J. Chem. SOC., Faraday Trans. 1, 1986, 82, 23‘11-2321 A Quasi-elastic Neutron Scattering Study of Water-in-oil Microemulsions stabilised by Aerosol-OT Effect of Additives including Solubilised Protein on Molecular Motions Paul D. I. Fletcher? and Brian H. Robinson* University Chemical Laboratory, University of Kent at Canterbury, Canterbury, Kent CT2 7NH James Tabony Departement de Physico-Chimie, Centre d’Etudes Nucleaires de Saclay, 91 191 Gif-sur- Yvette, France The quasi-elastic incoherent neutron scattering method has been used to investigate the mobility of surfactant and water molecules in single-phase oil-continuous microemulsions stabilised by sodium bis(2-ethyl hexy1)sulpho- succinate (AOT). Addition of benzyl alcohol, which is adsorbed at the interface, or toluene effects little change in the lateral translational diffusion of AOT within the interface.Replacement of the dispersed water by glycerol results in a two-fold reduction in surfactant mobility. Solubilisation of a-chymotrypsin withm the water droplet core of the microemulsion system causes no significant change in AOT mobility. However, spectra associated with water mobility in the enzyme-containing system clearly reveal the presence of 250-500 water molecules per protein molecule, which are ‘bound’ on the time scale of the experiment (6 x 1O1O rad s-l). The mobility of the remainder of the solubilised water in the system is unaffected by the presence of the enzyme. Quasi-elastic incoherent neutron scattering has recently been used to study molecular motions in micellar and microemulsion systems.3 For water-in-oil microemulsions stabilised by sodium bis(2-ethylhexyl)sulphosuccinate (AOT), such studies have indicated that the AOT molecules undergo lateral diffusion along the curved water/oil interface of the water droplets with a characteristic diffusion coefficient of 8 x m2 s-l. The motion of solubilised water within the droplet core is characterised by a diffusion coefficient of 1.2 x m2 s-l, which is comparable to the value obtained in high- ionic-strength bulk aqueous rnedia.l? * It is easily possible to prepare glycerol-in-oil microemulsions stabilised by AOT which are analogous to the more conventional water-in-oil type. The oil-continuous microemulsion system formed by glycerol-heptane-AOT, where the water core has been replaced by glycerol, has been extensively characterised using the techniques of photon correlation spectroscopy and viscometry and it behaves in a similar way to the corresponding water-containing microem~lsion.~ In the present study we compare quasi-elastic incoherent neutron scattering profiles obtained from both systems.By this means the local lateral molecular motions of AOT can be determined and it can be established whether these are controlled primarily by the viscosity of the non-continuous dispersed phase, the continuous apolar oil phase or the viscosity of the interfacial region. It is known that in the AOT-stabilised microemulsion system, water-solubilised hydrophilic reagents such as metal ions can readily be exchanged between water t Present address: Chemistry Department, University of Hull, Hidl HU6 7RX.2312 Neutron Scattering Study of Microemulsions droplets.4> This process occurs with a second-order rate constant of 106-107 dm3 mol-1 s-l and is thought to proceed via a transient coalescence of the water droplets, followed by reseparation of the droplets. This results in random exchange of species contained within the water droplets. This is an important kinetic process in microemulsions and is thought to be the mechanism whereby the microemulsion establishes its equilibrium properties (e.g.particle size, polydispersity etc.), which are then maintained in rapid, dynamic equilibrium. The kinetics of this exchange process are known to be affected by the addition of various additives to the system.For example, addition of 0.1 mol dm3 benzyl alcohol to the AOT-heptane-water system ([H,O]/[AOT] = 20; [AOT] = 0.1 mol dm-3) causes a 50% increase in the rate, whereas addition of 10% v/v toluene causes a 50% decrease in the rate.6 In particular, species located in the interface region, such as Ru(bipy)g+, are readily transferred between two droplets in c0ntact.j It is of interest, therefore, to determine whether the changes observed in the inter-droplet exchange rate are paralleled by changes in the local motions of the AOT molecules at the oil/water interface. Finally, there is much current interest in the solubilisation of enzymes in water-in-oil microemulsions, since these systems have interesting synthetic po~sibilities.