J. Phys. Chem. B 2004, 108, 9801-9810
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Changes in Microemulsion and Protein Structure in IgG-AOT-Brine-Isooctane Systems Natalia I. Gerhardt and Stephanie R. Dungan* Department of Food Science and Technology and Department of Chemical Engineering and Materials Science, UniVersity of California, DaVis, California 95616 ReceiVed: March 18, 2004
Changes in structure of protein molecules and water-in-oil (w/o) microemulsion aggregates were investigated using the large protein immunoglobulin G (IgG, MW 155,000) and an equivolume oil/water mixture composed of brine, sulfosuccinic acid bis[2-ethylhexyl]ester (sodium salt) (AOT), and isooctane. The protein solution in the microemulsion phase was metastable: over time this solution changed, as protein and w/o droplets aggregated and precipitated to the interface between aqueous and organic phases. Such factors as AOT concentration, temperature, and salt concentration were found to influence the protein and surfactant structures in the microemulsion. Protein conformation was probed using circular dichroism spectroscopy whereas the microemulsion structure was determined from dynamic light scattering measurements. Protein conformation and microemulsion structure were found to have significant effects on protein stability in the microemulsion. The stabilizing effects of clusters formed at higher salt and/or AOT concentrations are discussed. IgG adopts an intermediate denatured state in the microemulsion phase close to the alternatively folded state known as the A state, with well-defined contacts in the tertiary structure immediately after phase equilibration. The change in protein conformation with time accompanied by the cluster growth eventually leads to the protein and surfactant transfer into a third, solid middle phase from the organic solution.
Introduction Protein interactions with surfactants in self-assembled solutions have been an area of active research, both in aqueous solution1,2 and in organic solvents.2-7 This interest is motivated by the frequent combination of proteins with amphiphiles in cleaning, pharmaceutical, and food products, as well as in biological tissues, and by potential protein separation and biocatalytic applications. For hydrophobic surfactants that selfassemble to form water-in-oil microemulsions, early studies on solubilization of water-soluble proteins R-chymotrypsin and cytochrome c suggested that the protein could perturb the droplet structure, although there was considerable debate as to the qualitative nature of that perturbation8,9 (see ref 10 for review). More recent studies have shown that under the appropriate conditions, the presence of proteins can substantially alter the overall phase boundaries of the oil-water-surfactant mixture11-21 and induce droplet clustering.15,17-19,21,22 To understand these effects, we explore the connection between solution microstructure, protein conformation, and system properties in an oil/water system whose phase structure is slowly changing due to the presence of protein. The ability of proteins to alter surfactant self-assembly can be compared to the influence of polymers in surfactant solutions, a topic that has also been extensively studied. The addition of selected amphiphilic and large neutral polymers to an oilcontinuous microemulsion generates an association. Polymer can form either bridged or looped configurations with surfactant aggregates, where a significant hydrophobic portion of polymer is exposed to the organic solvent.23-25 The ability of small amounts of the large protein IgG to change the microstructure of AOT/isooctane microemulsions appears similar to that noted * Corresponding author. Telephone: 530-752-5447. Fax: 530-752-4759. E-mail:
[email protected].
with these polymers. The water soluble IgG also causes association of microemulsion droplets and bridging of these droplets, but because it has well-defined secondary and tertiary structure, the protein is perhaps more sensitive to exposure to organic AOT solution, resulting in precipitation to a third phase.22 We are interested in elucidating the differences between large and amphiphilic polymers and protein macromolecules in their interaction with microemulsions, and in particular the role that molecule size, hydrophobicity, and conformational effects play in inducing structural changes and the formation of a third, solid phase. Loss of solubilization of proteins in the microemulsion due to precipitation is a frequently observed phenomenon in the extraction of proteins into a reversed micellar phase. Protein precipitation is especially observed at low pH, low AOT concentration, and sometimes at low salt concentrations.12,26,27 Yet the mechanisms leading to a loss in protein solubility in the microemulsion phase remain poorly understood. The time scale of solubilization of the very large protein IgG in an AOTcontaining microemulsion seems to be significantly smaller in comparison to that of other proteins.11,22 This fact makes IgG a useful model protein for exploring the time-dependent behavior of the microemulsion in a comparatively short experimental time period. Results of our previous research show that the phase behavior of bovine IgG in water-in-oil (w/o) microemulsions is strongly dependent on salt concentration.22 At low salt concentration ([NaCl] < 0.2 M), where the surfactant tends to form droplets larger than IgG, the protein is not stable in the microemulsion phase, as manifested in protein and surfactant precipitation from the organic solution. Within this salt concentration range, we suggest that the protein resides within a single microemulsion droplet. At higher salt concentrations where the droplets formed in the absence of protein would be smaller than the protein
10.1021/jp040231i CCC: $27.50 © 2004 American Chemical Society Published on Web 06/10/2004
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Gerhardt and Dungan showing the proposed size effects on protein incorporation in the droplet. Protein stability at high salt concentration, as manifested through the absence of protein precipitation from the organic phase, appears to correlate with thermodynamic stability and protein activity in oil-continuous microemulsions noted in other studies. A microcalorimetry study of lysozyme, cytochrome c, and ribonuclease in a microemulsion phase shows that the thermodynamic stability of proteins in the organic solution tends to decrease with increasing wo.28 Here wo is the molar ratio of water-to-surfactant in the organic solution, a quantity that will decrease with increasing salt concentration. Proteins in microemulsions appear to exhibit their maximal enzymatic activity at levels of water content when the size of protein is close to that of the water pool of the microemulsion droplet.8,9 It has also been demonstrated that a catalytic human IgG antibody retains its activity when solubilized in a microemulsion.29 Maximum antibody activity was observed at a wo value of 28, a value consistent with the large molecular weight of IgG molecules. On the basis of geometric considerations, a wo value of 28 corresponds to a radius of approximately 55 Å, which is the size of an IgG molecule.30 Given these dependencies of protein stability, solubility, and activity on the protein-to-droplet size ratio, we are interested in exploring the role of protein/surfactant interactions on protein stability in the microemulsion phase. It is reported that at low AOT-to-protein loading cytochrome c tends to precipitate from the organic phase.11 This result was attributed to denaturation of cytochrome c followed by adsorption of more AOT molecules in the microemulsion. Interestingly, however, at higher AOTto-cytochrome c loading, cytochrome c did not precipitate from the organic phase within 2 weeks. The reason for this remains unclear; the surfactant may better shield the denatured cytochrome c at higher concentrations or changes in protein conformation may occur more slowly at higher [AOT]. The physicochemical properties of the entrapped protein have been studied using far- and near-UV circular dichroism (CD) and dynamic light scattering (DLS). Temperature effects on the protein conformation, on changing protein charge properties (pI) and on stability in the microemulsion phase are discussed. The role of surfactant in influencing protein stability in the microemulsion was explored by examining changes in protein conformation and microemulsion structure. To avoid any influence of precipitation on the CD and DLS measurements, conditions were chosen to ensure that protein did not precipitate during these measurements. These conditions corresponded to high AOT and NaCl conditions. The protein did not precipitate for at least 24 h after the phase equilibrium experiment.
