Each CD sample was prepared independently by mixingthe same volume of HSA stock solution with an appropriate amountof HTMA-PFP (depending on the desired polyelectrolyte final con-centration) and leading to a final volume of 1 mL, in all cases. A

Figure 1. Top left: Crystal structure of HSA indicating the threedifferent domains present and the location of its 18 Tyr residues (ingreen) and its single tryptophan residue (in red), Trp-214. Thestructure was obtained from the Protein Data Bank (PDB code 1HA2).Top right, in dark blue: Simulation by molecular dynamic of a tetramerof HTMA-PFP. Figures were rendered, on the same scale, usingPYMOL and CHEMDRAW, respectively. Bottom: HTMA-PFP chemi-cal structure.

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baseline was taken under the same conditions as those used for thesample and subtracted from each spectrum. Changes in the R-helicalcontent were calculated, as is described in Supporting Information (eqsS6-S8).

Thermal scans were collected at a fixed wavelength of 222 nm from25 to 90 °C in 0.1-cm-path length cells with a heating rate of 45 °C/h.Denaturation curves were fitted by nonlinear regression analysis usinga two-state model with the only purpose of determining the temperatureat the midpoint of denaturation and any variation produced by theaddition of polymer to HSA.

Results and Discussion

Changes in the Spectroscopic Properties of HTMA-PFPin the Presence of HSA. The effect of HSA on the absorptionspectrum of HTMA-PFP in aqueous solution was explored usinga constant polyelectrolyte concentration (3 µM, in terms ofrepeat units) and increasing concentrations of protein (Figure2). The addition of the protein, up to ∼0.35 µM, inducedsimultaneously a decrease in the absorbance intensity, a slightbroadening of the spectrum, and a shift of the maximum to thered, evidencing the interaction between both macromolecules.The spectra exhibit a well-defined isosbestic point at 392 nm,which suggests the establishment of an equilibrium between thefree polymer and the polymer complexed to HSA. For HSAconcentrations between 0.5 and 3.4 µM, an increase in the scatterof the sample was observed, which is indicative of aggregatesformation. Higher concentrations of HSA did not induce anychange in the scatter or in the absorption spectrum of HTMA-PFP. These results suggest that depending on the proteinconcentration at least two different types of polymer-proteincomplexes are formed: for lower protein concentration, a singlecomplex between HSA and HTMA-PFP is taking place,probably because of the electrostatic interactions between thepolyelectrolyte (having two positive charges by each monomerunit) and domains I and II of the protein, which show a negativenet charge under the experimental conditions. Higher concentra-tions of HSA, up to ∼3.4 µM, seem to induce additionalinteractions between the formed complexes, which because ofthe partial neutralization of their charges will exhibit lower watersolubility, giving place to aggregates with more complexmolecular architectures. Above ∼3.4 µM, the net charge ofHTMA-PFP is totally neutralized, and no more interactions areexpected among these aggregated and the excess of protein. Thisinterpretation is in agreement with the results obtained for thesame polyelectrolyte upon interaction with surfactants and

DNA.31,32 In both works, similar modifications in the absorptionspectra were observed, which were attributed to polymeraggregation and, in the case of DNA, to the formation of 1:1complexes between DNA and HTMA-PFP, for DNA concentra-tions lower than the concentration of polymer repeat units. Wealso consider that the complex formed between HTMA-PFP andHSA has a 1:1 stoichiometry at low HSA concentrations.32 Thisassumption can be supported taking into account that, on onehand, each single polymer chain contains on average around12 monomer units and, therefore, ∼24 positive charges, and,on the other hand, at neutral pH, each HSA molecule has anegative net charge of -19.36 The change in the absorptionspectrum of HTMA-PFP in the presence of low HSA concentra-tions was used to estimate the association constant of thiscomplex (KA). The inset in Figure 2 represents the decrease inthe absorbance intensity measured at the maximum of thespectrum (∆A ) AHTMA-PFP - AHTMA-PFP:HSA) versus HSAconcentration. Determination of KA can be made by fitting thesedata to the Benesi-Hildebrand equation37 (eq S9 in theSupporting Information). The solid line in the inset of Figure 2shows this fit, which yields a value for KA ) (9.6 ( 1.4) × 106

