ARTICLE pubs.acs.org/Langmuir
Influence of Nanoreactor Environment and Substrate Location on the Activity of Horseradish Peroxidase in Olive Oil Based Water-in-Oil Microemulsions Evangelia D. Tzika,† Maria Christoforou,‡ Stergios Pispas,§ Maria Zervou,^ Vassiliki Papadimitriou,† Theodore G. Sotiroudis,† Epameinondas Leontidis,‡,* and Aristotelis Xenakis*,† †
Institute of Biological Research and Biotechnology, §Theoretical and Physical Chemistry Institute, and ^Institute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, 11635 Athens, Greece ‡ Department of Chemistry, University of Cyprus, PO Box 20537, 1678 Nicosia, Cyprus
bS Supporting Information ABSTRACT: Oxidative enzymatic reactions using horseradish peroxidase (HRP) were carried out in water-in-oil (w/o) microemulsions composed of olive oil/lecithin/1-propanol/ water, a model biomimetic system. The substrates used (gallic acid, octyl gallate and 2,20 -azino-bis[3-ethylbenzo-thiazoline-6sulfonic acid] (ABTS)) have different hydrophobicities and possible locations in the microemulsion system. HRP reactivity with reference to substrate hydrophobicity and structural characteristics of the microemulsions is discussed. The nature of the enzyme microenvironments was examined using dynamic light scattering (DLS), differential scanning calorimetry (DSC) and diffusion NMR (DOSY) methodologies while the location of various enzymatic substrates in the microemulsion phase was assessed by solubility measurements and by taking pressure-area isotherms of mixed monolayers of the substrates with dipalmitoyl-phosphatidylcholine (DPPC), which is a major constituent of lecithin. In contrast to the bulk aqueous phase, in the severely restricted environment of the polar domains of the microemulsion HRP reacted faster with octyl gallate, a substrate that is solubilized at the lipid interfaces. HRP was deactivated in the olive oil microemulsions within a few hours, a phenomenon that has also been observed in other microemulsion systems.
’ INTRODUCTION Virgin olive oil (VOO), as a natural oil, is a mixture of triglycerides containing also a variety of minor amphiphilic and hydrophilic components and traces of water, inducing colloidal association within the lipophilic phase.1,2 Besides, freshly prepared VOO has been shown to contain, among other minor components (e.g., mono- and diacylglycerols, phospholipids, free fatty acids, phenolics, peptides), detectable amounts of oxidizing enzymatic activities such as lipoxygenase, polyphenol oxidase, and peroxidase.3-5 With respect to both the inner colloidal structure of VOO and the presence of oxidizing activities, formulations of biocompatible water-in-oil (w/o) microemulsions, composed of VOO as the continuous oil phase, have been introduced as model biomimetic media to carry out oxidative enzymatic reactions that may naturally occur in VOO.6,7 Biocatalysis in w/o microemulsions, is one of the earliest approaches introduced to solubilize enzymes in non polar organic solvents. In line with the general trend of employing enzymes in the production of pharmaceuticals, chemicals and food ingredients, biocatalysis in biocompatible microemulsions r 2011 American Chemical Society
has gained considerable interest mainly due to the unique properties of these systems as reaction media.8,9 The main aim of the present investigation was to further examine w/o microemulsions based on VOO as media for enzymatic reactions by focusing on horseradish peroxidase (HRP). In a previous study, phase diagrams, electrical conductivity and interfacial properties of these systems have already been examined.7 However, from an applied research perspective considering such systems as nanoreactors, a more detailed structural characterization is a prerequisite. To assess the size of the dispersed polar domains as a function of water content, dynamic light scattering (DLS) measurements were carried out. Diffusion NMR spectroscopy employing the DOSY technique was used to determine the diffusivity of each component and its location in the system.10 The existence of free and/or bulk-like water in the dispersed domains was examined using differential scanning calorimetry (DSC), a method that has been successfully used in the past to probe different water populations in microemulsions.11-14 Finally, activity of HRP toward three Received: May 10, 2010 Published: February 11, 2011 2692
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Langmuir reductant substrates of different hydrophilicities has been evaluated and related to substrate location in the system. HRP is a surfaceactive, relatively stable, monomeric, heme glycoprotein, which has been successfully incorporated in various microemulsion systems and its catalytic activity has been evaluated toward different substrates in the presence of hydrogen peroxide.15-22 As evidenced from these previous studies, HRP activity in reverse micelles was strongly affected by the structural characteristics and also the composition of the system used as reaction medium. In other words, water content, rigidity of the interface, the presence of additives, the nature of the surfactants and the cosurfactants, affected peroxidase’s behavior in various ways. More specifically, the oxidation of 2,20 -azino-bis[3-ethylbenzo-thiazoline-6-sulfonic acid] (ABTS) with H2O2 has been considered a typical reaction to characterize peroxidase activities both in aqueous and waterrestricted media, because of the hydrophilicity of ABTS and H2O2, which renders them easily accessible to the entrapped enzyme molecules. In the present study we evaluated HRP activity in olive oil based w/o microemulsions toward ABTS and two other substrates, namely gallic acid and its alkyl derivative, octyl gallate (Scheme 1). Gallic acid and octyl gallate are not often used in HRP activity studies, but they have a particular relevance for the microemulsions at hand. Gallic acid is a polyphenolic compound naturally occurring in VOO,23,24 and octyl gallate is a hydrophobic derivative of gallic acid, which is a synthetic antioxidant commonly used as a food preservative.25 The hydrophobicity of the enzymatic substrates was first calculated employing octanol/water partition coefficients, also known as log P values.26,27 However, in order to determine substrate location in the presence of lipid interfaces as in the case of microemulsions, mixed monolayers of the substrates with dipalmitoyl-phosphatidylcholine (DPPC), a major component of lecithin, were formed and pressure-area isotherms were obtained.28,29
