4710
Langmuir 1997, 13, 4710-4715
Interactions of Spread Lecithin Monolayers with Bovine Serum Albumin in Aqueous Solution Daechul Cho,† Ganesan Narsimhan,*,‡ and Elias I. Franses‡ Biochemical and Food Process Engineering, Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana 47907-1146, and Department of Chemical Engineering, Purdue University, West lafayette, Indiana 47907-1283 Received April 4, 1997. In Final Form: June 18, 1997X The dynamics of surface pressure (Π) and of surface concentration (Γ) of 14C radiolabeled bovine serum albumin (BSA) adsorbed onto spread lecithin monolayers at the air-water interface were measured. The adsorption of BSA onto spread lecithin monolayers of 107 and 64 Å2/molecule was enhanced at short times (within a few seconds), indicating synergism due to possible dissolution of BSA molecules into loosely or moderately packed lecithin layers. The surface concentration of BSA increased to 2-2.5 mg/m2 from about 1 mg/m2 and the surface pressure to 20-35 mN/m from about 10 mN/m in the presence of spread lecithin monolayer. At long times, monolayer composition was found, however, to be dominated by lecithin. BSA was expelled from the interface by close-packed lecithin monolayers, possibly because of the surface pressure and steric exclusion effects.
Introduction Protein-lipid systems are commonly found in many biological applications such as cell membranes, biosensors,1-3 and lung surfactants.4 ,5 Moreover, in food systems, emulsifiers are composed of biological polymers and low molecular weight surfactants or lipids.6-8 Even though these molecules affect foam and emulsion stability, the stabilizing mechanisms are not fully understood.8 Electrostatic and hydrophilic interactions may be important in mixed films. Protein-surfactant mixtures have been investigated extensively.9-19 Patino et al.13 found that addition of monostearin monolayers on preadsorbed protein films results in a rapid initial reduction of surface tension, * To whom correspondence should be addressed. Fax: 765-4961115. E-mail:
[email protected]. † Department of Agricultural and Biological Engineering. ‡ Department of Chemical Engineering. X Abstract published in Advance ACS Abstracts, August 1, 1997. (1) Castlden, J. A. J. Pharmacol. Sci. 1969, 58, 149. (2) Zhu, D. G.; Petty, M. C.; Ancelin, H. H.; Yarwood, J. Thin Solid Films 1989, 176, 151. (3) Okahata, O.; Tsuruta, T.; Ijiro, K.; Ariga, K. Thin Solid Films 1989, 180, 65. (4) Notter, R. H. In Surfactant Replacement Therapy; Shapiro, D. L., Notter, R. H., Eds.; A. R. Liss: New York, 1989; p 19. (5) Grotberg, J. B. BMES Bull. 1992, 16 (4), 51. (6) Darling, D. F.; Birkett, R. J. In Food Emulsions and Foams; Dickinson, E., Ed.; Royal Society of Chemistry: London, 1986; Chapter 1, pp 1-29. (7) Dickinson, E.; Stainsby, G. Colloids in Food; Applied Science: London,1982; Chapter 1, pp 7-8. (8) Dickinson, E.; Narhan, S. K.; Stainsby, G. J. Food Sci. 1989, 54, 77. (9) Nakamura, R.; Mizutani, R.; Masayo, Y.; Hayakawa, S. J. Agric. Food Chem. 1988, 36, 729. (10) Dickinson, E.; Iveson, G. Food Hydrocolloids 1993, 6 (6), 533. (11) Courthaudon, J.-L.; Dickinson, E. J. Agric. Food Chem. 1991, 39, 1365. (12) Fang, Y.; Dalgleish, D. G. J. Am. Oil Chem. Soc. 1996, 73 (4), 437. (13) Patino, J. M. R.; Nino, M. R. R. Colloid Surf. A 1995, 103, 91. (14) Wustneck, R.; Kretzschmar, G.; Zastrow, L. Colloid J. USSR 1987, 49, 207. (15) Wustneck, R.; Kretzschmar, G.; Zastrow, L. Colloid J. USSR 1985, 47, 387. (16) De Feijter, J. A.; Benjamins, J.; Tamboer, M. Colloid Surf. 1987, 27, 243. (17) Bos, M. A.; Nylander, T. Langmuir 1996, 12, 1791. (18) Aynie, S.; Meste, M. L.; Colas, B.; Lorient, D. J. Food Sci. 1992, 57 (4), 883. (19) Du, Y.-k.; An J.-y.; Tang, J.; Li, Y.; Jiang L. Colloids Surf. B 1996, 7, 129.
