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J. Phys. Chem. B 1999, 103, 6680-6689
Sugar Surfactant-Based Solutions as Host Systems for Enzyme Activity Measurements Anna Stradner,† Birgit Mayer,‡ Thomas Sottmann,§ Albin Hermetter,‡ and Otto Glatter*,† Institut fu¨ r Physikalische Chemie, UniVersita¨ t Graz, A-8010 Graz, Austria, Institut fu¨ r Biochemie und Lebensmittelchemie, Technische UniVersita¨ t Graz, A-8010 Graz, Austria, and Institut fu¨ r Physikalische Chemie I, UniVersita¨ t zu Ko¨ ln, D-50939 Ko¨ ln, Germany ReceiVed: February 11, 1999; In Final Form: June 4, 1999
We used fluorogenic substrates, namely, alkyldiacyl glycerols labeled with a fluorophore and a fluorescence quencher, to measure lipase activities. For this optical lipase assay it is necessary that the water-insoluble fluorogenic substrates are dispersed in an aqueous medium in the presence of appropriate detergents in order to obtain well-defined and reproducible systems with high optical transparency. The activity of lipases critically depends on the supramolecular organization of the substrate. Therefore, we tested different solutions containing nonionic surfactants and organic phases for their potential use as host systems for fluorogenic lipids. We then determined the pseudoternary phase diagram for the system buffer-alkyl polyglucoside-hexanol, which showed the highest apparent enzyme activities, in the buffer corner. We performed structural investigations on micellar sugar surfactant solutions within the L1 phase with and without hexanol using small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS) in order to investigate a possible correlation between lipase activity and the structure of the aggregates present in the host system.
Introduction Lipases are by definition enzymes, hydrolyzing long-chain acyl esters. Most of them are activated by the presence of lipidwater interfaces. In nature, lipases are required for the hydrolysis of exogenous triglycerides serving as a nutrient or for the mobilization of endogenous lipid stores. Working with lipases requires reliable and sensitive assays to measure enzyme activity. Availability of such lipase assays is essential not only for research but also for clinical chemistry for measuring lipase or phospholipase activities in body fluids in the biochemical diagnosis of various deseases.1 It is possible to use new fluorogenic substrates for the rapid determination of activities of lipolytic enzymes in biotechnology and medicine.1 The respective compounds are acyl and alkyl substituted glyceroand phosphoglycerolipids2,3 carrying an appropriate fluorophore and a quencher covalently bound to the hydrophobic acyl chains of the glycerolipid.1,2,4,5 The intact substrate shows only very low fluorescence due to intramolecular resonance energy transfer between fluorophore and quencher. Hydrolysis of the fluorogenic lipids by lipases releases the quencher fatty acid from the glycerol backbone. The fluorescence of the fluorophor is dequenched, and the emission intensity increases. From this time-dependent increase in fluorescence intensity the enzyme activity can be determined. The measurement of lipase activity using fluorescence techniques is a continuous optical experiment taking only a few minutes. For the enzyme-catalyzed reaction it is necessary that the fluorescent substrates are appropriately dispersed in an aqueous medium, leading to reproducible formation of supramolecular lipid structures. Neutral lipids such as triacyl glycerols have to be “solubilized“ in the presence of suitable detergents or proteins1 in order * To whom correspondence should be addressed. † Universita ¨ t Graz. ‡ Technische Universita ¨ t Graz. § Universita ¨ t zu Ko¨ln.
to obtain solutions that exhibit sufficiently high optical transparency and homogeneity. Depending on the detergent, different aggregates are obtained. The apparent enzyme activities depend to a large extent on the chemical composition of the “lipid“ aggregates and its supramolecular organization.4 The latter is in turn characterized by size, curvature, symmetry, and lamellarity of the aggregates. To understand the behavior of lipases in different detergent systems, many studies of surfactant-lipase interactions have been performed.6,7 Enzymes are sensitive to many kinds of surfactants. Cationic surfactants may form aggregates with the enzyme,8 anionic surfactants are known to promote denaturation of the protein, and unbranched nonionic alkyl polyglycol ethers, CiEj, may interfere with vital domains of the enzyme.9,10 Much work has been done on lipase-catalyzed reactions in water in oil (w/o) microemulsions or reverse micelles with varying water content and different surfactants and organic phases.9-15 In addition to lipase-catalyzed hydrolysis the w/o microemulsions can also be used for esterification16-19 and transesterification reactions.19 It was found that in general enzyme stability and activity in w/o microemulsions are better with very hydrophobic solvents. The rationale is that very hydrophobic solvents do not distort the essential water layer around the enzyme.13 The solvents of choice in w/o microemulsion systems are heptane, octane, isooctane, and nonane. If one works in an aqueous system using a water-insoluble fluorescent substrate, an efficient, well-defined, and tunable solubilization (i.e., micelles or microemulsions) is required. At the moment of the addition of the lipase, the measurement starts, and therefore, it is important to have an aqueous system ensuring a fast diffusion of the protein to the “lipid“ aggregates containing the fluorescent substrate. So, in contrast to the reversed micellar systems mentioned above in this study, we concentrate on systems with a continuous water phase as the lipase is dispersed in an aqueous phase (body fluids, etc.). The aim of the present
10.1021/jp9905171 CCC: $18.00 © 1999 American Chemical Society Published on Web 07/23/1999
