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Incorporation of β-lactoglobulin in monolayers of dioctadecyldimethylammonium bromide studied by Brewster angle microscopy
Colloids and Surfaces B: Biointerfaces 30 (2003) 259 /272 www.elsevier.com/locate/colsurfb
Incorporation of b-lactoglobulin in monolayers of dioctadecyldimethylammonium bromide studied by Brewster angle microscopy Ame´lia M. Gonc¸alves da Silva *, Rute I.S. Roma˜o, Suzana M. Andrade, Sı´lvia M.B. Costa Centro de Quı´mica Estrutural, Complexo I, Instituto Superior Te´cnico, 1049-001 Lisbon, Portugal Accepted 10 April 2003
Abstract The deposition of pure bovine b-lactoglobulin (bLG) monolayer or the incorporation in a dioctadecyldimethylammonium bromide (DODAB) monolayer, were studied by p /A measurements at the air /water interface and by direct visualization of the interface by BAM. The co-spreading of the monolayer material dissolved in a mixed volatile solvent (chloroform/ethanol) was selected based on reproducible data and minimum consumption of protein. Conformational changes induced by the mixed solvent on the native structure of bLG, investigated by circular dicroism, disappear after the solvent evaporation at the interface. The p /A isotherms suggest and BAM images confirm, that, at low surface pressures, bLG is incorporated in the liquid expanded monolayer of DODAB, while at the plateau near 30 mN m 1 the protein is squeezed out into micro-domains partially immersed in the subphase and strongly adsorbed under the DODAB layer. The topography of DODAB/y bLG mixed films changes with surface pressure and number of protein residues, y , incorporated per DODAB molecule. BAM observation shows that at low y DODAB molecules dominate the topography of monolayer at the interface. # 2003 Elsevier B.V. All rights reserved. Keywords: Monolayers; Incorporation of proteins; Lipid /proteins; BAM visualization
1. Introduction Lipids and proteins are very important in many fields because of their relevance in nature. They are widely used as emulsifiers or stabilizers in
* Corresponding author. Tel.: /351-218-41-9263; fax: / 351-218-46-4455. E-mail address: [email protected] (A.M. Gonc¸alves da Silva).
emulsions or foams, especially in the food industry. Thus, the behavior of proteins at interfaces [1 / 7] and the formation and characteristics of lipid / protein system have been studied with great interest in the last years [8 /12]. In particular, lipid /protein interactions in monolayers are of great importance to the understanding of processes in biological membranes and to the further design of functional biological surfaces [13 /15]. A Langmuir monolayer is in fact a rather simple way
0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7765(03)00102-4
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of modeling half a biomembrane. In spite of the great difference between such a simple model and the complex biological system, the information about molecular orientation and packing, selforganization and stability of organizates has proved to be valuable [16,17]. Incorporation of protein in the monolayer, or adsorption under the monolayer, depends on several parameters such as molecular weight of protein, surface pressure, state and charge of the monolayer, pH of the subphase, temperature, etc. [18 /21]. Furthermore, it has been shown that the adsorption or insertion of proteins in lipid monolayers involves an alteration of the native protein structure [22 /26]. Thus, modeling a complex (multicomponent) system such as a biological membrane, requires the understanding of the role of the individual lipid and protein constituents, in order to assess the synergistic effects in higher mixtures. Bovine b-lactoglobulin (bLG) is a major whey protein abundant in cow’s milk. bLG is a small globular protein (Mw 36 000) folded into a calyx formed by eight antiparallel b-strands that constitute 50% of the protein’s structure, and 3-turn ahelix (10%) located at the outer surface of the b-barrel [27]. This protein is known to have an intrinsic propensity to a-helix, as predicted by the amino acid sequence. This particular behavior led bLG to be recognized as an important model for exploring the mechanism of the transition between a and b structure suggested for several biologically important processes (e.g. involving prion and amylogenic diseases) [25,28,29]. In natural systems, the biological function of bLG is not well known but it is supposed that bLG acts as a carrier of small hydrophobic and amphiphilic compounds while the biomembrane acts as support of bLG. Hydrophobic, electrostatic and hydrogen-bonding interactions may occur simultaneously in the binding of bLG to the guest or host compounds like fatty acids, triglycerides and phospholipids forming lipid/ protein complexes. The first goal of this work is to study the interaction of bLG with the lipidic support at the air /water interface. This paper presents the study of the interaction of bLG with a lipid-like double-chained cationic
surfactant, dioctadecyldimethylammonium bromide (DODAB), in mixed monolayers. For pure and mixed films several methods of addition of bLG at the interface were compared, namely the effect of the spreading solvent on the protein. Pure DODAB was previously studied [30]. Floating monolayers of DODAB/bLG of variable composition were studied at the air /water interface by surface pressure/area measurements and by Brewster angle microscopy (BAM) visualization. At low content of bLG, the topography of mixed monolayers at the air /water interface, followed by BAM, compares well with the topography of pure DODAB.
2. Experimental section 2.1. Materials Bovine bLG AB mixture chromatographically purified and lyophilized with purity higher than 90% was purchased from Sigma (catalogue no. L3908). Dioctadecyldimethylammonium bromide (DODAB)]/98% purity was obtained from Fluka. The solvents chloroform and ethanol were of spectroscopic grade (Uvasol) from Merck. Amphiphiles and solvents were used without further purification. Water used in the subphase and in solutions was distilled twice and purified with the Millipore Milli-Q system in order to obtain a resistivity higher than 18 MV cm. pH 2 at the water subphase was reached by the addition of HCl. Stock solutions of DODAB were prepared in chloroform and stock solutions of bLG in ethanol/ water or in pure water. Mixed solutions of bLG and DODAB were prepared by adding precisely measured volumes of stock solutions and diluted to a final volumetric composition 9:9:1 v/v mixture of chloroform/ethanol/water. A drop of HCl was always added to solutions with protein. 2.2. Methods Because bLG is a water-soluble protein, the addition of bLG to the air /water interface was optimized by comparing the following procedures:
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spreading in a volatile solvent composed by ethanol, chloroform and water, 9:9:1 vol.; addition of an aqueous solution to the interface; injection of an aqueous solution in the subphase; and deposition of solid material at the water interface. Mixed monolayers of DODAB/bLG were formed by similar procedures to the ones above referred: co -spreading method consists of the spreading of the binary mixture in a common volatile solvent; injection of the aqueous solution of bLG in the subphase just before the spreading of DODAB solution at the interface was followed by 20 /30 min of waiting time for equilibration, and three compression /expansion cycles until 25 mN m 1, before the complete compression isotherm; deposition of the solid bLG, over a DODAB monolayer near its lift off, was followed by the same procedure used in the injection method before the complete compression isotherm. 2.3. Surface pressure /area measurements Surface pressure /area (p /A ) isotherm measurements were carried out on a KSV 5000 Langmuir / Blodgett system (KSV Instruments, Helsinki) installed in a laminar flow hood. Procedures for p / A measurements and cleaning care were described elsewhere [31]. The careful suction of the water surface was carried out before every measurement. The surface pressure increase lower than 0.15 mN m 1 when the area was reduced to one tenth warrants the absence of surfactants. The temperature of the subphase was controlled by water circulation from a thermostat within an error of 9/0.1 8C. p /A isotherms were measured several times and different spreading solutions were used. 2.4. Circular dicrosism (CD) spectroscopy CD spectra were obtained using a Jasco Model J-720 spectropolarimeter (Jasco, Hachioji City, Tokyo). The protein spectra were measured at controlled temperature (24.09/0.2 8C) using 10 mm (for near-UV) and 2 mm (for far-UV) cells. The solutions containing 2.5 mM (far-UV) or 14 mM (near-UV)*/obtained correctly by measuring 1 absorption at 280 nm where obLG 280 /17 600 M
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cm 1 */were scanned at 20 nm min 1 with a 0.2 nm step resolution, a 1 nm bandwidth and a sensitivity of 10 millidegrees. The average of four scans was recorded and corrected by subtracting a baseline spectrum. The CD signal was converted to mean residue ellipticity [u ] (deg cm2 dmol 1) defined as [u] /100 uobs(l c) 1, where uobs is the experimental CD value in degrees, c (residue mol dm 3) is the concentration, and l (cm) is the cell path length. 2.5. Brewster angle microscopy (BAM) The commercial BAM2, manufactured by NFT (Go¨ttingen, Germany) mounted on a KSV Langmuir balance was used to observe the morphologic patterns of the monolayer. The light reflected from the surface is collected by two achromatic lenses and detected with a CCD camera. The CCD camera converts the reflectivity signal from the sample into a video image. The spatial resolution is approximately 2 mm. Principles and detailed description of BAM have been described elsewhere [32,33].