S0743-7463(97)00348-X CCC: $14.00
followed by a gradual increase back to the value before monostearin was added. Thus, it appears that the protein was expelled or forced to desorb by the intruding lipid molecules. Human serum albumin and DPPC (dipalmitoylphosphatidylcholine) as a pulmonary surfactant mixture were investigated by Teneva et al.20 For the mixed monolayers, prepared by conventional spreading methods, Π-A isotherms indicated reliable transitions between pure monolayers.20 The area per molecule (at high surface compression, i.e., Π ≈45 mN/m) was less than the estimated area for complete packing in the monolayer, implying some partial expulsion of protein segments. Surface elasticity measurements supported this hypothesis and suggested that protein molecules were desorbed from the interface with mainly lipid molecules remaining. Desorption of macromolecules was analyzed by Stuart et al.21,22 using lattice models, with the Flory-Huggins parameter χs used for characterizing mixing in monolayers. Using this model with the segmental adsorption energy of the polymer in calculating the desorption free energy led to predictions in good agreement with data of poly(vinylpyrrolidone) on silica. Displacement of proteins by water-soluble or oil-soluble surfactants may occur because small surfactants have higher adsorption energies than proteins. Measurements of protein and surfactant adsorbed densities were used to probe how a surfactant above a critical surfactant concentration displaces a protein.16 Generally, reported mechanisms for protein-surfactant interactions involve either electrostatic interactions between ionic surfactant head groups and the charged macromolecules or hydrophobic interactions.23-30 . Conformational changes in macromolecules caused by complex (20) Teneva, S.; Panaiotov, I.; Ter-Minassian-Saraga, L. Colloid Surf., 1984, 10, 101. (21) Stuart, M. A. C.; Fleer, G. J.; Scheutjens, J. M. H. M. J. Colloid Interface Sci. 1984, 97, 515. (22) Stuart, M. A. C.; Fleer, G. J.; Scheutjens, J. M. H. M. J. Colloid Interface Sci. 1984, 97, 526. (23) Sovilj, V.; Djakovic, L.; Dokic, P. J. Colloid Interface Sci. 1993, 158, 483. (24) Malmsten, M.; Bergenstahl, B.; Masquelier, M.; Palsson, M.; Peterson, C. J. Colloid Interface Sci. 1995, 172, 485. (25) Cornell, D. G.; Patterson, D. L. J. Agric. Food Chem. 1989, 37 (6), 1455. (26) Malmsten, M., and Lassen, B. Colloid Surf. B 1995, 4 (3), 173. (27) Fang, Y.; Dalgleish, D. G. Langmuir 1995, 11, 75. (28) Cornell, D. G. J. Colloid Interface Sci. 1982, 88, 536. (29) Cornell, D. G.; Carrol, R. J. J. Colloid Interface Sci. 1985, 108, 226.
© 1997 American Chemical Society
Interactions of Lecithin Monolayers with BSA
(aggregate) formation may also be important in emulsion stability. For ionic surfactants in oil-water systems, both electrostatic and hydrophobic interactions appear to be important.23 Electrostatic interactions were investigated at varying pH conditions with a film balance,28,29 where the area per molecule (A h ) for each component was smaller in the mixed monolayer than in the single-component monolayer, suggesting net attractive interactions between the two components. Circular dichroism data suggested that there were more portions of R-helix and β-sheet structures in mixed monolayers of egg yolk phosphatidic acid and β-lactoglobulin than in the protein alone.28 Electron microscopy data on lipid-protein monolayers helped to clarify these structures.29 Lipids forming condensed films at the air-water interface may not mix well with proteins, leading to phase separation in the monolayer. By contrast, those exhibiting liquid-expanded monolayer behavior tend to produce well-mixed monolayers with proteins. The behavior of a model lipid and immunoglobulin G (IgG, an antibody), was studied at the air-water interface.30 Spreading of the lipid on aqueous protein substrates caused an increase in the surface pressure, due primarily to an irreversible hydrophobic binding. Lecithin with two long hydrocarbon (fatty acid) chains (lipophilic) packs naturally into bilayers. Egg lecithin, which contains a mixture of several lipids, some of which have unsaturated chains, forms with water at room temperature a dispersion of liquid microcrystallites (liposomes). It is used as