Sugar Surfactant-Based Solutions study was to find an alternative, adjustable, well defined, and reproducible system serving as a host system for lipase assays and to find a correlation between the microstructure of such systems and lipase activity. Since the nonionic alkyl monoglucoside octyl-β-D-glucopyranoside is known as a mild, nondenaturating solubilizer and is used in the extraction of membrane proteins, we tried to establish a sugar surfactant-based reaction medium for the lipase test using the much cheaper commercial alkyl polyglucoside (APG) Glucopon (trade name of Henkel KGaA, Du¨sseldorf).21 APGs are nonionic surfactants exclusively produced from renewable raw materials such as fatty alcohols from vegetable oil and sugars, for example, glucose from starch.21 They are nontoxic and readily biodegradable. In industrial processes mixtures of alkyl mono-, di-, tri-, and oligoglucosides are produced. Therefore, these industrial products are called alkyl polyglucosides, CiGj, and are characterized by the length of the alkyl chain i and the average number of glucose units j linked to it, the degree of polymerization.21 The critical micelle concentrations (cmc) are similar to those of other nonionic surfactants. They show a decrease with increasing alkyl chain length, whereas the degree of polymerization has a minor influence.22 The phase diagrams of APG-H2O mixtures using commercial products have been studied22-24 and compared with those of CiEj-H2O mixtures.25 In some cases, the properties of APGs differ clearly from those of the other big group of nonionic surfactants, namely, alkyl polyglycol ethers. For example, in aqueous solutions the dependence of the lower critical temperature Tβ of the upper loop on i and j is much stronger than with CiEj.25-27 The general effect of temperature on the solution behavior is smaller than for the CiEj surfactants. Their physicochemical properties are not markedly affected by electrolytes. It has been shown that it is possible to form microemulsions by using alkyl glucosides and APGs, in which a phase inversion can be induced by adding a suitable cosurfactant or cosolvent, such as alkanol, alkanediol, alkyl glycerol ether, or alkyl polyglycol ether.23,26,28-30 The reason for the need of cosolvents or cosurfactants is the hydrophilic nature of the glucose headgroup, which makes sugar surfactants extremely insoluble in many oils. The advantage of sugar surfactant-based over CiEj -based microemulsions lies in the fact that the stability of onephase microemulsions is less sensitive to temperature and salt.26 The insensitivity of CiGj systems to temperature arises from the strength of the hydrogen bonds between the hydroxy groups of the glucose unit and the water molecules, whereas the hydrate shell of the ethoxylate headgroup is very sensitive to temperature. In this work we study aqueous solutions of Glucopon, C8/10G1.5 (cmc ) 0.05 wt %),24 with small amounts of added hexanol. Hexanol acts as a “cosurfactant“ that dissolves mainly in the interfacial layer and changes its properties, such as curvature and rigidity.26 We established the ternary phase diagram in the water (buffer) rich corner. The micellar solutions within the L1 phase served as host systems for the lipase test, and we measured lipase activities on fluorescent substrates with respect to different hexanol content of the system. We have performed structural investigations on these sugar surfactant solutions with and without added hexanol using scattering methods, namely, small-angle neutron scattering (SANS), smallangle X-ray scattering (SAXS), and dynamic light scattering (DLS), to understand the correlation between lipase activity and the structure of the aggregates within the host system. We also checked lipase activities in these systems with additional small amounts of n-octane, which causes swelling of the micelles. In
J. Phys. Chem. B, Vol. 103, No. 32, 1999 6681 TABLE 1: Nomenclature and Composition of Buffer-APG-Hexanol-n-Octane Samplesa R [wt % γ [wt % wt % C6E0 per δ [wt % APG] hexanol] wt % APG n-octane]
sample A B C Boc Coc
pure APG low hexanol high hexanol low hexanol, with n-octane high hexanol, with n-octane
5 5 5 5
0.57 0.80 0.57
0.114 0.16 0.114
0.43
5
0.80
0.16
0.60
a R is the mass fraction in wt % of APG in the mixture of water (buffer) and APG, γ is that of hexanol in the water (buffer)-APGhexanol mixture, and δ is that of n-octane in the mixture of all four components, respectively.
addition, we performed activity measurements in an oil in water microemulsion (buffer-decane-C12E5) to compare them with the results in the sugar surfactant-based micellar systems. Materials and Methods Materials and Preparation. The technical grade alkyl polyglucoside C8/10G1.5 (Glucopon 215 CSUP) was kindly provided by Henkel, Germany. It was prepared by Fischer glycosidation31 with technical grade fatty alcohols. This commercial alkyl polyglucoside (APG) represents a mixture of Rand β-glucosides21 and is supplied as a 62-65 wt % solution in water with high pH value to avoid microbial attack. The surfactant was used without further purification. Pentaoxyethylene glycol mono-n-dodecyl ether (C12E5), 1-hexanol (purity, >99%), decane (purity, >98%), and n-octane (purity, 99%) were purchased from Fluka (Buchs, Switzerland) and used without further purification. D2O with isotopic purity of >99.75% was obtained from Merck (Darmstadt, Germany). The water used in this study was twice-distilled. The fluorogenic alkyldiacylglycerol, 1-trinitrophenylaminohexanoyl-2-pyrenebutanoyl-3-Ooctyl-sn-glycerol, was prepared from the corresponding 3-Oalkyl-1-O-triphenylmethyl-sn-glycerol as described.1 Crude Rhizomucor miehei lipase (RML) was obtained from Novo Nordisk (Denmark) and Pseudomonas species lipase (PSL) was from Nagase Biochemicals Ltd. (Japan). For lipase activity measurements, dynamic light scattering, and phase diagram determination, buffer (0.1 M Tris-HCl in H2O, pH 7.4) was used as solvent, while the surfactant solutions for SANS and SAXS experiments were made using D2O as solvent. An APG solution was prepared by weighing the surfactant into a glass bottle and diluting it to the desired concentration with D2O or buffer, respectively, by taking into account that the APG is a 62-65 wt % solution in water. Next, hexanol was weighed into glass bottles and diluted to the appropriate hexanol weight percent with the APG solution. Thus, hexanol concentration is on a total solution weight basis, whereas surfactant concentration is on a hexanol free basis (see Table 1). SANS and SAXS experiments were performed for the 5 wt % APG solution without added hexanol (sample A, pure APG) and with 0.57 and 0.80 wt % hexanol (sample B, low hexanol; sample C, high hexanol) at 22 °C. These three solutions are located in the L1 phase of the ternary phase diagram (see Figure 1). Small-angle scattering experiments were also performed on the hexanol-containing samples with additional concentrations of n-octane. Lipase activity measurements were performed in six different surfactant systems, namely, in the 5 wt % APG solutions without, with low and with high hexanol content, and with and without additional n-octane and in the microemulsion (2 wt % C12E5 and 3 wt % decane in Tris buffer). A solution of fluorogenic lipid [0.7 mM] in ethanol was diluted with a 350-
6682 J. Phys. Chem. B, Vol. 103, No. 32, 1999
Figure 1. Pseudoternary phase diagram of the buffer-C8/10G1.5-C6E0 system.