’ MATERIALS AND METHODS Materials. Soybean lecithin (Emulmetic 930), containing 92% phosphatidylcholine and a small amount of accompanying phospholipids, was supplied from Lucas Meyer Cosmetics SAS, France. Other lecithins used for Langmuir monolayer work were Epicuron 200 (a soybean lecithin from Lucas Meyer Cosmetics SAS, France, containing 96% phosphatidylcholine), L-R lecithin from egg yolk (from SigmaAldrich, containing 60% phosphatidylcholine), and 3-sn-phosphatidylcholine (from Fluka, containing >40% phosphatidylcholine). 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) 99%, was purchased from Avanti Polar Lipids. Chloroform (from Acros Organics, 99.9%) used as a solvent to prepare 1 mM solutions of mixtures of lecithins with enzyme substrates. Horseradish peroxidase (987 units/mg solid) was from Sigma-Aldich. 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) 98%, gallic acid, and octyl gallate 98% were from Sigma-Aldrich. Hydrogen peroxide (30% aqueous solution) was from Merck, Darmstadt, Germany. 1-Propanol was also from Merck. All substances were used without further purification. Extra virgin olive oil (VOO) and refined olive oil (ROO) were generously donated by ELAIS S.A.-Unilever, Greece. High-purity water was obtained either from a Millipore Milli Q Plus or from a Sartorius 611 reverse osmosis water purification system. All enzymatic reactions were carried in buffered solutions, which were a 0.2 M acetate buffer at pH = 4 for ABTS, and a 0.2 M phosphate buffer at pH = 6 for gallic acid and octyl gallate. These are the conditions for maximum activity of HRP in bulk water.30,31
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Methods. Preparation of Water-in-Oil Microemulsions. A typical VOO based w/o microemulsion was prepared by mixing virgin olive oil (VOO) or refined olive oil (ROO) with lecithin/1-propanol (1:3) at predetermined weight ratios (e.g., 90% VOO or ROO and 10% lecithin/ 1-propanol (1:3) etc.). Then appropriate amounts of water or aqueous buffer solution were added to obtain a clear reverse micellar solution. The composition of the microemulsions used was chosen to correspond to the monophasic area of the pseudoternary phase diagrams of the system determined at 25 C.7 Traces of water present in olive oil and lecithin were determined by Karl Fischer titration and taken into consideration in the calculation of the total water content of the system. Dynamic Light Scattering Measurements. Dynamic light scattering (DLS) measurements were performed in the angular range between 20 and 150, using a ALV/CGS-3 Compact Goniometer System (ALV GmbH, Germany) with a JDS Uniphase 22 mW He-Ne laser operating at 632.8 nm, and an avalanche photodiode detector. This was interfaced with a ALV-5000/EPP multitau digital correlator with 288 channels and a ALV/LSE-5003 light scattering electronics unit for stepper motor drive and limit switch control. All microemulsions were filtered through 0.5 μm PTFE filters before light scattering measurements. Autocorrelation functions from DLS measurements were collected five times at each observation angle for each sample, and were analyzed by the cumulant expansion method and the CONTIN routine. Apparent “spherical” hydrodynamic radii, Rh, at low dispersed phase concentrations were calculated from the mean diffusion coefficients using the Stokes-Einstein equation: kB T ð1Þ Rh ¼ 6πη0 Dapp where kB is the Boltzmann constant, T the absolute temperature, η0 the solvent viscosity, and Dapp the diffusion coefficient calculated from the analysis of the autocorrelation functions. NMR Spectroscopy. NMR spectra were recorded on a Varian INOVA 600 MHz spectrometer. A coaxial tube with D2O was used for lock purposes. The homonuclear 2D 1H-1H gCOSY (gradient enabled correlation spectoscopy) and 2D 1H-1H TOCSY (total correlation spectroscopy) as well as the heteronuclear 2D 1H-13C HSQC (heteronuclear single quantum coherence) and 2D 1H-13C HMBC (heteronuclear multiple bond correlation) experiments were run in order to facilitate the assignment of the peaks resonances. Experimental data were processed using VNMRJ routines (s/w platform of the V600 spectrometer). 1H detected 2D DOSY experiments were recorded at 298 K, using the bipolar pulse pair stimulated echo (Dbppste) pulse sequence of Varian library with a gradient duration of 2 ms, a diffusion delay of 200 ms and an array of 15 values for gradient strength varying between 1000 and 35 000 G cm-1. T1 measurements defined the relaxation delay between consecutive scans to 8 s. 1H spectra were collected with 256 transients. Spectra were processed using a Mnova Bayesian approximation. Microemulsion samples tested by NMR spectroscopy contained 84.1% VOO, 3.8% lecithin, 11.1% propanol, and 1% water. Differential Scanning Calorimetry (DSC) Measurements. A Q100 DSC instrument from TA Instruments was used for these measurements. A microemulsion containing 84.1% VOO, 15% lecithin/1propanol (1:3) and 0.9% water was prepared. A 6.6 mg sample of the microemulsion was introduced into the holder of the instrument. The temperature was raised at a rate of 2 C/min between -55 and þ30 C. Higher heating rates were also tried (5 and 10 C/min), but reproducible results were only obtained at 2 C/min. In the case of D2O the microemulsion contained 0.9% D2O instead of water, 7.4 mg of sample were introduced in the holder and the temperature was again changed at a rate of 2 C/min between -55 and þ30 C. Thermograms of the pure individual components of the microemulsions were also measured at a rate of 2 C/min between -55 and þ30 C. 2693
