Langmuir 1995,11, 75-79
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Studies on Interactions between Phosphatidylcholine and Casein Y. Fang and D. G. Dalgleish" Department of Food Science, University of Guelph, Guelph, Ontario, Canada N l G 2W1 Received June 16, 1994. In Final Form: October 19, 1994@ The interactions between casein and either egg phosphatidylcholine or dipalmitoylphosphatidylcholine were studied using dynamic light scattering and fluorescencepolarization. Casein was found not to adsorb to a suspension of phospholipid vesicles, but a stable complex was formed when casein and phospholipid were homogenized togetherto form the vesicles. The hydrodynamic thicknessofthe layer of casein associated with vesicles was close to twice that of the casein layer adsorbed to the surfaces of oil droplets in oil-inwater emulsions. The breakdown of incorporated casein molecules by trypsin caused aggregationor fusion ofthe vesicles, in contrast to the behavior ofemulsions, which remain stable under such treatment. Changes in the fluorescence polarization demonstrated that the association of casein with DPPC vesicles made the lipid bilayer more rigid in its liquid crystalline state, but little difference was detected in the gel state. The topological response of the vesicles to temperature change was significantlymodified by the presence of casein in the bilayer. The results suggest that the casein associated with the lipid bilayer is not simply adsorbed to the surface but penetrates deeper into the inner core of the bilayer, so that caseins appear to behave as transmembrane proteins.
Introduction The interactions between proteins and phospholipid bilayers have been extensively studied because of their biological importance. In a recent review,' proteins were classified into three categories, adsorbed protein, anchored protein, and embedded protein, depending on their position relative to the lipid bilayer. The protein complex in our study, casein, is not naturally associated with biological membranes, although caseins tend to adsorb from solution on to hydrophobic surfaces.2 The adsorption of individual caseins to polystyrene latices can be studied by monitoring the increase in the hydrodynamic radius of the latex particles as protein is added.3 This increase is a function of the amount of casein added until the latex surface is saturated with protein, a t which point the thickness of the adsorbed monolayer is about 10 nm. A comparable layer thickness has been found with oil-in-water emulsions stabilized by ~ a s e i n .However, ~ the structure of these interfacial caseinate layers in oil-in-water emulsions is modified by the presence of egg-yolk phosphatidylcholine (egg-PC).4,5To understand this in detail, it is necessary to understand not only the interactions between phospholipid and triglyceride but also between phospholipids and caseins. Casein from milk is a mixture of four proteins (a8', &a, /?, and K ) , which occur naturally in the approximate ratios 4:1:4:1. Under normal conditions in milk, these proteins are held together by calcium phosphate to form particles known as casein micelles. The proteins are all phosphorylated to different extents and have molecular weights from 19 600 Da (K-casein) to 25 400 Da (&a-casein) and isoelectric points close to 5.0. At neutral pH, all of the four proteins are negatively charged, especially the &I and fractions, which possess the greatest numbers of
