Counterion-Induced Changes to the Micellization of Surfactin-C16

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Counterion-Induced Changes to the Micellization of Surfactin-C16 Aqueous Solution Yi Li, Ai-Hua Zou, Ru-Qiang Ye, and Bo-Zhong Mu* Laboratory for AdVanced Materials and Institute of Applied Chemistry, East China UniVersity of Science and Technology, 130 Meilong Road, Shanghai 200237, P. R. China ReceiVed: July 3, 2009; ReVised Manuscript ReceiVed: October 3, 2009

The effects of counterions on surfactin-C16 micelle solution with its critical micelle concentration (cmc), microenvironment properties in micelles, micelle size distribution, and morphology were investigated by fluorescence, dynamic light-scattering, and freeze-fracture transmission electron microscopy measurements. Counterions enhanced the surface activity of surfactin-C16 and reduced the cmc. With the micellization of surfactin-C16, it adopted a β-sheet conformation, and univalent concentrations reduced micelle micropolarity, increased micelle microviscosity, and tended to cause formation of small and spherical micelles, while divalent counterions had a special effect. With low concentration of divalent cations, they had strong interaction with surfactin-C16 micelles and tended to form larger micelle aggregates. Introduction Lipopeptide is a kind of microbial biosurfactant. About 23 families of lipopeptides have been reported over the last two decades,1 of which 21 were found to be cyclic lipopeptides, including surfactin,2 Lichenysin,3 iturin,4 and fengycin.5 Surfactin, one of the principal representatives of the lipopeptide family, is produced by Bacillus subtilis. Their typical structure is characterized by a cyclic peptide loop bonded to a linear aliphatic acid chain. Surfactin has been reported to be the most effective lipopeptide due to its excellent surface activities.6 It can largely reduce the surface tension of water from 72 to 27 mN/m at the concentration of 1 × 10-5 mol/L.7,8 Besides the surface activities, surfactin has also shown ionophoric and sequestering properties.9,10 More important is that surfactin exhibits significant biological activities, such as antiviral,11,12 antibacterial,13,14 anti-HIV,15 antitumor,16,17 and hemolytic activities.17,18 These physicochemical activities and biological properties are closely related to the mode of molecular assembly of surfactin in micelles and the conformation of the surfactin molecules in the aggregates. The aggregation of surfactin, at interfaces and in solution, was researched by neutron reflectivity measurement and smallangle scattering experiments. It was found that the structure of the micelle is of the core-shell type with the hydrocarbon chain and the four hydrophobic leucines forming the core of the micelle.19 In the case of ionic surfactants, micellization characteristics of surfactin are affected by counterions. Surfactin can interact effectively with erythrocyte membrane by inorganic ions inducing conformational rearrangements probably due to ion-surfactin associations.10 The micellar shape and size of surfactin was discovered to change with the pH values and the addition of ions by electron cryotransmission electron microscopy (TEM).20 Some investigations indicated that Ca2+ had a special interaction with surfactin. Osman et al.21 studied the effects of pH, temperature, and Ca2+ on the microstructure of surfactin micelles by fluorometry. Vass et al.22 found surfactin had the ability of adopting strongly different conformations depending on the conditions, and its carboxyl groups are also * Corresponding author. Tel.: +86-21-64252063. Fax: +86-21-64252458. E-mail: [email protected].

Figure 1. Structure of surfactin-C16.