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Scheme 1. Chemical Structures of (a) Gallic Acid, and (b) Octyl Gallate
Pressure-Area Isotherms of Langmuir Monolayers. Isotherm measurements were carried out with a KSV 3000 Langmuir trough (KSV Instruments, Finland) equipped with a Wilhelmy plate for the determination of the surface pressure with an accuracy of (0.01 mN m-1. The effective trough surface area was 795 cm2 and the subphase volume was 1.2 L. All experiments were performed at (25.0 ( 0.1) C. The temperature of the subphase was maintained constant with a Julabo recirculating thermostat. Lipid monolayers were obtained by spreading on a painstakingly cleaned water surface 100 μL of a chloroform solution containing 1 mM of DPPC and X mM (where X e 0.2) mM of octyl gallate. After 15 min of evaporation time for the spreading solvent, the surface pressure (π) vs lipid molecular area (AL) isotherms were registered while compressing the monolayers at a constant speed of 10 mm/min. Different solvent evaporation times (10-30 min) and different compression speeds (2-10 mm/min) were examined as well, and were found to affect the isotherms very little. All isotherms were measured as many times as was necessary to examine monolayer stability or to obtain an accurate average isotherm. Monolayer expansion-compression experiments were also carried out in certain cases to verify the thermodynamic stability of the mixed monolayers. Enzymatic Activities in Water-in-Oil Microemulsions. Oxidation of ABTS. Enzymatic oxidation of ABTS by HRP started by adding a few microliters (μL) of a concentrated H2O2 solution in 1 g of a mixture of VOO with emulsifiers (VOO 85% w/w, lecithin/1-propanol 15% w/w) containing the enzyme and ABTS. Temperature was controlled at 25 C using a circulating water bath. The H2O2 concentration was determined by monitoring A240 using ε = 43.6 M-1 cm-1.32 Typical final concentrations (based on the total volume of the microemulsion) were [H2O2] = 0.08 mM, [ABTS] = 0.04 mM, and [HRP] = 0.14 μg/mL. The total water content of the system was adjusted by the addition of the appropriate amount of a buffered solution (0.2 M acetic buffer, pH 4) and kept constant, at 0.8% w/w, throughout the experiment. The oxidation was followed spectrophotometrically at 420 nm for several minutes. Oxidation of Gallic Acid and Octyl Gallate. Oxidation of gallic acid and octyl gallate by HRP started by adding a few microliters of a H2O2 solution in 1 g of a mixture of VOO with emulsifiers (VOO 85% w/w, lecithin/1-propanol 15% w/w) containing the enzyme and the substrate. Typical final concentrations (based on the total volume of the microemulsion) were [H2O2] = 0.065 mM, [gallic acid] = 0.058 mM, and [HRP] = 0.14 μg/mL. The total water content of the system was adjusted by the addition of the appropriate amount of a buffered solution (0.2 M phosphate, pH 6) and kept constant, at 0.8% w/w, throughout the experiment. Temperature was controlled at 25 C using a circulating water bath. The oxidation was followed spectrophotometrically at 400 nm for 9 min. When oxidation of octyl gallate was considered, the final concentration of the reactants in the system was the same as in the case of gallic acid. Initial velocities of the enzymatic oxidations in w/o microemulsions were calculated from the initial slope of the absorbance versus time, which was linear for the first few minutes. Enzymatic activity was expressed in enzymatic units per milliliter of reaction medium (EU/mL). One unit of the activity was defined as the amount of enzyme that caused an absorbance change of 0.001 per min under standard conditions.33
Figure 1. Hydrodynamic radii of water droplets versus water content in VOO/lecithin/1-propanol/water (filled squares), and ROO/lecithin/ 1-propanol/water (w/o) microemulsions (empty squares). HRP Stability in W/O Microemulsions. A 0.060 mL aliquot of a concentrated HRP solution (25 μg/mL) was added into 20 g of a mixture of VOO with emulsifiers (VOO 85% w/w, lecithin/1-propanol 15% w/w), which had been equilibrated in a water bath at 25 C for 30 min. Samples, 1 g each, were withdrawn every 60 min, and the appropriate substrates were added in the reaction mixture: (a) 2 μL of gallic acid solution (10 mM) and 2 μL of H2O2 solution (1 mM); (b) 2 μL of octyl gallate solution (10 mM) and 2 μL of H2O2 solution (1 mM). In both cases, the oxidation reactions were followed spectrophotometrically at 400 nm for 5 min. Final overall concentrations, referring to the total volume of the system, were [HRP] = 0.07 μg/mL, [gallic acid] = 0.018 mM, [octyl gallate] = 0.018 mM, [H2O2 ] = 0.002 mM. Solubility Studies of Gallic Acid and Octyl Gallate. Solubility studies of gallic acid and octyl gallate in water, 1-propanol, VOO and mixtures of VOO with lecithin/1-propanol (85%-15% w/w) were carried out by adding 5 mg of each compound to screw capped vials containing increasing amounts of each medium. The vials were shaken at 25 C for 48 h until equilibrium was reached. The experiments were performed in triplicate.
