Thermodynamic and Structural Changes Associated with the

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Thermodynamic and Structural Changes Associated with the Interaction of a Dirhamnolipid Biosurfactant with Bovine Serum Albumin Marina Sa´nchez,† Francisco J. Aranda,† Marı´a J. Espuny,‡ Ana Marque´s,‡ Jose´ A. Teruel,† ´ ngeles Manresa,‡ and Antonio Ortiz*,† A Laboratorio de Microbiologı´a, Facultad de Farmacia, UniVersidad de Barcelona, Joan XXIII s/n, E-08028, Barcelona, Spain and Departamento de Bioquı´mica y Biologı´a Molecular-A, Facultad de Veterinaria, UniVersidad de Murcia, E-30100, Murcia, Spain ReceiVed February 28, 2008. ReVised Manuscript ReceiVed April 1, 2008 The interaction of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa with bovine serum albumin was studied by means of various physical techniques. Binding of the biosurfactant to bovine serum albumin was first characterized by isothermal titration calorimetry, showing that one or two molecules of dirhamnolipid, in the monomer state, bound to one molecule of the protein with high affinity. These results were confirmed by surface tension measurements in the absence and presence of bovine serum albumin. As seen by differential scanning calorimetry, dirhamnolipid shifted the temperature of the thermal unfolding of bovine serum albumin toward higher values, thus increasing the stability of the protein on heating. The impact of dirhamnolipid on the structure of the native protein was low, since most of the secondary structure remained unaffected upon interaction with the biosurfactant, as shown by FTIR spectroscopy. However, 2D correlation infrared spectroscopy indicated that the sequence of temperatureinduced structural changes in native bovine serum albumin was modified by the presence of the biosurfactant. The consequences of these results in relation to possible applications of these dirhamnolipid biosurfactants for protein studies are discussed.

Introduction Biosurfactants constitute a group of surface-active compounds which are synthesized by a number of microorganisms, namely bacteria, yeasts, and fungi. These amphiphilic compounds present a wide structural diversity, including glycolipids, lipoaminoacids and lipopeptides, polymers, and phospholipids, mono- and diacylglycerols and fatty acids. Because of their special properties, low toxicity, biodegradable character, and effectiveness at extreme temperature and pH values, it is of great interest to characterize new biosurfactants to evaluate their use as potential alternatives to chemically synthesized compounds.1–4 Pseudomonas aeruginosa is an environmental microorganism and opportunistic pathogen that produces rhamnolipids when grown under the appropriate physicochemical conditions.5 Rhamnolipids are a group of glycolipid biosurfactants composed of a hydrophilic head, which is formed by one or two rhamnose molecules, called respectively monorhamnolipid and dirhamnolipid, and a hydrophobic tail containing one or two fatty acids (Figure 1). The type of the rhamnolipids produced depends on the bacterial strain, the carbon source used, and the culture conditions.6 Rhamnolipids represent one of the most important classes of biosurfactants because of various advantageous characteristics. Concerning its production, high yields are obtained * To whom correspondence should be addressed. Tel: +34-968-364788. Fax: +34-968-364147. E-mail: [email protected]. † University of Murcia. ‡ University of Barcelona.

(1) Desai, J. D.; Banat, I. M. Microbiol. Mol. Biol. ReV. 1997, 61, 47–64. (2) Cameotra, S. S.; Makkar, R. S. Appl. Microbiol. Biotechnol. 1998, 50, 520–529. (3) Singh, P.; Cameotra, S. S. Trends Biotechnol. 2004, 22, 142–146. (4) Rodrigues, L.; Banat, I. M.; Teixeira, J.; Oliveira, R. J. Antimicrob. Chemother. 2006, 57, 609–618. (5) Jarvis, F. G.; Johnson, M. J. J. Am. Chem. Soc. 1949, 71, 4124–4126. (6) Sobero´n-Cha´vez, G.; Le´pine, F.; De´ziel, E. Appl. Microbiol. Biotechnol. 2005, 14, 1–8.