fold excess of APG-solutions with different amounts of hexanol, n-octane, and the one-phase microemulsion to a final concentration of 2 µM. The obtained samples were stirred overnight at room temperature for equilibration before use. Methods. Phase Diagram Determination. To determine the phase diagram, various amounts of buffer (0.1 M Tris buffer, pH ) 7.4) and C8/10G1.5 (Glucopon 215 CSUP) were weighed into sample tubes and sealed with polyethylene stoppers. Subsequently, the samples were placed in a transparent water bath thermostated at 30.0 ( 0.02 °C. After temperature equilibration the stopper was removed, and the pseudo-twocomponent mixture was titrated with 1-hexanol (denoted as C6E0). The phase transitions were determined by visual inspection of the scattered and transmitted light as well as between crossed polarizers, to determine whether anisotropic phases were present. For identification of the individual two-phase regions, phase separation was awaited. Fluorescence Assay.4 Enzyme activities are simply determined from the time-dependent increase in fluorescence intensity, which is representative for substrate hydrolysis. The activity observed with our substrate is dependent on the enzyme used and the form of substrate solubilization. To solubilize the lipid substrate, well-defined amphiphilic aggregates with sufficiently high optical transparency and homogeneity are essential. Enzyme solutions (2.5-20 µL) were added to 2 mL of the substrate suspension. Rates of lipolysis intensity were determined from the increase in fluorescence intensity at 378 nm (excitation, 342 nm; slit widths, 3 nm each) on a Shimadzu spectrofluorophotometer RF-540 for several minutes. All activity measurements were performed at 30 °C with stirring. Small-Angle Scattering (SAS). SAS, in particular, small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS) probe the pertinent colloidal length scales of 1-100 nm and therefore are the methods of choice for determining size, shape, and internal structure of colloidal particles. In addition, because the two kinds of scattered radiation are sensitive to different physical properties of the scattering particle, these techniques can be used in parallel to obtain a rich variety of information.32 The intensity of neutrons scattered from an aggregate depends on the difference of the neutron scattering length density between the particles and the solvent. In comparison, the scattered intensity of X-rays depends on the difference of the electron density between the particles and the solvent.
Stradner et al. Small-Angle Neutron Scattering (SANS). SANS measurements have been performed using the D22 instrument of the Institut Laue Langevin (ILL), France.33 The range of scattering vectors 3.8 × 10-2 nm-1 e q e 4.8 nm-1 was covered by two different sample-to-detector distances (d ) 2 and 14 m). The length of the scattering vector q is given by q ) (4π/λ) sin(θ/2) where λ is the wavelength and θ is the scattering angle. The neutron wavelength was 0.6 nm for all experiments. The wavelength resolution was 10% (full width at half-maximum value). All experiments were done with a 38 cm detector offset. The samples were kept in stoppered quartz cells (Hellma, Germany) with a path length of 1 mm. All samples were measured at a temperature of 22 °C. The raw spectra were corrected for background from the solvent, sample cell, and electronic noise by conventional procedures. The two-dimensional scattering spectra were azimuthally averaged and corrected for detector efficiency by dividing by the incoherent scattering spectra of pure water. Data were not placed on an absolute scale. Small-Angle X-ray Scattering (SAXS). SAXS experiments were performed using an X-ray generator (Philips, PW 1730/ 10) operating at 45 kV and 45 mA with a sealed-tube Cu anode (KR wavelength of 0.154 nm). A Kratky compact camera equipped with a thermostated sample holder and a positionsensitive detector (MBraun, PSD-50M) with a metal wire was used. The resolution mode of the position-sensitive detector was 1K for all measurements with an effective total number of about 400 channels at a sample-to-detector distance of 217 mm (each channel corresponding to a spacing of ∼64 µm). All experiments were performed at 22 ( 0.5 °C. No monochromator was used, so the radiation contained KR and about 25% Kβ radiation. The Kratky camera was operated with an entrance slit of 40 µm. Sample and solvent were measured for 5 × 15 000 s, leading to a total of about 108 counts for every sample. Raw data were corrected for detector efficiency variations. Data were not placed on an absolute scale. Data Analysis. In principle, the analysis of SAS data can be performed in two steps. The extraction of the information contained in, and obtainable from, scattering curves are limited by experimental resolution, finite q-range, structural complexity, etc. The first step is a model-independent procedure. First, the background B, which is calculated from the slope of a Porod plot (Iq4 versus q4), is subtracted from the total scattered intensity. The resulting scattering curves are desmeared (correction of instrumental broadening from slit smearing in the Kratky camera and wavelength smearing for both X-rays and neutrons) and Fourier transformed. The Fourier transformation yields the pair distance distribution function p(r), which is a histogram of distances inside the particle weighted with the product of the scattering length density differences at the end points. The shape of the p(r) allows determination of the basic geometry (spherical, cylindrical, or planar) even for inhomogeneous particles. This methodology using indirect Fourier transformation (IFT) has been described in detail elsewhere.34-37 The second step is to compare the scattering data to calculated scattering curves of model structures.38 Dynamic Light Scattering. DLS measures the relaxation time of any fluctuation in the refractive index occurring within the length scale defined by the scattering vector q. We can distinguish between two different regimes, qRg , 1 and qRg . 1 with Rg being the radius of gyration. For qRg , 1, the fluctuation arises from the random diffusion of the particle. The characteristic relaxation time of this fluctuation is therefore proportional to the diffusion coefficient. Thus, for small particles in dilute solution the apparent diffusion coefficient Dapp is related
Sugar Surfactant-Based Solutions through the Stokes-Einstein equation Dapp ) kT/(6πηRH,app) to the apparent hydrodynamic radius RH,app, the size of an equivalent compact sphere, where k is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent. For qRg . 