an emulsion stabilizer in foods, where it may interact with proteins. Anderson and Pethica and others32-36 investigated in detail the behavior of the interfacial film of a synthetic lecithin. By comparing the surface pressure-area data of saturated 1,2-diacylphosphatidylcholines, Phillips and Chapman37 found that lecithins with shorter (C12-C14) hydrocarbon tails resulted in liquid expanded monolayers while those with longer hydrocarbon chains (C16-C18) tended to form liquidcondensed monolayers. Monolayers with phosphatidylcholine head groups were less dense than those with cephalin or phosphatidylamine head groups.38 Lecithin monolayers were more expanded as the substrate pH increased.38 From the surface pressures of monolayer mixtures of lecithin-serum albumin, it was inferred20 that some protein residues were expelled from the monolayer at surface pressures from 13 to 45 mN/m. Π-A isotherms of mixed monolayers were found to exhibit inflection points possibly due to monolayer phase transitions. The elasticity of mixed monolayers was found to have a minimum at a surface pressure of 23 ( 1 mN/m. They also observed hysteresis of mixed monolayers. In summary, previous studies of mixed protein-lipid monolayers measured the changes in surface pressure due to exchange of lecithin with BSA but did not directly probe the changes in surface concentration of the protein. In this paper, BSA concentrations in mixed monolayers with lecithin were measured using 14C-labeled BSA.39-41 The results point to enhancement of protein adsorption (30) Ivanova, M.; Panaiotov, I.; Trifonova, T.; Echkenazi, M.; Konstantinov, G.; Ivanova, R. Colloid Surf. 1984, 10, 269. (31) Anderson, P. J.; Pethica, B. A. In Biochemical Problems of Lipids; Popjak, G., Le Breton, E., Eds.; Butterworth: London, 1965; p 24. (32) Van Deenen, L. L. M.; Houtsmuller, U. M. T.; De Hass, G. H.; Mulde, E. J. Pharm. Pharmacol. 1962, 14, 429. (33) Demel, R. A.; Joos, P. Chem. Phys. Lipids 1968, 2, 35. (34) Cadenhead, D. A.; Demchak, R. J.; Phillips, M. C. Kolloid-Z. Z. Polym. 1967, 22, 59. (35) Shah, D. O.; Schulman, J. H. J. Colloid Interface Sci. 1967, 25, 107. (36) Watkins, J. C. Biochim. Biophys. Acta 1968, 152, 293. (37) Phillips, M. C.; Chapman, D. Biochim. Biophys. Acta 1968, 163, 301. (38) Minones, J.; Sandez Macho, M. I.; Iribarnegaray, E.; Sanz Pedrero, P. Colloid Polym. Sci. 1981, 259, 382.
Langmuir, Vol. 13, No. 17, 1997 4711
by relatively dilute lipid monolayer densities and a decline of adsorption with dense lipid monolayers. Materials and Methods Materials and Apparatus. Egg-yolk lecithin (with L-R- head group) with estimated average MW of 768 was used (Sigma Chemical Inc., St. Louis, MO). The fatty acid composition from gas chromatographic analysis (provided by Sigma) is known to be 30% oleic, 30% palmitic, 16% linoleic, 13% stearic acid, and 11% others. Reagent grade n-hexane and ethanol were purchased from Aldrich Chemical. Lecithin solutions were made in hexaneethanol (v/v ) 9/1) and were used within 2 days. Isotopes of [14C]formaldehyde and [14C]sodium acetate were purchased from Sigma Chemical Inc. Sodium cyanoborohydride (NaCNBH3, 95%) was purchased from Aldrich Chemical. A Langmuir minitrough (with dimensions of 330 × 75 × 6.5 mm) from KSV was used for both surface pressure and surface concentration measurements. A gas proportional detector (Ludlum model 120, with a 2 × 2 in Mylar window) with a digital scaler/counter (Ludlum model 520) was used for detecting radioactivity, in counts per minute (CPM), from the adsorbed monolayer at the air-water interface. Radioactivity was measured under P-10 gas (10% methane in argon) flowing at 55 mL/ min through the detector chamber. [14C]BSA was produced by methylating BSA with [14C]formaldehyde. [14C]BSA rather than [14C]stearic acid was used for calibration of the surface activity, since by using [14C]stearic acid one tends to underestimate the surface concentration because of its smaller size compared to BSA.39 Labeled sodium acetate was used for calibrating the background radioactivity. Further details of the calibration are given elsewhere.42 Methods. A 27 mg sample of BSA powder was dissolved in 12.5 mL of 0.05 M phosphate buffer (pH 7). The 1.25 mL of 0.1 M NaCNBH3 was added to the protein solution. 