Figure 1. Ribbon structure of native IgG, showing overall Y-shape and high degree of β-sheet secondary elements (Protein Data Bank). Protein structures are presented to scale with cartoons of (a) 3 nm, (b) 5 nm, and (c) 9 nm w/o microemulsion droplets, showing proposed arrangement of IgG with droplets.
molecule, a more stable IgG-containing microemulsion is obtained. In this salt concentration range, IgG is believed to promote attractive interactions between droplets, causing IgG to bridge more than one drop. Figure 1 presents a scale drawing of both protein and microemulsion droplets of different sizes,
Materials and Methods Materials. Purified bovine IgG (reagent grade) was obtained from Sigma Chemical Co. (St. Louis, MO). AOT (99% pure, sulfosuccinic acid bis[2-ethylhexyl] ester, sodium salt) was also purchased from Sigma. Karl-Fisher reagents were purchased from Allied Signal Inc. (Morristown, NJ). Isooctane and other chemicals (reagents for buffer, salts) were of reagent grade and obtained from Fisher Chemical Co. (Pittsburgh, PA). Equilibration Experiments. Aqueous protein solutions were prepared in doubly distilled water and adjusted to a desired pH and salt concentration by using a 0.05 M sodium acetate buffer over a pH range 4.8-5.8, by using a 0.05 M sodium phosphate buffer over a pH range of 5.8-7.0, and by adding sufficient NaCl to obtain the desired sodium ion concentration. Most experiments were carried out using a protein concentration of 1 g/L of water in the system.
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The aqueous IgG solution was mixed with an equal volume of an organic solution containing the anionic surfactant AOT in isooctane. Each solution had an initial volume of 1.0 mL. The solutions were mixed in a glass vial held at constant temperature in a jacketed vessel, with mixtures stirred at 500 rpm for 30 min with a magnetic stirrer. No further change in protein concentration was observed for contacting times longer than 30 min. The dispersion was then centrifuged for 10 min at a constant set temperature, as indicated. Samples were either analyzed immediately or held at temperatures as indicated for various times before analysis. In both cases the phases were separated just before the analysis. Analytical Techniques. Protein concentrations in each phase were determined by ultraviolet (UV) absorption at 280 nm on a UV-visible spectrophotometer (Shimadzu UV 160U, Kyoto, Japan) at 25 °C. Readings were corrected by subtraction of UV absorption at 310 nm.31 Standard curves were prepared for aqueous and organic phases. In the latter case, a small known amount of concentrated protein solution was injected into the organic phase. The water content of the microemulsion phase was determined on a Karl Fisher titrator DL18 (Mettler, Hightstown, NJ), and results are expressed as wo ) [H2O]/ [surfactant], where [H2O] and [surfactant] are the molar concentrations of water and AOT, respectively, in the organic phase. Dynamic Light Scattering. Dynamic light scattering (DLS) measurements were made on a Brookhaven instrument (Holtsville, NY) using a BI-200SM goniometer and the BI-9000 AT 155-channel digital correlator. The incident laser beam (λ ) 532 nm) was vertically polarized. All intensity autocorrelation functions g(2)(t) were measured at an angle of 90° and at a temperature of 24 °C. We accepted only those autocorrelation functions where the measured baseline, i.e., the averaged value of the correlation function at very long delay times, agreed with the computed baseline to within 0.1%. Samples were filtered through a 0.1 µm Millipore filter and/or centrifuged for 10-20 min in the scattering cell to remove any dust present. The measured scattered intensity correlation function g(2)(t) is related to the function g(1)(t) through the Seigert relation g(2)(t) ) 1 + b|g(1)(t)|2. Here b depends on the spatial and temporal coherence of the detected scattered light and is independent of time. The function g(1)(t) can then be related to the distribution G(Γ) of relaxation rates Γ as
g(1)(t) )
∫0∞G(Γ)e-Γt dΓ
(1)
or alternatively, to a distribution A(τ) of relaxation times τ:
g(1)(t) )
∫0∞A(τ)e-t/τ dτ ) ∫0∞ τA(τ)e-t/τ d ln τ
(2)
Laplace inversion of g(1)(t) then yields a potentially multimodal distribution of relaxation times, corresponding to the relaxation rates of particles in different size populations. Laplace inversion of the correlation curves was accomplished using the constrained regularization program (REPES)32 to obtain the distribution A(τ) of relaxation times, using the “probability to reject” parameter set at 0.5. Relaxation rates Γ can be obtained from the moments of the relaxation time distributions and related to the diffusion coefficient as D ) Γ/q2, where q ) 4πn/λ sin(θ/2) is the scattering vector. This requires knowledge of the refractive index n of the continuous phase (1.3915 for isooctane), the wavelength of light λ, and the scattering angle θ. Details of these experiments are described elsewhere.22