1, which can be found, for example, in the case of giant, polymer-like micelles, we are measuring relaxation times on the spatial length scale of q-1. For dilute solutions these relaxation times correspond to the internal modes of the polymer-like micelle and are independent of the overall coil size. In this case Dapp does not correspond any longer to the size of the particle and becomes strongly q-dependent. Also, for entangled wormlike micelles Dapp is no longer a measure of the micellar diffusion coefficient but reports on the dynamics of the transient network. Generally, in this case RH,app is then replaced by a hydrodynamic correlation length ξH. DLS measurements were carried out at a scattering angle of 90° and at a temperature of 22 °C. In addition we made a crosscheck measurement at 30 °C, the temperature for the activity tests, to be sure that the temperature dependence in this region can be neglected. The laboratory-built goniometer is equipped with an argon ion laser (Spectra Physics, model 2060-55, λ ) 514.5 nm) and an ALV-5000 correlator (ALV, Germany). The intensity autocorrelation functions were analyzed using a secondorder cumulant analysis39 yielding an apparent diffusion coefficient Dapp. From Dapp the hydrodynamic correlation length ξH was calculated using ξH ) kBT/(6πηDapp). Results and Discussion Phase Diagram. In a binary H2O-C8/10G1.5 system for temperatures above 20 °C an isotropic micellar phase (denoted as L1) up to a mass fraction of C8/10G1.5 equal to 0.95 is present. Above that fraction lyotropic phases occur.24,40 When H2O is replaced by buffer and C6E0 is added to the pseudobinary buffer-C8/10G1.5 system, a series of phases are observable in the water-rich corner. The phase behavior of this system was represented in a Gibbs triangle at T ) 30.0 °C (see Figure 1). By addition of C6E0, a transition into a two-phase region (denoted as L1′ + L1′′) is observable. At somewhat higher C6E0 concentrations LR (lamellar) and L3 (sponge) phases were observed. Apparently, the presence of medium- or long-chain alcohols induces the formation of the dilute bilayer phases LR and L3. This effect was observed first by Penders and Strey in the H2O-pentaethylene glycol mono-octyl ether (C8E5)-1octanol system, which does not show these phases in the absence of 1-octanol.41 In the pseudoternary buffer-C8/10G1.5-C6E0 system the LR and L3 phases extend far toward the buffer corner and are even observed for surfactant mass fractions of less than 0.02. As can be seen from Figure 1, the ratio of C6E0/C8/10G1.5 for which the L3 phase exists is nearly 0.375/0.625. The lamellar phase is identified by its characteristic anisotropy. The L3 phase is optically clear, increasingly opalescent upon dilution, and streaming birefringent. As customary in the literature, the 1-hexanol-rich phase was denoted as L2. In Figure 1 some of the two- and all three-phase regions are not drawn because of their small extension and for the sake of simplicity. The complex phase behavior of ternary mixtures of H2O-nonionic surfactant-alcohol is presented schematically in Figure 5 of ref 41. The formation of the LR and L3 phases at sufficiently high C6E0 concentrations can be explained by assuming that the C6E0 molecules partition favorably into the amphiphilic film and decrease the curvature of the two monolayers. At zero-curvature of the monolayers, a planar bilayer, i.e., the lamellar phase, should form. At the level of the midsurface the mean and Gaussian curvature are zero. By addition of more C6E0, the
J. Phys. Chem. B, Vol. 103, No. 32, 1999 6683 transition from the LR to the L3 is observed by passing the twophase coexistence of L3 and LR. This transition can be understood as a further decrease of curvature of the monolayers comprising the bilayer. Anderson et al.42 have argued that for the L3 phase the monolayers may satisfy their spontaneous curvature in a saddle-shaped bilayer so that the midsurface has a zero mean but in contrast to the LR phase a negative Gaussian curvature. This saddle-shaped structured L3 phase has previously been suggested43,44 and can be visualized by freeze fracture electron microscopy.45 Fluorescence Assay. We used fluorogenic 1,2-diacyl-3-Oalkyl-sn-glycerols for the determination of lipase activity. In water, lipase substrates are usually solubilized by additives such as proteins and/or detergents.4 Here, we tested two different nonionic surfactants, a commercial alkyl polyglucoside (APG) and an alkyl polyglycol ether (C12E5) containing different amounts of an organic phase (hexanol, n-octane, and decane). Activity of a microbial lipase (Pseudomonas species, PSL) and a crude fungus lipase (Rhizomucor miehei lipase, RML) were determined using the fluorescent lipids in the emulsions as substrates. Activities of the two lipases were analyzed in four different solubilization systems [5 wt % APG solution (A), 5 wt % APG solution with 0.57 wt % hexanol (B), 5 wt % APG solution with 0.80 wt % hexanol (C), and 2 wt % C12E5 solution with 3 wt % decane (D)]. The highest activities were measured for both enzymes in the system with low hexanol (B), whereas enzyme activity in the system containing more hexanol (C) and in the pure APG solution (A) significantly decreased (see Figure 2a). For both lipases, PSL and RML, no significant activity could be observed in the 2 wt % C12E5 solution with 3 wt % decane (D). Furthermore, we investigated the influence of various hexanol concentrations in the surfactant system on lipase activity. Figure 2b shows an increase of PSL activity at concentrations up to 0.9 wt % hexanol in the substratesurfactant solution. Between 0.4 and 0.7 wt % hexanol, lipase activities are constantly high and reach a plateau. Addition of more than 0.7 wt % hexanol to the substrate-surfactant system causes a decrease of enzyme activity. Lipase activities in the systems with low and high hexanol content (B and C) were compared to those in the same systems containing additional small amounts of n-octane (Boc and Coc). In Figure 2c one can see that in the case of PSL the activity drastically decreases on the addition of the oil. First tests with RML indicate that the influence of the addition of n-octane is less pronounced. However, additional experiments are needed to see if these differences for different enzymes are significant. Scattering Results. (1) Influence of Hexanol on OVerall and Local Structure: General Features. SANS. The q dependence of the scattered intensity measured with SANS is shown in Figure 3a for three micellar samples with different hexanol content. At high q values, where we obtain structural information on relatively short length scales, the data overlap within experimental resolution for all hexanol concentrations investigated. This indicates that the addition of hexanol results in negligible changes of the local structure, which is not that surprising in view of the small hexanol content and the correspondingly small volume ratio hexanol/surfactant. However, at low values of q the situation changes, and we observe a strong increase of the scattering intensity upon the addition of a small amount of hexanol. This indicates that the average molar mass and overall size of the micelles considerably increases. SAXS. The q dependence of the scattered intensity obtained from SAXS experiments for the same surfactant concentration
6684 J. Phys. Chem. B, Vol. 103, No. 32, 1999
Stradner et al.
Figure 3. SANS and SAXS intensity spectra after background subtraction for the sugar surfactant solutions without and with low and high hexanol content (A, 5 wt % APG, solid line; B, 5 wt % APG with 0.57 wt % hexanol, dashed line; C, 5 wt % APG with 0.80 wt % hexanol, dotted line). (a) SANS results. At high q values the data of all three samples overlap, indicating that the local structure of the aggregates is hardly affected by the hexanol content. At low q values the intensity increases upon addition of hexanol, indicating an increase of the average molar mass and overall size of the micelles. A slope of q-1 is indicated to highlight the region characteristic for scattering from rodlike micelles, which can be found in the sample with low hexanol content (B). (b) SAXS results. The nonmonotonic q dependence at high q values and the low intensity at small q values arise from the inhomogeneity and the very low overall contrast of the surfactant for X-rays. Figure 2. (a) Lipase activities (PSL [1.75 µg/mL] and RML [20 µg/ mL]) determined in different surfactant systems (A, 5 wt % APG; B, 5 wt % APG with 0.57 wt % hexanol; C, 5 wt % APG with 0.80 wt % hexanol; D, 2 wt % C12E5 with 3 wt % decane). (b) PSL activity [1.75 µg/mL] dependent on various concentrations of hexanol in the substrate-surfactant solution. (c) Comparison of PSL activity [1.75 µg/mL] in sugar surfactant-based solutions with and without n-octane (B, 5 wt % APG with 0.57 wt % hexanol; C, 5 wt % APG with 0.80 wt % hexanol; Boc, B + 0.43 wt % n-octane; Coc, C + 0.6 wt % n-octane).