3. Results and discussion 3.1. bLG at the air /water interface p /A compression isotherms of bLG on a pure water subphase, differing in the addition procedure of protein to the air /water interface, are compared in Fig. 1: spreading of a volatile solution (curve 1); addition of an aqueous solution on the interface (curve 2); injection of an aqueous solution (curve 3); and deposition of small crystals (curve 4). Although strongly separated in the area scale, all the p /A isotherms of Fig. 1A show similar shapes with two expanded regimes separated by an intermediate regime of lower slope at 20/30 mN m 1. Curve 1?, standing for the spreading procedure on the pH 2 subphase, will be discussed later. In spite of the solubility in water, bLG forms stable monolayers at the air /water interface due to the significant hydrophobic component of interaction. However, the presence of bLG in both
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Fig. 1. p /A isotherms of monolayers of bLG on a pure water subphase at 25 8C. Several methods of addition to the interface: spreading solvent (1); addition of an aqueous solution at the interface (2); injection of an aqueous solution in the subphase (3); sprinkling of small crystals (4). p /A isotherm of bLG on a pH 2 subphase at 25 8C (1?). A in the abscissas stands for the area per residue of bLG (A); the relative area in the abscissas stands for A (p )/A (28 mN m 1) (B).
regions, interface and bulk phase, should be considered. This can explain the deviation of p / A isotherms to smaller areas per residue when an aqueous solution was added at the interface (curve 2) when compared to curve 1 obtained by using a volatile solvent; an even higher deviation was
observed by injection of the aqueous solution into the subphase (curve 3). When the deposition method was used in the absence of solvent, (curve 4), the area occupied per residue at low surface pressures was nearly one tenth of the value obtained by spreading (curve 1). This seems to indicate that only a small fraction of the deposited solid spreads at the water interface and the majority precipitates before spreading, dissolves into the subphase or remains in a solid bulk phase at the interface. The p /A isotherms are reproducible and the monolayers are stable when bLG is added to the interface by using the spreading method. In fact, the second compression isotherm follows the first one after the compression /expansion cycle until 25 mN m1. The reproducibility was not so good with other methods. From the results described we consider the spreading of a volatile solution as the most reliable method to add bLG at the air /water interface. In fact, by injection or addition of an aqueous solution and by direct deposition of the solid, the surface concentration of bLG is not known, which is a serious limitation of these methods in some cases. On the contrary, by using the spreading method, the reproducibility of the p /A compression isotherms and the proximity of the compression /expansion cycles seems to indicate that dissolution is not significant during experience time and we can assume that the surface concentration of bLG is known. The p /A isotherm of bLG obtained by using the Trurnit spreading method [34] locates between curves 1 and 2 of Fig. 1A. This indicates a higher efficiency of this method than injection or addition methods used in this work but it still is less reliable than the spreading by a volatile solvent. Assuming a significant uncertainty on the amount of bLG at the interface obtained some of the methods used, the p /A curves were normalized at p /28 mN m 1 (near the inflection point of the curve, where the compressibility of the monolayers starts to decrease with p ). Each curve was re-plotted in a relative area scale, defined as A (p )/A (28 mN m 1), in Fig. 1B. In this scale, curves coincide at p/28 mN m 1. This representation will allow a prompt comparison of the monolayer compressibility. Curve 1 corresponds
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to the less compressible monolayer (for the same variation of surface pressure the variation of area is minimum for curve 1). Curves 2 and 3, obtained by deposition or injection of an aqueous solution evolve closely indicating similar conformational behavior at the interface. A solvent effect on the native structure of bLG may explain the differences observed in the expanded regimes at low surface pressures. The deviation of curves 2 and 3 to larger areas relatively to curve 1 can be ascribed to the enhancement of the hydrophilic character of bLG by the water solvent increasing the interaction with water subphase. On the contrary, the deviation of curve 1 to smaller areas relatively to curve 2 can be explained by the effect of the hydrophobic spreading solvent and its partial retention at the interface. 3.1.1. Solvent effect on the bLG conformation bLG precipitates in the spreading mixture chloroform/ethanol/water used for the addition at the interface. However, the solution turbidity disappears after the addition of a small drop of hydrochloric acid, for a chloroform content 5/ 50%. The effect of the mixed spreading volatile solvent and the addition of hydrochloric acid on the native structure of the protein in aqueous medium were investigated by CD measurements. The far-UV CD spectra give information on the secondary structure while the near-UV CD spectra inform about the tertiary structure of bLG. Fig. 2 shows the far-UV (A) and the near-UV (B) CD spectra of bLG in several media. The negative CD band found in the far-UV region around 216 nm, for the aqueous solution, pH 5.8, curve 1, was ascribed to the predominant b-sheet protein conformation in this medium, while the bands detected (293 and 286 nm) in the near-UV CD spectra are mainly due to the contribution of four tyrosines and two tryptophans (Trp) in a specific environment. The addition of a small drop of HCl (to a 5 ml volume) would be equivalent to a decrease in pH from 5.8 to 3.8 (curve 2), which means that the global charge of protein changes from slightly negative to positive (isoelectric pH, pI /5.1). However, this variation of pH does not seem to affect significantly the secondary or tertiary structures of bLG, as the proximity of
Fig. 2. Far-UV (A) and near-UV (B) CD spectra of bLG in aqueous solution at pH 5.8 (1) and pH 3.8 (2); bLG in acidified spreading mixture (3); bLG in acidified ethanol (4).
curves 1 and 2 in Fig. 2 suggests. Indeed, bLG is known to be very stable under acidic conditions, which has been explained by the strong stabilizing action of the two disulfide bonds [35]. In the acidified spreading mixture chloroform/ ethanol/water (curve 3), the CD spectra of bLG changed significantly in the far-UV and in the near-UV region. Similar changes were also found in acidified ethanol (curve 4). The analysis of the spectral changes allows some important conclusions. The more pronounced bands in the near-UV region (293 and 286 nm) disappeared suggesting that the environment of the Trp residues in this new conformation is much less specific */the residue side chains experience a more symmetrical environment than in the native molecular conformation and have a higher mobility. On the other hand, the behavior of bLG in acidified ethanol, observed in the far-UV is similar in the presence
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(curve 3) or in the absence (curve 4) of chloroform. This means that chloroform does not contribute to further changes in the secondary structure of bLG as occurs for the case of albumins [36]. In acidified ethanol (curve 4), two negative peaks at /208 and /222 nm, are obtained together with a stronger CD signal than in aqueous solution. This is usually connected with the increase in a-helix content, which has a stronger CD signal since helical structure facilitates a helical flow of charge [37]. In summary, CD results suggest that this spreading mixture chloroform/ethanol/water contributes to a certain loss of the rigid tertiary packing of the side chains, and promotes the transition from a b-sheet to a a-helix structure in bLG, thus contributing to a more compact secondary structure */a molten globule like state [38 / 40]. This has been proposed as a reversible intermediate state which can exist in all-b proteins and is induced e.g. by an increase in the solvent hydrophobicity (changes in dielectric constant of the surrounding medium and in the hydrogen bonding ability). In order to evaluate the remaining effect of the spreading solvent after the evaporation on the structure of bLG at the interface, the spreading mixture was deposited on a solid substrate [41] and the fluorescence of the Trp was measured. In the spreading solution the chloroform quenches the fluorescence of Trp. Thus, the significant emission recovered after the solvent evaporation at the solid substrate indicates that the structural changes in the tertiary structure of bLG are reversible. Then, the more compact structure of bLG at the interface, suggested by curve 1 (Fig. 1B), can be induced by traces of the hydrophobic spreading mixture retained in the inner structure of bLG. 3.1.2. Effect of pH in the subphase The p /A isotherm of bLG spread from a volatile solvent (spreading method) onto a subphase of pH 2 (curve 1?) differs significantly from the one formed on a pure water subphase (curve 1). Curve 1? deviates to larger molecular areas at low p , and a long plateau (folding/dissolution) starts near 28 mN m 1 until the maximum surface pressure at 33 mN m 1 is reached, instead of 49 mN m 1 as it does on the pure water subphase.
This means that the interaction of bLG with the water is enhanced at low pH. It is interesting to note that, in the relative area scale of Fig. 1B, curve 1? approximates to curves 2 and 3. At pH 2 the electrostatic interactions between bLG and the acid subphase prevail over the intramolecular hydrophobic interactions, promoting an open structure at low surface pressures as the one obtained by using aqueous solutions (curves 2 and 3). The effect of the hydrophobic solvent, suggested by curve 1, disappears at low pH 2. This confirms that the effect of the organic solvent on the bLG structure is reversible in agreement with the fluorescence measurements. The authors believe that the deviation of curves 2 and 3 from curve 1 (Fig. 1A) is mainly due to the loss of a significant amount of protein by dissolution into the subphase. Another possibility is that the organic spreading solvent promotes the unfolding of the protein, causing then the deviation of the p /A isotherms to larger areas. However, there are at least two arguments disfavoring the last explanation. First, the CD analysis indicates that the spreading solvent promotes a protein conformation nearly as compact as the native one. Second, the use of the spreading method at the acidic subphase gives a more expanded monolayer (curve 1?) than at the pure water (curve 1). This means that the electrostatic interactions prevails in the conformational changes at the acidic subphase promoting then a higher unfolding of the protein than that promoted by the spreading solvent. 3.2. Incorporation of bLG in a DODAB monolayer 3.2.1. Comparison of incorporation methods DODAB is a double-chained cationic surfactant which forms an expanded monolayer at 25 8C (approximately the critical temperature of the LE / LC transition) on a pure water subphase (Fig. 3, curve 1). Three methods of addition of bLG to the interface are compared in Fig. 3: co -spreading of a mixed solution with DODAB (curve 2), injection in the subphase before the spreading of DODAB (curve 3), and deposition of solid after the spreading of DODAB (curve 4) at the interface. The three curves are similar in shape, except that the
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gives evidence to the reversibility of each cycle and stability of the monolayer after three consecutive cycles (results not shown). Thus, from the above, we selected the co-spreading as the most reliable method for the incorporation of bLG in DODAB monolayers in the subsequent work.