10 mL of 2.12 × 10-4 M H14CHO stock solution (with a total radioactivity of 102 µCi) was mixed well with the protein solution. The methylation reaction was allowed to proceed at room temperature for 2 h, after which the mixture was dialyzed for 30 h at ∼4 °C. The dialyzed solution was concentrated using poly(ethylene glycol) (MW ) 8000). The BSA was found to have 3.1 labeled amide groups labeled per molecule on the average (2.14 µCi/mg of BSA), as analyzed with a scintillation counter (Tri-carb 4000, from Packard instruments);42 i.e., only about 0.5% of amino acid residues were methylated for radiolabeling. The difference in the surface activities of [14C]BSA and native BSA were found to be insignificant, as evidenced by the comparison of their spread monolayer Π-A isotherms (results not shown here). For spreading a lecithin monolayer, 25 mL of lecithin solution (1.0 mg/mL) in a hexane-ethanol mixture (v/v ) 9/1) was placed on the surface as several uniformly distributed droplets. About 10 min was allowed for solvent evaporation. For preparing spread monolayers of the protein, the Trurnit’s monolayer spreading method was used.43 Aliquots of 50 µL of a 0.0247 wt % protein solution were dripped from the top of a glass rod (5 mm diameter and 5 cm long) positioned across the air-water interface. The solution was spread uniformly on the interface. As detailed previously, there was negligible loss of protein to the bulk due to desorption.43 Consequently, it is reasonable to assume that all the proteins spread are adsorbed on the surface. For mixed monolayers, first, a BSA monolayer was formed with Trurnit’s method. Then, various aliquots of lecithin solution in hexane-ethanol were spread at several spots on the surface. The order of deposition (first lecithin, then protein, or vice versa) caused no effect on the results. In the first set of experiments, monolayers of BSA, lecithin, or mixtures were spread on the aqueous subphase. About 10 min was allowed for the monolayers to equilibrate. Surface areas were then reduced from 247.5 to 30 cm2 with the minitrough Teflon barriers at 15 cm2/min, with the surface pressure continuously recorded. Immediately after the end of the (39) Xu, S.; Damodaran, S. J. Colloid Interface Sci. 1993, 157, 485. (40) Anand, K.; Damodaran, S. J. Agric. Food Chem. 1996, 44, 1022. (41) Cho, D.; Narsimhan, G.; Franses, E. I. J. Colloid Interface Sci., in press. (42) Cho, D. Ph.D. Thesis, Purdue University, West Lafayette, Indiana, 1996. (43) Cho, D., Franses, E. I., and Narsimhan, G. Colloids Surf. A 1996, 117, 45.
4712 Langmuir, Vol. 13, No. 17, 1997
Cho et al.
Figure 2. Π - A h isotherms of mixed monolayers at 24 ( 2 °C and pH ) 7 for varied L/B mass ratio. (L/B > 1 or ) 1, per 10 µg of BSA; L/B < 1, per 10 µg of lecithin). The average molecular area of the mixture, A h ) A/(nL + nBSA) where A is the total area, nL and nBSA are the total number of molecules of lecithin and BSA, respectively.
Figure 1. (Top) Π - A h (area per molecule) isotherm of egg lecithin at 24 ( 2 °C (Bottom) Π - A h isotherm of BSA at 24 ( 2 °C. The subphase was 0.1 M phosphate buffer of pH 7. The 2/min. compression rate was 15 cm compression stage, the area was expanded at the same rate. The Π-A isotherms obtained by using this method were found to be reproducible. For pure lecithin monolayers, no difference between Π-A isotherms of repeated experiments was observed. For pure BSA monolayers, the Π-A isotherms of repeated experiments were identical in the expanded region of areas greater than 8000 Å2/molecule. In the condensed region of molecular areas less than 8000 Å2/molecule, the maximum difference in Π between repeated experiments was found to be 1 mN/m. In the second set of experiments, a BSA solution (bulk concentration, cb )1.25 or 2.5 ppm) was poured slowly into the Langmuir minitrough, and the surface was quickly aspirated to remove possible impurities. After that, a lecithin monolayer was spread on the nearly clean surface, and the surface pressure and surface concentration (via radioactivity measurements) were monitored for up to 6 h. Other experiments, without surface aspiration, were done in which lecithin was spread on top of adsorbed protein monolayers prepared by allowing BSA to adsorb for 0.5 or 2 h.