M. This constant suggests strong affinity between HTMA-PFPand HSA and is in the same range that the value obtained forthe HTMA-PFP:DNA complexes32 but higher than that deter-mined for HSA bound to other macromolecules, such asdendrimers25 or polyethyleneglycol.27

The interaction of HTMA-PFP with HSA is also evidentfrom the fluorescence spectra of the polyelectrolyte. Figure 3shows the effect of increasing concentration of HSA, up to 6µM, on the excitation and emission spectra. In this experiment,the concentration of HTMA-PFP was maintained constant, at1.5 µM in terms of repeat units (optical density <0.1), to avoidinner filter effects. The shape of the excitation spectrum, in theabsence of protein, was similar to that of the absorptionspectrum, showing a maximum around 378 nm. The additionof the protein induced a shift to higher wavelength, accompaniedby a broadening of the band, which is in agreement with thechanges observed in the absorption spectrum. Emission spectrumof HTMA-PFP in the absence of HSA showed a maximum peakaround 412 nm and two shoulders at 433 and 475 nm. Again,a clear red shift of the emission spectrum, followed by a higherresolution in the vibrational structure, was observed at increasingconcentrations of the protein (up to ∼1.5 µM), which suggestsa reduction in the number of degrees of freedom of the polymerchains and hence a decrease in the number of conformationspresent in the excited state. Such effects have also been reported

Figure 2. Effect of HSA on the absorption spectrum of HTMA-PFP(3 µM) in aqueous solution. Protein concentrations were: 0, 0.01, 0.02,0.04, 0.06, 0.08, 0.12, 0.24, 0.35, 0.5, 1.5, 3.4, and 9.4 µM. Inset:Benesi-Hildebrand nonlinear plot (∆A of HTMA-PFP, measured atthe maximum of the spectrum, versus HSA concentrations up to 0.5µM).

Figure 3. Effect of HSA on the normalized fluorescence excitationand emission spectra of HTMA-PFP (1.5 µM) in aqueous solution.Protein concentrations were: 0, 0.25, 0.5, 1, 1.5, 3, and 6 µM. Inset:Increase in the fluorescence intensity of HTMA-PFP, measured atthe maximum of the spectra, as a function of HSA concentration.

1496 Biomacromolecules, Vol. 11, No. 6, 2010 Martınez-Tome et al.

upon interaction of this polyelectrolyte with surfactants andDNA,31,32 and we also observed a similar behavior whenHTMA-PFP was dissolved in aqueous solution with variableionic strength, independently of the ionic strength source (datanot shown). This effect in the fluorescence spectrum wasaccompanied by an increase in Rayleigh scattering of the sample,supporting the hypothesis of the existence of nonespecificelectrostatic interactions between HTMA-PFP and any speciesof opposite charge to yield neutral complexes, which, uponcharge neutralization, aggregate in different supramolecularstructures.

In addition to the red shift in the absorption and emissionspectra, supramolecular self-assembly of conjugated polymersor their interaction with biomolecules is generally accompaniedby a decrease in the overall fluorescence quantum yield, due,among others, to an increase in the inter- and intrachain energytransport mechanisms. Recently, the Bunz group reported thatproteins such as histone, lysozyme, myoglobin, and hemoglobinquench the fluorescence of a series of anionic poly(p-phenyl-eneethynylene) derivatives.12 Similarly, Heeger and coworkersreported that the interaction of cytocrome c, myoglobin, andlysozyme with a sulfonated poly(paraphenylenevinylene) re-duced its fluorescence in a different extent.10 In the case ofHTMA-PFP, a fluorescence quenching was observed afterinteraction with DNA32 or with surfactants at concentrationsbelow its cmc,31 which was attributed to polymer aggregationfavored by the charge neutralization. In contrast, in the presentwork, a slight increase in the fluorescence intensity of HTMA-PFP was observed upon the addition of HSA (inset in Figure3). The same behavior has been reported for other polyelectro-lytes after interaction with bovine serum albumin (BSA), whichis a protein very similar to HSA in structure and biologicalfunction.12,38,39 There are several possible explanations, non-mutually exclusive, for this behavior. On one hand, theenhancement of fluorescence could be due to the existence ofhydrophobic interactions between the conjugated backbone ofthe polymer and the hydrophobic patches of HSA (suchinteractions have been reported to increase the fluorescenceintensity of hydrophobic and amphiphilic fluorophores).40-43