responsible for Ca2+ binding at low concentration. These research studies are very helpful in the further study of surfactin micelle properties and its biological applications. In this work, the [Asp1, Glu5] surfactin-C16 isoform consists of a heptapeptide head group with the sequence Asp-Leu-LeuVal-Glu-Leu-Leu closed to a lactone ring by a β-hydroxy fatty acid with 16 carbons, which is different in the sequence of peptide chain from those of traditional surfactins reported. The effect of counterions on the [Asp1, Glu5] surfactin-C16 micelle solution was investigated by fluorescence, dynamic lightscattering (DLS), and freeze-fracture transmission electron microscopy (FF-TEM) measurements. Materials and Methods Materials. The [Asp1, Glu5] surfactin-C16 sample was originally separated from the cell-free broth of Bacillus subtilis HSO121.23 Surfactin-C16 was obtained by extraction with methanol and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on a normal pressure ODS C18 column.24,25 The purity is about 90%, calculated as the purity by the area of the opposite corresponding peak of RPHPLC. The peptide part of surfactin was elucidated according to electrospray ionization quadruple-time-of-flight mass spectrometry (ESI Q-TOF MS), and the fatty acid part was analyzed by electroionization gas chromatography/mass spectrometry (EI GC/MS). Its structure is shown in Figure 1. The salts LiCl, KCl, MgCl2, and CaCl2 were AR grade, from Sinopharm Chemical Reagent Co., Led; tris(aminomethane) was from Gene-Tech with purity >99.95%; pyrene was from SigmaAldrich, 99 wt %. Sample Preparation. Tris buffer (0.05 mol/L) was prepared by dissolving the desired amount of tris(aminomethane) in water. The pH was adjusted to 8.5 by the addition of 0.1 mol/L HCl.

10.1021/jp9062862 CCC: $40.75  2009 American Chemical Society Published on Web 10/22/2009

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Figure 2. Effects of counterions on cmc of surfactin-C16 (K is the counterion binding degree).

Surfactin-C16 was dissolved in the Tris buffer solution and prepared as a series of concentrations for fluorescence measurement and accurate concentration (4 × 10-5 mol/L) for DLS and FF-TEM measurement. Fluorescence Spectra Measurement. The cmc value of surfactin-C16 in aqueous solution was determined by fluorometric measurements (Hitachi F-4500 spectrofluorometer, Japan), using pyrene as the probe. The fluorescence emission spectrum of pyrene has five peaks in the range of 350-500 nm for an excitation wavelength of 335 nm. Double distilled water previously saturated by pyrene (about 10-7 mol/L) was used to prepare the surfactin-C16 solutions for fluorescence measurement.21 Dynamic Light Scattering (DLS). DLS measurements were taken in a dynamic light-scattering spectrophotometer (NanoZS, Malvern Instruments Ltd. UK) at a 90° angle in 298 K. Freeze-Fracture Transmission Electron Microscopy (FFTEM). The microstructure of surfactin-C16 micelle samples were observed by FF-TEM. The sample was placed in a specimen holder and maintained at the temperature 77 K by liquid nitrogen. Then the sample was rapidly transferred into the vacuum chamber of the freeze-etching apparatus (BALZERS BAF-400D). Fracturing was achieved by displacing a microtome arm cooled by liquid nitrogen to 100 K, when the pressure was less than 1 × 10-6 Pa. The now-exposed fracture face was immediately shadowed by 2 nm platinum at an angle of 45° and 10-20 nm carbon at a vertical direction. After the specimens were washed in water, the replicas were observed with a transmission electron microscope (JEM-1400, JEOL, Japan) at 20 000 magnification. Results and Discussion Effect of Counterions on the cmc value of Surfactin-C16. The effect of different counterions on surfactin-C16 cmc is presented in Figure 2. The cmc value of surfactin-C16 decreases with the increasing of the counterions concentrations, while the degree of changes of cmc was obviously different with counterions. The logarithm of cmc and Cion has a linear relationship (Figure 2) as shown by the following equation

log cmc ) A - 2/n K log Cion

(1)

where n is the charge of counterion, K is the counterion binding degree, and A is the condition constant.26 The counterion binding degree K can be determined from the slope in Figure 2.