The diffusion coefficient was related to the correlation length (ζ) by using the Stokes-Einstein equation, in which D ) kT/(6πηζ). Here kT is the Boltzmann energy, and η is the continuous phase viscosity (η ) 0.474 cP at 25 °C). The correlation length was used instead of the hydrodynamic radius to describe our system to incorporate contributions from interparticle interactions, as has been done previously for the description of systems consisting of micelles and clusters.33 Isoelectric Focusing. The isoelectric point (pI) of the native IgG was determined by PhastGel isoelectric focusing (IEF) with PhastGel IEF 3-9. IEF was performed using a Phast System instrument (Pharmacia, KB Biotechnology, Uppsala, Sweden) consisting of a separation and control unit and a development unit. The Peltier element automatically cools and heats the separation bed to the programmed temperature. IEF was performed at 4, 15, 25, and 37 °C. Details of these experiments are described elsewhere.22 Circular Dichroism Spectroscopy. CD spectra were recorded with a JASCO J-600A spectropolarimeter at a protein concentration of ∼0.6-1.5 mg/mL in a 0.005 cm cell in the far-UV region and a 1 cm cell in the near-UV region. When the temperature was varied, a jacketed cylindrical cuvette of 0.005 light path length was used for the far-UV range measurements. Background circular dichroism was determined in each case by using a microemulsion of the appropriate composition but without IgG. The subtraction due to aqueous buffer solution was done at every pH using buffer solution without protein. After correction for the buffer spectrum, the results were converted into the mean molecular ellipticity using an IgG molecular weight of 155 000, and 1300 for the total number of amino acids. The deconvolution program CDsstr, which computes the secondary structure of a protein from its far-UV CD spectra, was provided by W. C. Johnson at Oregon State University.34 Results and Discussion Role of Surfactant Concentration. A 1 g sample of IgG per liter of water (6.5 × 10-3 mM) was added to a two-phase system containing approximately equal volumes of aqueous salt solution and w/o organic microemulsion. The protein distributes unequally between the two phases, and in some cases forms a third, solid phase at the interface between aqueous and organic solutions. As shown in Figure 2a, this distribution is a function of salt and surfactant concentration. At high surfactant concentrations ([AOT] > 0.2 M) the protein completely (0.1 M NaCl) or predominantly (0.25 M NaCl) favors the microemulsion phase. As surfactant concentration decreases below 0.2 M at low salt, protein increasingly moves from the organic microemulsion phase to the precipitate phase, with no increase in aqueous concentration. At 0.25 M NaCl, approximately equal fractions (∼16%) of protein are found in the aqueous and precipitate phases; this amount increases as the AOT concentration drops below 0.05 M (to ∼30%). Adachi and Harada11,26 also found that cytochrome c precipitated to the two-phase interface at low surfactant concentrations. The surfactant concentrations needed for protein to be maximally solubilized within the microemulsion phase were much lower, per mole of cytochrome c, than those shown in Figure 2a for IgG. Interestingly, however, the weight ratios of AOT-to-protein needed to reach maximum solubilization were similar for the two different proteins at a similar NaCl concentration. This suggests that the surfactant needed to retain the protein within the microemulsion droplets scales with the protein size, as well as with the number of protein molecules.
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Figure 3. Effect of AOT concentration on solubilization at 25 °C of IgG and water in the microemulsion phase as a function of time. Total protein concentration is 1 g/L of water (6.5 × 10-3 mM). pH ) 5.55, [NaCl] ) 0.1 M.
Figure 2. (a) Effect of AOT concentration on distribution at 25 °C of IgG between microemulsion (b, 9), aqueous (O, 0) and precipitate (+, ×) phases at different salt concentrations: (b, O, +) 0.1 M NaCl; (9, 0, ×) 0.25 M NaCl. (b) IgG concentrations in the organic phase from (a) and from ref 22, plotted versus estimated droplet radius. Total protein concentration is 1 g/L of water (6.5 × 10-3 mM). pH ) 5.55. Data taken immediately after centrifugation.