’ RESULTS AND DISCUSSION Characterization of the Nanoreactors. The phase diagrams of w/o microemulsions based on olive oil (either virgin or refined) as the continuous phase, lecithin as surfactant and 1-propanol as cosurfactant have been published before.7 The amount of water that can be solubilized in these systems is quite small (less than 2% by weight at best). As it has been shown in previous studies, the structure of the dispersed polar domains of lecithin-alcohol based w/o microemulsions depends on both the water content and the nature of the alcohol used as cosurfactant34,35 In the present study, the restricted environment of the polar domains in VOO w/o microemulsions has been examined using a combination of techniques such as DLS, DOSY-NMR, and DSC. 2694
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Figure 2. 2D DOSY map of the microemulsion. 1H resonance peak assignment is described as follows: (a) olefinic (VOO þ L); (b) >CHOH glyceryl group (sn-2) (VOO þ L); (c) residual water; (d) -CH2OH glyceryl group (sn-1) (VOO þ L); (e) -CH2OH glyceryl group (sn-3) (VOO þ L); (f) -CH2OH (P); (g) -N þ (CH3)3 (L); (h) bis-allylic -CH2- (VOO þ L); (i) -OCH(dO)-CH2- (VOO þ L); (j) allylic -CH2- (VOO þ L); (k) -OCH(dO)-CH2CH2- (VOO þ L); (l) -CH2CH2OH (P); (m) aliphatic -(CH2)n- (VOO þ L); (n) terminal -CH3 (VOO þ L þ P). The letters in parentheses denote the component of the microemulsion following the code: VOO, virgin olive oil; L, lecithin; P, propanol.
Dynamic Light Scattering (DLS) Studies. Dynamic light scattering measurements were carried out in order to evaluate the size of the dispersed polar domains in olive oil microemulsions as a function of water content. For both microemulsion series (using VOO or ROO as the continuous phase) the hydrodynamic radius of the droplets increases almost linearly with increasing water content in the microemulsions (Figure 1) and up to the highest possible water content (1.6 or 1.8% w/w depending on the type of oil). Radii in the range of 60 to 150 nm were observed leading to average diameters between 120 and 300 nm for the dispersed polar domains. An expected parallel increase in the scattering intensity was also evident as a result of the increase in the water concentration. The determined sizes of the dispersed polar domains fall within the range observed in a previous preliminary investigation.7 In another DLS study of similar systems based on limonene/lecithin/propanol/water, comparable sizes were also observed and correlated with the formation of structures with large effective diameters.34 Judging from the large apparent size of the droplets the formation of wormlike structures, such as those found in simpler lecithin-stabilized microemulsions is highly probable.36 It is also known that lecithin in fatty acid esters is able to form cylindrical aggregates upon the addition of small quantities of water.37 However, because of the complexity of the system (we are dealing with multicomponent mixtures, with more than four components, instead of the cleaner threecomponent system studied in refs 36 and 37) and the large observed polydispersity (from a cumulant analysis, the μ2/Γ2 values are in the range 0.4-0.6, while shape effects on light scattering data are known to be smeared out due to system polydispersity) we did not make any attempt to fit the data with more elaborate shape models. We regard the “hydrodynamic sizes” determined by DLS through eq 1 essentially as hydrodynamic correlation lengths that provide information on the structural changes taking place in these systems.36 In other words, the large hydrodynamic sizes observed may also be
correlated with the formation of, e.g., wormlike or cylindrical structures, with large effective diameters. It is worth mentioning that diffusing species were observed in the case of VOO/lecithin/propanol and ROO/lecithin/propanol mixtures even without added water. This may be attributed to the inherent humidity of the mixtures (0.15% w/w for VOO and 0.10% w/w for ROO based systems) and the possible participation of the cosurfactant 1-propanol in the formation of the dispersed domains. Another interesting observation from the DLS measurements in these microemulsion systems is that the size of the dispersed polar domains, at constant lecithin, propanol and water contents, is affected by the type of the oil used as the continuous phase. In other words, when refined olive oil was used, dispersed domains of larger sizes were observed than in the case of virgin olive oil microemulsions (Figure 1). Similar findings were also reported in a previous preliminary investigation of analogous systems.7 This may be attributed to the presence of endogenous amphiphilic molecules in the VOO matrix, which are not present in refined olive oil, having been removed during the refining process.1,2 These amphiphiles presumably contribute to the formation of a larger water/oil interface in the case of VOO thus decreasing the size of dispersed domains, since the later, at constant water content, is a reverse analogue of the systems total interface. ROO has not been further used in this study; it was used in the DLS measurements to illustrate the complexity of VOO. To conclude, DLS measurements of olive oil based w/o microemulsions, indicated the existence of diffusing species even in the absence of externally added water, showing a possible participation of the cosurfactant in the inner phase. The size of the dispersed polar domains increases almost linearly with increasing water content. The observed large sizes of the dispersed domains may be correlated with formation of wormlike or cylindrical structures, with large effective diameters. To further elucidate the propanol location in the system, DOSY-NMR measurements were carried out. 2695
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Table 1. log P Values of Gallic Acid and Octyl Gallate log P value
measurement method
2.11
distribution experiment using dialysis tubing44
3.72
TSAR 3D program45
4.63
chromatography retention time46
Octyl Gallate
Gallic Acid -0.53
Figure 3. Thermograms (heating curves at 2 C/min) of a waterlecithin-1-propanol-VOO microemulsion (dashed line) and the corresponding three-component mixture lecithin-1-propanol-VOO (solid line).