* To whom correspondence should be addressed. Abstract published in Advance A C S Abstracts, December 15, 1994. (1)Jain, M.K.;Zakim, D Biochim. Biophys. Acta 1987,906,33-68. (2)Graham,D. E.; Phillips, M. C. In The Theory and Practise of Emulsion Technology;Smith,A. L., Ed.;AcademicPress: London, 1976; pp 75-98. (3)Dalgleish, D. G. Colloids Sur$ 1990,46,141-155. (4)Fang, Y.;Dalgleish, D. G. J. Colloid Interface Sci. 1993,156, 329-334. ( 5 ) Fang, Y.; Dalgleish,D. G. Colloids Surf. B : Biointerfaces 1993, 1,357-364. @
phosphorylated residues.6 Of great relevance to their interfacial behavior is the distribution of amino acids in their sequences, which suggests that the proteins contain regions of high hydrophobicity. The favorable adsorption of these proteins to oil-water interfaces has been attributed to the possession of these hydrophobic regions, and in one case @-casein)it has been established that the hydrophobic portion of the molecule is indeed the cause of its adsorption to oil-water interfaces.' It has been reported that /?-casein can penetrate a phospholipid choline monolaye$ and that this interaction causes a lateral compression of the lipid monolayer. Similarly, it has been shown that K-casein can penetrate a monolayer of dimyrist~ylphosphatidylcholine.~The adsorption of a-lactalbumin to a phospholipid monolayer forms a protein shieldlo and prevents the enzymatic hydrolysis of this monolayer by phospholipase C. Glucagon has also been observed to compress lipid bilayers, and this interaction is affected by the phase transition of the lipid bilayer from the gel state to liquid crystalline state." A similar dependence on temperature of the interaction has also been found in other ~ y s t e m s , and ~~-~~ it is a general observation that the interaction between dipalmitoylphosphatidylcholine(DPPC)bilayers and protein is promoted by the liquid crystalline state. In many cases, the spontaneous association of protein with phospholipid vesicles has been found to cause aggregation and fusion of v e s i c l e ~ . ~ ~ The J ~ Jaggregation ~-~~ can be caused (6)Swaisgood, H. In Advanced Dairy Chemistry: 1-Proteins; Fox,
P.F., Ed.; Elsevier Applied Science: London and New York,1992;pp
63-110. (7)haver, J.; Dalgleish, D. G. Biochim. Biophys. Acta 1990,1041, 217-222. (8) Phillips, M. C.; Evans, M. T. A.; Hauser, H. ACSAdu. Chem. Ser. 1975,144,217-230. (9)Griffin, M . C. A.; Infante, R. B.; Klein, R. A. Chem. Phys. Lipids 1987,36,91-98. (10)Hanssens, I.: van Cauwelaert, F. H. Biochem. Biophrs. _ - Res. Commun. 1978,84,1088-1096. (11)Epand, R.M.;Epand, R. F.; Stewart, T. P.; Hui, S. W. Biochim. Biophy~.Acta 1981,649,608-615. (12)Campbell, I. M.;Pawagi,A.B. Can.J.Biochem. 1979,57,10991109. (13)Smith, R. Biochim. Biophys. Acta 1977,470,170-184. (14)Schenkman, S.;de Araujo, P. S.; Dijkman, R.; Quina, F. H.; Chaimovich, H. Biochim.Bwphys. Acta 1981,649,633-641. (15)Hanssens, I.; Houthuys, C.; Herreman, W.; van Cauwelaert, F. H. Biochim. Biophys. Acta 1980,602,539-557.
0743-7463/95/2411-0075$09.00/00 1995 American Chemical Society
76 Langmuir, Vol. 11, No. 1, 1995
by protein forming bridges between particles, and this effect can be reversed simply by adding more protein into the system. In addition to spontaneous association of protein with lipid vesicles, sonication of protein and phospholipid together can also induces the formation of a lipid-protein c o m p l e ~ . ~ ~ , ~ ~ In this work, we studied the association of casein with vesicles which had already been formed (to test for spontaneous interaction) and the interaction between casein and phospholipids when the vesicles were formed in the presence of the protein. We used dynamic light scatteringcombined with proteolysis of the protein to study the casein layers associated with the vesicles and labeled fluorescence polarization to study the effect of casein on the bilayer structure.