It is well-known that the water content of the microemulsion phase will vary with surfactant concentration, with most of that variation occurring for [AOT] < 0.1 M. Variations in wo occurring at low [AOT] should be accompanied by direct variation in individual droplet radius.35 Thus, the effect of surfactant concentration on protein solubilization may be at least partially attributed to the role of droplet size on limiting protein uptake. In fact, earlier work22 showed that when wo was varied by changing the aqueous salt concentration, protein solubility in the w/o microemulsion dropped significantly for an estimated radius 5 days), however, the protein begins to precipitate even at this higher surfactant concentration. These results indicate that increasing surfactant concentration slows the kinetics of the protein precipitation process, rather than eliminating the possibility of precipitation. The surfactant concentration in both cases is high enough to carry the protein from the aqueous phase into the organic solution, but a lower surfactant concentration reduces the stability of the protein within the microemulsion. The data presented in Figure 3 correspond to a system containing 0.1 M NaCl, i.e., to the condition where the protein size is less than the water pool of the microemulsion droplet and protein can be solubilized within a single droplet. At an AOT concentration of 0.1 M, IgG in these individual drops is likely subjected to a destabilizing environment, such as exposure to the organic solvent or to other protein molecules due to droplet-droplet collisions. At a surfactant concentration of 0.4 M, on the other hand, the higher volume fraction of droplets is known to cause droplet-droplet attraction and clustering, even in the absence of protein.36-40 These clusters may effectively shield the protein from aggregation or solvent-induced denaturation. As a result, in the presence of clusters more time is
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Figure 4. Relaxation time distributions for microemulsions with varying concentrations of AOT. Total protein concentration was 1 g/L of water (6.5 × 10-3 mM). pH ) 5.55, 0.25 M NaCl. Dashed curves indicate microemulsion in the absence of IgG.
required for protein-protein collisions to lead to protein precipitation, and the formation of clusters can stabilize the protein (Figure 1b). Results from dynamic light scattering clearly show that there is a bimodal distribution at higher salt concentrations, most likely corresponding to empty droplets and clusters, in which bovine IgG can reside.22 The influence of surfactant concentration on cluster formation was investigated, with results shown in Figure 4. Relaxation time distributions τA(τ) are shown for the organic microemulsion phase at 0.25 M NaCl, in the absence and presence of 1 g/L protein. Longer relaxation times τ correspond to smaller diffusion coefficients, and hence to larger correlation lengths. Plots of correlation times for a particular mode can in principle be plotted as a function of the volume fraction of the scattering particles, to quantify the first-order effects of interparticle interactions on the diffusion coefficient.41,42 We consider such an approach to get an estimate of the strength of interactions between droplets, using experimental results22 for a microemulsion (0.4 M AOT, 0.25 M NaCl, and 0.46 g/L IgG) whose droplet volume fraction was 0.26. The first moment of the second peak in the distribution data yields an average relaxation time for the slow mode, from which the effective diffusion coefficient D is determined. Diluting this microemulsion with isooctane (cf. Figure 9 in ref 22) and examining the scattering data as a function of low volume fractions φ, may allow us to evaluate the interaction parameter R from a fit to the equation
D ) D0(1 + Rφ)
(3)
Here D0 is the diffusion coefficient in the absence of forces between droplets.
As can be seen in Figure 9 of Gerhardt and Dungan,22 two relaxation time modes can only be clearly distinguished at the highest two concentrations (volume fractions above φ ) 0.1). At lower volume fractions a single broad peak was observed, which may indicate the presence of multiple droplet populations, with sizes that are too similar to be distinguished by DLS. Thus for φ < 0.1, where eq 3 is most appropriate, we were forced to use only the single broad mode to evaluate D, making these values more difficult to interpret. The extent of the decrease in D for φ > 0.06, which indicates the presence of attractive interactions between clusters, was quantitatively similar to that seen in other AOT/isooctane/brine microemulsions, in both the absence and presence of protein.15,43 However, our inability to identify two separated modes at low volume fractions does not permit the application of eq 3 to quantify D0. There is an additional reason we view the use of eq 3 for our system with some caution. Because of the evolving (time-dependent) and complex character of our solutions, it is quite possible that dilution of the clustering microemulsion can change the microstructure, and not simply decrease the particle concentration as intended. Consequently, we did not attempt to dilute successively each microemulsion described in the light scattering results below. Instead, results are discussed in terms of correlation lengths,33 which include effects of both particle size and interparticle interactions. Disentangling contributions of particle size changes and interparticle interactions on droplet diffusion is more straightforward in single-phase, equilibrium AOT microemulsions. In these systems the volume fraction of droplets can be readily varied by controlling the amount of surfactant and water added to the organic solvent. Previous research has employed dynamic light scattering in such solutions, to examine effects of protein incorporation on equilibrium droplet size and interaction. For microemulsions containing the water-soluble protein cytochrome c15 or myelin basic protein,44 with fewer than one protein per droplet, there is an increase in interdroplet attraction, which decreases the diffusion coefficient by up to 30% at higher droplet concentrations. No droplet clustering is seen at these conditions; however, at higher cytochrome c concentrations (more than one protein molecule per droplet), dimer formation is also observed in the presence of protein.19 Far more significant clustering was observed with DLS in systems containing the transmembrane protein Folch-Pi proteolipid,21,43 resulting in bimodal relaxation time distributions similar to that reported by us in Figure 4. The slow mode in the transmembrane protein system corresponds to a size that is an order of magnitude larger than that of the empty droplets. This result is quantitatively comparable to that seen in our IgG system, strongly suggesting that the slower mode found in our light scattering results is primarily caused by an increase in particle size due to clustering, rather than by the much smaller contribution due to attractive interactions. An increase in AOT concentration leads to higher droplet volume fractions in the microemulsion phase. An increase in the correlation length of clusters was observed with increasing AOT concentration (Figure 4), which may signal cluster growth or increasing attractive interactions between clusters. For comparison are shown the distribution functions for the microemulsion in the absence of IgG at the same surfactant concentration. The correlation length of these empty droplets remained constant (∼5 nm) at different AOT concentrations, although a broadening in the distribution function is observed at high AOT concentrations, which indicates increased interdroplet interaction.
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Figure 5. Relaxation time distributions for microemulsions as a function of time, for two concentrations of AOT. Total protein concentration was 1 g/L of water (6.5 × 10-3 mM). pH ) 5.55, 0.25 M NaCl.