DOSY-NMR Measurements. DOSY spectroscopy has been established as a method to distinguish the different components of a mixture along the chemical shift axis based on the different diffusion coefficients of the distinct components.10 Besides, its use has been expanded to detect the presence of assemblies or to investigate microemulsions structure.38,39 The 2D DOSY map is displayed in Figure 2 with the 1H spectrum shown in the horizontal projection. Resonance peaks assignment has been assisted by literature data and the use of homonuclear and heteronuclear 2D spectroscopy.40,41 A major component associated with a diffusion coefficient of (Ddiff = 6.44 10-8 cm2 s-1) includes traces corresponding to the resonance peaks of olive oil, lecithin, and propanol and confirms the formation of a microemulsion consisting of these components. Furthermore, a smaller component is associated with a phase including propanol and water and appears to move faster with a diffusion coefficient 1 order of magnitude larger (Ddiff = 7.59 10-7 cm2 s-1). From the DOSY-NMR measurements we can conclude that in the VOO based w/o microemulsions of the present study, propanol exists in two forms: (a) propanol that participates, together with lecithin, in the formation of the interface and (b) propanol mixed with water that participates in the formation of the dispersed polar domains. Differential Scanning Calorimetry (DSC) Studies. Thermograms of the individual components of the microemulsions (H2O, D2O, VOO, 1-propanol, Emulmetic B930 lecithin) in the range between -55 and þ30 C were measured first. The endothermic peaks for H2O and D2O were found at 1.3 and 4.3 C illustrating the small offset of the instrument used. The VOO, 1-propanol, and lecithin thermograms are appended as Supporting Information (Figures S1-S3). The thermogram of a microemulsion containing 84.1% VOO, 15% lecithin/propanol (1:3), and 0.9% water can be seen in Figure 3, where another thermogram for a system containing the three pseudocomponents, but without any added water, is added for comparison. An identical thermogram is also obtained when H2O is replaced by D2O to form the microemulsion (results not shown). The thermograms contain an endothermic peak at -14 C, a broad double endothermic peak with components at -11 and -6 C, and a broad endothermic peak at þ4 C. On the basis of the thermograms of the individual components, the peak at
distribution experiment using dialysis tubing44
0.43
chromatography retention time45
0.89
TSAR 3D program46
-14 C is due to 1-propanol, and the three other peaks are related to aliphatic chain ordering and appear in the thermograms of 1-propanol, lecithin, and VOO. No water peak is clearly visible close to zero, which implies the absence of bulk-like water in these systems. This is expected, since the microemulsion has a weight content of 3.8% lecithin and roughly 1% water. By assuming an average lecithin molecular weight of roughly 800 g/mol, this implies a molar ratio of water to lipid Nw/NL = (1/18)/(3.8/800) ≈ 11, which is at the lowest limits of the “full” hydration ratios expected for phospholipids, typically in the range of 10-30.42 For water-lecithin-cyclohexane microemulsions Willard et al. demonstrated the absence of free water for water to lipid ratios smaller than 5.43 No novel peak is found in the microemulsion thermogram, which is practically identical to that of the system without added water. It is not clear if the absence of other peaks is due to the low water content in these microemulsions, or if bound-water peaks are actually hidden under the broad peaks of the other (majority) components. The related literature is not very helpful since most DSC measurements have been carried out in microemulsions with water contents much higher (>10% w/w) than those in the present microemulsions.11-14 The clear message of the DSC measurements is that no bulk-like water exists in these systems. Enzyme Substrates: Hydrophobicity and Behavior in Mixed Monolayers with DPPC. Table 1 contains log P values of gallic acid and octyl gallate, as reported in the recent literature.44-46 The log P values for gallic acid are obviously pH-dependent since its ionization percentage changes with pH (the pKa for the -COOH group is equal to 4.4, although smaller values have also been reported,47 that of the phenolic -OH groups is roughly equal to 10.0). At the conditions of the present experiments (pH ≈ 6) the solute is expected to be almost 100% ionized at the carboxyl group. Gallic acid is thus expected to reside in the polar domains of the microemulsions. The available log P values for octyl gallate illustrate its very considerable hydrophobicity. The aqueous solubilities of gallic acid and octyl gallate are 11.5 and 0.072 g L-1 respectively.48,49 In parallel, solubility studies of gallic acid and octyl gallate in VOO w/o microemulsions at the experimental conditions of the oxidation reactions were carried out to better understand substrate’s location in the nanoreactors. The solubilities of octyl gallate in water (practically insoluble), in VOO (solubility of 2.1 mg/g), and in a mixture of 85% w/w olive oil, and 15% w/w 1:3 lecithin/propanol (solubility of 170 mg/g) at 25 C indicate that almost 99% w/w of the substrate is solubilized at the lipid-alcohol interface. When the same experiment was carried out for gallic acid, it was found practically insoluble in both VOO and the mixture of VOO and emulsifiers, thus indicating that the 2696
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Figure 4. Surface pressure/area isotherms at 298 K of mixed monolayers of octyl gallate and DPPC with variable molar composition (0% to 16.7% octyl gallate).
