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protein monolayer at the air–water interface
Colloids and Surfaces A: Physicochemical and Engineering Aspects 175 (2000) 77 – 82 www.elsevier.nl/locate/colsurfa
Stability investigation of the mixed DPPC/protein monolayer at the air–water interface Hengjian Zhang, Xiaoli Wang, Guangchen Cui, Junbai Li * International Joint Lab between Institute of Photographic Chemistry and Max Planck Institute of Colloid and Interface Sciences , Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, De Wai, Bei Sha Tan, Beijing 100101, People’s Republic of China
Abstract A subphase exchange method has been used to investigate the stability of the mixed L-a-dipalmitoylphosphatidylcholine (DPPC)/b-lactoglobulin monolayer formed via the protein adsorption into the spread lipid layer at the air–water interface by Brewster angle microscopy (BAM). The BAM images demonstrated that the adsorbed protein might compress the DPPC monolayer to enter a phase transition early close to a condensed phase. When protein concentration is very high the penetration of the protein is rather fast, and a larger domain was preferentially formed without compressing the DPPC monolayer. After the mixed DPPC/b-lactoglobulin monolayer was formed, the protein solution in subphase was totally washed out by pumping in buffer water for a long time. In the fluid phase and coexistence region of DPPC monolayer with penetrated protein layer the domains have been recorded by BAM images for comparing the variation before and after the exchange of subphase. The experimental results revealed that there was not significant change of the domains in size and shape. It indicates that the adsorbed protein has a strong interaction with DPPC and is more likely to remain at the interface. A relatively stable mixed lipid – protein monolayer can be constructed by this way. Hydrophobic and electrostatic interactions between DPPC and b-lactoglobulin as well as the conformation change of b-lactoglobulin at air – water interface have been taken into account to stabilize the mixed layers. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Brewster angle microscopy; Subphase exchange; Mixed L-a-dipalmitoylphosphatidylcholine/b-lactoglobulin monolayer
1. Introduction The system of mixed lipid – protein plays a significant role in many industrial and biomedical processes. For example, protein with lipid tends to accumulate at the air – water or oil – water interface in many foods where they serve to stabilize foams or emulsions [1]. In living systems, they * Corresponding author.
jointly compose biomembrane to separate one microenvironment from others. Various biological functions such as material metabolism, message transmission, energy conversion are accomplished by means of the biomembrane [2,3]. Thus, the formation and character of lipid–protein system as well as the interaction between lipid and protein have been studied with a great interest for last years [4,5]. To acquire more information, some theoretical models have been established.
0927-7757/00/$ - see front matter © 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 0 ) 0 0 4 8 3 - 0
78
H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
Among them two dimension model at air–water interface has been extensively accepted and used [6,7]. The traditional method constructing a lipid–protein monolayer at interface was by injecting protein solution into subphase after lipid has been spread on the subphase [8 – 10]. The mixed lipid – protein monolayer may be formed when the protein is adsorbed onto the interface from solution. By this way, the distribution of protein concentration in subphase can not be well controlled. L-a-dipalmitoylphosphatidylcholine (DPPC) as a defined system was widely investigated by different technique [10 – 12], its phase behavior and morphology of monolayer have been well understood [13,14]. In this work, it was used as a model lipid to study the property of mixed lipid – protein. With our experiments, we took use of adsorption kinetic curves of protein and compared Brewster angle microscopy (BAM) images to determine the formation of mixed lipid–protein. Then by using the method of subphase exchange to investigate the stability of the mixed DPPC/b-lactoglobulin at air – water interface by BAM. Many BAM images of monolayer
produced in the process of adsorption of protein and the subphase exchange were recorded for the comparison before and after the protein penetration into DPPC monolayer. The experimental results were discussed in terms of interaction between lipid and protein, composition and properties of monolayer.
2. Experimental
2.1. Material DPPC+99% and b-lactoglobulin obtained from Sigma was used without further purification. Chloroform with +99% purity was purchased from ACROS. The water used in all experiments was Millipore filtered. Protein buffer solutions, 10 mM phosphate, pH 7, have an identical ionic strength. All experiments were performed at a temperature of 209 0.1°C.