Fig. 3. p /A isotherms of mixed DODAB/y bLG monolayers on a pure water subphase at 25 8C obtained by different methods of addition of y residues of bLG, per DODAB molecule, to the interface: pure DODAB (1); spreading solvent for y/5.7 (2); injection of an aqueous solution in the subphase for y/23 (3); sprinkling of small crystals for y/33 (4).
monolayer formed by co-spreading (curve 2) has a final collapse at a surface pressure (pC /57 mN m 1) higher than those formed by powder deposition, or injection of aqueous solution (curves 3 and 4, pC /47 mN m 1). This indicates that the co -spreading method enhances the interaction lipid /protein at the interface. The reproducibility and accuracy is also higher for the co-spreading method. Finally, the efficiency of bLG incorporation in the DODAB monolayer can be evaluated by the number of bLG residue, y, per DODAB molecule in the binary mixture (DODAB/ybLG) used to reach the close p /A isotherms of Fig. 3. The efficiency of incorporation increases when y decreases: solid deposition (y/ 33) B/aqueous solution injection (y/23) B/cospreading (y /5.7). The lower efficiency of injection and deposition, when compared with that the co-spreading one, is naturally associated with the water solubility of bLG and the slow kinetic migration of such large molecules to the interface. Thus, injection or solid deposition methods present serious disadvantages and limitations when a quantitative analysis is required. Furthermore, by using the co-spreading method, the compression / expansion cycles performed until 40 mN m 1
3.2.2. Incorporation of increasing amounts of bLG by co-spreading The effect of the variation of the number of residues of bLG, y , added per DODAB molecule is shown in Fig. 4. Increasing y , the mixed isotherms (DODAB/ybLG) progressively deviate from the isotherm of pure DOBAB (curve 1) to larger areas at low surface pressures, while at high p the curves become very close. The transition regime near 30 mN m 1, become longer and flatter with the increase of y . This indicates that at low surface pressures, protein inserts in the expanded (LE) regime of the monolayer; at the plateau or transition regime the protein is expelled downwards from the monolayer; and at high surface pressures, the monolayer regime is probably dominated by the DODAB molecules, until the collapse near 55 mN m 1. Curve 3 (y/1.13)
Fig. 4. p /A isotherms of mixed DODAB/y bLG monolayers on pure water at 25 8C obtained by the spreading solvent method for different values of y residues of bLG, per DODAB molecule, to the interface: y/0 (1); y/0.45 (2), y/1.13 (3), y/4.8 (4), y/7.5 (5). Inset: variation of DA /DA0 as a function of y , at 10 mN m 1 (triangles) and at 25 mN m 1 (crosses).
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differs from the other curves at high surface pressures, with a new plateau (of low reproducibility) appearing near 48 mN m 1 and a very condensed regime forming at higher surface pressures until the final collapse at 63 mN m1. This second plateau, occurring near the collapse surface pressure of the pure DODAB, suggests that this monolayer collapses into a multilayered structure (or double layer) before the final collapse of the film. The increment of area introduced by the incorporation of y residues of bLG, DA , can be compared with the area occupied by the same amount of bLG in a pure monolayer, DA0, at the same surface pressure. The inset of Fig. 4 shows the variation DA /DA0 as a function of y , at two surface pressures below the plateau: 10 mN m 1 (triangles) and at 25 mN m1 (crosses). There is a positive deviation at low y (DA /DA0), while for y ]/3 it is slightly negative, the variation of area is nearly constant and approximates the value of pure bLG (DA 5/DA0). This suggests that at low y, the interaction with DODAB promotes a more open structure of bLG, while the behavior at high y is dominated by the major component bLG, masking the lipid /protein interaction. Thus, the adsorption/binding/insertion of bLG in the DODAB monolayer requires a significant change of the native structure as it was observed with similar systems [23]. The intermediate regime, or transition, clearly indicates that bLG inserts in the LE regime of the DODAB monolayer while it is squeezed out from the LC regime of the monolayer towards a sublayer. Cornec and Narsimham [34] also reported bLG being expelled from the monoglyceride monolayers at high surface pressures and a similar behavior was ascribed to the compression /expansion isotherms of the cytochrome c /dioleoylphosphatidylcholine system [10]. However, some arguments exclude the complete ‘‘squeezing out’’ mechanism. The collapse surface pressures of the mixed films are higher than the one of pure DODAB and the first and second compression /expansion cycles superpose (results not shown). These facts are indicative of strong interactions between bLG and DODAB retaining bLG adsorbed under the monolayer even at high surface pressures. Furthermore, the second
high-pressure regime, observed at curve 3 (y / 1.13) above the second plateau, shows clearly the important interactions between DODAB and bLG. The peculiar shape of this curve led to further investigation of this mixture behavior varying the pH and temperature of the subphase and the number of compression /expansion cycles. 3.2.3. Effect of the subphase pH The p /A isotherms of the mixed DODAB/ 1.13bLG, DODAB and bLG monolayers on a pure water subphase (curves 1 /3, respectively) and at a pH 2 subphase (curves 1?/3?) 25 8C are compared in Fig. 5A. A significant condensing effect is visible, when pH decreases, for DODAB/ 1.13 bLG (curves 1, 1?) and DODAB (curves 2, 2?) by contrast with the observed for the pure protein (curves 3, 3?). In particular, the pH variation particularly affects the mixed film (curves 1, 1?). At pH 2, the p /A isotherm deviates to smaller areas, the plateau appears at lower surface pressures flatter and longer than in the pure water subphase, and the condensed regime at surface pressures higher than 50 mN m 1 disappears. Curves 1? and 2? superimpose above 20 mN m 1. This seems to indicates that bLG is completely squeezed out of the DODAB monolayer at the plateau near 20 mN m 1. In fact, at pH 2, below its isoelectric pH, bLG has a positive net charge. Thus, the repulsive electrostatic interactions between positively charged bLG and the cationic DODAB monolayer can explain the deviation of curve 1? relatively to curve 1. This behavior agrees with the special stability of bLG in acidic conditions and its special affinity to anionic phospholipids [22]. However, the deviation of curve 1? to larger areas than curve 2?, below the plateau at 20 mN m 1, confirms that bLG can still interact with DODAB monolayer. The possible explanation resides on the contribution of the hydrophobic interactions in addition to the electrostatic ones [26]. In fact, many studies state that a conformation or orientational change of the protein structure might be necessary for the protein to interact with the lipid, resulting a protein /lipid complex [16,26]. This conformational change will allow the hydrocarbon chain of the lipid to interact with the hydrophobic outer surface of the a-helix while the
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Fig. 5. Surface pressure /area isotherms of DODAB/1.13 bLG monolayers. pH effect of the subphase at 25 8C (A): pure water at pH 5.8 (1) and pH 2 (1?). Also included for comparison are the correspondent isotherms of pure DODAB (2, 2?) and pure bLG (3, 3?). Temperature effect on a pure water subphase for the first compression (B): 15 8C (1), 20 8C (2) and 25 8C (3), and on the first compression /expansion cycles (C): 15 8C (1, 1?) and 20 8C (2, 2?).
hydrophilic part of the protein will orient to the headgroup. Thus, at pH 5.8, a negatively charged bLG interacts significantly with the cationic monolayer in the all range of p (curve 1), while at pH 2 a positively charged bLG interacts weakly with the cationic monolayer (curve 1?). The condensing effect also observed in the pure DODAB monolayer at pH 2 (HCl) can be explained by the shielding effect of the Cl counterion present in the acidic subphase.