only possible localization in a microemulsion is in the aqueous pseudophase. The log P index or the solubility do not always allow direct predictions about the location of a compound in a system containing lipid interfaces. We have therefore undertaken measurements of surface pressure/area isotherms of mixed monolayers of octyl gallate with lecithin. Gallic acid does not form stable monolayers by itself, and it does not affect DPPC monolayers in a measurable way, showing a strong propensity for water (results not shown). Unfortunately, the Emulmetic B930 lecithin used for the microemulsion formulation does not form stable monolayers at the air-water interface, as evidenced by the lack of reproducibility of the pressure-area isotherms, and by the shifting hysteresis loops in compression-decompression experiments (see Figures S4 and S5 in the Supporting Information). Other commercial lecithins, such as Epicuron 200, L-R-phosphatidylcholine from egg yolk, or 3-sn-phosphatidylcholine were also found to form unstable monolayers at the air-water interface (results not shown). It was therefore considered necessary to use the pure lipid dipalmitoyl-phosphatidyl choline (DPPC) as a stable monolayer forming lipid to emulate the actual monolayer of the microemulsion domains in a satisfactory way. Figure 4 contains pressure area isotherms of mixtures of DPPC with octyl gallate. The isotherms are stable and reproducible, proving that these two compounds mix well in the monolayers. The solutions, from which the octyl gallate/DPPC mixtures were spread as monolayers contained 1 mmol/L DPPC and from 0 to 0.2 mmol/L octyl gallate. A clear indication that octyl gallate expands the DPPC monolayer is already visible at an octyl gallate mole fraction of 2.4%. These mole fractions are considerably larger than those used experimentally (typically the octyl gallate/lecithin fraction used in the VOO microemulsions is less than 0.1%). However, the isotherms of Figure 4 clearly illustrate the affinity of octyl gallate for lipid monolayers. Unfortunately, octyl gallate does not form stable Langmuir monolayers by itself (results not shown), hence it is not possible to calculate the free energy of mixing of octyl gallate with DPPC in the mixed monolayers (the relevant formulas presuppose that each component by itself forms a stable insoluble monolayer28,29) and to obtain useful thermodynamic properties of mixing. HRP Activity in Olive Oil W/O Microemulsions. Oxidation of ABTS. Oxidation of ABTS catalyzed by HRP in the presence of
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Figure 5. Initial velocity of ABTS oxidation by HRP in the presence of H2O2 in VOO w/o microemulsions. [ABTS] = 0.04 mM, [H2O2] = 0.08 mM, [HRP] = 0.14 μg/mL, pH 4.0, and 25 C.
Table 2. Activity of HRP, Expressed in Enzyme Activity Units (EU) per mL, both in Aqueous Solution and VOO W/O Microemulsions, for Different Substrates at Specific Wavelengthsa enzyme activity (EU/mL) substrate ABTS gallic acid octyl gallate
aqueous buffer solution
VOO w/o microemulsions
56 ( 4
2.2 ( 0.6
(pH 4, λmax = 414 nm)
(pH 4, λmax = 420 nm)
2.4 ( 0.3
3.1 ( 0.1
(pH 6, λmax = 379 nm)
(pH 6, λmax = 400 nm) 4.1 ( 0.2 (pH 6, λmax = 400 nm)
[HRP] = 0.14 μg/mL, [ABTS] = [0.04] mM, [gallic acid] = 0.058 mM, [octyl gallate] = 0.058 mM, and 25 C.
a
H2O2 in VOO w/o microemulsions was studied by monitoring the increase of absorbance at 420 nm (Figure S6 in Supporting Information) due to the formation of the radical cation, ABTSþ. The reaction was followed for several minutes (Figure 5) and enzyme activity was found equal to 2.2 ((0.6) EU/mL (Table 2). HRP activity recorded in VOO microemulsions was lower than the corresponding activity measured in an aqueous buffer solution under identical experimental conditions (pH, temperature and reactant concentrations). In aqueous buffer solution, formation of the radical cation, ABTSþ•, was monitored at 414 nm and enzyme activity was found 56 ((4) EU/mL (Table 2). Similar behavior of HRP has been reported by Noritomi et al.50 when HRP activity toward hydroquinone oxidation in AOT-n-octane reverse micelles was considered. The optimal Vmax value in reverse micelles was twice lower than that observed in aqueous solution. In addition, circular dichroism measurements of HRP both in aqueous and reverse micellar environments indicated conformation changes of the protein as the water content of the reverse micelles decreased. In another study carried out by Jurgas-Grudzinska and Gebicka,19 HRP was entrapped in Ipegal reverse micelles at different water contents and its activity toward ABTS oxidation was measured. When the water content of the system was quite low, peroxidase activity was found lower in reverse micelles than in aqueous buffer solution. 2697
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Figure 6. Initial velocities of the oxidation reactions of gallic acid (empty circles) and octyl gallate (filled circles) by HRP in VOO w/o microemulsions. [Gallic acid] = 0.058 mM, [octyl gallate] = 0.058 mM, [H2O2] = 0.065 mM, pH 6.0, and 25 C.
Figure 7. Time-dependent stability of HRP in VOO/lecithin/1-propanol/water w/o microemulsions at 25 C for gallic acid (filled circles) and octyl gallate (empty cirles). The activity was expressed relative to the initial activity (100%). Experimental conditions: [HRP] = 0.07 μg/mL, [gallic acid] = 0.018 mM, [octyl gallate] = 0.018 mM, and [H2O2] = 0.002 mM.