The correlation length of the clusters was found to increase over time (Figure 5), most likely indicating slow growth in the cluster size. For a microemulsion having an initial AOT concentration of 0.1 M, the initial distribution is monomodal with a correlation length of 7.8 nm. This length is larger than that measured in the absence of IgG, and indicates the ability of the protein initially to increase the size of the droplets or to strengthen interdroplet attraction. Measurements taken 3.5 and 24 h after phase transfer indicate the presence of individual droplets and droplet clusters. The latter have correlation lengths of 25 and 50 nm for measurements taken at 3.5 and 24 h, respectively. As shown in Figure 5, the population of clusters grows at the expense of single droplets. At a higher surfactant concentration of 0.4 M, the initial distribution is clearly bimodal, in contrast to results at 0.1 M AOT under the same salt conditions (0.25 M [NaCl]). Growth of the slower cluster mode also appears to occur more rapidly at higher surfactant concentration. We therefore conclude that a higher surfactant concentration leads to more rapid cluster formation, and this tends to stabilize the protein from entering the interfacial precipitate phase. Interestingly, however, the distribution functions for two systems shown in Figure 5 are remarkably similar at 21-22 h. At 0.25 M NaCl, the proteincontaining microemulsion is able to attain a structure in which IgG can be stabilized for both AOT concentrations over 22 h. This accounts for the lack of observed precipitation at this salt concentration over 24 h.22 Coexistence of polymer-droplet aggregates with a large fraction (30%) of “free” droplets was found in the system amphiphilic graft polymer in AOT/cyclohexane microemulsion. The graft copolymer, which has a hydrophobic backbone and hydrophilic side chains, is soluble neither in organic solvent nor in water but is well-dissolved in an AOT microemulsion. The precipitation of a fraction of the graft copolymer upon dilution of microemulsion with cyclohexane was explained by the existence of some minimum of bound microemulsion droplets per copolymer molecule.25 Thus, microemulsion droplets shield polymer from significant exposure to organic solvent, as is the case with IgG. The precipitated IgG between the phases consists of aggregated protein partially coated with surfactant shells.22 The protein in this precipitate probably experiences conformational changes in select regions of the molecule and proteins are therefore not completely unfolded. Recent research on aggregation of lysozyme in aqueous solution reveals selective unfolding of the β-domains of the protein in these precipitates.45 The interplay between microemulsion structure and protein conformation will be discussed in detail below.
Figure 6. Effect of pH on partitioning of IgG between (a) aqueous, microemulsion and (b) precipitate phases at 4, 25, and 37 °C. Total protein concentration is 1 g/L of water (6.5 × 10-3 mM). 0.3 M [NaCl], 0.1 M [AOT].
Effect of Temperature. The influence of temperature on the IgG solubilization in the microemulsion at various pH values of the aqueous phase was also investigated. As can be seen in Figure 6a, as temperature increased, there was an increase in the threshold pH needed to effect protein solubilization/ precipitation (and removal from the water). Two factors present themselves as explanations for this behavior. Because temperature is expected to influence hydrophobic interactions more strongly than electrostatics, the temperature dependence shown in Figure 6a suggests that hydrophobic interaction of bovine IgG with the nonpolar tails of the anionic surfactant is an important driving force governing the interaction of this protein with the droplet. Thus, these results can be interpreted as indicating that as temperature goes up and hydrophobic interactions become stronger, the electrostatic interactions do not need to be as substantial to drive the protein partitioning. The protein needs to be less highly charged at higher temperatures. A second factor that must be considered is the effect of temperature on water content in the microemulsion, i.e., the effect of temperature on droplet size. Table 1 shows measured values of wo at various temperatures, and droplet radii estimated by using relations established by Rahaman and Hatton (1991).31 Because protein solubilization is known to decrease with decreasing droplet size, the trends shown in Figure 6a could be
Protein Structure in IgG-AOT-Brine-Isooctane Systems TABLE 1: Water-to-Surfactant Molar Ratio (wo) in the Microemulsion Phase as a Function of Temperature and Time pH 5.68
pH 6.02
temp. °C
10 min
24 h
10 min
24 h
4 25 37
18.5 25.5 29.0
18.5 24.5 12.5
18.5 26.0 31.0
18.5 25.5 14.0
a The system contained 1 g of IgG/L of water (6.5 × 10-3 mM) and 0.3 M NaCl, whereas the organic solution consisted of 0.1 mol AOT per L isooctane.