and hexanol content are shown in Figure 3b. The q range does not extend to very low values, and the SAXS measurements therefore do not allow us to determine structural properties on large length scales. When compared to the SANS results, the SAXS data exhibit a qualitatively different behavior, with low values for I(q) at low q and a nonmonotonic q dependence at high values of q. The major difference between the SAXS and SANS data is due to the different overall contrast of the surfactant for neutrons and X-rays. For SAXS, the surfactant has a very low overall contrast, which results in a vanishing intensity at low values of
q (I(qf0) ≈ 0), and the data become insensitive to the overall mass and size of the aggregates. Therefore, SAXS primarily yields information on the local micellar structure. The situation is quite different for SANS, where the surfactant has a high overall contrast against D2O, and combined with the large q range, the experiment is thus able to provide structural information on larger length scales. A qualitative inspection of the small-angle data indicates that small additions of hexanol cause a big increase of the micellar size, with negligible changes of the local arrangement. In the next step we can now obtain more quantitative information on the composition dependence of the micellar structure by performing a model-independent indirect Fourier transformation (IFT) of the scattering data. (2) Indirect Fourier Transformation (IFT). The IFT yields the p(r) function, which provides important real space information on the aggregate such as the maximum dimension and the geometry of the particle. OVerall Size and Shape. SANS. The p(r) functions obtained through IFT of the scattering data shown in Figure 4a indicate
Sugar Surfactant-Based Solutions
Figure 4. Pair distance distribution functions p(r) obtained from the IFT of the scattered intensities in Figure 3 (A, 5 wt % APG, solid line; B, 5 wt % APG with 0.57 wt % hexanol, dashed line; C, 5 wt % APG with 0.80 wt % hexanol, dotted line): (a) SANS data; (b) SAXS data.
a hexanol-induced evolution of the micellar size and shape from globular particles at [hex] ) 0 with a maximum dimension of approximately Dmax ≈ 8 nm to cylindrical micelles with Dmax ≈ 30-35 nm at [hex] ) 0.57 wt %. The constant cross section and the axial length of these cylinders are responsible for the linear region of p(r) for large r. The slope of the linear decay is directly related to the square of the average contrast between the particle and the solvent. At [hex] ) 0.80 wt %, the micelles are even larger, but the resulting p(r) is very different from the function expected for cylindrical aggregates. However, at short distances all p(r) functions overlap, which again indicates that the local micellar structure is very little influenced by the hexanol content. OVerall Size and Shape. SAXS. The p(r) functions (see Figure 4b) exhibit a qualitatively different behavior at low values of r when compared to those obtained from the SANS data. This is primarily due to the core-shell contrast of the micelle for X-rays, which leads to the observed oscillations in p(r) in the region 0 < r < Dmax,c with Dmax,c being the maximum dimension of the cross section of the aggregate. At higher values of r we see the same qualitative trend with increasing hexanol content as for SANS; however, the overall contrast for X-rays is close to zero. This fact, together with the too large minimum q value, makes a reliable determination of Dmax for the samples with higher hexanol content impossible. Local Structure of the Samples with Added Hexanol (B and C). Samples with hexanol (B and C) having almost identical local structure, combined with the fact that the solution with the low hexanol content (B) shows the clear evidence of
J. Phys. Chem. B, Vol. 103, No. 32, 1999 6685
Figure 5. Cross-sectional pair distance distribution functions pc(r) for the samples with added hexanol (sample B, dashed line; sample C, dotted line). (a) From SANS data the maximum cross section of the micellar core can be obtained, while SAXS data (b) provide information on the dimension of the micellar cross section including the headgroups.
cylindrical shape, allows a special transformation to be made, yielding the cross-sectional pair distance distribution function pc(r). This pc(r) is a weighted histogram of distances inside the particle cross section. This has, for example, also been used to investigate the local structure of polymer-like micelles.46,47 The r value for which pc(r) ) 0 provides an estimate of the maximum cross-sectional dimension of the aggregate. The pc(r) functions for the solutions with low and high hexanol content (B and C) obtained from SANS data (Figure 5a) indicate a nearly homogeneous cross section with a maximum dimension Dmax,c ≈ 3-3.5 nm. The pc(r) determined from the SAXS data shows very pronounced oscillations and a larger maximum distance of Dmax,c ≈ 4.5-5 nm (see Figure 5b). The oscillations are due to the core-shell contrast with a very low overall contrast caused by the different electron densities of the surfactant tail and headgroup, respectively. The second zero crossings of the pc(r) functions (about 2.5 nm) approximately correspond to the diameter of the micellar core. The different values of Dmax,c obtained from SANS and SAXS are due to the fact that for SANS the headgroups are almost matched because of the exchange of the hydrogens from the sugar OH groups with deuterium. Therefore, in the SANS experiment it is only the hydrocarbon chains that are fully visible, and we observe mostly the micellar core. The thus obtained values for the core diameter and overall diameter of the micellar cross section are in reasonable agreement with an estimate based on the known hydrocarbon chain length and the expected dimension of the sugar headgroup (according to Tanford,48 the maximum length
6686 J. Phys. Chem. B, Vol. 103, No. 32, 1999 of a saturated hydrocarbon chain with 10 carbon atoms can be assumed to be about 1.4 nm). In our IFT algorithm the solution is not forced to zero at r ) 0 by a mathematical constraint. The pc(r) functions for r ) 0 quite often show a nonzero value due to the limited resolution of the data. Deconvoluting pc(r) produces the contrast profile normal to the interface.32,35 This was possible for the pc(r) function obtained from the SANS data of the sample with high hexanol content (sample C) using, for example, a two-step model (results not shown). For the SAXS data the same procedure did not work. A possible explanation for the failure of deconvolution would be either the limited data quality caused by low contrast or a slightly elliptical cross section.49 Because of the different signs in electron density differences of the core and the shell of the micelles, SAXS data would be very sensitive to a possible asymmetry of the cross section. However, the quality of the current data is not high enough (low intensity at high q range) to distinguish between these two different possibilities. We thus obtain complementary structural information from the SANS and SAXS data, which allow us to interpret the structural changes induced by the addition of hexanol. The data suggest that the addition of 0.57 wt % hexanol results in a change from small globular to large cylindrical micelles. This is also supported by a direct inspection of the scattered intensity measured with SANS (Figure 3a), which indicates the typical 1/q dependence of I(q) expected for cylindrical particles with an overall length much larger than the cross-section diameter. At lower values of q we enter the Guinier regime from which one could obtain the radius of gyration Rg and the mass of the micelles. At higher values of q we see deviations from the 1/q dependence due to the influence of the cross-section form factor of the micelles. In the SAXS data this 1/q behavior is not visible because of the low overall contrast (see Figure 3b). However, the situation changes again for the sample with 0.80 wt % hexanol (C), where the SANS data at low values of q indicate an even larger molar mass of the micelles and much larger overall dimensions, although the minimum q measured is too large to observe a clear Guinier regime. Therefore, we are not able to determine the radius of gyration as a measure of the overall size of the micelles under these conditions. The deviations from the q dependence typical for cylindrical particles is not unexpected in view of previously published SANS data for large micelles with cylindrical cross section. In most of these studies the micelles had a high degree of flexibility, which resulted in polymer-like structures with a correspondingly different pattern for the q dependence of I(q).50 During the past years considerable progress has been made in the interpretation of scattering data of giant wormlike micelles.50,51 From the analogy to polymers and on the basis of an extensive Monte Carlo simulation study it has been possible to incorporate micellar growth, interactions, and flexibility, which is given in terms of either the persistence length lp or Kuhn length b, where b ) 2lp, in the interpretation of scattering data from wormlike micelles. Numerical expressions for the scattering functions and structure factor of wormlike micelles have been presented, which allow for the determination of the persistence length, contour length L, and cross section of wormlike micelles. An analysis of the scattering data from the sample with high hexanol content (C) using these scattering functions provides values for the Kuhn length (b ) 8.5 nm) and contour length (L ) 308 nm) (see Figure 6a). A two-shell model with a core radius R1 ) 0.8 nm and an outer cylinder radius R2 ) 1.7 nm of the cross section was used. However,
Stradner et al.