Figure 1. Chemical structure of the dirhamnolipid compounds produced by Pseudomonas aeruginosa. For Rha-Rha-C10-C10 m,n ) 6 and for Rha-Rha-C10-C12 m ) 8 and n ) 6.

as compared to other biosurfactants; furthermore, several raw materials, like used oils or wastes from the food industry, can be used as a source of carbon.6–8 Thus, the complete pathway of bacterial biosynthesis of rhamnolipids can be classified as a green process. Rhamnolipids are surface-active compounds which strongly reduce the surface tension of water.9 The CMC of pure rhamnolipids and its mixtures has been shown to depend greatly on the chemical composition of the various species.10 However, rhamnolipids have been also shown to present several interesting activities from a biological point of view. Monorhamnolipid and (7) Lang, S.; Wullbrandt, D. Appl. Microbiol. Biotechnol. 1999, 51, 22–32. (8) Banat, I. M.; Makkar, R. S.; Cameotra, S. S. Appl. Microbiol. Biotechnol. 2000, 53, 495–508. (9) Parra, J. L.; Guinea, J.; Manresa, A.; Robert, M.; Mercade, M. E.; Comelles, F.; Bosch, M. P. J. Am. Oil Chem. Soc. 1989, 66, 141–145. (10) Benincasa, M.; Abalos, A.; Oliveira, I.; Manresa, A. Antonie Van Leeuwenhoek 2004, 85, 1–8.

10.1021/la800636s CCC: $40.75  2008 American Chemical Society Published on Web 05/16/2008

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dirhamnolipid restricted the growth of Bacillus subtilis11,12 and had zoosporicidal activity on species of three genera of zoosporic phytopathogens.13 Several other biosurfactants, displaying similar biological actions, also affected the structure of phospholipid membranes,14,15 and a purified dirhamnolipid fraction produced by Pseudomonas aeruginosa was also shown to influence the physicochemical characteristics of phosphatidylcholine and phosphatidylethanolamine membranes.16–18 Thus, it is widely accepted for a number of biosurfactants that the majority of the mentioned activities are most likely related to its action on the lipid constituent of biological membranes. The biosurfactant produced by Pseudomonas aeruginosa is a heterogeneous mixture of rhamnolipids, which has been used for most of the published works. However, the evaluation of the individual contribution of each homologue to the biological properties of the whole mixture is of importance, since it would allow selecting a rhamnolipid compound with the desired properties for specific uses. Therefore, this work has been carried out with a dirhamnolipid fraction purified from the complete mixture. Surfactants are essential compounds for the study of the structure and function of membrane proteins.19 The studies on the specific and nonspecific interactions of surfactants and proteins are of wide relevance because of the various industrial and research laboratory applications of surfactant/protein systems. Thus, applications in cosmetics and detergency can be found, as well as in various biochemical laboratory methods.20 Albumins are extraordinary molecules because of their properties and applications, and bovine serum albumin (BSA) has been widely used as a general model to study this type of interactions of surfactants with globular proteins.21–25 Albumins, as transport proteins, can bind a wide number of organic compounds of medium size, including fatty acids, amino acids, steroids, or surfactants. BSA is a single-chain protein with 581 amino acids and a molecular weight of 66 300 Da, and its structure and physicochemical properties have been thoroughly described.26–30 The properties and efficacy of many pharmaceutical drugs can be modified on the basis of their interactions with BSA. Thus, in this work, we present a detailed molecular level study of the interactions of a purified dirhamnolipid biosurfactant produced (11) Lang, S.; Katsiwela, E.; Wagner, F. Fat Sci. Technol. 1989, 91, 363–366. (12) Lang, S.; Wagner, F. Biosurfactants, Dekker, New York, 1993. (13) Stanghellini, M. E.; Miller, R. M. Plant Dis. 1997, 81, 4–12. (14) Grau, A.; Go´mez-Ferna´ndez, J. C.; Peypoux, F.; Ortiz, A. Biochim. Biophys. Acta 1999, 1418, 307–319. (15) Grau, A.; Ortiz, A.; De Godos, A.; Go´mez-Ferna´ndez, J. C. Arch. Biochem. Biophys. 2000, 377, 315–323. (16) Ortiz, A.; Teruel, J. A.; Espuny, M. J.; Marque´s, A.; Manresa, A.; Aranda, F. J. Int. J. Pharm. 2006, 325, 99–107. (17) Sa´nchez, M.; Teruel, J. A.; Espuny, M. J.; Marque´s, A.; Aranda, F. J.; Manresa, A.; Ortiz, A. Chem. Phys. Lipids 2006, 142, 118–127. (18) Aranda, F. J.; Espuny, M. J.; Marque´s, A.; Teruel, J. A.; Manresa, A.; Ortiz, A. Langmuir 2007, 23, 2700–2705. (19) Ananthapadmanabhan, K. P. Interactions of Surfactants with Polymers and Proteins; CRC Press: London, 1993. (20) Lindman, B.; Karlstro¨m, G., Polymer Surfactant Systems, in: D.M. Bloor, E. Wyn-jones (Eds.), The Structure, Dynamics and Equilibrium Properties of Colloidal Systems, NATO ASI Ser. C; Vol. 324, Kluwer Academic: Dordrecht, 1990. (21) Vasilescu, M; Angelescu, D.; Almgren, M.; Valstar, A. Langmuir 1999, 15, 2635–2643. (22) Valstar, A.; Almgren, M.; Brown, W.; Vasilescu, M. Langmuir 2000, 16, 922–927. (23) Moriyama, Y.; Takeda, K. Langmuir 2005, 21, 5524–5528. (24) Singh, S. K.; Kishore, N. J. Phys. Chem. B 2006, 110, 9728–9737. (25) Orioni, B.; Roversi, M.; La Mesa, C.; Asaro, F.; Pellicer, G.; D’Errico, G. J. Phys. Chem. B 2006, 110, 12129–12140. (26) Vijai, K.; Forster, J. Biochemistry 1967, 6, 1152–1159. (27) Carter, D. C.; He, X. M. Science 1990, 249, 302–303. (28) He, X. M.; Carter, D. C. Nature 1992, 358, 209–215. (29) Carter, D. C.; Ho, J. X. AdV. Protein Chem. 1994, 45, 153–203. (30) Carter, D. C.; He, X.-M.; Munson, S. H.; Twigg, P. D.; Gernert, K. M.; Broom, M. B.; Miller, T. Y. Science 1989, 244, 1195–1198.