2.2. Methods 2.2.1. BAM and film balance A high resolution BAM (Optral, Germany) was mounted onto a commercial LB trough (R&K, Germany). The curves of pressure versus time were recorded automatically during the image being captured. The BAM images were stored on a video recorder for the later analysis. The selected images (600× 600 mm) were treated with special software to improve the contrast.
Fig. 1. Curves of surface pressure vs. time (…) during the adsorption of b-lactoglobulin from a buffer solution into DPPC monolayer at a surface pressure of 4 mN m − 1; ( —) during the subphase exchange after the mixed DPPC/b-lactoglobulin layer has been formed.
2.2.2. Preparation of the mixed monolayer After DPPC monolayer was spread on b-lactoglobulin buffer solution some time it was left to allow the chloroform to evaporate. To remove the protein from interface early, the initially formed monolayer was compressed to a position of 55 mN m − 1 for several min to squeeze out the adsorbed protein [2]. Then the barriers were expanded to the surface pressure of 4 mN m − 1 and fixed when the pressure was reached. In the status b-lactoglobulin was adsorbed from its buffer solution into the spread DPPC monolayer at the air–water interface. When a mixed DPPC/b-lactoglobulin layer has been confirmed to form, the
H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
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Fig. 2. BAM images of domains of DPPC monolayer caused by the penetration of b-lactoglobulin at the air – water interface in different surface pressure, (a) p= 4.0 mN m − 1; (b) p =7.9 mN m − 1; (c) p =12.3 mN m − 1; (d) p =15.7 mN m − 1.
protein buffer solution as a subphase was washed out by pumping in fresh phosphate buffer water without protein. In order to keep the DPPC monolayer less disturbed, a precisely controlled speed of pump is necessary.
3. Results and discussion Fig. 1 shows that surface pressure increases with time by the adsorption of b-lactoglobulin from a buffer solution into DPPC monolayer at surface pressure of 4 mN m − 1 and finally reaches to 15.7 mN m − 1, which is close to the equilibrium adsorption time after 195 min (the dot line of Fig. 1). The concentration of protein is 1.8 mg l − 1. This result is in a good agreement with that
reported earlier [2,15,16]. Fig. 2(a)–(d) shows the formation of liquid condensed domains of DPPC monolayer during the adsorption of b-lactoglobulin. The surface pressure increase caused by the penetration of protein is as what the monolayer is compressed by the trough barrier. A similar result has been obtained for a lipid–protein system in our previous work [17]. It can be seen that the domain density in Fig. 2 (bright part) was enlarged. At this stage, the mixed monolayer becomes relative viscose. Then the subphase was exchanged with buffer water by pumping to replace the protein solution. In 3 h, we did not find obvious change in shape and size of the domains (see Fig. 3(a)–(f)). As expected, the surface pressure was not changed with the exchange of subphase. Comparing these images, we can see in Fig.
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H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
2(d) the image of the condensed film is similar to that in the final status after the subphase replaced. This means that a rather stable mixed lipid– protein layer has been constructed. Otherwise, an
enriched dark part or big holes corresponding to the loss of protein during exchanging the subphase from the interface should appear. This suggests that DPPC molecules at interface interact
Fig. 3. BAM images of the mixed DPPC/b-lactoglobulin monolayer at the surface pressure of 15.7 mN m − 1 produced in the process of subphase exchanged with different time, respectively (a) t =0 min; (b) t =20 min; (c) t = 70 min; (d) t =120 min; (e) t= 150 min; (f) t = 180 min.
H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
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Fig. 4. BAM images of domains immediately formed after DPPC was spread on b-lactoglobulin solution with a higher protein concentration, Cb-lactoglobulin = 3.8 mg l − 1.