~ The hydro- philic protein a-chymotrypsin retains its activity when solubilised in water-in-oil microemulsions stabilised by AOT.Measurements of the catalytic activity of this enzyme in AOT microemulsions towards a variety of substrates have shown that the turnover number of the protein (kcat) is little affected upon solubilisation, but that the binding affinity between the enzyme and various substrates is reduced by a factor of ca. one-hundred-fold.8-11 A possible explanation of this effect for AOT is that it binds to the enzyme, thus reducing its substrate affinity. In this study, we compare the AOT spectra obtained from microemulsions with and without a-chymotrypsin in order to observe any changes in molecular mobility of the AOT caused by the presence of enzyme in the core. Also, selective deuteration permits a comparison to be made of the diffusion characteristics of the solubilised water in the two systems.Experimental AOT was obtained from Sigma (as sodium dioctylsulphosuccinate) and was used without further purification. Its purity and consistency were checked by interfacial tension measurements. The phase behaviour of the sample, in particular the upper temperature cloud point, was also checked. It contained negligible amounts of an acidic impurity which is often present in samples of AOT.12 Hydrolysis of AOT is known to produce a carboxylic acid and 2-ethylhexanol, the rate of hydrolysis being quite rapid under basic conditions. l2 However, under the conditions of sample preparation and recording of spectra such hydrolysis was negligible.The enzyme a-chymotrypsin was purchased from Sigma (bovine pancreas type 11). Deuterated octane and D,O were obtained from CEA, France. A sample of deuterated glycerol was generously donated by Drs J. C. Dore and A. Angell. Benzyl alcohol and toluene were of analytical grade and were used without further purification. Triply distilled water was employed throughout and all neutron measurements were made within 24 h of preparation of the microemulsions. Water-in-oil microemulsions were prepared by adding the correct volume of water to an AOT solution in octane. Clear solutions were obtained after gentle shaking for ca. 30 s. Glycerol microemulsions were prepared by weighing the required amount of glycerol into a flask, adding an octane solution of AOT, followed by gentle shaking.a-Chymotrypsin was weighed into the flask, the correct volume of water was added and the flask shaken to produce a solution. As before, addition of an AOT solution in octane followed by gentle shaking produced clear microemulsion solutions. Neutron measurements on the enzyme-contaning microemulsions were made within 3 h of preparation in order to obviate, as far as possible, the effects of enzyme inacti~ation.~? l1P. D. I. Fletcher, B. H. Robinson and J . Tabony 2313 Neutron incoherent quasi-elastic spectra were recorded at ambient temperature ( 1 9 _+ 2 "C) on the high-resolution, time focusing, time-of-flight spectrometer (IN6) located at the high-flux reactor of the Institut Laue-Langevin, Grenoble. The incident wavelength (A) was 5.1 A and counting times were of the order of 1 h.The samples were good scatterers, with ca. 20000 counts at the peak maximum, and ca. 4000 counts in the wings. Energy spectra at nineteen different scattering angles (6) between 12 and 112" were recorded for each sample. Spectra were taken with the sample at 45 and 135" to the incident beam. The energy resolution (half-width at half height) varied with both the scattering angle and the sample angle and was 40-80 peV. This resolution was determined using a vanadium sample. Samples were contained in flat aluminium holders of sample thickness 0.5 or 1 mm. Ca. 5 cm3 of the sample was required in order to obtain a spectrum. Neutron transmissions were greater than 90% in all cases. Data were corrected for transmission, detector efficiency and background scattering by means of standard procedures and programs available at the ILL.No corrections were made for multiple scattering. The incoherent neutron scattering spectrum S(Q, co) is related to diffusional motion (Fickian translation) by where Q = (412/A) sin 6 / 2 , D is the translation diffusion coefficient and w is the neutron energy. The quasi-elastic spectra are then described by Lorentzian curves with a full-width at half-height (2Aw) of 2DQ2. In contrast, for rotational motion, a superposition of a quasi-elastic peak, which is independent of Q, and an 'elastic' peak is observed. This approach has been used to obtain