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by Pseudomonas aeruginosa with bovine serum albumin, in order to consider potential future applications of these compounds.

Experimental Section Materials. All the reagents used were of the highest purity available. Purified water was deionized in a Milli-Q equipment from Millipore (Millipore, Bedford, MA, USA) and had a resistivity of 18 MΩ. BSA (essentially fatty acid and globulin free; min. 99%) was from Sigma Chemical Co. (St. Louis, MO). The buffer used throughout the work was 150 mM NaCl, 5 mM Hepes pH 7.4, unless otherwise indicated. Water and all of the solutions used in this work were filtered through 0.2 µm filters prior to use. Biosurfactant producer, strain 47T2 (NCIB 40044), was isolated from a contaminated soil sample and was selected for its capacity to accumulate surface-active rhamnolipids from hydrophobic substrates.31 This strain was maintained by fortnight cultures and preserved at -20 °C. From previous morphological and biochemical tests, the isolate was identified as Pseudomonas aeruginosa.32 Rhamnolipids were produced and purified as previously described.18 The composition of this dirhamnolipid component was determined by HPLC, as described before,33 and it consisted mainly of Rha-Rha-C10-C10 (50%) and Rha-Rha-C10-C12 (29%), with small contributions from the other three minor species, namely, Rha-Rha-C8-C10, Rha-Rha-C8-C12:1, and Rha-Rha-C10-C12: 1. Stock solutions of the dirhamnolipid were prepared in chloroform/ methanol (1:1) and stored at -80 °C. Sample Preparation. The dirhamnolipid aqueous samples were prepared by dispersion of the required amount of biosurfactant in the appropriate buffer, as indicated. Briefly, the desired amount of dirhamnolipid was dissolved in chloroform/methanol, and the solvent was gently evaporated under a stream of dry N2, to obtain a thin film at the bottom of a glass tube. The last traces of solvent were removed by a further minimum 3 h desiccation under high vacuum. The appropriate buffer was added to the dry samples, and these were shaken at room temperature until a visually homogeneous solution or suspension was obtained. BSA solutions were freshly prepared just before the experiments by dissolving the protein in the indicated buffer at the desired concentration. High Sensitivity Differential Scanning Calorimetry. The thermal denaturation of BSA was monitored by differential scanning calorimetry (DSC) using a VP-DSC high sensitivity differential scanning calorimeter from MicroCal (Northampton, MA, USA). Thermograms were recorded between 40 and 90 °C at a scan rate of 30 °C h-1. It is known that there is some dependence of the transition temperature upon scanning rate, particularly in the case of asymmetric endotherms. However, this effect did not affect our experiments since, on the one hand, the endotherms of pure BSA were symmetrical and, on the other hand, the same scan rate was used for pure BSA and BSA in the presence of dirhamnolipid, in order to make proper comparisons. Furthermore, the scan rate used here has been widely used for these types of studies with BSA and other proteins. The BSA concentration was 0.05 mM (MW 66 300) unless otherwise is indicated. The calorimetric data were analyzed using Origin software provided with the equipment, to obtain ∆H and Tmvalues. The curve fitting procedure carried out with some thermograms was performed with Origin software using a Gaussian function. Isothermal Titration Calorimetry. High sensitivity isothermal titration calorimetry (ITC) measurements were performed in a VPITC Titration Calorimeter from MicroCal (Northampton, MA, USA). The mixing cell had a volume of 1.442 mL. The data were processed using the Origin software provided with the equipment. The experiments were carried out as follows: The calorimeter syringe was filled with a concentrated solution (in 150 mM NaCl, (31) Robert, M.; Mercade´, M. E.; Bosch, P.; Parra, J. L.; Espuny, M. J.; Manresa, M. A.; Guinea, J. Biotechnol. Lett. 1989, 11, 871–874. (32) Haba, E.; Espuny, M. J.; Busquets, M.; Manresa, A. J. Appl. Microbiol. 2000, 88, 379–387. (33) Haba, E.; Pinazo, A.; Jauregui, O.; Espuny, M. J.; Infante, M. R.; Manresa, A. Biotechnol. Bioeng. 2003, 81, 316–322.