With the univalent cations, the cmc change caused by Li+ is largest. The counterion binding degree of Li+ is 0.068 (Figure 2), which is larger than that of K+. There are two competing tendencies in the formation of micelles of ionic surfactants: removal of hydrocarbon chains from water favors aggregation, and electrostatic repulsions between the ionic head groups oppose aggregation. Counterions stabilize ionic surfactant micelles by binding to the micelles and screening the electrostatic repulsions, and hence the binding affinity of the counterion influences the process of micellization. With the same charge and the smaller ionic radius, Li+ overcame less steric resistance to interaction with surfactin molecule. According to divalent cations, Mg2+ decreased the cmc value of surfactin-C16 more obviously than all univalent cations with counterion binding degree of 0.19. This result indicates that counterions effect on the surfactin micellization is related to their valence. The peptide ring of surfactin adopts a “horsesaddle” structure in aqueous solutions with the two charged residues forming a “claw”, which is a potential binding site for divalent cations. Thus, it has a strong interaction with Mg2+ and binds Mg2+ more tightly than the univalent cation in a low concentration. Effect of Counterions on Micropolarity of Surfactin-C16 Micelle. The fluorescence emission spectrum of pyrene has five peaks in the range of 350-500 nm for an excitation wavelength of 335 nm. Since the intensity of I1 (372 nm) is stronger in polar conditions and the intensity of I3 (383 nm) in nonpolar conditions, the I1/I3 ratio is used as a micropolarity index,27-29 which indicates the micropolarity in the micellar core and the degree of probe solubilized in micelles. The counterions effect on the I1/I3 ratio is shown in Figure 3. The value of I1/I3 changed a little when these univalent cations were in low concentrations and then decreased with the concentrations increasing. It was same as our previous work about the Na+ effect on surfactinC16.30 It means that the micropolarity around the pyrene molecules descended with the addition of enough ions. This result agreed well with that obtained from the effect of univalent cations on cmc values. Since the univalent cations caused decreases in the cmc of surfactin-C16, surfactin-C16 molecules are easier to form micelles at the same concentration. Therefore, more fatty acid chains of surfactin-C16 assembled in a micelle core, and the closer packing associated with tighter binding, which would reduce water penetration into the interfacial region. So, the micropolarity around pyrene molecules decreased, and correspondingly the values of I1/I3 decreased. To the divalent cations, the divalent ions had a lower micropolarity than univalent ions at the same concentration of all the counterions, which means the divalent ions enhance a tighter molecular packing and stronger structure of surfactinC16 micelles. For Mg2+, the I1/I3 value had a little decrease with the increase of Mg2+ concentration, and this change was not as obvious as the univalent ions. However, it was found that the I1/I3 value strongly increased to reach a crest value at the low Ca2+ concentration and was almost unchanged at other concentrations (Figure 3), which was quite different from the results of univalent ions. The Ca2+ effect on the I1/I3 value of surfactinC16 micelle agreed with the studies of Osman et al.,21 which found that the I1/I3 value reached a peak in the presence of 2 × 10-4 mol/L of Ca2+. This result means that the Ca2+ ions had special interaction with the surfactin-C16 even at low concentration compared with the univalent ions and Mg2+. This was in agreement with Thimon et al.’s results that surfactin was nonselective in binding univalent cations but was slightly more effective in binding Ca2+ than Mg2+ or Ba2+.9 With the same

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Figure 3. Effects of counterions on micropolarity of surfactin-C16 micelle (Csurfactin: 4 × 10-5 mol/L).

Figure 4. Effects of counterions on microviscosity of surfactin-C16 micelle (Csurfactin: 4 × 10-5 mol/L).