3.2.4. Temperature effect The changes on the p /A isotherms of DODAB/ 1.13 bLG with temperature are shown in Fig. 5B. At low surface pressures, the effect of temperature on mixtures is similar to the one observed on a pure DODAB monolayer [30]. As the temperature increases, the LE /LC transition occurs at increasing surface pressures (6 /7 mN m 1 and 11 /13 mN m 1 at 15 and 20 8C, respectively), disappearing near 25 8C. The squeezing out of bLG at the
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plateau near 30 mN m 1 is not affected by the temperature. Additionally, the plateau near 50 mN m 1, only visible at 25 8C, is probably related with the collapse of DODAB domains in the mixed monolayer. The absence of this plateau at lower temperatures, 15 and 20 8C, should result from the low mobility of molecules in stiff monolayers. 3.2.5. Hysteresis The compression /expansion cycles of the mixed DODAB/1.13 bLG protein monolayer (Fig. 5C) show a significant hysteresis in the transition regime, i.e. during the expansion, the transition occurs at surface pressures lower than during the compression run: ptcomp /ptexp. Additionally, this deviation (ptcomp/ptexp) is enhanced when the temperature decreases. This is probably related with the ionic nature of the monolayer and the role of the repulsive interactions on the reorganization of charges during compression versus expansion. 3.3. BAM observation 3.3.1. Pure bLG Image of bLG at the interface, added by the spreading method is completely homogeneous at low surface pressures (Fig. 6, image a). Upon compression, some bright spots (microdomains) appear near 14 mN m 1 very far apart from each other (b) and some bright stripe-like morphologies appeared in the plateau region during compression/expansion as shown in image (c). Microheterogeneity is visible at the end of the plateau near 29 mN m 1 (d), disappearing during the next expansion. The reversibility of p /A curve was confirmed by BAM images. 3.3.2. DODAB/y bLG Morphologies of mixed monolayers strongly depend on the amount of bLG incorporated per DODAB molecule. Fig. 6 (e and f) shows the BAM observation for a large amount of bLG (y/ 5.7). Very bright images were observed with some stripes observed also in monolayers of pure bLG (c). Image (e), obtained at low surface pressures (p/5.6 mN m 1), shows several small irregularshaped bright domains in addition to the long straight line-like domain. With the increase of p,
the stripe-like domains, similar to the ones observed in the pure bLG, appear at the plateau region (image f, p /28 mN m 1). The intense brightness of the image is probably related with a thick monolayer dominated by the protein excess. These images mainly present characteristics of the major component bLG and the small domains observed at low p are likely related with phase separation of the minor component, DODAB. However, for a lower content of bLG, y /1.13 (curve 3 of Fig. 4) the same morphologies observed in pure DODAB monolayers [30] were also observed in the presence of bLG (Fig. 7). At low surface pressures, in the range of temperature observed (15 /25 8C), the BAM images are homogeneous (a). First domains appear near 3, 10 and 20 mN m 1 at 15, 20 and 25 8C (image b), respectively, in good agreement with LE /LC transition shown in Fig. 5B. The density of domains and the brightness of the image increase with p until the plateau occurring near 30 mN m1 (c). Then, as the compression progresses, dark holes appear in the bright background (d). This image obtained above the plateau, near 40 mN m1, looks like the negative of image (c) at the beginning of the plateau: dark holes in a very bright matrix (image d) while bright domains appear in a dark matrix (image c). The dark holes are probably originated by the partial immersing of the bLG into the subphase (squeezing out of the bLG into a downlayer), and the bright background can be ascribed to a dense monolayer of DODAB. During expansion (after compression until 40 mN m 1), the dark holes disappear progressively as bLG downlayer is being reinserted in the monolayer, and bright domains in fractallike aggregates (image e) appear as in pure DODAB monolayers. This topography vanished as the surface pressure decreased (f) until reaching the monolayer homogeneity at the maximum expansion. The topography of the monolayer above 40 mN m1 depends on the temperature. The compression of the condensed films at 15 and 20 8C (curves 1 and 2) leads to the very bright and microheterogeneous image with dark holes dispersed in the bright image until very high p (image g, p /58 mN m 1). Near the collapse at 60 mN m 1 the
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Fig. 6. Visualization of bLG (a /d) and DODAB/5.7bLG (e, f) by BAM at 25 8C on a pure water subphase during the compression of monolayers: p/1 mN m 1 (a); p/20 mN m 1 (b); p/24 mN m 1 (c); p/29 mN m 1 (d); p/6 mN m 1 (e); p/28 mN m 1 (f). Image size was 470/630 mm2.
microheterogeneity disappears and a very bright image remains (h). The grain boundary of DODAB domains disappears at high surface pressures in the presence of bLG. The close packing of DODAB domains in the upper layer probably
causes the increasing homogeneity and brightness with bLG being squeezed into the downlayer. The absence of heterogeneity near the collapse suggests a collapse mechanism into the subphase. By compressing the monolayer at 25 8C, a different
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Fig. 7. Visualization of DODAB/1.13bLG monolayers by BAM on pure water subphase. Images obtained at 25 8C during the compression until 40 mN m 1: p/3 mN m 1 (a); p/20 mN m 1 (b); p/30 mN m 1 (c); p/40 mN m 1 (d); and during expansion: p/22 mN m 1 (e); p/16 mN m 1 (f). Images obtained during compression above 40 mN m 1: p/58 mN m 1, 20 8C (g); p/60 mN m 1, 20 8C (h); and p/53 mN m 1, 25 8C (i). Image size was 470 /630 mm2.
topography was observed (i). Very bright domains form, in the presence of the dark holes, during the plateau near 50 mN m 1. These brighter domains are probably originated by the nucleation and upgrowth of multilayer structures of DODAB (collapse of DODAB domains at the plateau).
The change of topography with temperature, at high p, corroborates the temperature dependence of p /A isotherms of Fig. 5B. When an even smaller amount of bLG, y/0.44, is incorporated in the monolayer, the DODAB topography dominates at low surface pressures
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before the plateau, while a bright image of lower density in black holes, in agreement with the smaller amount of bLG present in the mixture, forms at the plateau (images not shown). The observation of the fractal-like structures of pure DODAB monolayers in DODAB/ybLG mixed films, suggests that the bLG mainly locates in the liquid expanded phase during the LE /LC transition regime and does not affect the LC domains of pure DODAB. This is in good agreement with the results of Heckl et al. [42]. They observed by fluorescence microscopy that proteins in membranes were mainly located in the fluid membrane phase, which coexisted with solid lipid domains of pure phospholipids. The general findings from the above p /A measurements and BAM visualization indicate that the mechanism of interaction of bLG with DODAB is similar to the one proposed to the interaction with other lipids. Lefe`vre and Subirade [22] postulated that the mechanism of interaction of bLG with a phospholipid monolayer consisted of a three-step process: first, the bLG adsorbs at the monolayer; second, the protein binds to the phospholipids; and finally, some parts of the protein insert into the phospholipid monolayer. They also found that the effect of bLG on some lipid membranes was different whether the lipids were in the gel or in the liquid /crystalline phases, suggesting that the order and packing of the lipid chain can influence the strength of the interactions. Accordingly, we can assume from the present data that bLG is incorporated in the expanded monolayer of DODAB and the threestep mechanism is completed before the monolayer compression. Upon compression of the mixed monolayer, the bLG is expelled along the plateau, without breaking the binding to the DODAB molecules. This permanent binding can explain the monolayer stabilization at high surface pressures. During the following expansion, bLG reinserts in the LE regime until recovering the initial state. The compression /expansion behavior of the mixed monolayers and the appearing /disappearing of dark holes in the BAM images seem to confirm the reversibility of the insertion /expelling third step.
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4. Summary The (co-)spreading of bLG in a volatile solution is the most efficient and reliable method of addition of bLG onto the interface. The conformational change induced by the spreading mixture in the metastable structure of bLG disappears after its evaporation at the interface. Pure bLG adopts a more open structure on a pH 2 subphase than on a pure water subphase. On the other hand, the interaction of bLG with the cationic monolayer is stronger on a pure water subphase than at pH 2. The incorporation of bLG in the LE state of the monolayer is shown by the deviation of p /A isotherms to larger areas and by the homogeneous image at low p. The plateau region of isotherm and the black domains in the BAM images indicate that the protein is squeezed out from the LC regime of DODAB into a sublayer. The p /A isotherm recovering during the second compression run corroborates the strong binding between DODAB and bLG. The remaining characteristics of pure DODAB in the p /A isotherms and BAM images of the mixed films become evident by the presence of the LE /LC transition and by the temperature effect on the transition surface pressure. This confirms that small amounts of bLG can be incorporated in the liquid expanded phase of DODAB monolayer without a significant effect on the topography of pure DODAB condensed domains. On the other hand, the positive deviation of the experimental isotherm relatively to the calculated one, observed at low content of bLG, confirms that the protein in the presence of DODAB adopts a more open conformation than the native one, as was observed in the presence of other lipids.
Acknowledgements This work was supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT) of Portugal through project (POCTI) 35398/2000. R. Roma˜o acknowledges a BIC Grant from project (POCTI) 35398/ 2000. FCT and FSE (III Quadro Comunita´rio de Apoio) are gratefully acknowledged.