Materials and Methods Egg-yolk phosphatidylcholine(egg-PC),dipalmitoylphosphatidylcholine (DPPC),TPCK-trypsin,and imidazolewere purchased from Sigma Chemicals, St. Louis, MO, and they were usedwithout further purification. All experiments were performed using imidazole buffer at a concentration of 20 mM a t pH 7. Whole casein was prepared by precipitating skim milk at pH 4.6 and washing and redissolving the washed precipitate in NaOH at pH 7,after which the solution was freeze-dried. Vesicle Formation. Vesicles were made at room temperature by homogenizing dispersions of both egg-PC and DPPC in imidazole buffer using a Model 110smicrofluidizer (Microfluidics Corp., Newton, MA) with an input pressure of 0.3 MPa which corresponds to a pressure drop of 42 MPa in the homogenizing chamber. During this process, there was a rise in temperature of 10 "C, so that the vesicles after formation were a t temperatures of about 35 "C. Amounts of PC in the range of 20-50 mg were weighed out and dispersed in 10 mL of imidazole buffer (20mM, pH 71, and the homogenization was carried out immediately. Casein was incorporated in the dispersion either before or after homogenization, the amount of casein being varied between 5 and 30 mg mL-l. When the vesicles were made in the presence of casein, the phospholipid was dispersed first in the casein solution before homogenization. After preparation, the dispersions of vesicles were stored for up to 1 week either at room temperature or at 4 "C. Becauseusing homogenization to prepare phospholipid vesicles is rather unconventional, we also prepared for comparison eggPC vesicles usingan extrusion method,21where the PC dispersion was forced through a filter (pore size 0.2 pm) five times under nitrogen a t a temperature of 50 "C and was then cooled t o room temperature. These vesicles were treated identically to the vesicles prepared by homogenization, using identical measuring techniques. Light Scattering. Photon correlation spectroscopy (PCS) experiments used a Malvern 4700 optical system attached t o a 7032 Multi-8 correlator (Malvern Instruments, Southboro, MA) to study the hydrodynamic dimensions of vesicles. Diffusion coefficients of the particles were calculated by the method of cumulants, and the average hydrodynamicdimensionswere then calculated, assuming that the vesicles obey the Stokes-Einstein law. Vesicle dispersions were diluted in imidazole buffer (200 pL in 3 mL), which had previously been filtered through a 0.2 pm filter. To investigate the casein-PC interaction, a method based on proteolysiswas used, which had been initially developed to study the protein layer adsorbed to the lipid droplets of oilin-water emulsion^.^^^^^ Trypsin solution was dissolved to a (16)Schenkman, S.; de Araujo, P. S.; Sesso, A.; Quina, F. H.; Chaimovich, H. Chem. Phys. Lipids 1981,28,165-180. (17) Haagsman, H. P.; Elfring, R. H.; van Buel, B. L. M.; Voorhout, W.F. Biochim. J. 1991,275,273-276. (18)Farias, R. N.;Vinals, A. L.;Morero, R. D. Biochim. Biophys. Res. Commun. 1985,128,68-74. (19) Huang, L.; Kennel, S . J. Biochemistry 1979,18,1702-1707. (20) Brown, E. M.; Carroll, R. J.;Heffer, Ph. E.; Sampugna, J.Lipids 1983,18,111-118. (21) Hallett, F. R.; Watton, J.; Krygsman, P.Biophys. J . 1991,59, 357-362. (22) Dalgleish, D. G.;Leaver, J. J . Colloid Interface Sci. 1991,141, 288-294.
Fang and Dalgleish concentration of 1mg mL-1 in imidazole buffer. After the initial size of vesicles was measured, trypsin (1pL) was added to the suspension in the light scattering cell and the proteolysis process was followed by measuring the diameters of the particles every minute for up to 4 h. Fluorescence Polarization. To study the effect of the presence of casein on the organization of the lipid bilayer, labeled fluorescencepolarization measurements were carried out using a Shimadzu W 2 4 0 spectrofluorimeter(Shimadzu,Tokyo,Japan). DPPC vesicles were labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH). A2% solution ofDPH in dihydrofuran (DHF)was diluted in an equal volume of 20 mM imidazole buffer, and the diluted DPH solution was stirred vigorously for 1 h to evaporate the DHF. Dispersions of DPPC vesicles were then incubated with the DPH solution for half an hour before measurements of the fluorescencepolarization were made. The vesicle dispersion was diluted to give a final phospholipid concentration of 4.3 x M. Preparations of vesicles contained from 0 to 2.7 x loT5M casein, and in pairs of samples containing casein, one had the protein added before and one after vesicle formation. The temperature of the fluorimeter cell was controlled with a circulating water bath to h0.2 "C. The polarization value^^^^^^ were determined from 25 to 50 "C a t intervals of 5 "C in a single temperature cycle. Polarization is expressed as the ratio p = (41-ZJ(Z11+ Zl),where thezvalues refer to fluorescence intensities where the input and output polarizers are parallel and perpendicular to one another.