Although HRP is an enzyme which has been shown to exhibit, in general, increased activity when solubilized in reverse micelles, this “superactivity” is always related to high water contents of the microheterogeneous system.16,17,21,51 In other words, there seems to be a correlation between enzyme activity and the nature of water present in the interior of reverse micelles. When the water content is low, there is not enough free water inside the micelle to hydrate the enzyme since water molecules are mostly bound to the surfactant polar heads. In that case, the enzyme exhibits lower activity than in bulk water. On the contrary, when there is enough water to hydrate both surfactant polar heads and protein molecules, enzyme superactivities can be observed. In other words, HRP must be sufficiently hydrated to be fully active.20 In the present study, water in VOO microemulsions, contain remarkably low amounts of water and as verified from the DSC experiments no bulk water can be detected. This finding is therefore in accordance with the decreased enzymatic activity of HRP in VOO reverse micelles. Unfortunately, in these systems, it was not possible to study the effect of different water contents on enzyme activity since the ability of the system to incorporate water was further decreased when buffer, enzyme, and substrate solutions were added. In addition, VOO w/o microemulsions contain almost 11% w/w propanol which partially serves as cosurfactant but also participates in the formation of the inner phase as indicated from the DOSY-NMR studies. In this regard, apart from possible conformational changes induced to the protein molecules due to the absence of free water and the presence of propanol, substrate solubility in the media should be also reconsidered. ABTS, although highly soluble in water (50 mg/mL), is practically insoluble in pure propanol and olive oil. From this point of view, the observed decrease in HRP activity also stem from the reduced solubility of ABTS in the interior of the reverse micelles. Oxidation of Gallic Acid and Octyl Gallate. The oxidation reaction of both gallic acid and octyl gallate in VOO w/o microemulsions was followed by monitoring the increase in absorbance at 400 nm for a few minutes. The initial velocities of the oxidation reactions were calculated from the slopes of the corresponding plots of absorption versus time (Figure 6). Enzyme activity was found equal to 3.1 ((0.1) EU/mL for gallic acid and 4.1 ((0.2) EU/mL for octyl gallate (Table 2). These values are comparable to the ABTS value in VOO
microemulsions, which is not expected, since ABTS is in a way an “ideal” substrate for HRP in bulk water. For comparison reasons, HRP-catalyzed oxidation of gallic acid was also carried out in aqueous buffer solution under the same experimental conditions as in reverse micelles. Product formation was followed at 379 nm and enzyme activity was found equal to 2.4 ((0.3) EU/mL (Table 2). This value is considerably lower than the initial rate of ABTS oxidation in aqueous solution. These results for HRP activity in aqueous solutions are not surprising, since plant peroxidases (class III) e.g olive peroxidase, are characterized by similar high rates for ABTS oxidation and remarkably lower activity for gallic acid and other polyphenolic substrates.5 From the activity studies of HRP toward gallic acid and octyl gallate in comparison with ABTS both in aqueous solution and in VOO microemulsions the following observations were made. In aqueous solution, HRP is more active toward ABTS than gallic acid which is not surprising since, as mentioned above, polyphenolic substrates are less specific peroxidase substrates.5 Nevertheless, in the restricted environment of VOO w/o microemulsions, HRP is less active toward ABTS than the two polyphenolic substrates. In other words, there seems to be a change of peroxidase specificity induced by the absence of bulk water and the presence of considerable amounts of propanol. In a study of HRP activity in water-alcohol mixtures, it was shown that the enzyme retained catalytic activity although conformation changes affected the course of the enzymatic reaction.52 On the other hand, HRP is less active toward gallic acid than toward its amphiphilic alkyl derivative, octyl gallate. From the monolayer results of Figure 4 a strong affinity of octyl gallate for the lipid interfaces is evidenced. On the contrary, both ABTS and gallic acid, even in the presence of the DPPC monolayer, prefer the aqueous phase. When water-in-oil microemulsions are considered as a medium for enzymatic catalysis, substrate polarity is of great importance since it affects solubility, partitioning and transport of the substrate to the entrapped enzyme molecules.53,54 Octyl gallate possesses a lipophilic alkyl chain that allows the molecule to anchor at the lecithin-alcohol membrane with its three -OH groups oriented toward the water core. These functional groups are apparently more accessible to the HRP active center compared to the -OH groups of gallic acid. It is also possible that the confined space hinders the mobility of the enzyme, producing frequent 2698
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Langmuir collisions with the walls, and thus enhanced reactivity toward substrates solubilized at the interface. Finally, since the presence of propanol in the inner phase of VOO microemulsions was verified, substrate solubility in pure propanol was tested. A significant variation of solubilities was found (1 g/mL for octyl gallate, 130 mg/mL for gallic acid, and ABTS practically insoluble) indicating a correlation between substrate solubility and enzyme activity. To conclude, although polyphenols with two or three -OH in the benzoic ring are less specific substrates than ABTS for measuring peroxidase activity in aqueous solution, in the restricted environment of VOO microemulsions these differences were considerably reduced. In other words, substrate specificity of HRP is strongly affected by the water-restricted and propanolrich microenvironment of lecithin-based reverse micelles in olive oil, in favor of the more hydrophobic substrates. HRP Stability in the Olive Oil Based W/O Microemulsions. The time course of horseradish peroxidase (HRP) activity has been investigated in VOO w/o microemulsions for the oxidation of gallic acid and octyl gallate in the presence of hydrogen peroxide. Formation of the oxidation products was followed, in both cases, at 400 nm. Initial velocities were calculated at 1 h time intervals. The results are shown in Figure 7. As it can be observed in Figure 7, HRP was inactivated within few hours of incubation in olive oil/lecithin/propanol/water microemulsions. In the case of gallic acid, roughly 70% of HRP activity was already lost within 1 h of incubation and complete inactivation was found after 3 h of total incubation time. On the contrary, when octyl gallate was used as enzyme substrate, 93% of the initial enzyme activity was retained after 1 h of incubation in the microemulsion system. In this case, enzyme inactivation was slower and total loss of activity occurred after 4 h of total incubation time. According to the literature, enzyme stability in microemulsions depends mostly on the composition of the phase.55 In other words, the water content of the system, the nature of the organic solvent, the nature of the surfactants, and also the presence of substrates and other additives, strongly affect the stability of the entrapped enzyme molecules. More specifically, protein hydration, among other parameters, seems to play an important role in the catalytic activity and conformational stability of the enzyme. In the present study, the strong ionic interactions between the lecithin head groups and the enzymes, and also the presence of alcohol at the droplet interfaces and in the polar domains may lead to a rapid protein denaturation process. Interestingly, the degree of peroxidase’s deactivation in VOO reverse micelles is much less when a substrate solubilized at the lipid interfaces, namely octyl gallate, was considered.