On the other hand, the formation of the complex could inducein the polymer a conformational change in which chains becomeslightly more extended, minimizing the polymer-polymerinteraction. The changes in the fluorescence intensity of HTMA-PFP in the presence of HSA were not used to estimate KA

because these data were not sufficiently reproducible: we notedthat the fluorescence intensities of the polymer were slightlyperturbed not only by HSA addition but also after each agitation,

even if no solute was added to the sample, probably becauseagitation induces polymer aggregation. Independently of the lowreproducibility, it was observed, in all fluorescence experiments,that protein concentrations higher than ∼1.5 µM did not modifyeither the shape or the intensity of the emission spectrum ofHTMA-PFP. Parallel to these experiments, we recorded theRayleigh scatter of the sample at 430 nm as a function of HSAconcentration. This scatter signal increased to ∼1.5 µM tobecome steady at higher concentrations (data not shown). Allof these results, together with those obtained from absorptionspectra of HTMA-PFP, support the hypothesis that whenpolymer charge is totally neutralized, there are no moreinteractions between the aggregates formed and HSA.

To test if electrostatic interactions were the only onesresponsible for polymer-protein complex stabilization, theeffect of low pH on the fluorescence spectra of HTMA-PFP(1.5 µM), in the absence and in presence of HSA (6 µM), wasanalyzed (Figure S1 in the Supporting Information). Concentra-tions were selected to be sure that all polymer was bound toHSA. Results show that the addition of HCl to the samples, toreach pH 2, practically did not modify the fluorescence emissionspectrum of the polymer/HSA complexes but shifted to the redthat of the polymer alone. Because at pH 2 all protein basicgroups are completely protonated and the diameter of HSA doesnot differ from those at neutral pH ranges,44 these results indicatethat not only electrostatic interaction but also hydrophobicinteraction between polymer and protein is necessary for theformation of complex and aggregate, as was previously sug-gested from the increase observed in the HTMA-PFP fluores-cence intensity. The fact that the emission spectrum of thepolymer alone was shifted to the red at pH 2 can be explainedby the presence of chloride anion in the sample, which inducespolymer aggregation through charge neutralization.

We evaluated the thermal stability of the complex formedby recording the emission spectrum of HTMA-PFP in theabsence and presence of HSA at different temperatures (Figure4). Thermal scans were recorded simultaneously for bothsamples, one containing HTMA-PFP alone and the othercontaining the same concentration of HTMA-PFP (1.5 µM) butwith HSA in excess (6 µM) to be sure that all polymer wasbound to the protein. The increase in temperature, up to 88 °C,induced an effect totally different in both systems. In the absenceof protein, fluorescence intensity decreased, as was reported forother conjugated polymers,45 and the spectrum became slightlyless resolved, suggesting the increase in the degree of freedomof the polymer chains with temperature (Figure 4A,B). Incontrast, for the polymer bound to HSA, fluorescence intensity

Figure 4. Effect of temperature on the normalized spectra of HTMA-PFP (1.5 µM) (λexc ) 380 nm) (A) in the absence and (C) in the presenceof HSA (6 µM). (B) Temperature dependence of the fluorescence intensity of HTMA-PFP, measured as the area under the non-normalizedspectrum in the absence (9) and in the presence (b) of HSA.