partly caused by the expansion in the droplets at higher temperatures. A third possibility is that temperature is influencing the isoelectric point of IgG, resulting in the shift in the curves shown in Figure 6a. To explore this issue further, we measured the isoelectric point for IgG at 4, 15, 25, and 37 °C using isoelectric focusing. These measurements indicate that the pI of bovine IgG varied over a wide range, with most of the distribution found in the pH range 6.6-9.0 at 4, 15, and 25 °C. The pI range decreased to 6.6-8.5 at 37 °C. For human IgG, the pI range of Fab fragments (MW 50 000) is 8.4-9.8, and the pI range of the Fc fragment (MW 50 000) is 6.2-7.0.46 According to electrophoretic mobility of the protein, the structural changes at 37 °C observed by isoelectric focusing probably occurred in the more basic fragments (Fab). This result is consistent with CD data that show that Fab fragments are more sensitive to temperature.47 These isoelectric focusing results indicate that a shift in pI with temperature is not responsible for the shift in curves in Figure 6a. Temperature also influences the relative tendency of IgG to remain in the microemulsion or to move to form a third, precipitate phase (Figure 6a,b). We observed a pH of maximum microemulsion solubilization, which shifted to higher values as temperature increased. These measurements indicate that the processes leading to protein precipitation are more sensitive to pH at higher temperatures. At the lowest pH, this trend results in greater precipitation at 37 °C than at the two lower temperatures. Temperature affects several mechanisms that may be implicated in the protein aggregation process. As discussed above, hydrophobic interactions become stronger at higher temperatures. Protein conformational changes are also more rapid and more significant at higher temperatures (see below). In addition, as temperature goes up, the interdroplet collision rate increases and droplets tend to cluster and fuse, resulting in more rapid exposure of protein to the bulk organic phase and/ or other protein molecules. The change in the parameter wo with temperature and time is shown in Table 1. We compared the behavior of the organic microemulsion solution in the presence of protein and in its absence. wo varied to the same degree with temperature and time in both sets. The parameter wo increased with temperature, most likely indicating an increase in the droplet radius as noted above. The water content was measured immediately following a 10 min centrifugation and after a 24 h incubation. With these data, it is thus possible to estimate the extent of precipitation of water droplets (i.e., with surfactant and protein) at different temperatures and over time. The water content in the microemulsion phase remained constant at 4 °C, decreased slightly over time at 25 °C, and decreased significantly after 24 h at 37 °C. These observations suggest that AOT precipitates more substantially with the protein at higher temperatures in the system.
J. Phys. Chem. B, Vol. 108, No. 28, 2004 9807 Protein Conformation. An IgG molecule is composed of four polypeptide chains that are connected by disulfide bonds and noncovalent forces (Figure 1). The four chains can be divided into two groups: two heavy and two light chains. All four polypeptide chains are grouped in different domains: two Fab fragments and one Fc fragment, together forming a “Yshaped” conformation. The presence of “hinges” that are located between the Fab and Fac fragments creates flexibility in the IgG molecule. These different domains within the IgG molecule individually fold to create compact globular structures. For native IgG at pH 6 in 5 mM phosphate buffer, analysis of the far-UV CD spectrum yields approximately 75% β-sheet-type secondary structure in the polypeptide.48 Much lower contributions from β-turn or random coil conformations, and no R-helix fraction, were observed in the same system.48 The predominant β-sheet component in immunoglobulin domains is consistently observed in all extant immunoglobulin protein structures. However, the optical rotatory dispersion and CD spectra of some IgG antibodies always have some individual spectral features, with analysis of the far-UV spectra generally underpredicting the extent of secondary structural features identified by X-ray diffraction.49 Some variation in the amplitude of the far-UV CD spectra of different immunoglobulins, as well as spectral differences in the near-UV region, has been attributed to contributions of aromatic amino acid side chains such as tryptophan and tyrosine, which are positioned at the immunoglobulin interdomain surfaces.49 At pH 7 and 5.5 in aqueous phosphate buffer solution, farUV CD spectra show very small differences in secondary structure of IgG with pH (Figure 7). Within this pH range, the spectra exhibit a negative band at around 217 nm (representative of β-pleated sheet structure), an inflection at 228 nm (due to aromatic amino acids), and a positive band at around 202 nm. These values agree well with those previously observed in the CD spectra of other IgG antibodies.49-51 Changes in the CD spectra observed when IgG is incorporated within the microemulsion phase are a shift of the positive maximum from 202 to 194 nm, an overall increase in the ellipticity and the absence of inflection. The CD spectrum of IgG solubilized in the microemulsion reveals a shape similar to that of the protein in aqueous solution at pH 2-3 (Figure 7b). This shift in the spectrum suggests that, upon incorporation into the microemulsion phase, the IgG molecule undergoes transformations similar to those in the aqueous phase at low pH. As shown in Figure 8, the tertiary structure of IgG in phosphate buffer solution at pH 2 and in the organic phase (5 min after separation and centrifugation) is also similar, with the near UV spectra exhibiting a distinct maximum at 260 nm. This maximum was not observed in aqueous solutions of native IgG in phosphate buffer at pH 7 or acetate buffer at pH 5.5. Instead, we observe two peaks in the range 285-300 nm. These peaks decrease in intensity for IgG in the microemulsion solution and in the aqueous phase at pH 2. It should be noted that a positive feature located approximately between 285 and 300 nm does not occur in all subclasses of IgG antibodies and can be attributed to changes in tryptophan residues and disulfide bonds.52 The absence of the maximum at 260 nm in the native state and its presence in the oil-continuous microemulsion was also observed in spectra for lysozyme53 and for human pancreatic lipase.54 The contributions to the CD spectra in the 260 nm region are also from aromatic amino acid side chains49 and sulfide chromophore.53
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Figure 8. Variation of the mean molecular ellipticity with wavelength in the aromatic region at 25 °C. The organic phase contained 0.4 M AOT and was contacted with an aqueous solution containing 0.25 M NaCl, and 100 mM acetate buffer (pH 5.55). Aqueous spectra were taken at 25 °C in a 50 mM phosphate buffer containing 0.1 M NaCl.
Figure 7. Variation of the mean molecular ellipticity with wavelength in the peptide region at 25 °C. Shown are spectra taken in an aqueous buffer solution at different pH (50 mM phosphate buffer, 0.1 M NaCl) and a spectrum taken in the organic phase after phase equilibration using initial conditions of 0.1 M AOT, 50 mM sodium phosphate buffer, pH 5.55, 0.1 M NaCl. The spectrum in microemulsion is compared with aqueous solution spectra at 0.1 M NaCl and (a) pH 5.55 and 7 or (b) 2 < pH < 3.