with b-lactoglobulin [9]. Both hydrophobic effect and electrostatic interaction are considered to play important roles to make the complex stable [5]. By this way, the surface activity of the complex will be increased compared to the native protein. Fig. 4(a) and (b) show the BAM images of domains initially formed in a fluid phase after DPPC was spread on b-lactoglobulin solution with a higher protein concentration (Cb-lactoglobulin = 3.8 mg l − 1). In this case, the adsorption rate of the protein becomes extremely quick [2]. The bright parts can be regarded as the domain of protein or mixed lipid – protein. They do not obviously change their size and shape within a shorter adsorption time. It indicates that the existence of protein molecule at the interface limits the growth of DPPC domains. In one case, the hydrophobic interaction between protein and DPPC molecules restricts the lipid molecular mobilization. Additionally, the electrostatic interaction between headgroup of lipid molecules and the hydrophilic region of protein molecules may prevent DPPC molecules from motion or orientation. The binding between DPPC and b-lactoglobulin can also lead to such a huge domain formation as the protein preferentially takes a space at the interface. From the above fact, it indicates that the protein adsorption is obviously different from the physical adsorption of small surfactant molecules
from solution which will spontaneously leave the surface within a short interval of time. The surface activity of simple aqueous surfactant molecules are relative to its bulk concentration, the surface pressure rises with the increase of bulk concentration until the surface pressure reaches the equilibrium value. In principle, surface pressure should decrease when the surfactant molecules were washed out from the bulk solution. However, in this experiment the pressure did not change when subphase exchange was carried out to wash out the protein molecules in bulk. This means that the desorption rate of protein molecule from interface is rather small or the adsorption process is irreversible. To maintain this property, the globular conformation of b-lactoglobulin molecules may undergo extensively unfolding and rearranging of conformation when they are adsorbed from bulk to the interface. After protein has been adsorbed onto interface and accumulate to a higher surface concentration, the adsorbed protein will denature and interact with the lipid molecules at the interface. This makes the formed lipid/b-lactoglobulin monolayer rather stable.
4. Conclusions Mixed DPPC/b-lactoglobulin monolayer has been constructed by the adsorption of protein into
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H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
a spread DPPC monolayer. The surface pressure increase caused by the adsorption of protein could obligate the DPPC monolayer enter a relatively condensed state from a liquid expanded phase. BAM images of the mixed films obtained in the coexistence region of DPPC monolayer confirm the complex stability through the exchange of subphase with buffer phosphate water. Hydrophobic and electrostatic interactions between DPPC and b-lactoglobulin molecules as well as the conformation change of b-lactoglobulin are considered to stabilize the mixed layers at the air – water interface.
Acknowledgements The authors would like to thank the project support of the Max-Planck-Gesellschaft, Germany and the Chinese Academy of Sciences. Junbai Li wants to acknowledge the financial support by the President Fund of the Chinese Academy of Sciences and the National Personal Department.
References [1] D.G. Cornell, D.L. Patterson, N. Hoban, J. Colloid Interface Sci. 140 (1990) 428.
.
[2] M.A. Bos, T. Nylander, Langmuir 12 (1996) 2791. [3] G. Gabrielli, G. Caminati, M. Puggelli, A. Gilardoni, B. Margheri, J. Phys. IV 3 (1993) 279. [4] A. Watts (Ed.), Protein – Lipid Interactions, New Comprehensive Biochemistry, vol. 25, Elsevier, Amsterdam, 1993. [5] M.A. Bos, T. Nylander, T. Arnebrant, D.C. Clark, Food Emulsifiers and Their Applications, 1995. [6] R. Verger, F. Pattus, Chem. Phys. Lipids 30 (1982) 189. [7] F. MacRitchie, A.E. Alexander, J. Colloid Sci. 18 (1963) 453. [8] D.G. Cornell, D.L. Patterson, J. Agric. Food Chem. 37 (1989) 1455. [9] W.M. Heckl, B.N. Zaba, H. Mo¨hwald, Biochim. Biophys. Acta 903 (1987) 166. [10] W.M. Heckl, M. Thompson, H. Mo¨hwald, Langmuir 5 (1989) 390. [11] O. Albrecht, H. Gruler, E. Sackmann, J. Phys. 39 (1978) 301. [12] D. Ho¨nig, D. Mo¨bius, J. Phys. Chem. 95 (1991) 4590. [13] C. Weidemann, D. Vollhardt, Colloids Surf. 100 (1995) 187. [14] T.M. Fischer, R.F. Bruinsma, C.M. Knobler, Phys. Rev. E 50 (1994) 213. [15] R. Wu¨stneck, J. Kraegel, R. Miller, V.B. Fainerman, P.J. Wilde, D.K. Sarker, D.C. Clark, Food Hydrocolloids 10 (1996) 395. [16] E. Tornberg, J. Colloid Interface Sci. 64 (1978) 391. [17] V.B. Fainerman, J. Zhao, D. Vollhardt, A.V. Makievski, J.B. Li, J. Phys. Chem. B (in press).