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was much more stable, decreasing slightly up to ∼55 °C andincreasing from this temperature to reach a stable value, ∼80°C (Figure 4B). This change in the fluorescence intensity wasaccompanied by a clear blue shift of the emission spectrum,followed by a lower resolution in its vibrational structure (Figure4C), suggesting that the increase in temperature induces changesin polymer conformation but not the dissociation of theHSA-polymer complex. It seems more likely that severalprocesses with opposite effects are taking place. On one hand,fluorescence intensity tends to decrease because of the expectedeffect of temperature on the fluorescence quantum yield ofHTMA-PFP (as is observed for the polymer alone). On the otherhand, simultaneously, the early events in the protein thermalunfolding process induce the HTMA-PFP chains to becomemore extended, disrupting backbone interactions and increasingthe polymer conjugation length and, therefore, its fluorescenceintensity. In addition, exposure of hydrophobic amino acids dueto protein unfolding probably results in stronger hydrophobicinteractions between HTMA-PFP and the protein, which alsoproduces an increase in the fluorescence intensity. It has beenreported that thermal denaturation of HSA takes place throughmultiple steps: native f expanded f intermediate f un-folded.46-48 Increasing temperature to 50 °C results in thereversible separation of domain I and domain II (expandedform). In the intermediate state, above 60 °C, the unfolding ofdomain II starts, but domain I remains intact. Finally, increasingtemperatures above 70 °C results in irreversible unfolding ofdomain I. Sinha et al. have reported that during these processesthe protein undergoes a considerable change in its nativeglobular structure, increasing its hydrodynamic diameter from10.1 nm, in the native state at 25 °C, to 38.7 nm at 75 °C.43

Because HTMA-PFP probably interacts with domains I and IIof the protein because of their negative charge, it is logical tothink that such changes affect the conformational state ofHTMA-PFP and that the complex thermal behavior of thepolymer fluorescence reflects the different steps involving HSAunfolding.

Changes in the Intrinsic Fluorescence of HSA in Pres-ence of HTMA-PFP. To get more insight into the nature ofthe HSA-HTMA-PFP interaction, the effect of polyelectrolyteon the fluorescence spectrum of the protein was explored. HSAcontains a single tryptophan, Trp-214, and 18 tyrosine residuesthat are responsible for its fluorescence emission. Trp-214 islocated in subdomain IIA within a hydrophobic pocket, whereastyrosines are distributed along the whole polypeptide chain. (SeeFigure 1.) Upon excitation at 280 nm, both tryptophan andtyrosine are readily excited, but most of the fluorescence comesfrom Trp-214 because of the efficient resonance energy transfer

(RET) from tyrosine to tryptophan.49 Figure 5A shows thefluorescence emission spectrum (λexc ) 280 nm) of a 6.0 µMHSA solution in water at different HTMA-PFP concentrations.In the absence of the polyelectrolyte, the spectrum displayed arather broad fluorescence band, characteristic of tryptophan, witha maximum at ∼335 nm, which indicates that this residue isrelatively buried inside the HSA.35 The addition of HTMA-PFP resulted in substantial change of Trp214 fluorescence.Because polymer concentration was increased from 0 to 77 µM,the intensity of the emission maximum decreased gradually, tobe practically abolished, and a second peak, corresponding tothe tyrosines emission, appears at 304-306 nm. These resultssuggest that because of the interaction of HTMA-PFP with HSA,the polyelectrolyte strongly quenches the fluorescence ofTrp214, allowing the tyrosine emission to be more visible,contributing to the total protein emission band. It supports theidea of domain II being one of the possible interaction sites ofHTMA-PFP. The changes of fluorescence spectrum could bealso interpreted as in terms of the conformational change ofHSA upon the interaction with HTMA-PFP. The addition ofpolymer could induce the initial steps of protein unfolding,which imply separation of domains I and II, causing the increasein the distance between tyrosine and tryptophan. Therefore, theRET process between these residues becomes less efficient, andtyrosine emission is enhanced. However, if this was the onlymechanism responsible for the fluorescence quenching, twopeaks will be observed in the emission spectrum of the protein:one corresponding to the tyrosines emission (∼305 nm) andanother one corresponding to the tryptophan emission, whichshould be of lower intensity and probably shift to the red withrespect to its initial position.49

