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Formation of Complexes between the Conjugated Polyelectrolyte Poly{[9,9-bis(6′-N,N,Ntrimethylammonium)hexyl]fluorene-phenylene} Bromide (HTMA-PFP) and Human Serum Albumin Maria Jose´ Martı´nez-Tome´, Rocı´o Esquembre, Ricardo Mallavia, and C. Reyes Mateo* Instituto de Biologı´a Molecular y Celular, Universidad Miguel Herna´ndez 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]fluorenephenylene} bromide (HTMA-PFP) and human serum albumin (HSA) has been investigated from changes observed in both the spectroscopic properties of HTMA-PFP and the intrinsic fluorescence of HSA. Absorption and fluorescence spectra of HTMA-PFP suggest that HTMA-PFP and HSA form polymer-protein complexes due to electrostatic interactions between the cationic side chains of HTMA-PFP and the negatively charged surface of the protein. Interaction between both macromolecules induces an increase in the fluorescence signal of HTMAPFP, which suggests that hydrophobic forces also contribute to the polymer-protein complex stabilization. In addition, this interaction causes a decrease in the HSA fluorescence, partially due to static quenching and energy transfer between both macromolecules. Effects of HTMA-PFP on the thermal stability and protein conformation were explored from CD experiments. Results indicate that as polymer is added it binds to HSA and initiates unfolding. This unfolding process induces HTMA-PFP chains to become more extended, disrupting backbone interactions and increasing polymer fluorescence intensity.
Introduction Fluorescent conjugated polyelectrolytes (CPEs) constitute an interesting class of materials with a wide range of properties. They are π-conjugated polymers, their rigid backbone consisting of an alternation of simple and double bonds with water-soluble side chains containing cationic or anionic groups. Electron delocalization facilitates rapid intra- and interchain exciton migration, conferring collective optical responses and amplified signals when comparing with conventional fluorophores.1,2 In addition, these new materials can undergo spontaneous selfassembly through reversible, electrostatic, and/or hydrophobic interactions with some other species, generally of opposite charge, resulting in supramolecular structures with interesting optical and material properties.3 Given these properties, CPEs have received great attention during the last years because of their potential applications in areas such as chemical and biological sensing.2-8 The use of CPEs as biomolecule sensors is based on changes suffered by the polymer upon interaction with the biomolecules. This interaction can cause different conformational transitions of the polyelectrolyte backbone, aggregation or separation of the polyelectrolyte chains, and energy or electron transfer processes between polymer and biomolecule, which yield important changes in the polymer intrinsic fluorescence.9,10 Several authors have reported the existence of nonspecific interactions between CPEs and biomolecules such as proteins that perturb the fluorescence of the polyelectrolytes.10-12 These interactions generate a distinct fluorescence response pattern for a given protein and therefore have opened opportunities for the development of new biosensors.2,13,14 However, the existence of such interactions becomes a double-edge sword because many * Corresponding author. Fax: +34 966 658 758. E-mail:
[email protected].
of these sensors are designed with the aim of being used in living cells or human biological fluids that contain a complex mixture of proteins and other biomolecules. For instance, human serum contains more than 20 000 proteins with an overall protein content >1 mM. Interactions between such proteins and the polyelectrolyte should result in a nonspecific background that could be very high and obscure any fluorescence specific signal.12 Therefore, it should be of great interest to know the fluorescence response of CPEs to human serum proteins to better define the real-world sensing applications of this class of polymers. Human serum albumin (HSA) is the most abundant and principle extracellular protein, forming ∼60% of the mass of human plasma proteins, with a typical concentration of 5 g/100 mL in the bloodstream.15 It is a globular protein composed of a single polypeptide of 585 amino acids with three R-helical domains I-III, each containing two subdomains A and B (Figure 1). Its isoelectric point is 4.9;16 therefore, the protein displays a negative net charge at neutral pH. However, the charge distribution is not homogeneous through the three domains; domains I and II more acidic than domain III.17 The protein contains multiple binding sites that allow its interaction with many organic and inorganic compounds and make this protein an important regulator of intercellular fluxes.17 Its 3D structure, determined through X-ray crystallographic measurements, indicates that the principal regions of ligand binding sites in HSA are located in hydrophobic cavities in subdomains IIA and IIIA and the sole tryptophan residue (Trp-214) of the protein is located in subdomain IIA.18,19 The molecular interactions between HSA and many compounds have been successfully investigated, including drugs,18,20 surfactants,21,22 lipids and liposomes,23,24 and macromolecules such as dendrimers,25 polyoxometalates,26 or polethyleneglycol.27 However, to our
10.1021/bm100123t 2010 American Chemical Society Published on Web 04/27/2010
Formation of Complexes between HTMA-PFP and HSA
Figure 1. Top left: Crystal structure of HSA indicating the three different domains present and the location of its 18 Tyr residues (in green) and its single tryptophan residue (in red), Trp-214. The structure was obtained from the Protein Data Bank (PDB code 1HA2). Top right, in dark blue: Simulation by molecular dynamic of a tetramer of HTMA-PFP. Figures were rendered, on the same scale, using PYMOL and CHEMDRAW, respectively. Bottom: HTMA-PFP chemical structure.