the translational diffusion coefficient (D) of AOT and water in oil-continuous microemulsions in cyclohexane by means of plots of the half width Aco us.Q2.1 The spectrum corresponding to one particular chemical component in the microemul- sion solutions was obtained by subtraction of the various experimental spectra. For example, the spectrum due to AOT motion in the system D,O-AOT-D(octane) was obtained by subtracting the deuterated octane solvent spectrum from the spectrum of the AOT microemulsion containing D20. (Necessary corrections were made in this procedure for the volume fraction of dispersed material.) The resulting spectrum contains a negligible scattering contribution from D 2 0 (for the concentrations used here where the volume fraction of D 2 0 is of the order of 2%) in comparison with the scattering arising from the proteated AOT. A check was in any case made that the integrated intensities were proportional to the concentration of proteated material.The resulting spectra was then fitted to a convolution of a Lorentzian curve with the experimentally determined resoluton function. Results and Discussion Effect on Surfactant Motion of the Substitution of Water by Glycerol in the Droplet Core Spectra were recorded for the following samples: (i) 0.2 mol dm-3 AOT, 4 mol dm-3 D,O in deuterated octane and (ii) 0.2 mol dm-3 AOT, 0.550 mol dm-3 deuterated glycerol in deuterated octane. The spectra corresponding to AOT alone were obtained by subtraction of the solvent [D(octane)] spectrum. Both microemulsion systems are known to contain discrete, approximately spherical droplets of dispersed phase [D,O or D(glycerol)] surrounded by an interfacial layer of the surfactant.A small amount of the total surfactant may be present in the form of reversed mi~e1les.l~ Both microemulsion systems have been2314 Neutron Scattering Study of Microemulsions -0.5 0 0.5 - 0.5 0 0.5 O / 0 0 0 0003000000300 -0.5 0 0.5 energy/meV 1.250 h 3 9 0.625 0.0 -1.0 -0.5 0 0.5 1 .o energylmev Fig. 1. Quasi-elastic incoherent spectra due to AOT in (a) the water microemulsion, (b) the glycerol microemulsion and the (c) resolution function. The scattering angle 8 was 97.3", sample at 45" to incident beam, continuous phase is n-octane. ( d ) Matching of the experimentally determined curve to the theoretical line. AOT in the aaueous microemulsion at 8 = 97.3".P. D. I. Fletcher, B. H. Robinson aiid J .Tabony 200 r 2315 0 2 4 Q2/8-2 Fig. 2. Plots of linewidth us. Q2 for AOT spectra in the water microemulsion (0) and the glycerol microemulsions (a). The dashed and solid lines refer to the resolution function width for sample mounting at 135 and 45", respectively. Error is ca. 15% in Am. All points refer to spectra recorded at 45" except Q2 values in the range 1-2 k2. Continuous phase is n-octane. characterised by a variety of methods including small-angle neutron scattering and photon correlation spectro~copy.~~ 14-16 The droplet core radii (ie. not including the surfactant shell) of the water and glycerol samples are 3.5 and 2.4 nm, respectively. The overall hydrodynamic radii (i.e. including the surfactant shell and any associated solvent) are of the order of 5.0 and 4.1 nm.Therefore, for the quasi-elastic neutron scattering experiments reported in this paper the spectra of the interfacial AOT in the water and glycerol dispersions are compared in droplet systems of comparable particle sizes and hence curvature of the surfactant interface. We do not expect a significant variation with droplet size or droplet concentration, based on previous experience with the water droplet dispersion.lt Fig. 1 shows the AOT spectra in both systems and the resolution function obtained under identical conditions. It can be clearly seen that the energy broadening is considerably reduced for the glycerol system [fig. 1 (b)] as compared with the water system (a). In addition, there is clearly some broadening in (b) as compared with the instrument resolution (c).The spectra were analysed as described previously to obtain a quasi-elastic scattering width (Am) as a function of the scattering vector Q. A typical fit of the experimental data to the theoretical curve based on eqn (1) is shown as fig. 1 (d). Plots of Am us. Q2 for both systems are shown in fig. 2. For the dispersed water system, the plot closely resembles that previously observed for AOT motion when cyclohexane was used as the oil-continuous so1vent.l There is an approximately linear relationship between Am and Q2 and from the slope the diffusion coefficient D is obtained. (The gap in