To speculate about the fluorescence quenching mechanisms,a Stern-Volmer analysis was applied to the fluorescencequenching data for HSA (Figure 5B). In the range of concentra-tion used, the Stern-Volmer plot showed an upward curvatureat high quencher concentrations. The linear part of the plot,corresponding to the lower concentrations of HTMA-PFP, wasused to estimate the Stern-Volmer constant, KSV (eq S1 andFigure S2 in the Supporting Information), and a value KSV )6.4 × 104 M-1 was obtained from the slope of the plot. Toinvestigate the nature of the quenching process (dynamic orstatic), an apparent quenching constant kq ) 1013 M-1 s-1 wasestimated from KSV, assuming τ0 ) 6.4 ns.49 This value is higherthan the maximum diffusion collision rate constant of variousquenchers with the biopolymer (2.0 × 1010 M-1 s-1),50

suggesting that a static quenching mechanism is operative inthe present complexes and supporting the idea that both

Figure 5. (A) Fluorescence emission spectra of HSA (6 µM) (λexc ) 280 nm) in the presence of increasing concentrations of HTMA-PFP (from0 to 77 µM). (B) Stern-Volmer curve for quenching of HSA fluorescence by HTMA-PFP.

1498 Biomacromolecules, Vol. 11, No. 6, 2010 Martınez-Tome et al.

macromolecules are complexed through electrostatic interac-tions. The existence of a static mechanism was supported fromthe Stern-Volmer analysis carried out at different temperatures.Quenching experiments were performed at 15, 25, and 35 °C,well below the protein thermal unfolding to avoid that confor-mational changes could modify the protein fluorescence lifetime.Results show that an elevation of temperature does not increasethe quenching efficiency (Figure S2 in the Supporting Informa-tion), which would be expected if dynamic quenching waspresent, suggesting that this type of mechanism is not predomi-nant, at least at low polyelectrolyte concentrations (up to 10µM).

The changes observed in the fluorescence emission of HSAas well as the upward curvature of the Stern-Volmer plotsuggest that protein quenching by HTMA-PFP cannot simplybe through a static process and that more than one quenchingmechanism is simultaneously taking place in the system.35

Because there is good overlap region between the fluorescencespectrum of HSA and the absorption spectrum of HTMA-PFP(Figure S3 in the Supporting Information), one of thesemechanisms could be RET from singlet excited state oftryptophan to HTMA-PFP. Such mechanism is not uncommonand has been described in many occasions for the ensemble ofHSA and its ligands.26,43,49 Different methods were used toverify the existence of RET. On one hand, the fluorescencespectra (from 290 to 500 nm) of HSA and HTMA-PFP/HSAsolutions after excitation at 280 nm were recorded and compared(Figure 6). This excitation wavelength was selected to minimizedirect absorption of the excitation light by the acceptor. BecauseHTMA-PFP still absorbs at this wavelength, emission spectrawere corrected using eq S5. (See the Supporting Information.)Figure 6 shows that the decrease in tryptophan fluorescence inHSA is followed by an increase in the fluorescence of HTMA-PFP and that the blank HTMA-PFP (dashed line in Figure 6),recorded under the same conditions, displays a fluorescenceemission that was three times lower than that recorded in thepresence of the protein. This result suggests the existence ofenergy transfer from the protein to the bound HTMA-PFP. TheRET process was further sustained by recording the excitationspectrum of the HTMA-PFP/HSA complex at a fixed concentra-tion of HSA and the excitation spectrum of the polyelectrolytealone at λem ) 444 nm, where HSA does not fluoresce. Thesubtraction of both spectra, with and without protein, gave a