Stability investigation of the mixed DPPC/protein monolayer at the air–water interface Hengjian Zhang, Xiaoli Wang, Guangchen Cui, Junbai Li * International Joint Lab between Institute of Photographic Chemistry and Max Planck Institute of Colloid and Interface Sciences , Center for Molecular Science, Institute of Chemistry, Chinese Academy of Sciences, De Wai, Bei Sha Tan, Beijing 100101, People’s Republic of China
Abstract A subphase exchange method has been used to investigate the stability of the mixed L-a-dipalmitoylphosphatidylcholine (DPPC)/b-lactoglobulin monolayer formed via the protein adsorption into the spread lipid layer at the air–water interface by Brewster angle microscopy (BAM). The BAM images demonstrated that the adsorbed protein might compress the DPPC monolayer to enter a phase transition early close to a condensed phase. When protein concentration is very high the penetration of the protein is rather fast, and a larger domain was preferentially formed without compressing the DPPC monolayer. After the mixed DPPC/b-lactoglobulin monolayer was formed, the protein solution in subphase was totally washed out by pumping in buffer water for a long time. In the fluid phase and coexistence region of DPPC monolayer with penetrated protein layer the domains have been recorded by BAM images for comparing the variation before and after the exchange of subphase. The experimental results revealed that there was not significant change of the domains in size and shape. It indicates that the adsorbed protein has a strong interaction with DPPC and is more likely to remain at the interface. A relatively stable mixed lipid – protein monolayer can be constructed by this way. Hydrophobic and electrostatic interactions between DPPC and b-lactoglobulin as well as the conformation change of b-lactoglobulin at air – water interface have been taken into account to stabilize the mixed layers. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: Brewster angle microscopy; Subphase exchange; Mixed L-a-dipalmitoylphosphatidylcholine/b-lactoglobulin monolayer
1. Introduction The system of mixed lipid – protein plays a significant role in many industrial and biomedical processes. For example, protein with lipid tends to accumulate at the air – water or oil – water interface in many foods where they serve to stabilize foams or emulsions [1]. In living systems, they * Corresponding author.
jointly compose biomembrane to separate one microenvironment from others. Various biological functions such as material metabolism, message transmission, energy conversion are accomplished by means of the biomembrane [2,3]. Thus, the formation and character of lipid–protein system as well as the interaction between lipid and protein have been studied with a great interest for last years [4,5]. To acquire more information, some theoretical models have been established.
0927-7757/00/$ - see front matter © 2000 Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 5 7 ( 0 0 ) 0 0 4 8 3 - 0
78
H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
Among them two dimension model at air–water interface has been extensively accepted and used [6,7]. The traditional method constructing a lipid–protein monolayer at interface was by injecting protein solution into subphase after lipid has been spread on the subphase [8 – 10]. The mixed lipid – protein monolayer may be formed when the protein is adsorbed onto the interface from solution. By this way, the distribution of protein concentration in subphase can not be well controlled. L-a-dipalmitoylphosphatidylcholine (DPPC) as a defined system was widely investigated by different technique [10 – 12], its phase behavior and morphology of monolayer have been well understood [13,14]. In this work, it was used as a model lipid to study the property of mixed lipid – protein. With our experiments, we took use of adsorption kinetic curves of protein and compared Brewster angle microscopy (BAM) images to determine the formation of mixed lipid–protein. Then by using the method of subphase exchange to investigate the stability of the mixed DPPC/b-lactoglobulin at air – water interface by BAM. Many BAM images of monolayer
produced in the process of adsorption of protein and the subphase exchange were recorded for the comparison before and after the protein penetration into DPPC monolayer. The experimental results were discussed in terms of interaction between lipid and protein, composition and properties of monolayer.
2. Experimental
2.1. Material DPPC+99% and b-lactoglobulin obtained from Sigma was used without further purification. Chloroform with +99% purity was purchased from ACROS. The water used in all experiments was Millipore filtered. Protein buffer solutions, 10 mM phosphate, pH 7, have an identical ionic strength. All experiments were performed at a temperature of 209 0.1°C.
2.2. Methods 2.2.1. BAM and film balance A high resolution BAM (Optral, Germany) was mounted onto a commercial LB trough (R&K, Germany). The curves of pressure versus time were recorded automatically during the image being captured. The BAM images were stored on a video recorder for the later analysis. The selected images (600× 600 mm) were treated with special software to improve the contrast.