the region of Q2 = 1-2 k2 is due to the fact that for samples mounted at 45" to the beam the scattering is attenuated by the sample container edge. Mounting the sample at 135" yields a full data set, but the energy resolution is less favourable.For certain samples, measurements were made at both 45 and 135".) The measurements made at 135" show a plateau in the plots of Am us. Q2 for values of Q2 between 1 and 2 k2, This could indicate that the observed motion is more complex than a simple translational motion. However, the plateau could also result from the fitting procedure used. The random error in the derived broadening is ca. 15 % , and this covers most of the difference between the values of the broadening plotted and a linear least-squares fit through the experimental points. In the plateau region the broadening is comparable with the instrument resolution, which may introduce some difficulties into the fitting procedure.When the broadening is significantly different from the instrument resolution, no major problems are encountered with the fitting routine. Broadenings less than the resolution are detected (as indicated for example in fig. 2) because a Lorentzian broadening has wings which extend much further out than those of the instrument resolution, which is Gaussian-shaped [fig. 1 (c)]. However, when the broadening is comparable with the resolution, it may be2316 Neutron Scattering Study of Microemulsions that the fitting criteria used cannot so readily distinguish between slightly different broadenings. Hence, for broadenings which are slightly different the derived value could be essentially invariant, which might account for the plateau behaviour observed between Q2 = 1-2 k2.The discussion in this paper will be concentrated on possible interpretations of the Q-dependent diffusion parameter that we associate with an essentially translational diffusion mode, which for AOT represents lateral molecular diffusion within the interface. Drawing straight lines passing through the origin for the Am z's. Q2 plots yields values for the diffusion coefficient of AOT in the water and glycerol systems of 6.1 and 3.8 x 10-lo m2 s-l, respectively, with n-octane as dispersed phase. The value determined in the corresponding AOT-water-cyclohexane system was 8 x 10-lo m2 s-l. It is important to note that this was essentially independent of the amount of water in the microemulsion system.l As the water concentration is increased at constant AOT concentration the size of the droplets increa~es.~ Since a different fitting procedure was used in the cyclohexane work, the difference between the two water-containing systems as the solvent is changed is not thought to be of significance.However, there is a very clear reduction in translational diffusion when glycerol is substituted for water. The observed translational motion of AOT may arise from motion of AOT around a microemulsion droplet or from motion of the whole microemulsion droplet through the solution. The translational diffusion coefficient (DT) of the total microemulsion droplet may be calculated from the known droplet hydrodynamic radius and the solvent viscosity using the Stokes-Einstein equation.2 It is readily determined experimentally, for low concentrations of droplets, using photon correlation spectroscopy : D, = kT16nrq.(2) In eqn (2), r is the droplet hydrodynamic radius and q is the solvent viscosity (5.42 x kg m-l s-l for octane at 20 "C). Using eqn (2), values of the translational diffusion coefficient for the water and glycerol droplets of 0.8 x loplo and 1 .O x m2 s-l, respectively, may be calculated. Thus the overall translational motion of the droplets can account at most for only 1&20% of the observed effect. The surface motion of the AOT surfactant molecules may arise from two causes. First, the AOT molecules may diffuse laterally along the interface. Secondly, since the characteristic length scale of our neutron scattering observation is in the range 2 to 10 A (0.2-1 nm), dimensions much smaller than the droplet radius (ca.3.5 nm) are probed. On this length scale, the overall rotation of the droplet would cause the AOT molecules, if rigidly located on the surface of the sphere, to move with an apparent translational motion. The magnitude of this effect may be calculated using eqn (3) for the rotational diffusion coefficient (8) of a sphere:17 8 = kT/8nyr3. (3) The apparent diffusion coefficient caused by rotation of the whole particle [Dr(app)] is then given by the product of 8 and the mean-square jump distance. Taking the