The conformation of IgG in the microemulsion and in acidic pH solutions is not represented by an unfolded, random coil. Instead, immunoglobulins are known to adopt a particular, stable, folded structure at low pH values (pH < 3) termed the A state.50,55 This alternatively folded state exhibits a high degree of secondary structure, increased exposure of hydrophobic residues, and a tendency to aggregate slowly. For the antibody murine MAK 33, subtype IgG1 at pH 2, the CD spectrum of the A state (at pH 2 in phosphate buffer) in the near-UV range does not show pronounced features of tertiary structure, although well-defined contacts are present.50 At pH 2, rabbit IgG keeps a well-defined tertiary structure in the domains that are least sensitive to pH, accompanied by extensive retained secondary structure.55 Similar near-UV features were observed in the IgG structure in the microemulsion phase for at least several minutes after solubilization. This alternatively folded state exhibits certain characteristics of the so-called molten globule, but differs distinctly from it by its greater structural stability that is more characteristic of native protein structures.50 For example, the well-defined tertiary contacts shown in the near-UV CD spectra are not typically observed with proteins that adopt the molten globule state.16
IgG in the microemulsion phase exhibits a structure close to the A state but unfolds into more denatured structures over time in the microemulsion setting (Figure 8). The spectra of IgG in the microemulsion phase (0.1 M AOT) in the far-UV region taken 15 min, 3 h, and 24 h after phase separation of the dispersion is shown in Figure 9a. Figure 9b gives similar results after 0, 5, and 21 days in a 0.4 M AOT organic solution. An increase in the ellipticity is observed over time as protein sits in the microemulsion solution. This increase is less prominent for 0.4 M AOT over an equal period of time, because the protein is better stabilized in the microemulsion at higher surfactant concentration. These results for secondary structure are consistent with time-dependent changes in the tertiary structure. Conformational changes in the secondary protein structure over time were also observed for cytochrome c.26 It has also been reported that back-extraction of IgG from the microemulsion phase into an aqueous phase is less effective when the microemulsion was incubated for several days, as compared to the back-extraction of IgG from fresh solution.56 The data from dynamic light scattering experiments show that the size of the IgG-containing clusters increases over time in the microemulsion. This may indicate that even as the protein is well-stabilized within droplet clusters, further exposure of IgG hydrophobic domains due to changes in protein conformation occur over time within the microemulsion. Conformational analysis of the spectra using the deconvolution program CDsstr reveals that the native protein in phosphate buffer at pH 7 has no detectable R-helix contribution. The calculated amount of β-strands and β-turns was 34% and 12%, respectively. Compared with the results obtained for another IgG antibody,48 the percent of R-helix is in good agreement, whereas the β-sheet content seems to be underestimated due to effects of aromatic amino acid side chains. Analysis of the CD spectra in the far-UV region at pH 2 and also in the organic phase clearly shows a reduction in the β-sheet content. Upon solubilization in the microemulsion, the R-helix content increases from 0% to 4%. The same change was observed in aqueous solution at pH 2.5. The formation of R-helixes is attributed to the formation of intramolecular hydrogen bonds. A strong R-helix induction in IgG structure has been observed due to aggregation, adsorption on a hydro-
Protein Structure in IgG-AOT-Brine-Isooctane Systems
J. Phys. Chem. B, Vol. 108, No. 28, 2004 9809
Figure 10. Temperature dependence of the mean molar ellipticity spectra of bovine IgG in the far-UV region. Spectra in the organic phase were taken 24 h after the phase equilibration experiment. The organic phase contained 0.4 M AOT and was contacted with an aqueous solution containing 0.15 M NaCl and 100 mM acetate buffer (pH 5.55).
Figure 9. Time dependence of the mean molecular ellipticity spectra of bovine IgG in the far-UV region at 25 °C. Spectra in the organic phase were taken at various times after the phase equilibration experiment. Aqueous solution in contact with the organic phase contained 0.25 M NaCl and 50 mM phosphate buffer (pH 5.55). Organic phase contained (a) 0.1 M AOT or (b) 0.4 M AOT.
phobic surface or interaction with sodium dodecyl sulfate at temperatures higher than 35-40 °C.48,57,58 These changes are consistent with the notion that hydrophobic interactions play an important role in phase structural changes in the IgG-AOToil-water system. There are different structural transformations prompted by acidic pH and thermal transitions. The denaturation of a multidomain protein can be described by the denaturation of the individual domains, with the order in which different domains are affected depending on the type of denaturation process.47 Kats and co-workers observed different conformational changes in immunoglobulin when the protein is aciddenatured and when it is reversibly heat-denatured; both conformations differ from the structure of the irreversibly heatdenatured state.59 Under acidic conditions the main conformation changes appear in the hinge region of the molecule55,60 and/or in the Fc fragments,47,60 whereas heat denaturation may involve changes in interdomain areas separating the constant and the variable domains of the Fab parts.60 The changes in the Fab fragments47 and in the hinge region of the molecule61 were observed at temperatures close to or coinciding with the onset of aggregation. After both heat- and low-pH-induced denaturation, a significant fraction of the secondary structure remains.