Results and Discussion Vesicle Sizes and Changes during Trypsin Treatment. Vesicles made by microfluidization or by the extrusion method in the absence of casein were stable; this was tested using centrifugation at lOOOOOg for 1.5 h, aRer which no pellet of sedimented material was observed, and by PCS, which showed that the sizes of the vesicles remained constant for at least 7 days. In fact, in all of the experiments, no time dependent changes were found in the behavior of the vesicle-caseinate system. The addition of casein solution to the vesicles after they were formed did not change their sizes over time periods of about 1 h, neither did subsequent addition oftrypsin have any effect on the size of the vesicles (Figure 1A). Adsorption of casein to polystyrene latices causes the particles immediately to increase in size, and then addition of trypsin reduces the diameter toward its original value in the absence of protein.26 Therefore, the insensitivity of the size of the PC vesicles to the addition of casein and trypsin indicates that casein has very little afinity for the vesicle surface. A recent study2' on p-casein binding to liposomes of PC or phosphatic acid (PA)showed similar results to Figure 1Awhen the liposomes were made in the absence of NaCl solution, but adsorption of protein was found when 0.9% (154 mM) of NaCl was used in the dispersing phase. We used a similar concentration of NaCl in the same comparative experiments, but no adsorption was detected using our preparations of vesicles; it is probable that these differences reflect different methods of preparing vesicles in the two studies. The lack of adsorption of casein to the intact vesicles is substantiated by the behavior of oil-in-water emulsions stabilized by mixtures of casein and PC.4,5 Emulsions can be prepared in the absence of PC with a surface concentration of as little as 0.9 mg m-2 of adsorbed casein, but this can be simply increased to a maximum saturated (23) Leaver, J.; Dalgleish, D. G. J . Colloid Interface Sci. 1992,149, 49-55. (24) Marangoni, A. G. Colloids Surf. B : Biointerfaces 1993,1,167176. ~. (25) Nealon, D. G.;Sorensen, E. M. B.; Acosta, D. J . Tiss. Cult.Meth. 1984,9,11-17. (26) Dalgleish, D. G . Colloids Surf. B : Biointerfaces 1993,1, 1-8. (27) Brooksbank,D. V.; Leaver, J.;Horne, D. S. J . Colloid Interface Sci. 1993,161,38-42.
Langmuir, Vol. 11, No. I , 1995 77
Interactions between Phosphatidylcholine and Casein 200
,
,
,
I
,
,
I
,
,
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20
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1 0 2 0 4 0 6 0 8 0 time (min)
Figure 1. Changes in the hydrodynamic diametersof egg-PC vesicles measured by PCS as functions of time. (A) Diameters of vesicles, showing the lack of change in the diameters when casein and trypsin are added t o the vesicles after formation. (B)Change of diameter of the vesicles formed when egg-PCand casein are homogenized together and trypsin is added later.
value (3 mg m-2) by adding more casein into the emulsion. However, in the presence of egg-PC, the addition of extra casein to the emulsions made with small amounts of casein does not change the casein layer thickness. It is reasonable to suggest that egg-PC is adsorbed on the oil droplets in the form of a monolayer between the adsorbed casein molecules, and this monolayer demonstrates a similar surface property as the vesicular surface relative to casein adsorption. This nonadsorption of casein can be explained since the layer of egg-PC is not charged at neutral pH and is hydrophilic in nature, whereas casein tends to adsorb preferentially to hydrophobic surfaces. When vesicles of PC were made in the presence of casein, the particles once again were stable, since their diameters did not change with time, but there were extensive changes when those composite vesicles were treated with trypsin. For vesicles of egg-PC, the average vesicle size decreased at first, during hydrolysis of the casein molecules by trypsin, as has been found previously in oil-in-water emulsions and model latex-protein complexes.26 However, after a minimum value was reached (Figure lB),the average size began to increase again, with the rate of increase and the final size attained by the particles depending on the amount of trypsin used. This is very different from the behavior of emulsion droplets which remain stable after they are treated with trypsin even though the adsorbed layer of the protein is destroyed by the proteinase. It was also apparent that the average change in diameter of the vesicles between the original and the minimum value after trypsin treatment was much larger than the analogous change for casein adsorbed to an oil-water interface, which is about 20 nm for a saturated casein m ~ n o l a y e r . ~ The magnitude of the decrease in diameter during trypsin treatment depended on the concentration of casein present during the vesicle preparation. Figure 2 shows that the average diameter change during trypsinolysis increased with the casein concentration up to about 1.5% casein and then leveled off to a value of about 37 nm at higher concentrations of casein. This measurement was
~
0.5 1 1.5 2 2.5 3 casein concentration (%)
Figure 2. Decrease of hydrodynamic diameter of egg-PC vesicles associated with casein, taken as a difference between the size measured before the addition of trypsin and the minimum size obtained during the trypsin treatment. The M, and concentration of vesicles was maintained at 6.8 x casein concentration was varied. Each point is the average of at least two separate experiments.