latter as being approximately the droplet radius, gives : (4) kT Dr(app) = - 8nqr. As might be expected, this diffusion coefficient is very similar to that derived on the basis of the Stokes-Einstein law and yields values for Dr(app) similar to those calculated for D,.For both translational and rotational displacement of the droplet, the diffusion rate will vary with the inverse of the radius. Previous experiments of AOT dispersions in cyclohexane, in which the droplet size was increased by a factor of three, resulted in no change in the Q-dependent broadening with increasing droplet size.l The sum of the evidence therefore suggests that our observed diffusion effect is dominated by theP. D. I. Fletcher, B. H. Robinson and J . Tabony 300 r 200 I l o o t 0 o o 2 Q2/A-2 2317 Fig. 3. Plots of linewidth us. Q2 for the AOT spectra in AOT-octane-water microemulsions containingno additive (O), 10% v/v deuterated toluene (0) and 0.2 mol dmP3 benzyl alcohol (A).Error is ca. 15 % in Am. Continuous phase is n-octane. translational diffusion of the surfactant molecules laterally along the interface. Moreover since the water and glycerol droplets have sizes which differ by only 20%, and the glycerol droplet is smaller than the water droplet, the reduction in the AOT line broadening when glycerol is substituted for water must arise from a reduction in the translational mobility of the AOT molecules in the interface. Since the viscosity of glycerol is ca. 1500 times greater than that of water, this would imply little or no ‘anchoring’ of the AOT within the dispersed phase in either system. Effect of Surfactant Motion of Addition of Benzyl Alcohol and Toluene Spectra were recorded for the following samples : (iii) 0.2 mol dm3 AOT, 4 mol dmP3 D20, 10% v/v deuterated toluene in deuterated octane and (iv) 0.2 mol dmP3 AOT, 4 mol dm-3 D20, 0.2 mol dm-3 benzyl alcohol in deuterated octane.As discussed previously, for sample solution (iii) the spectrum of the AOT alone was obtained by subtraction of the solvent (deuterated octane) spectrum. Toluene, being deuterated, made a negligible contribution to the observed scattering. For sample solution (iv), deuterated benzyl alcohol was not available, hence the difference spectrum will contain a contribution from both AOT and the alcohol. The ratio of integrated intensities from AOT and the alcohol is approximately given by the ratio of protons in each molecule; this ratio is 37: 8. Plots of Aco us. Q2 for samples (i), (iii) and (iv) are shown in fig.3. Values of the apparent diffusion coefficient calculated from the slopes for samples (i), (iii) and (iv) are (6.1, 7 and 8) x 10-lo m2 s-I, respectively. Since the benzyl alcohol-containing microemulsion spectra contain contributions from the alcohol, it seems that the AOT mobility is essentially unchanged with and without the additives. Certainly there appears to be no correlation between the local AOT mobility and the rate of solubilisate exchange, which involves droplet coalescence. In addition, the upper phase-transition temperature is affected differently by the two additives : benzyl alcohol induces phase separation at a lower temperature, whereas toluene increases the tem- perature corresponding to instability of the single-phase microemulsion. The implication is that the interactions are more attractive in the benzyl alcohol containing system.6 It has been observed previously that addition of pentanol to aqueous micelles of tetradecyltrimethylammonium bromide causes no change in the quasi-elastic broadening of the surfactant spectrum.2 Also, in the case of glycerol microemulsions, in which we have now shown that the AOT mobility is reduced considerably, recent measurements2318 Neutron Scattering Study of Microenzulsions 0 2 I, @/A -2 Fig.4. Plots of linewidth us. Qz for AOT spectra in microemulsions with (@) and without (0) a-chymotrypsin. Error is ca. 15 % in Am. Continuous phase is n-octane. of the kinetics of exchange of ions confined within the glycerol droplet indicate that there is no large reduction in the rate of solubilisate exchange between glycerol droplets as compared with water droplets.lR This again would seem to imply little or no correlation between the local AOT mobility and the kinetics of droplet exchange.Effect of Solubilised a-Chymotrypsin in Water Droplets on the Dynamics of Surfactant Motion and Motion of Solubilised Water within the Droplet Core a-Chymotrypsin (aCT) is a highly water-soluble hydrolytic enzyme with a molecular weight of 24800 daltons. Its external dimensions are ca. 4.0 x 4.0 