Some authors suggest that the observed conformational transformations are the result of changes in interactions and dispositions of invariably folded domains rather than in structural changes within the domains.55,60 Changing the temperature of an aqueous solution of IgG in acetate buffer at pH 5.55 and letting the solution equilibrate at that certain temperature for approximately 15 min did not cause changes in the protein structure. This observation is consistent with literature reports that temperature-induced protein transformations were observed above 50-60 °C.47,61 However, prolonged temperature exposure can cause protein transformation at a lower temperature.47 In the case of IgG solubilization in the AOT-containing microemulsion, the protein did not change conformation in the aqueous phase during the short time of equilibration of initial phases at the specified temperature and consequent emulsification of the phases. There are no differences in the spectra of the IgG in the aqueous phase after phase equilibration and native IgG in the buffer before phase contact in the far-UV region at 25 °C. However, changes in the protein structure in the microemulsion phase after 24 h were observed to depend on temperature (Figure 10). IgG is more unfolded in the microemulsion phase at higher temperature. This phenomenon can account for the more rapid IgG aggregation and precipitation at 37 °C, especially at lower pH values. The higher temperature may enhance the binding of surfactant to the protein due to stronger hydrophobic interactions, which in turn results in a change in the protein conformation. The interdroplet collision rate also increases at higher temperature, resulting in IgG denaturation in an unfavorable organic environment during the breakdown of the complex. The significant shift in the minimum from 217 nm to lower wavelengths with increasing temperature (Figure 10) was not observed during time-dependent changes in protein conformation in the microemulsion (Figure 9) or when pH was varied in aqueous solution (Figure 7). In these two last cases, only a significant increase in the negative ellipticity was observed. This observation supports the idea that in the microemulsion phase, as in aqueous solution, different structural transformations in the IgG molecule are promoted by higher temperature, acidic pH, or residence time of protein in the surfactant structures.
9810 J. Phys. Chem. B, Vol. 108, No. 28, 2004 Conclusion The presence of the large protein IgG within a w/o microemulsion phase leads to changes in phase structures as well as in phase behavior, signaled by the emergence of a third, solid, middle phase. Protein promotes droplet aggregation into large clusters, which stabilize the partially denatured protein better than does an individual droplet and which slow the formation of the third, precipitate phase. Cluster formation occurs more readily with higher surfactant or salt concentration, when the protein and droplet are comparable in size. There are two opposing effects of temperature on the solubilization of protein. Increasing temperature is favorable for protein solubilization within the microemulsion, due to the increasing of hydrophobic interactions and/or an increase in droplet size. However, the protein at higher temperature is more denatured in the microemulsion, which results in a greater protein precipitation, especially at low pH. When solubilized in the microemulsion phase, IgG does not unfold immediately after phase equilibration but adopts a specific stable structure close to that adopted at low pH in water (A state). As in the case of structural transformations prompted by acidic pH and thermal transitions in aqueous solution, the nature of conformational changes in IgG in the microemulsion depends on whether it is challenged with changes in temperature, pH of the aqueous phase, or time spent in the microemulsion environment. The various globular domains of the IgG structure are sensitive to certain conditions in the microemulsion phase, most likely including their positioning in the surfactant monolayer at the droplet interface. Acknowledgment. We gratefully acknowledge funding from Dairy Management, Inc., the California Dairy Research Foundation, and an NSF Young Investigator Award CTS-93-58508 to S.R.D. This work was supported in part by the CPIMA MRSEC Program of the National Science Foundation under Award Number DMR-9808677 for CPIMA II. References and Notes (1) Ananthapadmanabhan, K. P. Interactions of Surfactants with Polymers and Proteins; Goddard, E. D., Ananthapadmanabhan, K. P., Eds.; CRC Press: Boca Raton, FL, 1993; pp 319-365. (2) Lawrence, M. J.; Rees, G. D. AdV. Drug DeliVery ReV. 2000, 45, 89. (3) Ono, T.; Goto, M. Curr. Opin. Colloid Interface Sci. 1997, 2, 397. (4) Paul, B. K.; Moulik, S. P. J. Dispersion Sci. Technol. 1997, 18, 301. (5) Pires, M. J.; Aires-Barros, M. R.; Cabral, J. M. S. Biotechnol. Prog. 1996, 12, 290. (6) Carvalho, C. M. L.; Cabral, J. M. S. Biochimie 2000, 82, 1063. (7) Stamatis, H.; Xenakis, A.; Kolisis, F. N. Biotechnol. AdV. 1999, 17, 293. (8) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson, B. H. Biochim. Biophys. Acta 1988, 947, 209. (9) Hirai, M.; Takizawa, T.; Yabuki, S.; Kawai-Hirai, R.; Oya, M.; Nakamura, K.; Kobashi, K.; Amemiya, Y. J. Chem. Soc., Faraday Trans. 1995, 91, 1081. (10) (a) Luisi, P. L.; Majid, L. J. CRC Crit. ReV. Biochem. 1986, 20, 409. (b) Leodidis, E. B.; Hatton, T. A. Structure ReactiVity in ReVerse Micelles; Pileni, M. P., Ed.; Elsevier: Amsterdam, 1989; pp 288-294. (c) Gupte, A.; Nagarajan, R.; Kilara, A. Food FlaVours: Generation, Analysis and Process Influence; Charalambous, G., Ed.; Elsevier: Amsterdam, 1995; pp 1-74. (11) Adachi, M.; Harada, M. J. Colloid Interface Sci. 1994, 165, 229. (12) Lye, G. J.; Asenjo, J. A.; Pyle, D. L. Biotechnol. Bioeng. 1995, 47, 509. (13) Kawakami, K.; Harada, M.; Adachi, M.; Shioi, A. Colloids Surf. AsPhysicochem. Engin. Aspects 1996, 109, 217. (14) Ichikawa, S.; Imai, M.; Shimizu, M. Biotechnol. Bioeng. 1992, 39, 20. (15) Huruguen, J. P.; Authier, M.; Greffe, J. L.; Pileni, M. P. Langmuir 1991, 7, 243.
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