Results Surface Isotherms. For reference, the Π-A isotherms of egg lecithin and BSA monolayers are shown in Figure 1. Lecithin monolayers showed small surface pressures for A h ranging from 126 to about 80 Å2/molecule. Upon further compression, Π increased fast until it reached a plateau at A h ) 36 Å2/molecule and Πmax ) 47 mN/m. The isotherm suggests that the lecithin monolayer was gaslike in the first range, and liquid-like in the second. Moreover, collapse and hysteresis were observed. The BSA monolayers were gas-like at areas greater than 10 000 Å2/molecule, beyond which an apparent phase transition was noted to liquid-like with much lower monolayer compressibility [(-(1/A h ))(∂A h /∂Π)]. Another pronounced transition, probably to a more condensed monolayer phase, seems to occur for A h ranging from 7000 to 3000 Å2/molecule, Π ≈ 18-30 mN/m. This suggests some expulsion of molecular segments into the subphase layer and closer packing. The value of A h ≈ 1500
Å2/molecule at the maximum observed surface pressure (30 mN/m) is close to the theoretical minimum area, A h min ≈ 1256 Å2/molecule (cross sectional area of prolate spheroid of dimensions, 4 × 4 × 14 nm), where the protein molecules are expected to be close-packed and oriented with their major axis normal to the surface. Even though the qualitative features of the isotherm for BSA (Figure 1b) agreed well with our previously reported isotherms,44 the actual area per molecule for the isotherm obtained by continuous compression is less than the earlier reported values. For example, at Π of 20 mN/m, A h is 5000 Å2/ molecule for continuous compression whereas A h is 7000 Å2/molecule by stepwise compression. The earlier isotherm44 was obtained by compression of the monolayer in small steps, and the monolayer was allowed to equilibrate after each compression. The smaller molecular areas for continuous compression is therefore due to the fact that not sufficient time is allowed for the monolayer to relax during compression. Mixed monolayer isotherms are shown in Figure 2 for weight ratios of lecithin to BSA (RLB) from 2 to 1/2. Although the compression curves of the mixed layer look similar to that of the BSA monolayer, there are two differences: (i) the specific area at which each monolayer transition occurs depends on the composition, and (ii) the condensed phase is observed for a wider range of molecular areas for lower RLB ratios. Whereas lecithin-rich monolayers exhibit smoother phase transitions, BSA-rich monolayers exhibit sharper transitions with smaller slopes. Expansion at the same speed results in a strong monolayer hysteresis behavior (Figures 1 and 2). Since the Πmax values in all tested mixed monolayers were about the same, and closer to that of the lecithin monolayer than of the BSA monolayer, lecithin seems to be the main contributor to the surface pressure. This is more pronounced at the close-packing limit, at which BSA may be expelled from the surface layer. The average areas per molecule were usually smaller for the mixed monolayers (43-22 Å2/molecule) than for compressed lecithin monolayers (33 Å2/molecule). Monolayer hysteresis and relaxation were further tested for RLB ) 13/12 (12 µg of BSA).42 The spread monolayer was compressed at 37 cm2/min until it reached Πmax ≈ 48 (44) Cho, D.; Narsimhan, G.; Franses, E. I. J. Colloid Interface Sci. 1996, 178, 348.
Interactions of Lecithin Monolayers with BSA
Langmuir, Vol. 13, No. 17, 1997 4713
Table 1. Results of the Exchange Experiments exp. no. conc of BSA amt of lecithin time change in Γ2 (fig. no.) solution (ppm) (µg) in solventa delay (h) (mg/m2 for BSA) 1 (3) 2 (3) 3 (-) 4 (4)
1.25 1.25 1.25
50 solvent only 50
no no
negligible ∼0.4
∼12 ∼10 a small overshoot 15 f 21
5 (-)
2.5
50
no
∼0.05
17 f 19
6 (6)
1.25
50
1/2
0.8 f 2.2 f 2
1 f 20
7 (7)
1.25
50
2
1 f 0.7
6 f 22
8 (8) 9 (9) 10 (10)
1.25 1.25 1.25
80 30 30,20b
2 2 2, 2.25b
1 f 0.1 1f2 1 f 2.5 f 0.6
6 f 43 f 35 6 f 21 6 f 20 f 24
a
∼1
change in Π (mN/m)
remarks gradual increase in Π, reaching 12 after 12 h sudden increase in Π with a small overshoot no significant interaction immediate desorption after spreading; steady afterwards almost complete desorption right after spreading rapid increase in Γ2 from 0.8 to 2.2 followed by slower desorption slow desorption leading to slow decrease in Γ2 from 1 to 0.7 sudden decrease in Γ2 and continuous drop of Π steady Γ2 and Π after sharp increases rapid enhancement in adsorption followed by desorption
Hexane-ethanol (v:v ) 9:1). b 30 µg first and 20 µg later
Figure 3. Evolution of surface pressure for lecithin monolayer, pure BSA solution and BSA solution with spread lecithin monolayer. In the case of lecithin monolayer, time refers to the time after spreading. In the case of BSA solution, time refers to the time elapsed after aspiration of the interface. Curves a and b denote Π(t) of BSA or lecithin, respectively. Curve c is for the mixture (from Figure 4). Curve d is the sum of curves a and b.