Fig. 1. Curves of surface pressure vs. time (…) during the adsorption of b-lactoglobulin from a buffer solution into DPPC monolayer at a surface pressure of 4 mN m − 1; ( —) during the subphase exchange after the mixed DPPC/b-lactoglobulin layer has been formed.
2.2.2. Preparation of the mixed monolayer After DPPC monolayer was spread on b-lactoglobulin buffer solution some time it was left to allow the chloroform to evaporate. To remove the protein from interface early, the initially formed monolayer was compressed to a position of 55 mN m − 1 for several min to squeeze out the adsorbed protein [2]. Then the barriers were expanded to the surface pressure of 4 mN m − 1 and fixed when the pressure was reached. In the status b-lactoglobulin was adsorbed from its buffer solution into the spread DPPC monolayer at the air–water interface. When a mixed DPPC/b-lactoglobulin layer has been confirmed to form, the
H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
79
Fig. 2. BAM images of domains of DPPC monolayer caused by the penetration of b-lactoglobulin at the air – water interface in different surface pressure, (a) p= 4.0 mN m − 1; (b) p =7.9 mN m − 1; (c) p =12.3 mN m − 1; (d) p =15.7 mN m − 1.
protein buffer solution as a subphase was washed out by pumping in fresh phosphate buffer water without protein. In order to keep the DPPC monolayer less disturbed, a precisely controlled speed of pump is necessary.
3. Results and discussion Fig. 1 shows that surface pressure increases with time by the adsorption of b-lactoglobulin from a buffer solution into DPPC monolayer at surface pressure of 4 mN m − 1 and finally reaches to 15.7 mN m − 1, which is close to the equilibrium adsorption time after 195 min (the dot line of Fig. 1). The concentration of protein is 1.8 mg l − 1. This result is in a good agreement with that
reported earlier [2,15,16]. Fig. 2(a)–(d) shows the formation of liquid condensed domains of DPPC monolayer during the adsorption of b-lactoglobulin. The surface pressure increase caused by the penetration of protein is as what the monolayer is compressed by the trough barrier. A similar result has been obtained for a lipid–protein system in our previous work [17]. It can be seen that the domain density in Fig. 2 (bright part) was enlarged. At this stage, the mixed monolayer becomes relative viscose. Then the subphase was exchanged with buffer water by pumping to replace the protein solution. In 3 h, we did not find obvious change in shape and size of the domains (see Fig. 3(a)–(f)). As expected, the surface pressure was not changed with the exchange of subphase. Comparing these images, we can see in Fig.
80
H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
2(d) the image of the condensed film is similar to that in the final status after the subphase replaced. This means that a rather stable mixed lipid– protein layer has been constructed. Otherwise, an
enriched dark part or big holes corresponding to the loss of protein during exchanging the subphase from the interface should appear. This suggests that DPPC molecules at interface interact
Fig. 3. BAM images of the mixed DPPC/b-lactoglobulin monolayer at the surface pressure of 15.7 mN m − 1 produced in the process of subphase exchanged with different time, respectively (a) t =0 min; (b) t =20 min; (c) t = 70 min; (d) t =120 min; (e) t= 150 min; (f) t = 180 min.
H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
81
Fig. 4. BAM images of domains immediately formed after DPPC was spread on b-lactoglobulin solution with a higher protein concentration, Cb-lactoglobulin = 3.8 mg l − 1.