Figure 6. Comparison of experimental SANS data for wormlike micelles (O, 5 wt % APG with 0.80 wt % hexanol) with theoretical results based on the wormlike chain model with excluded volume effects. (a) Model fit (solid line) with Kuhn length b ) 8.5 nm, contour length L ) 308 nm, core radius R1 ) 0.8 nm, and outer cylinder radius R2 ) 1.7 nm. (b) Demonstration of micellar flexibility in the Holtzer representation: model fit with Kuhn length b ) 8.5 nm (solid line) and b ) 11 nm (dashed line).
for an optimum characterization of these wormlike micelles a q range extending to smaller values would be necessary to get information on the structure factor and the overall size.52 Figure 6b shows the scattered intensity in the so-called bending rod or Holtzer plot given by qI(q) versus q.53,54 This kind of plot is suitable for testing the agreement between theoretical and experimental scattered intensity, since it is very sensitive to all deviations between data and the theoretical curve. Figure 6a shows that the experimental data agree quite well with the scattering functions of wormlike micelles. The sensitivity with respect to the choice of the value of the persistence length is also demonstrated in Figure 6b. A value of b ) 8.5 nm appears to be quite small when compared to previously published data of the flexibility of wormlike micelles. We are thus currently performing a detailed investigation of the Kuhn length as a function of the hexanol content of APG solutions.52 (3) Structural Changes on the Addition of n-Octane. The addition of small amounts of n-octane to the sugar surfactantbased solutions with low and high hexanol content (B and C) leads to distinct structural changes, as can be seen in the p(r) functions of the small-angle neutron and X-ray scattering data (Figure 7). SANS. The p(r) functions of the sample with low hexanol content (B, solid line) and the sample that additionally contains 0.43 wt % n-octane (Boc, dashed line) are shown in Figure 7a.
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J. Phys. Chem. B, Vol. 103, No. 32, 1999 6687
Figure 8. Hydrodynamic correlation length ξH of solutions with different amounts of added hexanol at varying total concentrations obtained from DLS measurements (pure APG (O); C6E0/APG ) 0.114 (0); C6E0/APG ) 0.16 (4)). c* is the so-called entanglement threshold or overlap concentration.
Figure 7. Comparison of the pair distance distribution functions p(r) of the sugar surfactant-based solutions with and without added n-octane (sample B, solid line, 5 wt % APG with 0.57 wt % hexanol; sample C, dotted line, 5 wt % APG with 0.80 wt % hexanol; sample Boc, dashed line, B + 0.43 wt % n-octane; sample Coc, dashed-dotted line, C + 0.6 wt % n-octane): (a) SANS data; (b) SAXS data.
The clear evidence of the existence of anisotropic particles in the sample without n-octane remains, and the feature of the p(r) for the sample with oil indicates cylindrical shaped aggregates of different but shorter lengths. The cross-sectional dimension increases because of the swelling of the micelles, while the overall dimension decreases. The same tendency can be observed for the sample with [hex] ) 0.80 wt % (C, dotted line) and the sample which in addition contains 0.6 wt % of the oil (Coc, dashed-dotted line); the size of the aggregates becomes smaller and the wormlike micelles turn into cylindrical micelles. Again, the cross section extends because of swelling. The contrast for neutrons increases because of the protons of the added oil. SAXS. The p(r) functions obtained from SAXS data (Figure 7b) exhibit the same structural evolution on adding oil as observed from neutron data. Again, we can see that the cylindrical aggregates in the sample with low hexanol content (B) and the wormlike micelles at high hexanol content (C) transform into smaller particles with larger cross section. As a consequence of the low electron density of n-octane the total contrast for X-rays decreases, which makes it impossible to get information on the overall dimension. On the other hand, owing to the different sign of the electron density of the hydrocarbon core and the headgroups of the aggregates, the changes in the cross-sectional dimension are more easily observed compared to neutron data.