mN/m, and after about 2 s it was expanded. The next compression cycle was initiated as soon (∼2 s) as the expansion was completed. The monolayer transition occurred at lower area per molecule than at the first compression. The surface pressure did not recover to its initial value Π0 after complete expansion, for up to 30 min. Adsorption and Exchange for BSA-Lecithin Mixtures. A number of experiments (summarized in Table 1) were designed to investigate the interactions of protein and spread lecithin monolayers (Figures 3-10). The first three control experiments probed the effects of bulk concentration of [14C]BSA, amount of spread lecithin, and time delay for the lecithin spreading. The control experiments revealed that the spreading solvent did not significantly affect the BSA adsorption or spreading behavior of lecithin. Spread lecithin was found to remain irreversibly on the surface, or not to experience desorption, since Π remained constant after spreading. Therefore, no attempt was made to separately monitor lecithin surface concentration, which was assumed to be constant. Experiments 1, 2, and 4 showed some initial synergism of the surface pressure in the mixed system (Figure 3), since the sum of the surface pressures for lecithin or BSA alone was less than that of the mixture at short times and gradually approached the mixture curve at longer times. Immediately following spreading of lecithin (50 µg; 64 Å2/molecule) on a nearly clean surface of [14C]BSA solution (experiment 4, Figure 4), the surface pressure jumped to 15 mN/m within seconds. Then, Π increased gradually
Figure 4. Time-dependent changes of Π and Γ when a solution of [14C]BSA (cb ) 1.25 ppm) was poured in the trough, followed by immediate spreading of lecithin (50 µg or 64 Å2/molecule). The substrate was a phosphate buffer (pH ) 7 and I ) 0.5 M) at 24 ( 2 °C (symbols: (s) Π; (b) Γ of BSA). Inset, time-expanded Π(t) plot.
Figure 5. Comparison of dynamic adsorption densities of [14C]BSA (cb ) 1.25 ppm) from protein solution (open circles) or from protein solution in the presence of a spread lecithin monolayer (closed circles). The lines indicate the initial adsorption rate, dΓ/dt.
until it reached a plateau at 21 mN/m in ca. 6 h. The surface concentration of [14C]BSA also increased rapidly to ∼0.4 mg/m2 within 1 min for BSA bulk concentration of 1.25 ppm (Figure 4) (and to ∼0.1 mg/m2 for BSA bulk concentration of 2.5 ppm; results not shown)42 and showed no further increase. The initial adsorption rate, dΓ/dt, as estimated from the slopes of Figure 5, was 0.25 (mg/m2)/ min, or about 11 times faster than that when BSA only was present. Since the control experiment with solvent alone (experiment 3) showed the absence of any specific interaction between BSA and the solvent, one infers that the lecithin monolayer enhances adsorption of BSA at short times.
4714 Langmuir, Vol. 13, No. 17, 1997
Figure 6. Time-dependent changes of Π and Γ for 1.25 ppm [14C]BSA solution. At t ) 30 min, 50 µg (64 Å2/molecule) of lecithin was spread on the surface (symbols: (s) Π; (b) Γ for BSA).
Figure 7. Time-dependent changes of Π and Γ at the same conditions as in Figure 6. At t ) 2 h, 50 µg (64 Å2/molecule) of lecithin was spread on the surface (symbols: (s) Π; (b) Γ for BSA).
Figure 8. Time-dependent changes of Π and Γ for spreading of 80 µg (39 Å2/molecule) at 2 h. All the other conditions are the same as in Figure 6 (symbols: (s) Π; (b) Γ for BSA).