with b-lactoglobulin [9]. Both hydrophobic effect and electrostatic interaction are considered to play important roles to make the complex stable [5]. By this way, the surface activity of the complex will be increased compared to the native protein. Fig. 4(a) and (b) show the BAM images of domains initially formed in a fluid phase after DPPC was spread on b-lactoglobulin solution with a higher protein concentration (Cb-lactoglobulin = 3.8 mg l − 1). In this case, the adsorption rate of the protein becomes extremely quick [2]. The bright parts can be regarded as the domain of protein or mixed lipid – protein. They do not obviously change their size and shape within a shorter adsorption time. It indicates that the existence of protein molecule at the interface limits the growth of DPPC domains. In one case, the hydrophobic interaction between protein and DPPC molecules restricts the lipid molecular mobilization. Additionally, the electrostatic interaction between headgroup of lipid molecules and the hydrophilic region of protein molecules may prevent DPPC molecules from motion or orientation. The binding between DPPC and b-lactoglobulin can also lead to such a huge domain formation as the protein preferentially takes a space at the interface. From the above fact, it indicates that the protein adsorption is obviously different from the physical adsorption of small surfactant molecules
from solution which will spontaneously leave the surface within a short interval of time. The surface activity of simple aqueous surfactant molecules are relative to its bulk concentration, the surface pressure rises with the increase of bulk concentration until the surface pressure reaches the equilibrium value. In principle, surface pressure should decrease when the surfactant molecules were washed out from the bulk solution. However, in this experiment the pressure did not change when subphase exchange was carried out to wash out the protein molecules in bulk. This means that the desorption rate of protein molecule from interface is rather small or the adsorption process is irreversible. To maintain this property, the globular conformation of b-lactoglobulin molecules may undergo extensively unfolding and rearranging of conformation when they are adsorbed from bulk to the interface. After protein has been adsorbed onto interface and accumulate to a higher surface concentration, the adsorbed protein will denature and interact with the lipid molecules at the interface. This makes the formed lipid/b-lactoglobulin monolayer rather stable.
4. Conclusions Mixed DPPC/b-lactoglobulin monolayer has been constructed by the adsorption of protein into
82
H. Zhang et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 175 (2000) 77–82
a spread DPPC monolayer. The surface pressure increase caused by the adsorption of protein could obligate the DPPC monolayer enter a relatively condensed state from a liquid expanded phase. BAM images of the mixed films obtained in the coexistence region of DPPC monolayer confirm the complex stability through the exchange of subphase with buffer phosphate water. Hydrophobic and electrostatic interactions between DPPC and b-lactoglobulin molecules as well as the conformation change of b-lactoglobulin are considered to stabilize the mixed layers at the air – water interface.
Acknowledgements The authors would like to thank the project support of the Max-Planck-Gesellschaft, Germany and the Chinese Academy of Sciences. Junbai Li wants to acknowledge the financial support by the President Fund of the Chinese Academy of Sciences and the National Personal Department.
References [1] D.G. Cornell, D.L. Patterson, N. Hoban, J. Colloid Interface Sci. 140 (1990) 428.
.
[2] M.A. Bos, T. Nylander, Langmuir 12 (1996) 2791. [3] G. Gabrielli, G. Caminati, M. Puggelli, A. Gilardoni, B. Margheri, J. Phys. IV 3 (1993) 279. [4] A. Watts (Ed.), Protein – Lipid Interactions, New Comprehensive Biochemistry, vol. 25, Elsevier, Amsterdam, 1993. [5] M.A. Bos, T. Nylander, T. Arnebrant, D.C. Clark, Food Emulsifiers and Their Applications, 1995. [6] R. Verger, F. Pattus, Chem. Phys. Lipids 30 (1982) 189. [7] F. MacRitchie, A.E. Alexander, J. Colloid Sci. 18 (1963) 453. [8] D.G. Cornell, D.L. Patterson, J. Agric. Food Chem. 37 (1989) 1455. [9] W.M. Heckl, B.N. Zaba, H. Mo¨hwald, Biochim. Biophys. Acta 903 (1987) 166. [10] W.M. Heckl, M. Thompson, H. Mo¨hwald, Langmuir 5 (1989) 390. [11] O. Albrecht, H. Gruler, E. Sackmann, J. Phys. 39 (1978) 301. [12] D. Ho¨nig, D. Mo¨bius, J. Phys. Chem. 95 (1991) 4590. [13] C. Weidemann, D. Vollhardt, Colloids Surf. 100 (1995) 187. [14] T.M. Fischer, R.F. Bruinsma, C.M. Knobler, Phys. Rev. E 50 (1994) 213. [15] R. Wu¨stneck, J. Kraegel, R. Miller, V.B. Fainerman, P.J. Wilde, D.K. Sarker, D.C. Clark, Food Hydrocolloids 10 (1996) 395. [16] E. Tornberg, J. Colloid Interface Sci. 64 (1978) 391. [17] V.B. Fainerman, J. Zhao, D. Vollhardt, A.V. Makievski, J.B. Li, J. Phys. Chem. B (in press).