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[21] J. Sa´nchez-Gonza´lez, M.A. Cabrerizo-Vı´chez, M.J. Ga´lvez-Ruiz, Colloids Surf. B: Biointerf. 21 (2001) 19. [22] T. Lefe`vre, M. Subirade, Biochim. Biophys. Acta 1549 (2001) 37. [23] J. Zhao, D. Vollhardt, G. Brezesinski, S. Siegel, J. Wu, J.B. Li, R. Miller, Colloids Surf. A: Physicochem. Eng. Aspects 171 (2000) 175. [24] H. Zhang, X. Wang, G. Cui, J. Li, Colloids Surf. A: Physicochem. Eng. Aspects 175 (2000) 77. [25] Y. Fang, D.G. Dalgleish, J. Colloid Interf. Sci. 196 (1997) 292. [26] M.A. Bos, T. Nylander, Langmuir 12 (1996) 2791. [27] S. Brownlow, J.H. Morais Cabral, R. Cooper, D.R. Flower, S.J. Yendall, I. Polikarpov, A.C.T. North, L. Sawyer, Structure 5 (1997) 481. [28] N. Sreerama, M. Hoshino, S.Y. Venyaminov, R.W. Woody, Protein Sci. 8 (1999) 370. [29] K. Kuwata, M. Hoshino, V. Forge, S. Era, C.A. Batt, Y. Goto, Protein Sci. 8 (1999) 2541. [30] A.M. Gonc¸alves da Silva, R.S. Roma˜o, A.L. Caro, J.M. Rodrı´guez Patino, submitted for publication. [31] A.M. Gonc¸alves da Silva, J.C. Guerreiro, N.G. Rodrigues, T.O. Rodrigues, Langmuir 12 (1996) 4442. [32] D. Ho¨nig, D. Mo¨bius, Thin Solid Films 210/211 (1992) 64. [33] J.M. Rodrı´guez Patino, C.C. Sa´nchez, M.R. Rodrı´guez Nin˜o, Langmuir 15 (1999) 4777. [34] M. Cornec, G. Narsimhan, Langmuir 16 (2000) 1216. [35] S. Iametti, B. De Gregori, G. Vecchio, F. Bonomi, Eur. J. Biochem. 237 (1996) 106. [36] S.K. Patra, M.K. Pal, Eur. J. Biochem. 246 (1997) 658. [37] C.R. Cantor, P.R. Schimmel, Biophysical Chemistry Part II: Techniques for the Study of Biological Structure and Function, W.H. Freeman, New York, 1980. [38] V.N. Uversky, N.V. Narizhneva, S.O. Kirschstein, S. Winter, G. Lo¨ber, Folding Des. 2 (1997) 163. [39] X.L. Qi, C. Holt, D. Mcnulty, D.T. Clarke, S. Brownlow, G.R. Jones, Biochem. J. 324 (1997) 341. [40] E. Dufour, C. Bertrand-Harb, T. Haertle´, Biopolymers 33 (1993) 589. [41] The very low emission from a LB monolayer could not be measured. [42] W.M. Heckl, B.N. Zaba, H. Mo¨hwald, Biochim. Biophys. Acta 903 (1987) 166.
Incorporation of b-lactoglobulin in monolayers of dioctadecyldimethylammonium bromide studied by Brewster angle microscopy Ame´lia M. Gonc¸alves da Silva *, Rute I.S. Roma˜o, Suzana M. Andrade, Sı´lvia M.B. Costa Centro de Quı´mica Estrutural, Complexo I, Instituto Superior Te´cnico, 1049-001 Lisbon, Portugal Accepted 10 April 2003
Abstract The deposition of pure bovine b-lactoglobulin (bLG) monolayer or the incorporation in a dioctadecyldimethylammonium bromide (DODAB) monolayer, were studied by p /A measurements at the air /water interface and by direct visualization of the interface by BAM. The co-spreading of the monolayer material dissolved in a mixed volatile solvent (chloroform/ethanol) was selected based on reproducible data and minimum consumption of protein. Conformational changes induced by the mixed solvent on the native structure of bLG, investigated by circular dicroism, disappear after the solvent evaporation at the interface. The p /A isotherms suggest and BAM images confirm, that, at low surface pressures, bLG is incorporated in the liquid expanded monolayer of DODAB, while at the plateau near 30 mN m 1 the protein is squeezed out into micro-domains partially immersed in the subphase and strongly adsorbed under the DODAB layer. The topography of DODAB/y bLG mixed films changes with surface pressure and number of protein residues, y , incorporated per DODAB molecule. BAM observation shows that at low y DODAB molecules dominate the topography of monolayer at the interface. # 2003 Elsevier B.V. All rights reserved. Keywords: Monolayers; Incorporation of proteins; Lipid /proteins; BAM visualization
1. Introduction Lipids and proteins are very important in many fields because of their relevance in nature. They are widely used as emulsifiers or stabilizers in
* Corresponding author. Tel.: /351-218-41-9263; fax: / 351-218-46-4455. E-mail address: [email protected] (A.M. Gonc¸alves da Silva).
emulsions or foams, especially in the food industry. Thus, the behavior of proteins at interfaces [1 / 7] and the formation and characteristics of lipid / protein system have been studied with great interest in the last years [8 /12]. In particular, lipid /protein interactions in monolayers are of great importance to the understanding of processes in biological membranes and to the further design of functional biological surfaces [13 /15]. A Langmuir monolayer is in fact a rather simple way
0927-7765/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0927-7765(03)00102-4
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of modeling half a biomembrane. In spite of the great difference between such a simple model and the complex biological system, the information about molecular orientation and packing, selforganization and stability of organizates has proved to be valuable [16,17]. Incorporation of protein in the monolayer, or adsorption under the monolayer, depends on several parameters such as molecular weight of protein, surface pressure, state and charge of the monolayer, pH of the subphase, temperature, etc. [18 /21]. Furthermore, it has been shown that the adsorption or insertion of proteins in lipid monolayers involves an alteration of the native protein structure [22 /26]. Thus, modeling a complex (multicomponent) system such as a biological membrane, requires the understanding of the role of the individual lipid and protein constituents, in order to assess the synergistic effects in higher mixtures. Bovine b-lactoglobulin (bLG) is a major whey protein abundant in cow’s milk. bLG is a small globular protein (Mw 36 000) folded into a calyx formed by eight antiparallel b-strands that constitute 50% of the protein’s structure, and 3-turn ahelix (10%) located at the outer surface of the b-barrel [27]. This protein is known to have an intrinsic propensity to a-helix, as predicted by the amino acid sequence. This particular behavior led bLG to be recognized as an important model for exploring the mechanism of the transition between a and b structure suggested for several biologically important processes (e.g. involving prion and amylogenic diseases) [25,28,29]. In natural systems, the biological function of bLG is not well known but it is supposed that bLG acts as a carrier of small hydrophobic and amphiphilic compounds while the biomembrane acts as support of bLG. Hydrophobic, electrostatic and hydrogen-bonding interactions may occur simultaneously in the binding of bLG to the guest or host compounds like fatty acids, triglycerides and phospholipids forming lipid/ protein complexes. The first goal of this work is to study the interaction of bLG with the lipidic support at the air /water interface. This paper presents the study of the interaction of bLG with a lipid-like double-chained cationic
surfactant, dioctadecyldimethylammonium bromide (DODAB), in mixed monolayers. For pure and mixed films several methods of addition of bLG at the interface were compared, namely the effect of the spreading solvent on the protein. Pure DODAB was previously studied [30]. Floating monolayers of DODAB/bLG of variable composition were studied at the air /water interface by surface pressure/area measurements and by Brewster angle microscopy (BAM) visualization. At low content of bLG, the topography of mixed monolayers at the air /water interface, followed by BAM, compares well with the topography of pure DODAB.
2. Experimental section 2.1. Materials Bovine bLG AB mixture chromatographically purified and lyophilized with purity higher than 90% was purchased from Sigma (catalogue no. L3908). Dioctadecyldimethylammonium bromide (DODAB)]/98% purity was obtained from Fluka. The solvents chloroform and ethanol were of spectroscopic grade (Uvasol) from Merck. Amphiphiles and solvents were used without further purification. Water used in the subphase and in solutions was distilled twice and purified with the Millipore Milli-Q system in order to obtain a resistivity higher than 18 MV cm. pH 2 at the water subphase was reached by the addition of HCl. Stock solutions of DODAB were prepared in chloroform and stock solutions of bLG in ethanol/ water or in pure water. Mixed solutions of bLG and DODAB were prepared by adding precisely measured volumes of stock solutions and diluted to a final volumetric composition 9:9:1 v/v mixture of chloroform/ethanol/water. A drop of HCl was always added to solutions with protein. 2.2. Methods Because bLG is a water-soluble protein, the addition of bLG to the air /water interface was optimized by comparing the following procedures:
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spreading in a volatile solvent composed by ethanol, chloroform and water, 9:9:1 vol.; addition of an aqueous solution to the interface; injection of an aqueous solution in the subphase; and deposition of solid material at the water interface. Mixed monolayers of DODAB/bLG were formed by similar procedures to the ones above referred: co -spreading method consists of the spreading of the binary mixture in a common volatile solvent; injection of the aqueous solution of bLG in the subphase just before the spreading of DODAB solution at the interface was followed by 20 /30 min of waiting time for equilibration, and three compression /expansion cycles until 25 mN m 1, before the complete compression isotherm; deposition of the solid bLG, over a DODAB monolayer near its lift off, was followed by the same procedure used in the injection method before the complete compression isotherm. 2.3. Surface pressure /area measurements Surface pressure /area (p /A ) isotherm measurements were carried out on a KSV 5000 Langmuir / Blodgett system (KSV Instruments, Helsinki) installed in a laminar flow hood. Procedures for p / A measurements and cleaning care were described elsewhere [31]. The careful suction of the water surface was carried out before every measurement. The surface pressure increase lower than 0.15 mN m 1 when the area was reduced to one tenth warrants the absence of surfactants. The temperature of the subphase was controlled by water circulation from a thermostat within an error of 9/0.1 8C. p /A isotherms were measured several times and different spreading solutions were used. 2.4. Circular dicrosism (CD) spectroscopy CD spectra were obtained using a Jasco Model J-720 spectropolarimeter (Jasco, Hachioji City, Tokyo). The protein spectra were measured at controlled temperature (24.09/0.2 8C) using 10 mm (for near-UV) and 2 mm (for far-UV) cells. The solutions containing 2.5 mM (far-UV) or 14 mM (near-UV)*/obtained correctly by measuring 1 absorption at 280 nm where obLG 280 /17 600 M
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cm 1 */were scanned at 20 nm min 1 with a 0.2 nm step resolution, a 1 nm bandwidth and a sensitivity of 10 millidegrees. The average of four scans was recorded and corrected by subtracting a baseline spectrum. The CD signal was converted to mean residue ellipticity [u ] (deg cm2 dmol 1) defined as [u] /100 uobs(l c) 1, where uobs is the experimental CD value in degrees, c (residue mol dm 3) is the concentration, and l (cm) is the cell path length. 2.5. Brewster angle microscopy (BAM) The commercial BAM2, manufactured by NFT (Go¨ttingen, Germany) mounted on a KSV Langmuir balance was used to observe the morphologic patterns of the monolayer. The light reflected from the surface is collected by two achromatic lenses and detected with a CCD camera. The CCD camera converts the reflectivity signal from the sample into a video image. The spatial resolution is approximately 2 mm. Principles and detailed description of BAM have been described elsewhere [32,33].