nearly double the thickness of the casein layer which is adsorbed to emulsion droplets under the same treatment (although at a considerably higher concentration of lipid). We can suggest several possible explanations for this large value. Possibly, only small parts of the casein molecules are involved in the interaction with the bilayer, so that a large portion of the rather flexible polypeptide chains protrude into the solution to give a larger hydrodynamic dimension. Alternatively, the casein molecules may form multilayers which will of course be thicker, although we have little evidence for their formation in oil-in-water emulsions. Third, some casein molecules may interact with more than one vesicle, so that clusters are formed via casein bridging; when the bridging protein is broken down by proteolysis, there is a change in quaternary structure as well as in the diameters of the particles. At the present time, none of these can be ruled out with certainty, although the last may be unlikely since bridging is generally associated with low, rather than high, concentrations of protein.13 The differencesbetween parts A and B of Figure 1clearly indicate Werent associations between casein and vesicles, depending on whether or not the casein is present at the time of vesicle formation. The fact that the vesicles prepared in the absence of casein were stable and did not show changes in their light scattering when casein was added to the vesicle dispersion and then treated with trypsin means that casein is not associated with these vesicle surfaces. Conversely, the response of casein-PC vesicles to trypsin treatment indicates that a complex is formed between the PC and casein. We have shown using electron microscopyz8that proteolysis of the casein caused the vesicles not only to aggregate but to fuse to give large, multilamellar particles, showing that casein was not simply interacting with the vesicle surface (as, for example, it does in oil-in-water emulsions), and we can conclude that particles of casein molecules were incorporated into the hydrophobic core of the bilayer. Like the egg-PC vesicles, DPPC vesicles were also found not to absorb casein added after the vesicle was formed. These DPPC vesicles were also stable in size during PCS measurement and when treated with trypsin. However, DPPC-casein vesicles showed a response to trypsin. Indeed, they showed different behavior when they were (28) Fang, Y.;
Dalgleish, D. G. Food Struct., in press,
Fang and Dalgleish
78 Langmuir, Vol. 11, No. 1, 1995 45 A
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time (min) Figure 4. Variation of light scattering intensity at an angle of 90" of DPPC vesicles (m), and DPPC-casein vesicles (0 and dotted line) with temperature (solid line). The same sample was heated from 25 t o 45 "C in steps of 5 "C and then cooled from 45 to 25 "C. This temperature cycle was repeated three times for each sample.
time (min) 250 Figure 3. Changes in hydrodynamic diameter of DPPC vesicles, homogenized with casein, during proteolysis of the casein by trypsin: (A) measured at 25 "C; (B) measured at 45 2 200 c "C. L B treated with trypsin at temperatures below and above 150 the phase transition temperature (42 "C) of the DPPC bilayer. At room temperature (25"C) where the bilayer e .-0 s is in the gel state, the average size of the vesicles increased 100 immediately after trypsin was added to the system (Figure p! 3 * 3A), but at 45 "C where the DPPC bilayer is in the liquid c crystalline state, the vesicles behaved similarly to egg60 PC-casein vesicles (Figure 3B) in that trypsin treatment 40 caused a decrease in diameter followed by an increase. 20 IThe result a t 45 "C was in fact more complex: at this temperature the diameters ofthe particles increased with 0 0 time in the absence of trypsin, so that the treatment took 0 5 10 15 20 25 30 place on an ascendingbase line (Figure 3B). These suggest Time (min) that a rearrangement of casein molecules relative to the Figure 5. Change in the hydrodynamic diameter of DPPC DPPC bilayers resulted from the phase transition, possibly vesicles (0 and dotted line),and DPPC-casein vesicles (H and causing the trypsin to attack different sites in the proteins. full line)as a result of temperature change (lower full line).The In the liquid crystalline state, some diffusion within the temperature cycle is identical to that described in Figure 5, the bilayer is possible because the hydrophobic core is in a sample being recycled from 25 t o 45 to 25 "C three times. liquid state; this would be difficult in a