knowledge there has been no study exploring the nature of the interactions of CPEs with HSA. In this work, we present a spectroscopy study of the interaction 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 previously demonstrated to interact with surfactants and DNA.28-32 The interaction was characterized from spectroscopic changes observed in both macromolecules. Effects of polymer on the thermal stability and conformation of the protein were also explored from CD experiments to get more insight into the nature of this interaction.
Experimental Section Materials. HSA was purchased from Sigma-Aldrich Chemical (Milwauke, WI). HSA was dissolved in deionized doubly distilled water. j ) 8340 g/mol, repeat unit molecular The cationic CPE HTMA-PFP (M weight, 694.71 g/mol; n′ ) 12 based on polyfluorene calibration) was obtained and characterized in our laboratory, as was previously described.33,34 In brief, a low-molecular-weight batch of the neutral polymer, poly[9,9-bis(6′-bromohexyl)fluorene phenylene], was synthesized by Suzuki coupling reaction with Pd(II) as catalyst and treated with gas-phase trimethylamine to obtain the corresponding cationic polyelectrolyte. Other chemicals were of analytical or spectroscopic reagent grade. The pH of all aqueous solutions was controlled, and variations were always lower than (0.15, even at the maximal concentrations of HSA and HTMA-PFP used in this study.
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Preparation of HTMA-PFP Solutions. When CPEs are dissolved in water, they self-assemble into aggregates through the interaction between the charged side chains and the hydrophobic backbone. The degree 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 aggregates facilitating their solubilization. In this work, we have used DMSO as cosolvent of HTMA-PFP, with the exception of the CD experiments, in which dioxane was selected because of the strong absorption of DMSO in the far UV region. In all cases, the proportion of the cosolvent in the water mixture was always lower than 1% (v/v). For absorption and fluorescence experiments, stock solutions of HTMA-PFP (3.65 × 10-4 M, in repeat units) were prepared in DMSO. Aliquots of this stock solution were dissolved in deionized doubly distilled water, obtaining a clear and transparent solution, which suggests the nonexistence of aggregates. Absorption and Steady-State Fluorescence Measurements. Absorption measurements were carried out at room temperature using a Shimadzu spectrophotometer (UV-1603, Tokyo, Japan). Fluorescence measurements were performed in a PTI-QuantaMaster spectrofluorometer interfaced with a Peltier cell. Excitation wavelengths at 280 and 380 nm for HSA and HTMA-PFP, respectively, were utilized. Thermal scans were collected from 25 to 88 °C with a heating rate of 10 °C/ min. Spectra were taken 10 sec after the temperature equilibrium was reached. The experimental samples were placed in 10 × 10 mm path length quartz cuvettes. Background intensities were always checked and subtracted from the sample when it was necessary. Quenching Experiments. Fluorescence emission of HSA was studied in the absence and presence of different concentrations of HTMA-PFP. Analysis was applied to the fluorescence quenching data, according to the standard Stern-Volmer equation (eq S1 in the Supporting Information) to obtain the Stern-Volmer constant, KSV. In this work, samples were excited at 280 nm to minimize polymer absorption, and the emission intensity was measured as the area of the emission spectrum, calculated from 340 to 380 nm, to avoid the tyrosine emission. The significance of KSV depends on the nature of the quenching process: it may represent the association constant for complex formation or the rate of dynamic quenching (KSV ) kqτ0), where kq is the bimolecular rate constant of the quenching process, and τ0 is the lifetime of the biomolecule. Dynamic or static quenching can be distinguish, among others, by their differing dependence on temperature and by the different alterations induced in the fluorescence lifetime or in 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 tryptophan excitation. Data analysis was carried out using the Fo¨rster equations, as is described in the Supporting Information (eqs S2-S4). As for quenching experiments, samples were excited at 280 nm to minimize polymer absorption, and the emission intensity was measured as the area of the emission spectrum, calculated from 340 to 380 nm, to avoid the tyrosine emission. Artifacts, such as the effect of the acceptor (HTMA-PFP) absorption at the donor (tryptophan) excitation wavelength (280 nm), were corrected according to eq S5 in the Supporting Information. Circular Dichroism Experiments. CD measurements of HSA and its polymer complexes were carried out with a Jasco spectropolarimeter, model J-815 (JASCO, Easton, MD), interfaced with a PTC-423S/15 Peltier-type cell holder for temperature control. Spectra were collected at 25 °C with a scan speed of 50 nm per min, response time of 4 s, and a bandwidth of 1 nm. For each spectrum, four scans were accumulated and averaged to improve the signal-to-noise ratio. Spectra were recorded from 260 to 197 nm using 0.1 cm quartz cells. HSA concentration was kept constant (3.0 µM) while each polymer concentration was varied (1.5, 9, 12 µM). Each CD sample was prepared independently by mixing the same volume of HSA stock solution with an appropriate amount of HTMA-PFP (depending on the desired polyelectrolyte final concentration) and leading to a final volume of 1 mL, in all cases. A
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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 at the maximum of the spectrum, versus HSA concentrations up to 0.5 µM).