To test how an already adsorbed BSA monolayer would respond to spreading of a lecithin monolayer, the spreading was delayed by 30 min (experiment 6, Figure 6). Similar to the earlier observations, adsorption of BSA was initially enhanced (from 0.8 to 2.2 mg/m2) by the spreading of lecithin, to values over twice that of the steady-state adsorption of BSA. Subsequently, some BSA was apparently desorbed but remained at about 2 mg/m2 at long times. On the other hand, Π surged up to 22.5 mN/m and relaxed to about 20 mN/m. This enhancement of protein adsorption by lecithin may be due either to hydrophobic interactions between the lecithin chains and BSA45 or to electrostatic interactions of BSA with the lecithin head groups.28,29 A similar experiment (experiment 7) was done with a time delay of 2 h (Figure 7), when the surface monolayer was nearly at steady state.42 No further (45) Lebaron, F. N. In Structural and functional aspects of lipoprotein in living systems; Tria, E., Scanu, A. M., Eds.; AP: London, 1969; Chapter B3, pp 201-239.
Cho et al.
Figure 9. Time-dependent changes of Π and Γ for spreading of 30 µg (107 Å2/molecule) at 2 h. All the other conditions are the same as in Figure 6 (symbols: (s) Π; (b) Γ for BSA).
Figure 10. Time-dependence of Π and Γ at the same conditions as in Figure 6. Lecithin was spread twice, first at t ) 2 h, 30 µg (107 Å2/molecule) and then at t ) 2.25 h, 20 µg (160 Å2/ molecule) (symbols: (s) Π; (b) Γ for BSA).
adsorption of BSA was observed. Instead, desorption started immediately, with protein surface concentration dropping slowly from 1.2 to 0.76 mg/m2 within 2 h. The steady Π value was 22 mN/m. It is to be noted that the surface concentration of BSA depends on the time of spreading lecithin even though the same amount was spread. In experiments 4, 6, and 7, 50 µg of spread lecithin corresponded to molecular area of 64 Å2/molecule, which was much larger than close packed area of 33 Å2/molecule (see Figure 1a), thus leaving sufficient sites for BSA to adsorb. In experiments 4 and 7 (Figures 4 and 7), BSA molecules may adsorb at the interface in side-on orientation in an expanded state. Even though BSA molecules may adsorb in an expanded state up to the time (2 h) at which lecithin was spread in experiment 6 (Figure 6), occupation of many sites by spread lecithin may result in further adsorption of BSA in endon orientation. This possible difference in the orientation as well as extent of unfolding of BSA before and after lecithin spreading may explain this behavior. Experiments 8 and 9 were done for testing how the enhancement of BSA adsorption depends on the amount of spread lecithin. Using 80 µg rather than 50 µg of lecithin, decreased the lecithin area from 64 to 39 Å2/ molecule and increased Π from 5 to 45 mN/m (see Figure 1a). Spreading of more lecithin caused faster and nearly complete desorption of BSA with no adsorption enhancement of BSA (Figure 8). This may be due to the fact that the low molecular area corresponds to a close-packed or even collapsed lecithin monolayer. When 30 µg (107 Å2/ molecule) of lecithin was spread on a saturated BSA monolayer (experiment 9, Figure 9), BSA adsorption was enhanced. In summary, whereas small amounts of lecithin seem to enhance adsorption of BSA, large amounts cause displacement of the protein.16,21 Spreading in two doses (experiment 10, Figure 10), at 2 and 2.25 h, results first in enhancement of BSA adsorption (to ∼2.5 mg/m2)
Interactions of Lecithin Monolayers with BSA
Figure 11. Illustrations of protein-phospholipid interactions at the air-water interface. (a) Initially, with no lecithin monolayer, only few protein molecules adsorb. (b) Once lecithin spreads on the surface the attractive interactions (upward arrow) between lipid molecules and subphase protein molecules enhance protein adsorption. (c) Spread lecithin molecules rearrange resulting in displacement of some protein molecules. (d) If the spread lecithin monolayer is close-packed, then protein molecules are apt to be expelled from monolayer (downward arrow) because of high surface pressure or demixing effects. The total interaction energy for the protein-lipid monolayer can be expressed by Ead ) Esp + Eel + Ehy, where the energy due to surface pressure is repulsive while the others are believed to be attractive (see text for more details).