Interaction of a Dirhamnolipid Biosurfactant with BSA 5 mM Hepes, pH 7.4 buffer) of the dirhamnolipid in the micellar state (usually 2-3 mM, i.e., about 20× larger than the CMC). The injection of 10 µL aliquots of this solution into a 0.1 mM BSA solution, in the same buffer, contained in the calorimeter cell, resulted in the release of heat upon binding of the dirhamnolipid to the protein. The calorimeter cell was constantly stirred at a speed of 260 rpm. The data were corrected for dirhamnolipid heats of dilution, which were determined in a separate set of experiments. Surface Tension Measurements. Equilibrium surface tension (γ) was measured at 25 °C using a Kru¨ss K9 digital tensiometer (Kru¨ss, Helsinki, Finland) by the ring method. The instrument was calibrated against ultrapure water (γ ) 72 mN m-1), pure ethanol (γ ) 22.7 mN m-1), and 30.5% ethanol/water mixture (γ ) 34.4 mN m-1) to ensure accuracy over the entire range of surface tension. Prior to use, the platinum ring and all of the glassware were sequentially washed with chromic acid, deionized water, and acetone, and finally flamed in a Bunsen burner. Infrared Spectroscopy. The samples for the infrared measurements were prepared essentially as described above in a D2O buffer containing 100 mM NaCl, 100 mM phosphate pD 7.4 (pH 7.0). The use of Hepes buffer was avoided for these measurements because it presented infrared bands that could interfere with those of the protein. In any case, the use of phosphate buffer did not have any influence on BSA structure and its interaction with dirhamnolipid. An aliquot of the sample (approximately 40 µL) was placed between two CaF2 windows using 50 µm Teflon spacers, and the set was mounted in a thermostatted Symta cell holder. Infrared spectra were acquired in a Nicolet 6700 Fourier-transform infrared spectrometer (FTIR) (Madison, WI). Each spectrum was obtained by collecting 256 interferograms with a nominal resolution of 2 cm-1. The equipment was continuously purged with dry air to minimize the contribution peaks of atmospheric water vapor. The sample holder was thermostatted using a Peltier device (Proteus system from Nicolet). Spectra were collected at 2 °C intervals, allowing 5 min equilibration between temperatures. The D2O buffer spectra taken at the same temperatures were subtracted interactively using either Omnic or Grams (Galactic Industries, Salem, NH) software. Derivation and Fourier self-deconvolution were applied in order to resolve the component bands of the amide I′ region of the spectrum.34,35 The secondary structure of the protein was quantified by curve-fitting analysis of the components of the amide I′ band using Grams software. During the fitting procedure, the maxima of the bands, determined from deconvolution as explained above, were allowed to move ( 2 cm-1. 2-D Correlation Infrared Spectroscopy. The analysis by 2-D correlation infrared spectroscopy was carried out using the 2DShige software written by S. Morita and Y. Ozaki (Kwansei-Gakuin University, Japan) downloaded from their webpage (http:// sci-tech.ksc.kwansei.ac.jp/∼ozaki/e_2D.htm). 2D FTIR synchronous and asynchronous plots were constructed for the spectral region between 1600 and 1700 cm-1, corresponding to the amide I′ band. The intensity of a synchronous 2D correlation spectrum represented the simultaneous changes of spectral intensity variations measured at two different frequencies, during an interval of the external variable. The intensity of the asynchronous spectrum represented sequential changes of spectral intensities measured at two frequencies. In our study, temperature was the external perturbation, and the sequence of events along temperature changes was characterized according to the signs of the peaks as explained before.36