charges, the ionic radius of Ca2+ was between those of Mg2+ and Ba2+. Maybe the size of Ca2+ ions was fitted to the surfactinC16 structure, and they had the strongest chelation. So at low Ca2+, this great interaction leads to the intercalation of Ca2+ ion in the head group region of the micelles and may prevent the fluorescence probe from solubilizing into the micelles unceasingly. So the I1/I3 value first increased. With more Ca2+ ion addition, there should be reorganization of the surfactinC16 micelle and then decrease of the I1/I3 value. Effect of Counterions on Microviscosity of Surfactin-C16 Micelle. It is known that there will be an excimeric emission at the band around 470 nm when the concentration of pyrene is high enough.31 This means that more pyrene probes are solubilized into the micelles and become associated. The microviscosity of micelles is usually expressed as the ratio of emission fluorescence intensities of excimers (Ie) to those of monomers (Im) of a proper fluorescent probe.21,32 The term microviscosity relates to the spatial restraints, which affect the conformation and molecular assembly of micelles over very short periods of time. The values of microviscosity are of particular importance for the understanding of the micellar phase, especially the solubilizing properties of the micellar core. The values of Ie/Im with different counterions are shown in Figure 4. The ratio Ie/Im of pyrene increases as the univalent ions concentration increases, which indicates that univalent ions were helpful to solubilize more pyrene into the micelles from the bulk solution. Therefore, the microviscosity of the surfactinC16 solution increases with the increase of univalent counterions. The addition of counterions affects the conformation of the head groups in the micelles, consequently leading to a tighter rearrangement of tail groups and affecting microviscosity. This change of microviscosity agreed with our previous work30 about Na+ effect on the surfactin-C16 micelle system.

With divalent cations, the micelle microviscosity increased at low concentrations of ions and fell with high concentrations in both Mg2+ and Ca2+ systems. Ca2+ brought an increase of microviscosity a little higher than Mg2+. This also indicated that divalent cations cause surfactin-C16 micelles to form some dense structures, which is quite different from univalent ions. Effect of Counterions on Morphology of Surfactin-C16 Micelles. In order to know the effects of counterions on size distribution and morphology of surfactin-C16, we performed DLS and FF-TEM measurement. The surfactin-C16 micelle size distribution by relative volume is shown in Figure 5, measured by dynamic light-scattering spectrophotometer Nano-ZS with different counterions. In terms of the relative scattered volume, the distribution of micelle size was bimodal, with one peak at around 100 nm and another peak between 100 and 1000 nm. This result is as similar to Yu-Chun Han’s results,33 which had two peaks in DLS measurements and showed that surfactin had strong self-assembly ability to form micelles and the micelles tend to form larger aggregates. The distribution of two peaks changed with different counterions (Figure 5). Figure 5a is the surfactin-C16 micelle size distribution without counterions, where the peak around 100 nm was 54.6%, and the 600 nm peak was 45.4%. In the Li+ system, the peak around 80 nm was 72.6%, and the other higher peak was 27.4%. But in the presence of Ca2+, the peak at 1000 nm is 76.1%. Therefore, the univalent ions helped surfactin-C16 to form little micelles, and the divalent ions enhance to become large aggregates. FF-TEM is a powerful tool to characterize the actual morphology of colloids in the liquid state. The morphologies of surfactin-C16 micelles with different counterions were observed by FF-TEM, as shown in Figure 6. It was clear that irregular droplet micelles of about 50 nm and some larger micelle reaggregates were observed. In the univalent counterions

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Figure 5. Surfactin-C16 micelle size distribution by volume with different counterions (Csurfactin: 4 × 10-5 mol/L). (a) No counterions, (b) Li+ (0.1 mol/L), (c) K+ (0.1 mol/L), (d) Mg2+ (2 × 10-4 mol/L), and (e) Ca2+ (2 × 10-4 mol/L).

system, surfactin-C16 micelles tended to be uniform similar spherical micellar particles. The micelle was the smallest in the Li+ solution and was more like the spherical particle in K+. When the divalent cations existed, they obviously formed the large sheet aggregates about 200 nm wide and more than 500 nm length. Surfactin is a kind of acidic lipopeptide, with a peptide loop of seven amino acid residues and a β-hydroxy hydrophobic fatty acid chain. The β-sheet is the second common form of regular secondary structure in proteins. With some work reported previously, surfactin formed micelles with β-sheet structure. Ishigami et al.7 found that the surfactin formed large rod micelles with β-sheet structure at low concentration just around the cmc and also showed a possible organization between molecules due to β-sheet structure at the air-water interface under forced experimental conditions. Osman et al.34 studied the effects of extrinsic environmental conditions on the conformation of surfactin to indicate that both surfactin monomers and surfactin micelles had β-sheet conformation at a neutral pH value. Han et al.33 found that surfactin can display different secondary structures at different concentrations. It mainly adopts the β-turn conformation at relatively low micelle concentration, whereas it adopts the β-sheet conformation at higher micelle concentration. In this work, surfactin-C16 concentration was 4 × 10-5