3. Results and discussion 3.1. bLG at the air /water interface p /A compression isotherms of bLG on a pure water subphase, differing in the addition procedure of protein to the air /water interface, are compared in Fig. 1: spreading of a volatile solution (curve 1); addition of an aqueous solution on the interface (curve 2); injection of an aqueous solution (curve 3); and deposition of small crystals (curve 4). Although strongly separated in the area scale, all the p /A isotherms of Fig. 1A show similar shapes with two expanded regimes separated by an intermediate regime of lower slope at 20/30 mN m 1. Curve 1?, standing for the spreading procedure on the pH 2 subphase, will be discussed later. In spite of the solubility in water, bLG forms stable monolayers at the air /water interface due to the significant hydrophobic component of interaction. However, the presence of bLG in both
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Fig. 1. p /A isotherms of monolayers of bLG on a pure water subphase at 25 8C. Several methods of addition to the interface: spreading solvent (1); addition of an aqueous solution at the interface (2); injection of an aqueous solution in the subphase (3); sprinkling of small crystals (4). p /A isotherm of bLG on a pH 2 subphase at 25 8C (1?). A in the abscissas stands for the area per residue of bLG (A); the relative area in the abscissas stands for A (p )/A (28 mN m 1) (B).
regions, interface and bulk phase, should be considered. This can explain the deviation of p / A isotherms to smaller areas per residue when an aqueous solution was added at the interface (curve 2) when compared to curve 1 obtained by using a volatile solvent; an even higher deviation was
observed by injection of the aqueous solution into the subphase (curve 3). When the deposition method was used in the absence of solvent, (curve 4), the area occupied per residue at low surface pressures was nearly one tenth of the value obtained by spreading (curve 1). This seems to indicate that only a small fraction of the deposited solid spreads at the water interface and the majority precipitates before spreading, dissolves into the subphase or remains in a solid bulk phase at the interface. The p /A isotherms are reproducible and the monolayers are stable when bLG is added to the interface by using the spreading method. In fact, the second compression isotherm follows the first one after the compression /expansion cycle until 25 mN m1. The reproducibility was not so good with other methods. From the results described we consider the spreading of a volatile solution as the most reliable method to add bLG at the air /water interface. In fact, by injection or addition of an aqueous solution and by direct deposition of the solid, the surface concentration of bLG is not known, which is a serious limitation of these methods in some cases. On the contrary, by using the spreading method, the reproducibility of the p /A compression isotherms and the proximity of the compression /expansion cycles seems to indicate that dissolution is not significant during experience time and we can assume that the surface concentration of bLG is known. The p /A isotherm of bLG obtained by using the Trurnit spreading method [34] locates between curves 1 and 2 of Fig. 1A. This indicates a higher efficiency of this method than injection or addition methods used in this work but it still is less reliable than the spreading by a volatile solvent. Assuming a significant uncertainty on the amount of bLG at the interface obtained some of the methods used, the p /A curves were normalized at p /28 mN m 1 (near the inflection point of the curve, where the compressibility of the monolayers starts to decrease with p ). Each curve was re-plotted in a relative area scale, defined as A (p )/A (28 mN m 1), in Fig. 1B. In this scale, curves coincide at p/28 mN m 1. This representation will allow a prompt comparison of the monolayer compressibility. Curve 1 corresponds
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to the less compressible monolayer (for the same variation of surface pressure the variation of area is minimum for curve 1). Curves 2 and 3, obtained by deposition or injection of an aqueous solution evolve closely indicating similar conformational behavior at the interface. A solvent effect on the native structure of bLG may explain the differences observed in the expanded regimes at low surface pressures. The deviation of curves 2 and 3 to larger areas relatively to curve 1 can be ascribed to the enhancement of the hydrophilic character of bLG by the water solvent increasing the interaction with water subphase. On the contrary, the deviation of curve 1 to smaller areas relatively to curve 2 can be explained by the effect of the hydrophobic spreading solvent and its partial retention at the interface. 3.1.1. Solvent effect on the bLG conformation bLG precipitates in the spreading mixture chloroform/ethanol/water used for the addition at the interface. However, the solution turbidity disappears after the addition of a small drop of hydrochloric acid, for a chloroform content 5/ 50%. The effect of the mixed spreading volatile solvent and the addition of hydrochloric acid on the native structure of the protein in aqueous medium were investigated by CD measurements. The far-UV CD spectra give information on the secondary structure while the near-UV CD spectra inform about the tertiary structure of bLG. Fig. 2 shows the far-UV (A) and the near-UV (B) CD spectra of bLG in several media. The negative CD band found in the far-UV region around 216 nm, for the aqueous solution, pH 5.8, curve 1, was ascribed to the predominant b-sheet protein conformation in this medium, while the bands detected (293 and 286 nm) in the near-UV CD spectra are mainly due to the contribution of four tyrosines and two tryptophans (Trp) in a specific environment. The addition of a small drop of HCl (to a 5 ml volume) would be equivalent to a decrease in pH from 5.8 to 3.8 (curve 2), which means that the global charge of protein changes from slightly negative to positive (isoelectric pH, pI /5.1). However, this variation of pH does not seem to affect significantly the secondary or tertiary structures of bLG, as the proximity of
Fig. 2. Far-UV (A) and near-UV (B) CD spectra of bLG in aqueous solution at pH 5.8 (1) and pH 3.8 (2); bLG in acidified spreading mixture (3); bLG in acidified ethanol (4).
curves 1 and 2 in Fig. 2 suggests. Indeed, bLG is known to be very stable under acidic conditions, which has been explained by the strong stabilizing action of the two disulfide bonds [35]. In the acidified spreading mixture chloroform/ ethanol/water (curve 3), the CD spectra of bLG changed significantly in the far-UV and in the near-UV region. Similar changes were also found in acidified ethanol (curve 4). The analysis of the spectral changes allows some important conclusions. The more pronounced bands in the near-UV region (293 and 286 nm) disappeared suggesting that the environment of the Trp residues in this new conformation is much less specific */the residue side chains experience a more symmetrical environment than in the native molecular conformation and have a higher mobility. On the other hand, the behavior of bLG in acidified ethanol, observed in the far-UV is similar in the presence
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(curve 3) or in the absence (curve 4) of chloroform. This means that chloroform does not contribute to further changes in the secondary structure of bLG as occurs for the case of albumins [36]. In acidified ethanol (curve 4), two negative peaks at /208 and /222 nm, are obtained together with a stronger CD signal than in aqueous solution. This is usually connected with the increase in a-helix content, which has a stronger CD signal since helical structure facilitates a helical flow of charge [37]. In summary, CD results suggest that this spreading mixture chloroform/ethanol/water contributes to a certain loss of the rigid tertiary packing of the side chains, and promotes the transition from a b-sheet to a a-helix structure in bLG, thus contributing to a more compact secondary structure */a molten globule like state [38 / 40]. This has been proposed as a reversible intermediate state which can exist in all-b proteins and is induced e.g. by an increase in the solvent hydrophobicity (changes in dielectric constant of the surrounding medium and in the hydrogen bonding ability). In order to evaluate the remaining effect of the spreading solvent after the evaporation on the structure of bLG at the interface, the spreading mixture was deposited on a solid substrate [41] and the fluorescence of the Trp was measured. In the spreading solution the chloroform quenches the fluorescence of Trp. Thus, the significant emission recovered after the solvent evaporation at the solid substrate indicates that the structural changes in the tertiary structure of bLG are reversible. Then, the more compact structure of bLG at the interface, suggested by curve 1 (Fig. 1B), can be induced by traces of the hydrophobic spreading mixture retained in the inner structure of bLG. 3.1.2. Effect of pH in the subphase The p /A isotherm of bLG spread from a volatile solvent (spreading method) onto a subphase of pH 2 (curve 1?) differs significantly from the one formed on a pure water subphase (curve 1). Curve 1? deviates to larger molecular areas at low p , and a long plateau (folding/dissolution) starts near 28 mN m 1 until the maximum surface pressure at 33 mN m 1 is reached, instead of 49 mN m 1 as it does on the pure water subphase.