Results and Discussion Rhamnolipids constitute one of the most important groups of biosurfactants to date. The presence of one or two rhamnose rings in the polar head, and usually two fatty acid chains in the hydrophobic region, confers to rhamnolipids a marked am(34) Kauppinen, J. K.; Moffatt, D. J.; Mantsch, H. H.; Cameron, D. G. Anal. Chem. 1981, 53, 1454–1457. (35) Cameron, D. G.; Moffatt, D. J. J. Test. EVal. 1984, 12, 78–85. (36) Noda, I.; Dowrey, A. E.; Marcott, C.; Story, G. M.; Ozaki, Y. Appl. Spectrosc. 2000, 54, 236A–248A

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Figure 2. ITC determination of the binding of dirhamnolipid to BSA. Panel A: representative raw heat flow plot obtained upon injection of a dirhamnolipid suspension into a BSA solution at 25 °C. The experiment was carried under the conditions explained in Materials and Methods. Panel B: the heats of injection per mole of injected dirhamnolipid (δhi/ δnDiRL) as a function of the dirhamnolipid/BSA molar ratio in the calorimeter cell, CDiRL0/CBSA0. The data correspond to CBSA0 ) 0.02 mM (O) and CBSA0 ) 0.1 mM (b). Data of a representative experiment are shown. The solid lines are the theoretical fits.

phiphilic character. The aim of this work was to describe the interactions of dirhamnolipids with a well-characterized model protein, such as BSA, to obtain information for possible applications of these biosurfactants in experimental protocols involving proteins. Binding of Dirhamnolipid to BSA. Apart from the indirect determination of the binding constant from the data of the DSC thermograms of BSA/dirhamnolipid mixtures (see below), binding of dirhamnolipid to BSA was quantitatively and directly studied, in a precise way, by constructing the binding isotherms obtained either by isothermal titration calorimetry or by surface tension measurements. Figure 2 shows the raw results of a typical ITC experiment of binding of dirhamnolipid to BSA. Upon injecting aliquots of a 2.5 mM dirhamnolipid solution into a 0.1 mM BSA solution, the heat evolution peaks (Panel A) indicated an exothermic interaction. Once the protein became saturated with the biosurfactant, a series of endothermic peaks was obtained. These peaks were due to the heat of dilution, and allowed determination of the heat of dilution per mole of dirhamnolipid, for the proper correction of the binding isotherms. The integrated data were plotted in panel B, which represented the heat per mole of total rhamnolipid added and, as expected, tended to zero as the ligand (dirhamnolipid) concentration was increased. Experiments were conducted at BSA concentrations of 0.02 and 0.1 mM. When the concentration of BSA was 0.02 mM BSA, all of the dirhamnolipid

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Table 1. Thermodynamic Parameters for the Interaction of Dirhamnolipid and BSA at Two Different BSA Concentrations, As Obtained By ITC at 25 °C a

BSA 0.1 mM BSA 0.02 mMb

n

K (M-1)

∆H (kcal mol-1)

∆S (cal mol-1K-1)

1.3 ( 0.3 1.4 ( 0.06

3.9 × 104 ( 1.2 × 104 2.0 × 105 ( 1.5 × 105

-4.1 ( 0.4 -3.7 ( 0.8

7.4 ( 1.9 8.6 ( 0.1

a The value of the parameters corresponds to the average of 5 independent experiments ( SE. b The value of the parameters corresponds to the average of 3 independent experiments ( SE.