fluorescence excitation spectrum that contains the proteintryptophan band (inset in Figure 6). Additional experiments wereperformed to better support the existence of RET, and resultsare included in the Supporting Information. Fluorescenceexcitation spectra of the HTMA-PFP/HSA complex were againrecorded, fixing the emission wavelength at 444 nm, but, inthis case, the concentration of polymer was maintained constantwhile increasing concentrations of protein were added to thesample. The background signal (excitation spectrum of HTMA-PFP in the absence of protein) was subtracted after normalizationto the intensity at 330 nm. Results show that protein additionclearly increases the HSA excitation band (Figure S4 in theSupporting Information). Because HSA fluorescence at 444 nmwas negligible, these results only can be explained by theincrease in the energy transfer efficiency from Trp-14 to HTMA-PFP. The inset in Figure S4 represents the fluorescence intensityat the maximum of the spectrum versus HSA concentration.The curve shows a behavior that was similar to the bindingcurve recorded from changes in the absorption spectra ofHTMA-PFP (inset in Figure 2). This similitude suggests thatchanges in the energy transfer efficiencies are proportional tothe fraction of polyelectrolyte bound to the protein.

From the spectral overlap between the HSA emission andHTMA-PFP absorption bands, the overlap integral, J, for theHTMA-PFP:HSA system was found to be 4.35 × 1014 nm4 cm-1

M-1. By taking values of the local refractive index, n ) 1.4,26

κ2 ) 2/3, and a quantum yield, ΦF ) 0.1 (determined in ourlaboratory for HSA at λexc ) 280 nm, using NATA as reference),the computed R0 value was ∼30 Å. Because RET is onlysignificant at distances r < 1.5R0,

35 these experiments indicatethat the average distance between HTMA-PFP and Trp-214 ofHSA is e45 Å. It should be possible to determine with moreprecision this distance by calculating the efficiency of the energytransfer (eq S4 in the Supporting Information). However, giventhat energy transfer is not the only mechanism involved in theHSA fluorescence quenching, this information could not beavailable from our experiments.

Effect of HTMA-PFP on the Conformation and ThermalStability of HSA. When a substance interacts with a protein,the binding processes can alter the intramolecular forcesresponsible for maintaining its secondary and tertiary structure,resulting in a conformational change of the protein. Suchchanges are reflected in the circular dichroism (CD) spectrumof the protein. In the present work, preliminary results of CDexperiments have provided an alternative approach to explorethe interaction between HTMA-PFP and HSA. Figure 7 shows

Figure 6. Emission spectra (λexc ) 280 nm) of HSA (6 µM) as afunction of the indicated HTMA-PFP concentrations: 0, 0.75, 3, 6,and 9 µM, showing the fluorescence of the polymer due to the RET.Spectra are compared with that of HTMA-PFP 0.75 µM in the absenceof HSA excited at the same wavelength (dotted line). Inset: Differenceexcitation spectrum between the HTMA-PFP (3 µM) in the presenceand in the absence of protein, measured at λem ) 444 nm, whereHSA does not fluoresce.

Figure 7. Effect of HTMA-PFP on the CD spectrum of HSA (3.0 µM).Polyelectrolyte concentrations were: 0, 1.5, 9, and 12 µM. Inset:Thermal denaturation of HSA monitored by the CD signal at 222 nmin the absence (continuous line) and the presence (dotted line) ofHTMA-PFP (12 µM).