x 5.1 nm, which corresponds approximately to a sphere of radius 2.2 nm.9 The enzyme was solubilised in a droplet system in which the radius of the water cores before solubilisation is ca, 3.5 nm and which contains ca. 6000 water molecules. Ultracentrifugation studies, together with studies by small-angle neutron scattering and photon correlation spectros~opy,~~ have shown that a-chymotrypsin is solubilised into this microemulsion system with no appreciable change in droplet size.In contrast, in some systems, there is evidence that large droplets are formed, containing the majority of the protein molecules, and these are in equilibrium with a population of smaller dr0p1ets.l~~ 2o Ultracentrifuge results, confirmed by small-angle neutron results, show that at the solution composition used here the size and stoichiometry of the AOT-stabilised droplets is not significantly affected by the presence of a-chymotrypsin.lg The overall concentration of a-chymotrypsin used was 0.81 x lop3 mol dm-". The initial concentration of water droplets, measured using a fluorescence technique, is 0.82 x lop3 mol drnP3.,l Therefore, the solutions used in this work are thought to correspond to a situation in which each water droplet contains approximately one a-chymotrypsin molecule.The following samples were prepared and spectra recorded : (v) 0.2 mol dmV3 AOT, 4 rnol dm-3 D20, 0.81 x lop3 mol dmP3 aCT in deuterated octane; (vi) 0.2 mol dmP3 AOT, 4 mol H,O, 0.81 x lop3 mol dm-3 aCT in deuterated octane; and (vii) 0.81 x The spectrum due to aCT was obtained by subtraction of the D,O solvent spectrum from the spectrum of (vii). The spectrum of AOT was obtained by subtraction of the 'elastic' aCT spectrum and the octane solvent spectrum from spectrum (v). The spectrum of the water in the aCT-containing microemulsion was obtained by the subtraction of spectrum (v) from spectrum (vi).The water spectrum of the non-aCT-containing microemulsion was obtained by a similar H,O/D,O difference procedure. Fig. 4 shows the results obtained for the AOT linewidths in microemulsions with and without the protein. The results are virtually identical indicating that the protein has no significant effect on the surfactant mobility. Fig. 5 shows the spectrum due to the solubilised water in the microemulsion droplets mol dm-3 aCT in D,O.P. D. I. Fletcher, B. H. Robinson and J . Tabony > 3 d g 2 0 0 . . 2319 w/meV Fig. 5. Spectra due to water at 8 = 11 1.6” for (a) microemulsion containing aCT and (b) microemulsion without xCT. Continuous phase is n-octane.300 a 0 0 0 0. 0 'oat***@. , , , , 0 2 L Q2/A-2 Fig. 6. Plots of linewidth us. Q2 for the water spectra (quasi-elastic portion) in microemulsions with (0) and without (0) aCT. Continuous phase is n-octane. in the presence of aCT (a) and in the absence of aCT (b). The total integrated intensities are equal under both peaks, but an ‘elastic’ peak can clearly be observed in the spectrum from the enzyme-containing solution, suggesting that some fraction of the total solubilised water has been ‘iimmobilised’ or has a drastically reduced mobility. The energy resolution of the IN6 experiment is such that any translational diffusion slower than 3 x loplo m2 s-l and rotational motions slower than loplo s will appear as ‘immob- lised’ and it should be borne in mind that the term ‘immobilised’ can only be considered with reference to the resolution of our experiment (i.e.40 peV or 6 x 1O1O rad s-l). The fact that this ‘elastic’ peak is only observed when enzyme is present indicates the presence of some ‘immobilised’ water associated with the enzyme. The analysis procedure involved fitting the spectrum in fig. 5(a) to the sum of an ‘elastic’ peak and2320 Neutron Scattering Study of Microemulsions a Lorentzian curve. Linewidths were derived from the quasi-elastic portions of the curves. Fig. 6 shows a comparison of the quasi-elastic linewidths as a function of Q2 for water in microemulsions with and without aCT. As observed previously, the plot is linear and a value for the diffusion coefficient of 1.3 x lop9 m2 s-l is obtained.This value is approximately half that observed for water self-diffusion in bulk water (D = 2.5 x m2 s-l), but is comparable to that observed in ca. 5 mol dm+ lithium chloride in aqueous solution.22 This result is not unexpected in view of the high concentration of Na+ counter ions in the water pools. For [H,O]/[AOT] = 20, “a+],, z 1.4 mol dm-3. The result for the enzyme-containing droplet system shows that there is virtually no change in the mobility of a major fraction of