baseline was taken under the same conditions as those used for the sample and subtracted from each spectrum. Changes in the R-helical content were calculated, as is described in Supporting Information (eqs S6-S8). Thermal scans were collected at a fixed wavelength of 222 nm from 25 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 using a two-state model with the only purpose of determining the temperature at the midpoint of denaturation and any variation produced by the addition of polymer to HSA.
Results and Discussion Changes in the Spectroscopic Properties of HTMA-PFP in the Presence of HSA. The effect of HSA on the absorption spectrum of HTMA-PFP in aqueous solution was explored using a constant polyelectrolyte concentration (3 µM, in terms of repeat units) and increasing concentrations of protein (Figure 2). The addition of the protein, up to ∼0.35 µM, induced simultaneously a decrease in the absorbance intensity, a slight broadening of the spectrum, and a shift of the maximum to the red, 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 the free polymer and the polymer complexed to HSA. For HSA concentrations between 0.5 and 3.4 µM, an increase in the scatter of the sample was observed, which is indicative of aggregates formation. Higher concentrations of HSA did not induce any change in the scatter or in the absorption spectrum of HTMAPFP. These results suggest that depending on the protein concentration at least two different types of polymer-protein complexes are formed: for lower protein concentration, a single complex between HSA and HTMA-PFP is taking place, probably because of the electrostatic interactions between the polyelectrolyte (having two positive charges by each monomer unit) and domains I and II of the protein, which show a negative net charge under the experimental conditions. Higher concentrations of HSA, up to ∼3.4 µM, seem to induce additional interactions between the formed complexes, which because of the partial neutralization of their charges will exhibit lower water solubility, giving place to aggregates with more complex molecular architectures. Above ∼3.4 µM, the net charge of HTMA-PFP is totally neutralized, and no more interactions are expected among these aggregated and the excess of protein. This interpretation is in agreement with the results obtained for the same polyelectrolyte upon interaction with surfactants and
Martı´nez-Tome´ et al.
Figure 3. Effect of HSA on the normalized fluorescence excitation and 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 at the maximum of the spectra, as a function of HSA concentration.
DNA.31,32 In both works, similar modifications in the absorption spectra were observed, which were attributed to polymer aggregation and, in the case of DNA, to the formation of 1:1 complexes between DNA and HTMA-PFP, for DNA concentrations lower than the concentration of polymer repeat units. We also consider that the complex formed between HTMA-PFP and HSA has a 1:1 stoichiometry at low HSA concentrations.32 This assumption can be supported taking into account that, on one hand, each single polymer chain contains on average around 12 monomer units and, therefore, ∼24 positive charges, and, on the other hand, at neutral pH, each HSA molecule has a negative net charge of -19.36 The change in the absorption spectrum of HTMA-PFP in the presence of low HSA concentrations was used to estimate the association constant of this complex (KA). The inset in Figure 2 represents the decrease in the absorbance intensity measured at the maximum of the spectrum (∆A ) AHTMA-PFP - AHTMA-PFP:HSA) versus HSA concentration. Determination of KA can be made by fitting these data to the Benesi-Hildebrand equation37 (eq S9 in the Supporting Information). The solid line in the inset of Figure 2 shows this fit, which yields a value for KA ) (9.6 ( 1.4) × 106 M. This constant suggests strong affinity between HTMA-PFP and HSA and is in the same range that the value obtained for the HTMA-PFP:DNA complexes32 but higher than that determined for HSA bound to other macromolecules, such as dendrimers25 or polyethyleneglycol.27 The interaction of HTMA-PFP with HSA is also evident from the fluorescence spectra of the polyelectrolyte. Figure 3 shows the effect of increasing concentration of HSA, up to 6 µM, on the excitation and emission spectra. In this experiment, the concentration of HTMA-PFP was maintained constant, at 1.5 µM in terms of repeat units (optical density