Formation of Complexes between HTMA-PFP and HSA Biomacromolecules, Vol. 11, No. 6, 2010 1499

the far-UV CD spectra of HSA (3.0 µM) in the absence andpresence of different concentrations of HTMA-PFP (up to 12µM). Higher concentrations of HTMA-PFP were not studiedbecause of the turbidity of the samples. In the absence ofpolyelectrolyte, the spectrum exhibited two negative bands, at 208and 222 nm, characteristic of the R-helical structure. HTMA-PFPdid not contribute to the CD signal in the range of 200-260 nm,and thus the observed CD was solely due to the peptide bonds ofprotein. As polyelectrolyte concentration was increased, the CDsignal of HSA decreased slightly. Changes in the R-helical contentwere calculated from these curves using eqs S6-S8, as is describedin the Supporting Information. Results indicate that free HSA hasa high R-helix content that decreases by ∼0.7, 1, and 1.2% in thepresence of 1.5, 9, and 12 µM HTMA-PFP, respectively. Reductionin the R-helix content suggests, as was pointed out previously fromthe quenching experiments, that as polymer is added, it binds tothe protein and initiates unfolding. Similar behavior was recentlyfound in the CD spectra of HSA and BSA in the presence ofmacromolecules such as surfactants, cationic lipids, or dendri-mers,22,23,25,51 which was also attributable to a partial proteinunfolding on account of its interaction with the macromolecules.

CD experiments were also performed to explore the effectof HTMA-PFP on the thermal unfolding of HSA. As waspreviously discussed, the folding process for a multidomainprotein, such as HSA, is complex because each domain unfoldsindependently at different temperatures. In the case of HSA,the thermal unfolding is sequential, occurring over a widetemperature range, from 55 to 85 °C, with a midtemperature of∼67 °C.26 This result is in agreement with the value obtainedin this work by monitoring the far-UV CD signal at 222 nm, asa function of temperature (inset in Figure 7). The addition ofHTMA-PFP induced a decrease in the midtransition temperatureof ∼10 °C confirming that the molecular interaction betweenHTMA-PFP and HSA produces changes in the stability andsecondary structure content of the protein.

Conclusions

This study reveals the strong interactions that can existbetween CPEs and human serum proteins and that must be takeninto account to define the real-world sensing applications ofthis class of polymers. In particular, the work uses differentapproaches to explore the interaction between the cationic CPEHTMA-PFP and the protein HSA in aqueous solution. Interac-tion was confirmed through alterations of the absorption andfluorescence spectra of HTMA-PFP and the quenching of theintrinsic fluorescence of the protein, which is predominantlydue to static quenching and energy transfer mechanism betweenprotein and polymer. Results indicate that polymer-proteincomplexes are formed with high affinity because of a combina-tion of electrostatic interaction between the cationic side chainsof HTMA-PFP and the negatively charged surface of the proteinas well as hydrophobic interactions between the conjugatedpolymer backbone and the hydrophobic patches of HSA. Thefact that domains I and II of the protein are more acidic thatdomain III suggests that the electrostatic interaction occursmainly between the polymer and the domains I and II. Thishypothesis is supported from the thermal behavior of thepolymer fluorescence, which is highly sensitive to the differentsteps involving HSA unfolding as well as from the proteinquenching experiments, given the location of Trp-214 in domainII. These results were complemented by CD experiments of theHSA and its polymer complexes, which indicate that themolecular interaction between HTMA-PFP and HSA decreases

the stability of the protein, producing a loss of helical secondarystructure content. Such changes seem to induce an alteration inpolymer conformation that is signaled by an enhancement inits fluorescence intensity.

Acknowledgment. We thank the Spanish Ministerio deCiencia e Innovacion (MICINN) for grant MAT2008-05670.R.E. acknowledges the support of a predoctoral fellowship fromMICINN. We are grateful for the useful insights obtained indiscussions with Drs. Javier Gomez and Pilar Lillo. We alsothank Dr. Jesus Sanz for his useful comments and for help inFigure 1 preparation.

Supporting Information Available. Equations that describethe data analysis of fluorescence quenching, energy transfer,CD, and absorption experiments. Effect of low pH on theHTMA-PFP fluorescence spectra, Stern-Volmer plots at dif-ferent temperatures, the spectral overlap between HSA emissionand HTMA-PFP absorption, as well as the excitation spectraof HTMA-PFP/HSA complex in the presence of increasingconcentrations of protein. This material is available free ofcharge via the Internet at http://pubs.acs.org.

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