the solubilised water on addition of aCT. From the integrated areas of the elastic and quasi-elastic portions of the scattering curves, it can be concluded that ca. 5-10% of the solubilised water is ‘immobilised’ in the vicinity of the enzyme. This corresponds to 250-500 water molecules for each solubilised protein molecule, or 0.2-0.4 g of water per gram of protein. This level of ‘bound’ water is comparable with that observed for many proteins, as determined by various techniques including dielectric rnea~urements,~~~ 24 i.r.spectros~opy~~ and calorimetry.26 [For a fuller discussion, see ref. (27).] Thus, when the enzyme aCT is solubilised within the microemulsion water cores, there is clearly an interaction with the core water. Since only a small amount of water is present, it is easily possible to distinguish between ‘free’ and ‘immobilised ’ or bound water. In conclusion, the results described in this paper suggest that the mobility of the AOT surfactant in oil-continuous microemulsions is not much affected by addition of toluene or benzyl alcohol.Likewise, solubilisation of a-chymotrypsin within the water pools has no effect. Substitution of the water by glycerol, however, produces a significant reduction in AOT mobility. Measurements of the solubilised-water mobility clearly reveal the presence of 5-10% of ‘ bound’ water around the solubilised enzyme in the microemulsion system. The mobility of the remaining 90-95% of the solubilised water in the core is apparently unaffected by the addition of the enzyme. We thank the ILL, Grenoble and the S.E.R.C. (Neutron Beams) for provision of beam time and financial support. We also thank Dr A. J. Dianoux for his assistance on IN6, and Drs A. Angel1 and J. C . Dore for a gift of a sample of deuterated glycerol. B. H. R. and P. D. I. F. thank the S.E.R.C.(Biotechnology Directorate) and Tate and Lyle (Reading) for support of this work through a Cooperative award. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 J. Tabony, A. Llor and M. Drifford, Colloid Polym. Sci., 1983, 261, 938. J. Tabony, Chem. Phys. Lett., 1985, 113, 75. P. D. I. Fletcher, M. F. Gala1 and B. H. Robinson, J. Chem. Sac., Furuduy Trans. I , 1984, 80, 3307. P. D. I. Fletcher and B. H. Robinson, Ber. Bunsenges. Phys. Chem., 1981, 85, 863. S. S. Atik and J. K. Thomas, J. Am. Chem. Soc., 1981, 103, 3543. P. D. I. Fletcher, A. M. Howe and B. H. Robinson, J. Chem. Soc., Furuduy Trans. I , in press. P. L. Luisi, Angew. Chem., Znt. Ed. Engl., 1985, 24, 439. F. M. Menger and K. Yamada, J. Am. Chem. Soc., 1979, 101, 6731. S. Barbaric and P. L. Luisi, J. Am. Chem.SOC., 1981,103, 4239. K. Martinek, A. V. Levashov, N. L. Klyachko, V. I. Pantin and I. V. Berezin, Biochim. Biophys. Acta, 1981, 657, 277. P. D. I. Fletcher, R. B. Freedman, J. Mead, C. Oldfield and B. H. Robinson, Colloids Surf., 1984, 10, 193. P. D. I. Fletcher,N. M. Perrins, B. H. RobinsonandC. ToprakciogluinReverseMicelles,ed. P. L. Luisi and B. E. Straub (Plenum Press, New York, 1984), p. 69. P. D. I. Fletcher, A. M. Howe, B. H. Robinson, J. C. Dore, N. M. Perrins and C. Toprakcioglu, in Surfuctunts in Solution, ed. K. Mittal and B. Lindman (Plenum Press, New York, 1983), vol. 3, p. 1745. M. Zulauf and H. F. Eicke, J. Phys. Chem., 1979, 83, 480.P. D. I . Fletcher, B. H. Robinson and J . Tabony 2321 15 B. H. Robinson, C. Toprakcioglu, J. C. Dore and P. Chieux, J. Chem. Soc., Faraday Trans. 1, 1984, 16 J. D. Nicholson and J. H. R. Clarke in Surfactants in Solutions, ed. K. Mittal (Plenum Press, New York, 17 K. E. van Holde, Physical Biochemistry, (Prentice-Hall, New Jersey, 1971). 18 N. Z. Atay and B. H. Robinson, unpublished results. 19 P. D. I. Fletcher, R. B. Freedman and B. H. Robinson, to be published. 20 P. L. Luisi and L. J. Magid, Solubilisation of Enzymes and Nucleic Acids in Hydrocarbon Micellar 21 N. J. Bridge and P. D. I. Fletcher, J . Chem. SOC., Furaday Trans. I , 1983, 79, 2161. 22 J. W. White, Ber. Bunsenges. Phys. Chem. 1971, 75, 379. 23 E. H. Grant, R. J. Sheppard and G. P. South, Dielectric Behazjiour of Biological Molecules in Solution 24 J. T. Koide and E. L. Carstensen, J. Phys. Chem., 1976, 80, 2526. 25 P. L. Poole and J. L. Finney, Biopolymers, 1983, 22, 255. 26 P-H. Yang and J. A. Rupley, Biochemistry, 1979, 18, 2654. 27 G. NCmethy, W. J. Peer and H. A. Scheraga, Annu. Rev. Biophys. Bioeng., 1981, 10, 459. 80, 13. 1984), Vol. 3. Solutions, in CRC Critical Reviews in Biochemistry (CRC Press, Boca Raton, Florida), in press. (Oxford University Press, Oxford, 1978). Paper 5/1232; Received 19th July, 1985