In contrast to the addition of hexanol, which hardly changes the local structure of the aggregate because of incorporation into the surface layer, small amounts of n-octane in APGhexanol systems lead to swelling of the micelles, resulting in enlarged cross sections. Dynamic Light Scattering. DLS experiments were carried out for varying total concentrations covering the range from 1 wt % APG to 25 wt % APG but with constant surfactant-tohexanol ratios according to the ratios in the samples without and with low and high hexanol content. The solubility of hexanol in water or buffer was not taken into account. A strong dependence of the measured hydrodynamic correlation length on the total concentration and also on the hexanol content can be observed (see Figure 8). On the basis of the results of the SAS experiments, we expect relatively small and globular micelles to be present without hexanol. Under these conditions we indeed measure an apparent hydrodynamic radius of approximately 3.5 nm at low concentrations. With increasing concentration RH,app first decreases, which might be due to the effects of intermicellar interactions. For hard spheres one would indeed expect a decrease of RH,app following RH,app ≈ RH/(1 + 1.45φ) with φ being the volume fraction.55 At higher concentration we then find a slight increase of RH,app, which is most likely due to a concentration-induced micellar growth that more than compensates for the interaction effects. The SAS experiments have indicated a hexanol-induced transition from globular to large and locally cylindrical micelles. The DLS results are indeed consistent with these findings. At low hexanol content (C6E0/APG ) 0.114) the apparent hydrodynamic radius at low concentrations is larger (RH,app ) 5 nm) and increases first with increasing surfactant concentration, in agreement with the behavior generally found for surfactant systems where anisotropic micellar aggregates form. At higher concentrations it then reaches a maximum and decreases again, which is most likely due to the onset for the formation of an entangled network predicted to occur at some overlap threshold c* for cylindrical particles. The same behavior can be observed at high hexanol content (C6E0/APG ) 0.16), although the micellar growth is now much more pronounced, which consequently leads to a lower value for c* and a clear power-law decrease for the hydrodynamic correlation length at c > c*. The different features observed for these samples can be explained qualitatively by making an analogy to polymer solutions as previously done for a variety of surfactant systems that exhibit pronounced micellar growth and the formation of giant polymerlike aggregates. At
6688 J. Phys. Chem. B, Vol. 103, No. 32, 1999 low concentrations to about 6 wt %, the solutions correspond to a dilute regime where the coils of the wormlike micelles do not overlap, the measured ξH corresponds to the size of the micelles, and the increasing value is due to micellar growth. Under these conditions an additional difficulty arises from the fact that the micelles close to c* are too large in order to meet the criterion of qRg , 1, and ξH also contains contributions from the internal motion of the wormlike micelles. At concentrations c > c*, the micelles overlap and form an entangled network. In this semidilute regime, DLS yields no information on the size of the individual micelles but reports on the dynamics of the transient network; i.e., the decrease in ξH is a consequence of the decreasing screening length for hydrodynamic interactions in the network and not the result of a decreasing micellar size. Such a concentration dependence for the hydrodynamic correlation length has been found in a variety of surfactant systems, and it has been demonstrated that the effects of micellar growth and intermicellar interactions can indeed be decoupled using polymer theory.50 We are currently performing a detailed investigation using static light scattering at low minimum q value and SANS in order to fully analyze the structure, flexibility, and size distribution of these anisotropic micelles as a function of hexanol and APG concentrations. Conclusion We were able to demonstrate that micellar solutions of alkyl polyglucosides and small amounts of hexanol represent an ideal host system for the investigation of enzyme activity. Moreover, the structure and overall size of the micellar aggregates can be tuned by the amount of hexanol added, and the influence of the micellar size and structure on enzyme activity can thus be systematically investigated. We indeed found clear evidence that enzyme activity strongly depends on the supramolecular structure of the lipid aggregates. The water-insoluble fluorogenic substrate, which allows an optical determination of lipase activity, is assumed to be located in the surface layer of the aggregates. The distinct increase of the activity for both PSL and RML on the addition of small amounts (0.57 wt %) of hexanol to a micellar APG solution might be due to the incorporation of hexanol into the surface layer, which may cause a change in the chemical environment and the availability and conformation of the substrate in the hydrophobic-hydrophilic interface. These changes could subsequently lead to a better solubilization state of the substrate. However, the composition dependence of the enzyme activity could also be directly related to the structural change from globular to cylindrical “lipid aggregates” occurring upon the addition of hexanol. In this context it is particularly interesting to note that further addition of hexanol (0.80 wt %), which gives rise to a structural transition to wormlike micelles, is accompanied by a decrease of enzyme activity. Enzyme activity in this system is thus lower than under solution conditions where much shorter cylindrical aggregates form, although the local micellar structure as well as the chemical environment are almost identical (these structural changes are caused by only very small variations of the hexanol content). Therefore, we believe that this decrease is mainly due to the corresponding changes in the physical properties of the solution. In the case of relatively short cylindrical micelles, the protein has good access to the substrate, whereas at higher hexanol content the aggregates grow into giant and flexible micelles that can then form a transient network analogous to semidilute polymers. Under these conditions the mobility of the enzyme molecules will be strongly reduced, and the diffusion to intact substrate molecules will be considerably hindered. As
Stradner et al. a consequence, the apparent concentration of substrate for the enzyme within this “microenvironment” in which it is partially trapped is lower, which would then result in a lower activity. Addition of n-octane or decane is a standard means for the creation of a nonionic microemulsion. Addition of n-octane to our sugar surfactant system is only possible in small amounts (less than 1 wt %). This leads to swelling of the micelles, i.e., an increase of the diameter of the cross section, and at the same time to a strong decrease of the enzyme activity of PSL. This change cannot be attributed to the small structural changes and must therefore be understood as a denaturating effect of the oil. It is clear that there is a need for additional investigations of the detailed structural evolution of the micellar aggregates upon addition of hexanol. However, we believe that we have a very useful model system for a detailed and quantitative study of the relationship between local and global host structure and enzyme activity that allows us to address numerous aspects of enzyme reactivity in complex host-guest systems. Acknowledgment. We acknowledge the Institut LaueLangevin, Grenoble, France, for providing the neutron research facilities, and we gratefully acknowledge the expert help of our local contact at the instrument D22, Dr. Stefan Egelhaaf. We thank Prof. Peter Schurtenberger for inspiring discussions and important help in the interpretation of scattering data from wormlike micelles. A.S. also gratefully acknowledges the support and the hospitality of Prof. Reinhard Strey and his crew during her stay at the Department of Physical Chemistry I, University of Cologne, where the phase diagram determination was performed. In addition we thank Dr. Wolfgang von Rybinski from Henkel company, who supplied us with the alkyl polyglucoside C8/10G1.5 used in this study. This work was supported by the O ¨ sterreichischer Fonds zur Fo¨rderung der wissenschaftlichen Forschung under Grants P11778-CHE and F0107. References and Notes (1) Duque, M.; Graupner, M.; Stu¨tz, H.; Wicher, I.; Zechner, R.; Paltauf, F.; Hermetter, A. J. Lipid Res. 1996, 4, 868. (2) Paltauf, F.; Hermetter, A. Methods Enzymol. 1991, 197, 134. (3) Blencowe, C.; Hermetter, A.; Kostner, G. M.; Deigner, H. P. J. Biol. Chem. 1995, 270, 31151. (4) Zandonella, G.; Haalck, L.; Spener, F.; Faber, K.; Paltauf, F.; Hermetter, A. Eur. J. Biochem. 1995, 231, 50. (5) Hermetter, A. Appl. Fluoresc. Technol. 1990, 2, 1. (6) Skagerlind, P.; Folmer, B.; Jha, B. K.; Svensson, M.; Holmberg, K. Prog. Colloid Polym. Sci. 1998, 108, 47. (7) Folmer, B.; Holmberg, K.; Svensson, M. Langmuir 1998, 13, 55864. (8) Holmberg, K. AdV. Colloid Interface Sci. 1994, 51, 137. (9) Holmberg, K.; O ¨ sterberg, E. Prog. Colloid Polym. Sci. 1990, 82, 181. (10) Skagerlind, P.; Holmberg, K. J. Dispersion Sci. Techn. 1994, 15, 317. (11) Stark, M.-B.; Skagerlind, P.; Holmberg, K.; Carlfors, J. Colloid Polym. Sci. 1990, 268, 384. (12) Sonesson, C.; Holmberg, K. J. Colloid Interface Sci. 1991, 141, 239. (13) Holmberg, K. In Industrial Applications of Microemulsions; Solans, C., Kunieda, H., Eds.; Marcel Dekker: New York, 1997. (14) Komives, C. F.; Osborne, D. E.; Russell, A. J. J. Phys. Chem. 1994, 98, 369. (15) Carlile, K.; Rees, G. D.; Robinson, B. H.; Steer, T. D.; Svensson, M. J. Chem. Soc., Faraday Trans. 1996, 92, 4701. (16) Stamatis, H.; Xenakis, A.; Menge, U.; Kolisis, F. N. Biotechnol. Bioeng. 1993, 42, 103 (17) Stamatis, H.; Xenakis, A.; Provelegiou, M.; Kolisis, F. N. Biotechnol. Bioeng. 1993, 42, 931. (18) Stamatis, H.; Xenakis, A.; Bornscheuer, U.; Scheper, T.; Menge, U.; Kolisis, F. N. Biotechnol. Lett. 1993, 15, 703. (19) Xenakis, A.; Valis, T. P.; Kolisis, F. N. Prog. Colloid Polym. Sci. 1991, 84, 508.