mol/L, much more than the cmc, so the surfactin-C16 micelles and some kinds of large aggregations formed by β-sheet structure. The univalent counterions stabilize ionic surfactant micelles by binding to the micelles and screening the electrostatic repulsions, which favors the micellization of surfactants. Therefore, the addition of univalent counterions decreases the electrostatic repulsions between the surfactin-C16 head groups and enhanced the formation of micelle monomers. So surfactinC16 tended to form regular and smaller micelle aggregations in univalent counterions systems. Previous work indicated that surfactin would have some special properties in the Ca2+ system. Hosono et al.35 observed an increase in the inhibitory activity of the deactivated surfactin on cAMP phosphodiesterase by Ca2+. Osman et al.32 indicated that Ca2+ and other molecules may function as directing templates in the assembly and conformation of surfactin in micelles. Vass et al.22 found surfactin had the ability to adopt strongly different conformations depending on Ca2+ binding at low concentration. In this work, both Ca2+ and Mg2+ induced surfactin-C16 to form large aggregations. The divalent counterions enhance the β-sheet conformation and intensified the interaction of β-sheet between micelles. So surfactin-C16 tended to form some larger micelle aggregations and also cause the

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Figure 6. FF-TEM images of surfactin-C16 micelles, where (a)-(e) correspond to the five samples shown in Figure 5. The images are observed at 20 000 magnification, and the scale bar is 200 nm.

special phenomenon in the microenvironments of micelles in divalent systems. Conclusions In this work we investigated the effect of counterions on surfactin-C16 micelle solution by fluorescence, DLS, and FFTEM measurements. Counterions enhanced the surface activity of surfactin-C16 and reduced its cmc. Surfactin-C16 formed the micelles with β-sheet structure. The addition of univalent concentrations reduced micelle micropolarity, increased the microviscosity, and tended to cause formation of small and spherical micelle particles. Li+ had the largest binding degree and formed the smallest micelles. However, divalent counterions had a special effect. With low concentration of divalent cations, they had strong interaction with surfactin-C16 micelles, enhanced the β-sheet effect, and tended to form larger micelle aggregates. Acknowledgment. We thank Shu-Feng Sun for his help in making FF-TEM samples in the Center for Biological Electron Microscopy, the Institute of Biophysics. Dr. A.-H.Z. gratefully

acknowledges the support of this work by the Research Fund for the New Teacher of the Doctoral Program of Higher Education of China (200802511024). This work is also supported by the National Natural Science Foundation of China (50574040) and by the Department of Science and Technology Shanghai (071607014). References and Notes (1) Liu, X. Y.; Yang, S. Z.; Mu, B. Z. Biotechnol. Bull. 2005, 4, 18– 26. (2) Arima, K.; Kakinuma, A.; Tamura, G. Biochem. Biophys. Res. Commun. 1968, 31, 488–494. (3) Yakimov, M. M.; Abraham, W. R.; Meyer, H.; Giuliano, L.; Golyshin, P. N. Biochim. Biophys. Acta 1999, 1438, 273–280. (4) Peypoux, F.; Guinand, M.; Michel, G.; Delcambe, L.; Das, B. C.; Lederer, E. Biochemistry 1978, 17, 3992–3996. (5) Vanittanakom, N.; Loeffler, W.; Koch, U.; Jung, G. J. Antibiot. 1986, 30, 888–901. (6) Deleu, M.; Razafindralambo, H.; Popineau, Y.; Jacques, P.; Thonart, P.; Paquot, M. Colloids Surf., A 1999, 152, 3–10. (7) Ishigami, Y.; Osman, M.; Nakahara, H.; Sano, Y.; Ishiguro, R.; Matsumoto, M. Colloids Surf., B 1995, 4, 341–348.

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