This means that the interaction of bLG with the water is enhanced at low pH. It is interesting to note that, in the relative area scale of Fig. 1B, curve 1? approximates to curves 2 and 3. At pH 2 the electrostatic interactions between bLG and the acid subphase prevail over the intramolecular hydrophobic interactions, promoting an open structure at low surface pressures as the one obtained by using aqueous solutions (curves 2 and 3). The effect of the hydrophobic solvent, suggested by curve 1, disappears at low pH 2. This confirms that the effect of the organic solvent on the bLG structure is reversible in agreement with the fluorescence measurements. The authors believe that the deviation of curves 2 and 3 from curve 1 (Fig. 1A) is mainly due to the loss of a significant amount of protein by dissolution into the subphase. Another possibility is that the organic spreading solvent promotes the unfolding of the protein, causing then the deviation of the p /A isotherms to larger areas. However, there are at least two arguments disfavoring the last explanation. First, the CD analysis indicates that the spreading solvent promotes a protein conformation nearly as compact as the native one. Second, the use of the spreading method at the acidic subphase gives a more expanded monolayer (curve 1?) than at the pure water (curve 1). This means that the electrostatic interactions prevails in the conformational changes at the acidic subphase promoting then a higher unfolding of the protein than that promoted by the spreading solvent. 3.2. Incorporation of bLG in a DODAB monolayer 3.2.1. Comparison of incorporation methods DODAB is a double-chained cationic surfactant which forms an expanded monolayer at 25 8C (approximately the critical temperature of the LE / LC transition) on a pure water subphase (Fig. 3, curve 1). Three methods of addition of bLG to the interface are compared in Fig. 3: co -spreading of a mixed solution with DODAB (curve 2), injection in the subphase before the spreading of DODAB (curve 3), and deposition of solid after the spreading of DODAB (curve 4) at the interface. The three curves are similar in shape, except that the
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gives evidence to the reversibility of each cycle and stability of the monolayer after three consecutive cycles (results not shown). Thus, from the above, we selected the co-spreading as the most reliable method for the incorporation of bLG in DODAB monolayers in the subsequent work.
Fig. 3. p /A isotherms of mixed DODAB/y bLG monolayers on a pure water subphase at 25 8C obtained by different methods of addition of y residues of bLG, per DODAB molecule, to the interface: pure DODAB (1); spreading solvent for y/5.7 (2); injection of an aqueous solution in the subphase for y/23 (3); sprinkling of small crystals for y/33 (4).
monolayer formed by co-spreading (curve 2) has a final collapse at a surface pressure (pC /57 mN m 1) higher than those formed by powder deposition, or injection of aqueous solution (curves 3 and 4, pC /47 mN m 1). This indicates that the co -spreading method enhances the interaction lipid /protein at the interface. The reproducibility and accuracy is also higher for the co-spreading method. Finally, the efficiency of bLG incorporation in the DODAB monolayer can be evaluated by the number of bLG residue, y, per DODAB molecule in the binary mixture (DODAB/ybLG) used to reach the close p /A isotherms of Fig. 3. The efficiency of incorporation increases when y decreases: solid deposition (y/ 33) B/aqueous solution injection (y/23) B/cospreading (y /5.7). The lower efficiency of injection and deposition, when compared with that the co-spreading one, is naturally associated with the water solubility of bLG and the slow kinetic migration of such large molecules to the interface. Thus, injection or solid deposition methods present serious disadvantages and limitations when a quantitative analysis is required. Furthermore, by using the co-spreading method, the compression / expansion cycles performed until 40 mN m 1
3.2.2. Incorporation of increasing amounts of bLG by co-spreading The effect of the variation of the number of residues of bLG, y , added per DODAB molecule is shown in Fig. 4. Increasing y , the mixed isotherms (DODAB/ybLG) progressively deviate from the isotherm of pure DOBAB (curve 1) to larger areas at low surface pressures, while at high p the curves become very close. The transition regime near 30 mN m 1, become longer and flatter with the increase of y . This indicates that at low surface pressures, protein inserts in the expanded (LE) regime of the monolayer; at the plateau or transition regime the protein is expelled downwards from the monolayer; and at high surface pressures, the monolayer regime is probably dominated by the DODAB molecules, until the collapse near 55 mN m 1. Curve 3 (y/1.13)
Fig. 4. p /A isotherms of mixed DODAB/y bLG monolayers on pure water at 25 8C obtained by the spreading solvent method for different values of y residues of bLG, per DODAB molecule, to the interface: y/0 (1); y/0.45 (2), y/1.13 (3), y/4.8 (4), y/7.5 (5). Inset: variation of DA /DA0 as a function of y , at 10 mN m 1 (triangles) and at 25 mN m 1 (crosses).
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differs from the other curves at high surface pressures, with a new plateau (of low reproducibility) appearing near 48 mN m 1 and a very condensed regime forming at higher surface pressures until the final collapse at 63 mN m1. This second plateau, occurring near the collapse surface pressure of the pure DODAB, suggests that this monolayer collapses into a multilayered structure (or double layer) before the final collapse of the film. The increment of area introduced by the incorporation of y residues of bLG, DA , can be compared with the area occupied by the same amount of bLG in a pure monolayer, DA0, at the same surface pressure. The inset of Fig. 4 shows the variation DA /DA0 as a function of y , at two surface pressures below the plateau: 10 mN m 1 (triangles) and at 25 mN m1 (crosses). There is a positive deviation at low y (DA /DA0), while for y ]/3 it is slightly negative, the variation of area is nearly constant and approximates the value of pure bLG (DA 5/DA0). This suggests that at low y, the interaction with DODAB promotes a more open structure of bLG, while the behavior at high y is dominated by the major component bLG, masking the lipid /protein interaction. Thus, the adsorption/binding/insertion of bLG in the DODAB monolayer requires a significant change of the native structure as it was observed with similar systems [23]. The intermediate regime, or transition, clearly indicates that bLG inserts in the LE regime of the DODAB monolayer while it is squeezed out from the LC regime of the monolayer towards a sublayer. Cornec and Narsimham [34] also reported bLG being expelled from the monoglyceride monolayers at high surface pressures and a similar behavior was ascribed to the compression /expansion isotherms of the cytochrome c /dioleoylphosphatidylcholine system [10]. However, some arguments exclude the complete ‘‘squeezing out’’ mechanism. The collapse surface pressures of the mixed films are higher than the one of pure DODAB and the first and second compression /expansion cycles superpose (results not shown). These facts are indicative of strong interactions between bLG and DODAB retaining bLG adsorbed under the monolayer even at high surface pressures. Furthermore, the second
high-pressure regime, observed at curve 3 (y / 1.13) above the second plateau, shows clearly the important interactions between DODAB and bLG. The peculiar shape of this curve led to further investigation of this mixture behavior varying the pH and temperature of the subphase and the number of compression /expansion cycles. 3.2.3. Effect of the subphase pH The p /A isotherms of the mixed DODAB/ 1.13bLG, DODAB and bLG monolayers on a pure water subphase (curves 1 /3, respectively) and at a pH 2 subphase (curves 1?/3?) 25 8C are compared in Fig. 5A. A significant condensing effect is visible, when pH decreases, for DODAB/ 1.13 bLG (curves 1, 1?) and DODAB (curves 2, 2?) by contrast with the observed for the pure protein (curves 3, 3?). In particular, the pH variation particularly affects the mixed film (curves 1, 1?). At pH 2, the p /A isotherm deviates to smaller areas, the plateau appears at lower surface pressures flatter and longer than in the pure water subphase, and the condensed regime at surface pressures higher than 50 mN m 1 disappears. Curves 1? and 2? superimpose above 20 mN m 1. This seems to indicates that bLG is completely squeezed out of the DODAB monolayer at the plateau near 20 mN m 1. In fact, at pH 2, below its isoelectric pH, bLG has a positive net charge. Thus, the repulsive electrostatic interactions between positively charged bLG and the cationic DODAB monolayer can explain the deviation of curve 1? relatively to curve 1. This behavior agrees with the special stability of bLG in acidic conditions and its special affinity to anionic phospholipids [22]. However, the deviation of curve 1? to larger areas than curve 2?, below the plateau at 20 mN m 1, confirms that bLG can still interact with DODAB monolayer. The possible explanation resides on the contribution of the hydrophobic interactions in addition to the electrostatic ones [26]. In fact, many studies state that a conformation or orientational change of the protein structure might be necessary for the protein to interact with the lipid, resulting a protein /lipid complex [16,26]. This conformational change will allow the hydrocarbon chain of the lipid to interact with the hydrophobic outer surface of the a-helix while the
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Fig. 5. Surface pressure /area isotherms of DODAB/1.13 bLG monolayers. pH effect of the subphase at 25 8C (A): pure water at pH 5.8 (1) and pH 2 (1?). Also included for comparison are the correspondent isotherms of pure DODAB (2, 2?) and pure bLG (3, 3?). Temperature effect on a pure water subphase for the first compression (B): 15 8C (1), 20 8C (2) and 25 8C (3), and on the first compression /expansion cycles (C): 15 8C (1, 1?) and 20 8C (2, 2?).
hydrophilic part of the protein will orient to the headgroup. Thus, at pH 5.8, a negatively charged bLG interacts significantly with the cationic monolayer in the all range of p (curve 1), while at pH 2 a positively charged bLG interacts weakly with the cationic monolayer (curve 1?). The condensing effect also observed in the pure DODAB monolayer at pH 2 (HCl) can be explained by the shielding effect of the Cl counterion present in the acidic subphase.