Sugar Surfactant-Based Solutions (20) Holmberg, K.; O ¨ sterberg, E. Prog. Colloid Polym. Sci. 1987, 74, 98. (21) Eskuchen, R.; Nitsche, M. In Alkyl Polyglycosides; Hill, K., v. Rybinski, W., Stoll, G., Eds.; VCH: Weinheim, 1997 (22) Nickel, D.; Nitsch, C.; Kurzendo¨rfer, P.; v. Rybinski, W. Prog. Colloid Polym. Sci. 1992, 89, 249. (23) Fukuda, K.; So¨derman, O.; Lindman, B.; Shinoda, K. Langmuir 1993, 9, 2921. (24) Platz, G.; Po¨like, J.; Thunig, C.; Hofmann, R.; Nickel, D.; v. Rybinski, W. Langmuir 1995, 11, 4250. (25) Balzer, D. Langmuir 1993, 9, 3375. (26) Kahlweit, M.; Busse, G.; Faulhaber, B. Langmuir 1995, 11, 3382. (27) Nickel, D.; Fo¨rster, T.; v. Rybinski, W. In Alkyl Polyglycosides; Hill, K., v. Rybinski, W., Stoll, G., Eds.; VCH: Weinheim, 1997. (28) Kahlweit, M.; Busse, G.; Faulhaber, B. Langmuir 1996, 12, 862. (29) Ryan, D. L.; Schubert, K.-V.; Kaler, E. W. Langmuir 1997, 13, 1510. (30) Stubenrauch, C.; Paeplow, B.; Findenegg, G. H. Langmuir 1997, 13, 3652. (31) Fischer, E. Ber. Dtsch. Chem. Ges. 1895, 28, 1145. (32) Iampietro, D. J.; Brasher, L. L.; Kaler, E. W.; Stradner, A.; Glatter, O. J. Phys. Chem. B 1998, 102, 3105. (33) The Yellow Book: Guide to Neutron Research Facilities at the ILL; Bu¨ttner, H. G., Ed.;ILL: Grenoble, France, 1997. For further information, see http:/www.ill.fr/d22/d22.html. (34) Glatter, O. J. Appl. Crystallogr. 1977, 10, 415. (35) Glatter, O. J. Appl. Crystallogr. 1981, 14, 101. (36) Glatter, O.; Strey, R.; Schubert, K.-V.; Kaler, E. W. Ber. BunsenGes. Phys. Chem. 1996, 100, 323.
J. Phys. Chem. B, Vol. 103, No. 32, 1999 6689 (37) Strey, R.; Glatter, O.; Schubert, K.-V.; Kaler, E. W. J. Chem. Phys. 1996, 105, 1175. (38) Pedersen, J. S. AdV. Colloid Interface Sci. 1997, 70, 171. (39) Koppel, D. E. J. Chem. Phys. 1972, 57, 4814. (40) v. Rybinski, W. Curr. Opin. Colloid Interface Sci. 1996, 1, 587. (41) Penders, M. H. G. M.; Strey, R. J. Phys. Chem. 1995, 99, 6091. (42) Anderson, D. M.; Wennerstro¨m, H.; Olsson, U. J. Phys. Chem. 1989, 93, 4243. (43) Porte, G.; Marignan, J.; Bassereau, P.; May, R. J. Phys. (Paris) 1988, 49, 511. (44) Gazeau, D.; Belloq, A.; Roux, D.; Zemb, T. Europhys. Lett. 1989, 9, 447. (45) Strey, R.; Jahn, W.; Porte, G.; Bassereau, P. Langmuir 1990, 6, 1635. (46) Pedersen, J. S.; Schurtenberger, P. J. Appl. Crystallogr. 1996, 29, 646. (47) Schurtenberger, P.; Jerke, G.; Cavaco, C.; Pedersen, J. S. Langmuir 1996, 12, 2433. (48) Tanford, C. J. Phys. Chem. 1972, 76, 3020. (49) Bergstro¨m, M.; Pedersen, J. S. Langmuir 1999, 15, 2250. (50) Jerke, G.; Pedersen, J. S.; Egelhaaf, S. U.; Schurtenberger, P. Phys. ReV. E 1997, 56, 5772. (51) Pedersen, J. S.; Schurtenberger, P. Macromolecules 1996, 29, 7602. (52) Stradner, A.; Glatter, O.; Schurtenberger, P. Manuscript in preparation. (53) Holtzer, A. J. Polym. Sci. 1955, 17, 432. (54) Denkinger, P.; Burchard, W. J. Polym. Sci., Part B: Polym. Phys. 1991, 29, 589. (55) Olsson, U.; Schurtenberger, P. Langmuir 1993, 9, 3389.