concentrations injected into the cell kept below its CMC of 0.11 mM37 for the whole range used in the experiment, and thus ensured that dirhamnolipid was in the monomer state in all cases. At a BSA concentration of 0.1 mM, the concentrations of dirhamnolipid in the calorimeter cell were mostly above the CMC, therefore allowing the study of binding of aggregated (micellar, or even vesicular) biosurfactant in this case.37 The interaction of both monomer and micellar dirhamnolipid with BSA was exothermic. The experimental calorimetric data were analyzed using the Origin software provided with the equipment. The data were best fitted to a model with one single binding site for both BSA concentrations, and the fitting parameters n (number of dirhamnolipid molecules bound per mole of BSA), ∆H (binding enthalpy) and ∆S (binding entropy) were close in the two cases (Table 1), indicating that aggregation of the protein was not affecting these experiments. A larger variation was observed for K (binding constant), which might indicate a slight influence of the protein concentration on the binding affinity. Regardless, in both cases, ∆S > 0, meaning that the process was entropy driven, as it happened for the binding of other organic ligands to BSA.38 These data showed that BSA became saturated with two molecules of monomer dirhamnolipid bound per molecule of BSA, in an essentially uncooperative manner. It has been reported that various detergents bind BSA in a highly cooperative manner39 what constitutes an essential difference with respect to our dirhamnolipid. Micellar or vesicular dirhamnolipid did not further increase binding to BSA as compared to the monomer compound. This low binding ratio (n ) 2) could be the result of the strong tendency of dirhamnolipid to self-aggregation,37 but it could also be due to the net negative charge that BSA bears at pH 7,40 producing electrostatic repulsions with the negatively charged molecules of dirhamnolipid. Surfactant binding to BSA is mainly driven either by electrostatic or hydrophobic interactions, and the former result suggests that it is more likely that binding of dirhamnolipid is driven by hydrophobic interactions. Binding sites in albumin were initially classified in two principal categories Site I and Site II.41 The general consensus now is that albumin has up to six dominant areas of ligand association, which can allocate different types of amphiphilic molecules.27,29,39 It has been reported24 that BSA can bind up to four molecules of Triton X-100 in each of two binding sites, and tens of smaller anionic surfactants like n-decyl phosphate42 or sodium dodecyl sulfate.43 Furthermore, the ∆H of the interaction of these anionic surfactants was 70 Kcal mol-1 which, when compared to the values in the order of -4 Kcal mol-1 that we have obtained here (Table 1), suggested a different interaction in the case of dirhamnolipid. Despite dirhamnolipid should be negatively charged, since its pKa, which is essentially determined by the free carboxyl group, (37) Sa´nchez, M.; Aranda, F. J.; Espuny, M. J.; Marque´s, A.; Teruel, J. A.; Manresa, A.; Ortiz, A. J. Colloid Interface Sci. 2007, 307, 246–253. (38) Aki, H.; Yamamoto, M. J. Pharm. Pharmacol. 1989, 41, 674–679. (39) De, S.; Girigoswami, A.; Das, S. J. Colloid Interface Sci. 2005, 285, 562–573. (40) Peters, T. AdV. Protein Chem. 1985, 37, 161–245. (41) Sudlow, G.; Birkett, D. J.; Wade, D. N. Mol. Pharmacol. 1975, 11, 824– 832. (42) Jones, M. N.; Skinner, H. A.; Tipping, E. Biochem. J. 1975, 147, 229– 234. (43) Deep, S.; Ahluwalia, J. C. Phys. Chem. Chem. Phys. 2001, 3, 4583–4591.

has been reported to be 5.6,44 it is more likely that the biosurfactant was behaving more like a nonionic surfactant. It is interesting to note that, despite the fact that the amount of dirhamnolipid bound per mole of BSA was low (n ) 2), the affinity constant of 2 × 105 M-1 was similar to that reported for other surfactants,39 and indicated that the binding affinity of dirhamnolipid was strong, both in the monomer and micellar state. Surface tension measurements were also used as an indirect method to study binding of dirhamnolipid to BSA. Experiments were carried out for BSA concentrations between 0.025 and 0.2 mM, and it was carefully checked that the protein did not affect the surface tension of the solutions within this range. Thus, since the only surface-active species is the free biosurfactant, the Szyszkowski equation:

γ ) γ0- RTΓ∞log(1 + ks[S])

(1)

can be used as a mathematical relationship between surface tension and free surfactant concentration.45 In this equation, γ is the surface tension, γ0 is the surface tension of the solution in the absence of all solutes, Γ∞ is the single maximum surface excess concentration, ks is the Langmuir equilibrium adsorption constant, and [S] is the concentration of free surfactant. According to the ITC binding results shown above, dirhamnolipid binding to BSA was well-fitted using the classical Scatchard independent-bindingsite treatment.46 Thus, assuming this model, it can be easily obtained that the free surfactant concentration is the solution to the following quadratic equation:

[S]2 + (n[BSA] - [S]T + K - 1)[S] - K - 1[S]T ) 0 (2) where n is the number of surfactant molecules bound to one BSA molecule, [BSA] is the total BSA concentration, [S] is the free surfactant concentration, [S]T is the total surfactant concentration added, and K is the binding constant for one single site. Thus, a combination of eqs 1 and 2 allows fitting of the experimental values of surface tension as a function of the total surfactant concentration, and the determination of n and K. Figure 3 shows plots of the surface tension as a function of the total dirhamnolipid concentration in the absence and presence of increasing concentrations of BSA. The presence of the protein shifted the plots toward higher dirhamnolipid concentrations in such a way that there was a concentration range for which the surface tension remained essentially unchanged. Thus, within this range, binding of dirhamnolipid monomers to BSA was taking place, and therefore, the concentration of free biosurfactant remained essentially zero, the surface tension being unaffected. Upon increasing dirhamnolipid concentration, the protein became saturated with the biosurfactant, and the surface tension started to decrease as a result of increasing the free concentration of the biosurfactant. As the concentration of BSA was increased, the plots were shifted to higher biosurfactant concentrations, since more dirhamnolipid was required to saturate the protein. A (44) Ishigami, Y.; Gama, Y.; Nagahora, H.; Yamaguchi, H.; Ankara, H.; Kamata, T. Chem. Lett. 1987, 5, 763–766. (45) Bell, C. G.; Breward, C. J. W.; Howell, P. D.; Penfold, J.; Thomas, R. K. Langmuir 2007, 23, 6042–6052. (46) Scatchard, G. Ann. N.Y. Acad. Sci. 1949, 51, 660–672.

Interaction of a Dirhamnolipid Biosurfactant with BSA

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Figure 3. Binding of dirhamnolipid to BSA as determined by surface tension measurements. A plot of the surface tension, γ, as a function of the dirhamnolipid concentration (log scale) for a series of BSA solutions prepared in the buffer mentioned in Figure 2 at 25 °C. (O) Pure dirhamnolipid, (b) BSA 0.025 mM, (0), BSA 0.05 mM, (9) BSA 0.075 mM, (∆) BSA 0.1 mM, (2) BSA 0.15 mM, and (1) BSA 0.2 mM.

Figure 5. Panel A: a typical fitting of a DSC thermogram for the thermal denaturation of BSA in the presence of 0.2 mM dirhamnolipid. The area of each endotherm (O, high T endotherm; b, low T endotherm) was quantified and plotted as a function of dirhamnolipid concentration (Panel B).

Figure 4. DSC thermograms for the thermal denaturation of BSA in the absence and presence of dirhamnolipid. The experiment was carried out in the buffer mentioned in Figure 2. The concentration of BSA was 0.05 mM. Numbers on the curves give the concentration of dirhamnolipid (mM). Scan rate was 30 °C h-1.

qualitatively similar behavior has been shown to occur for SDS/ BSA systems.47,48 The experimental surface tension isotherms were fitted to the model explained above (Figure 3, solid lines). This fitting procedure yielded a value for n larger than 1, and K ) 3.33 × 104 ( 7 × 103 confirming, in very good agreement with the results obtained from ITC experiments, that probably two dirhamnolipid molecules bound to one single site of BSA with high affinity. Similar experiments were performed using denatured BSA (not shown). The plots of denatured BSA were shifted to lower dirhamnolipid values as compared to normal BSA, indicating (47) Ghosh, S.; Banerjee, A. Biomacromolecules 2002, 3, 9–16. (48) Santos, S. F.; Zanette, D.; Fischer, H.; Itri, R. J. Colloid Interface Sci. 2003, 262, 400–408.

lower binding. Furthermore, the dependence on the biosurfactant concentration was essentially the same irrespective of the BSA concentration, and the data could not be fitted to the model used above. Altogether, these results indicated that dirhamnolipid binding to denatured BSA was very low or even totally negligible. This supported the idea that, upon denaturation, BSA released bound dirhamnolipid as discussed above, and it was also in good agreement with the FTIR data shown below. Effect of Dirhamnolipid Binding on the Thermal Denaturation of BSA. Throughout the work, dilute solutions of BSA (