3.2.4. Temperature effect The changes on the p /A isotherms of DODAB/ 1.13 bLG with temperature are shown in Fig. 5B. At low surface pressures, the effect of temperature on mixtures is similar to the one observed on a pure DODAB monolayer [30]. As the temperature increases, the LE /LC transition occurs at increasing surface pressures (6 /7 mN m 1 and 11 /13 mN m 1 at 15 and 20 8C, respectively), disappearing near 25 8C. The squeezing out of bLG at the
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plateau near 30 mN m 1 is not affected by the temperature. Additionally, the plateau near 50 mN m 1, only visible at 25 8C, is probably related with the collapse of DODAB domains in the mixed monolayer. The absence of this plateau at lower temperatures, 15 and 20 8C, should result from the low mobility of molecules in stiff monolayers. 3.2.5. Hysteresis The compression /expansion cycles of the mixed DODAB/1.13 bLG protein monolayer (Fig. 5C) show a significant hysteresis in the transition regime, i.e. during the expansion, the transition occurs at surface pressures lower than during the compression run: ptcomp /ptexp. Additionally, this deviation (ptcomp/ptexp) is enhanced when the temperature decreases. This is probably related with the ionic nature of the monolayer and the role of the repulsive interactions on the reorganization of charges during compression versus expansion. 3.3. BAM observation 3.3.1. Pure bLG Image of bLG at the interface, added by the spreading method is completely homogeneous at low surface pressures (Fig. 6, image a). Upon compression, some bright spots (microdomains) appear near 14 mN m 1 very far apart from each other (b) and some bright stripe-like morphologies appeared in the plateau region during compression/expansion as shown in image (c). Microheterogeneity is visible at the end of the plateau near 29 mN m 1 (d), disappearing during the next expansion. The reversibility of p /A curve was confirmed by BAM images. 3.3.2. DODAB/y bLG Morphologies of mixed monolayers strongly depend on the amount of bLG incorporated per DODAB molecule. Fig. 6 (e and f) shows the BAM observation for a large amount of bLG (y/ 5.7). Very bright images were observed with some stripes observed also in monolayers of pure bLG (c). Image (e), obtained at low surface pressures (p/5.6 mN m 1), shows several small irregularshaped bright domains in addition to the long straight line-like domain. With the increase of p,
the stripe-like domains, similar to the ones observed in the pure bLG, appear at the plateau region (image f, p /28 mN m 1). The intense brightness of the image is probably related with a thick monolayer dominated by the protein excess. These images mainly present characteristics of the major component bLG and the small domains observed at low p are likely related with phase separation of the minor component, DODAB. However, for a lower content of bLG, y /1.13 (curve 3 of Fig. 4) the same morphologies observed in pure DODAB monolayers [30] were also observed in the presence of bLG (Fig. 7). At low surface pressures, in the range of temperature observed (15 /25 8C), the BAM images are homogeneous (a). First domains appear near 3, 10 and 20 mN m 1 at 15, 20 and 25 8C (image b), respectively, in good agreement with LE /LC transition shown in Fig. 5B. The density of domains and the brightness of the image increase with p until the plateau occurring near 30 mN m1 (c). Then, as the compression progresses, dark holes appear in the bright background (d). This image obtained above the plateau, near 40 mN m1, looks like the negative of image (c) at the beginning of the plateau: dark holes in a very bright matrix (image d) while bright domains appear in a dark matrix (image c). The dark holes are probably originated by the partial immersing of the bLG into the subphase (squeezing out of the bLG into a downlayer), and the bright background can be ascribed to a dense monolayer of DODAB. During expansion (after compression until 40 mN m 1), the dark holes disappear progressively as bLG downlayer is being reinserted in the monolayer, and bright domains in fractallike aggregates (image e) appear as in pure DODAB monolayers. This topography vanished as the surface pressure decreased (f) until reaching the monolayer homogeneity at the maximum expansion. The topography of the monolayer above 40 mN m1 depends on the temperature. The compression of the condensed films at 15 and 20 8C (curves 1 and 2) leads to the very bright and microheterogeneous image with dark holes dispersed in the bright image until very high p (image g, p /58 mN m 1). Near the collapse at 60 mN m 1 the
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Fig. 6. Visualization of bLG (a /d) and DODAB/5.7bLG (e, f) by BAM at 25 8C on a pure water subphase during the compression of monolayers: p/1 mN m 1 (a); p/20 mN m 1 (b); p/24 mN m 1 (c); p/29 mN m 1 (d); p/6 mN m 1 (e); p/28 mN m 1 (f). Image size was 470/630 mm2.
microheterogeneity disappears and a very bright image remains (h). The grain boundary of DODAB domains disappears at high surface pressures in the presence of bLG. The close packing of DODAB domains in the upper layer probably
causes the increasing homogeneity and brightness with bLG being squeezed into the downlayer. The absence of heterogeneity near the collapse suggests a collapse mechanism into the subphase. By compressing the monolayer at 25 8C, a different
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Fig. 7. Visualization of DODAB/1.13bLG monolayers by BAM on pure water subphase. Images obtained at 25 8C during the compression until 40 mN m 1: p/3 mN m 1 (a); p/20 mN m 1 (b); p/30 mN m 1 (c); p/40 mN m 1 (d); and during expansion: p/22 mN m 1 (e); p/16 mN m 1 (f). Images obtained during compression above 40 mN m 1: p/58 mN m 1, 20 8C (g); p/60 mN m 1, 20 8C (h); and p/53 mN m 1, 25 8C (i). Image size was 470 /630 mm2.
topography was observed (i). Very bright domains form, in the presence of the dark holes, during the plateau near 50 mN m 1. These brighter domains are probably originated by the nucleation and upgrowth of multilayer structures of DODAB (collapse of DODAB domains at the plateau).
The change of topography with temperature, at high p, corroborates the temperature dependence of p /A isotherms of Fig. 5B. When an even smaller amount of bLG, y/0.44, is incorporated in the monolayer, the DODAB topography dominates at low surface pressures
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before the plateau, while a bright image of lower density in black holes, in agreement with the smaller amount of bLG present in the mixture, forms at the plateau (images not shown). The observation of the fractal-like structures of pure DODAB monolayers in DODAB/ybLG mixed films, suggests that the bLG mainly locates in the liquid expanded phase during the LE /LC transition regime and does not affect the LC domains of pure DODAB. This is in good agreement with the results of Heckl et al. [42]. They observed by fluorescence microscopy that proteins in membranes were mainly located in the fluid membrane phase, which coexisted with solid lipid domains of pure phospholipids. The general findings from the above p /A measurements and BAM visualization indicate that the mechanism of interaction of bLG with DODAB is similar to the one proposed to the interaction with other lipids. Lefe`vre and Subirade [22] postulated that the mechanism of interaction of bLG with a phospholipid monolayer consisted of a three-step process: first, the bLG adsorbs at the monolayer; second, the protein binds to the phospholipids; and finally, some parts of the protein insert into the phospholipid monolayer. They also found that the effect of bLG on some lipid membranes was different whether the lipids were in the gel or in the liquid /crystalline phases, suggesting that the order and packing of the lipid chain can influence the strength of the interactions. Accordingly, we can assume from the present data that bLG is incorporated in the expanded monolayer of DODAB and the threestep mechanism is completed before the monolayer compression. Upon compression of the mixed monolayer, the bLG is expelled along the plateau, without breaking the binding to the DODAB molecules. This permanent binding can explain the monolayer stabilization at high surface pressures. During the following expansion, bLG reinserts in the LE regime until recovering the initial state. The compression /expansion behavior of the mixed monolayers and the appearing /disappearing of dark holes in the BAM images seem to confirm the reversibility of the insertion /expelling third step.
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4. Summary The (co-)spreading of bLG in a volatile solution is the most efficient and reliable method of addition of bLG onto the interface. The conformational change induced by the spreading mixture in the metastable structure of bLG disappears after its evaporation at the interface. Pure bLG adopts a more open structure on a pH 2 subphase than on a pure water subphase. On the other hand, the interaction of bLG with the cationic monolayer is stronger on a pure water subphase than at pH 2. The incorporation of bLG in the LE state of the monolayer is shown by the deviation of p /A isotherms to larger areas and by the homogeneous image at low p. The plateau region of isotherm and the black domains in the BAM images indicate that the protein is squeezed out from the LC regime of DODAB into a sublayer. The p /A isotherm recovering during the second compression run corroborates the strong binding between DODAB and bLG. The remaining characteristics of pure DODAB in the p /A isotherms and BAM images of the mixed films become evident by the presence of the LE /LC transition and by the temperature effect on the transition surface pressure. This confirms that small amounts of bLG can be incorporated in the liquid expanded phase of DODAB monolayer without a significant effect on the topography of pure DODAB condensed domains. On the other hand, the positive deviation of the experimental isotherm relatively to the calculated one, observed at low content of bLG, confirms that the protein in the presence of DODAB adopts a more open conformation than the native one, as was observed in the presence of other lipids.
Acknowledgements This work was supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT) of Portugal through project (POCTI) 35398/2000. R. Roma˜o acknowledges a BIC Grant from project (POCTI) 35398/ 2000. FCT and FSE (III Quadro Comunita´rio de Apoio) are gratefully acknowledged.
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