and then desorption of BSA (to ∼0.6 mg/m2). The difference in the enhanced BSA surface concentration upon spreading 30 µg of lecithin at 2 h between experiment 9 (Γ ∼2 mg/m2, see Figure 9) and experiment 10 (Γ ∼2.5 mg/m2, see Figure 10) can be attributed to possible convection effects caused by spreading lecithin. Such results on exchange of BSA could not be inferred from the measurements of surface pressure alone, and demonstrate the need for parallel surface concentration measurements. Discussion Certain effects of spread lecithin monolayers on the adsorption behavior of BSA at the air-water interface are shown schematically in Figure 11. When lecithin is spread at the air-water interface, it may initially form a physical complex9,19,46 with the protein molecules because of interactions between the hydrocarbon chains and hydrophobic patches of BSA. This complex formation may explain the enhancement in the adsorption of BSA soon after lecithin is spread (see Figures 4-6 and 9). Because such interactions are weak (∼10 kcal/mol),46 they may yield to lecithin-lecithin interactions in the monolayer, leading to demixing in the monolayer and protein desorption.29 When the lecithin molecules are established at the surface and expose their head groups to water, they may generate near the interface a hydrophilic environment that may prevent further adsorption of BSA.24 The formation of such a physical complex may be described in terms of energies as follows. The adsorption (46) Castleden, M. J. Food Sci. 1973, 38, 756.
Langmuir, Vol. 13, No. 17, 1997 4715
energy Ead of a BSA molecule can be written as the sum of three terms, Ead ) Esp + Eel + Ehy: (a) Esp, which is expected to be positive, is the work that needs to be done by an adsorbing BSA molecule to anchor itself at the interface and acts against the surface pressure or against the steric interactions of the other already adsorbed molecules. Esp increases with surface concentration of lecithin or BSA. (b) Eel is the electrostatic energy. (c) Ehy is the hydrophobic energy due to the exposure of hydrophobic groups of BSA into air.44,47 Ehy, being negative, would promote adsorption. The larger the surface concentrations of lecithin the more favorable the interaction energy Ehy is, and possibly Eel also. BSA may form a complex with lecithin when Eel and Ehy are negative and stronger than Esp. When the lecithin monolayer is close packed, the contribution from Esp may be dominant, and hence BSA may be excluded or expelled from the interface. For BSA, based on average surface hydrophobicity of 2.8 × 10-3 J/m2 44 and molecular dimensions of 4 × 4 × 14 nm, the estimated value Ehy is of the order of 60 kcal/mol. For a loosely packed lecithin monolayer (Π ∼ 6 mN/m) consisting of adsorbed BSA of Γ ∼0.5 mg/m2, the Esp is of the order of 10 kcal/mol and Eel is of the order of 20 kcal/ mol. Consequently, for a loosely packed lecithin monolayer Ehy > Eel + Esp. On the other hand, for a close-packed lecithin monolayer without any BSA (Π ∼ 40 mN/m), the estimated value of Esp is of the order of 70 kcal/mol and that of Eel is negligible since the interface is not charged. Therefore, Ehy < Eel + Esp for a close-packed lecithin monolayer. Conclusions Π-A isotherms of spread monolayers of egg-yolk lecithin, BSA, and their mixtures were determined with a Langmuir minitrough. The isotherms of lecithin-BSA mixtures were closer to that of BSA alone for weight ratios of lecithin to BSA of 0.5-2.0. Lecithin-rich monolayers exhibited hysteresis and showed much smoother phase transitions than BSA-rich monolayers. The dynamic adsorption of 14C-labeled BSA for different amounts (0.8-2.0 mg/m2) and different time delays of lecithin spreading after the initiation of BSA adsorption (0.5-2 h) was investigated through the measurements of surface pressure and BSA surface concentration using a radiotracer technique. Synergism was observed between the two molecules at short times leading to surface pressures greater than the sum of the surface pressures for lecithin and BSA separately. Spreading of lecithin monolayers with A h values of 107 and 64 Å2/molecule was found to enhance adsorption of BSA at short times, leading to a surface concentrations of BSA (2-2.5 mg/m2) greater than the steady state value in the absence of lecithin (∼1 mg/m2). This implies complexation of BSA molecules with dilute lecithin monolayers. At longer times, BSA desorbed slowly. Spreading of a close-packed lecithin monolayer (of A h ) 33 Å2/molecule), however, caused eventually desorption of most of the previously adsorbed monolayers of BSA, apparently because of the surface pressure and steric exclusion effects produced by the spread lecithin monolayers. This information on lecithin-BSA interactions would be useful in developing guidelines for the range of emulsifier concentrations to be employed for emulsion stability. Acknowledgment. Authors would like to acknowledge the National Science Foundation (Grants No. BCS-9112154, CTS-93-04328, and CTS-96-15649) and Purdue Research Foundation for partial support of this research. LA970348G (47) Narsimhan, G.; Uraizee, F. Biotechnol. Prog. 1992, 8, 187.