’ CONCLUSIONS The dispersed polar domains of olive oil-based w/o microemulsions serve as nanoreactors for oxidative enzymatic reactions involving the surface active horseradish peroxidase and three reductant substrates of different hydrophilicities. HRP has been found active in these microemulsion media. HRP was more active toward octyl gallate, a compound that is almost exclusively solubilized at the lipid interfaces of the microemulsions (as evidenced by solubility and surface monolayer studies) than toward gallic acid or ABTS, compounds solubilized exclusively in the polar domains. This must be attributed to the very restricted space in the water pools and the absence of free water (as evidenced by DSC), to the local enhancement of octyl gallate at
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the interfaces (as evidenced by pressure-area isotherms of mixed monolayers), and also to the presence of propanol in the inner phase of the reverse micelles (as verified by DOSYNMR). The same factors that modify the selectivity of the enzyme could also lead to its rapid inactivation. The size of the dispersed polar domains in VOO w/o microemulsions was evaluated by dynamic light scattering (DLS) and the existence of structures with large effective diameters was shown. In the present study, VOO based w/o microemulsions were further structurally characterized using a variety of techniques in a complementary way. Observations made could of interest in the perspective of future biotechnological applications.
’ ASSOCIATED CONTENT
bS
Supporting Information. Thermograms of each component of the microemulsion (virgin olive oil, lecithin, 1-propanol), isotherms of lecithin Langmuir monolayers, and the timedependent spectra following the kinetics of ABTS reaction with HRP in VOO based w/o microemulsuions. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION *Telephone: þ302107273762. Fax: þ302107273758. E-mail:
[email protected].
’ ACKNOWLEDGMENT The authors acknowledge funding from a Greece-Cyprus bilateral research grant issued by the General Secretariat of Research and Technology, Greece (06CYP-78), and the Research Promotion Foundation of Cyprus (Greece-Cyprus bilateral research project, Contract KY-EΛ/0406/65). E.L. and M.C. would also like to acknowledge additional financial support from the University of Cyprus. ’ REFERENCES (1) Sotiroudis, T. G.; Sotiroudis, G. T.; Varkas, N.; Xenakis, A. J. Am. Oil Chem. Soc. 2005, 82, 415–420. (2) Xenakis, A.; Papadimitriou, V.; Sotiroudis, T. G. Curr. Opin. Colloid Interface Sci. 2010, 15, 55–60. (3) Georgalaki, M. D.; Sotiroudis, T. G.; Xenakis, A. J. Am. Oil Chem. Soc. 1998, 75, 155–159. (4) Georgalaki, M. D.; Bachmann, A.; Sotiroudis, T. G.; Xenakis, A.; Porzel, A.; Feussner, I. Fett/Lipid 1998, 100, 554–560. (5) Tzika, E. D.; Sotiroudis, T. G.; Papadimitriou, V.; Xenakis, A. Eur. Food. Res. Technol. 2009, 228, 487–495. (6) Papadimitriou, V.; Sotiroudis, T. G.; Xenakis, A. J. Am. Oil Chem. Soc. 2005, 82, 1–6. (7) Papadimitriou, V.; Sotiroudis, T. G.; Xenakis, A. Langmuir 2007, 23, 2071–2077. (8) Xenakis, A.; Papadimitriou, V.; Stamatis, H.; Kolisis, F. N. In Microemulsions. Properties and Applications; Fanun, M., Ed.; Surfactant Science Series; Taylor & Francis CRC Press: Boca Raton, FL, 2009; Vol. 144, p 349. (9) Garti, N. Curr. Opin. Colloid Interface Sci. 2003, 8, 197–211. (10) Thrippleton, M. J.; N. M. Loening, N. M.; Keeler, J. Magn. Reson. Chem. 2003, 41, 441–447. (11) Garti, N.; Aserin, A.; Ezrahi, S.; Tiunova, I.; Berkovic, G. J. Colloid Interface Sci. 1996, 178, 60–68. (12) Garti, N.; Aserin, A.; Tiunova, I.; Fanun, M. Colloids Surf. A 2000, 170, 1–18. (13) Podlogar, F.; Gasperlin, M.; Tomsic, M.; Jamnik, A.; Bester Rogac, M. Int. J. Pharm. 2004, 276, 115–128. 2699
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