gel state. The different, being larger and having considerably less overall lateral diffusion of the hydrophobic peptides from the temperature dependence (Figure 4). This is very different protein breakdown into the core of the bilayers in the from the interaction between polylysine and DPPC, which liquid crystal state could explain the difference in the showed a large increase in absorbance (turbidity) over aggregation behavior of the vesicles at different temthe transition from gel to liquid crystalline state of the perature. Temperature Dependence of DPPC Vesicles. It DPPC bilayer. This was originally explained as a consequence of the aggregation of vesicles caused by has been reported12that temperature is a key factor of the polylysine, with the aggregates being reversibly dissociinteraction between poly-L-lysineand DPPC vesicles.That ated when the temperature was brought back to below conclusion was drawn from absorbance measurements of DPPC vesicles in the presence and absence of poly-L-lysine the phase transition, (i.e., no fusion of the vesicles as functions of temperature. We performed a parallel occurred). The relatively stable scattering intensity of study on the DPPC/casein system using measurements of the complex between casein and DPPC indicates that total light scattering at an angle of go", using the PCS casein did not induce aggregation of DPPC vesicles during equipment to measure the integrated light scattering. The the phase transition of the bilayers. intensity of light scattered from DPPC vesicles alone varied PCS was used to measure how the apparent average inverselywith the change in temperature (Figure 4),which sizes of the vesicles in the dispersion changed with is in good agreement with the published work on absortemperature (Figure 5 ) . As the temperature was cycled from 25 to 45 "C, two maximum and two minimum sizes bance.12 The addition of casein to DPPC vesicles did not were observed for DPPC vesicles in the absence of casein, change the behavior of the system with temperature, confirming that no interaction occurs between casein and this process being completely reversible as the temperaDPPC vesicles, in contrast to the behavior of poly-L-lysine ture was cycled. The vesicles prepared with casein showed and DPPC vesicles. However, the scattering intensity of only one maximum and one minimum, the maximum at the DPPC vesicles prepared in the presence of casein was 30 "C during the cooling process being inhibited by the v
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Langmuir, Vol. 11, No. 1, 1995 79
Interactions between Phosphatidylcholine and Casein 0.25
-3
B .-0
.I -2
0.2 0.15 0.1
0.05 - 0 20 25 30 35 40 45 50 55 Temperature ('C)
Figure 6. Fluorescence polarization values for the fluorescent probe bound to DPPC vesicles (O),DPPC vesicles with added casein (A),and DPPC vesicles homogenizedtogetherwith casein (0). Each point was the average of two separate experiments, made using a single increase in temperature; i.e., the temperature was not cycled for the same sample. presence of casein. It has been r e p ~ r t e dthat ~ ~ the ~~~ heating of phospholipid vesicles causes a dilatational stress between the inner and outer layer which leads to a topological change in the vesicles, such as vesicle budding when a larger vesicle forms a number of smaller ones connected by narrow tubes. This cluster of vesicles remains topologically as one surface and the process is reversible when the conditions are reversed. Are-entrant shape change has also been demonstrated, both theoretically and experimentally, when the vesicles are subjected to temperature change.30 These kinds of changes will affect the PCS, and it is clear that the incorporation of casein intothe bilayer modifies significantlythe topological response of DPPC bilayer to temperature change. Fluorescence Polarization. The fluorescence polarization measurement was performed only with DPPC vesicles because of their very accessible phase transition temperature. The variations of fluorescence polarization with temperature are shown in Figure 6. For all three samples (vesicles, vesicles with added casein, and vesicles incorporating casein), there was a rapid decrease in polarization between 35 and 45 "C, marking the transition of the DPPC bilayer from gel state to liquid crystalline state about 42 "C. The incorporation of casein into the DPPC bilayer did not suppress this phase transition, nor did it alter the transition temperature. It has been reported24that the binding ofRhizopusarrhizus lipase on DPPC vesicles abolishes the gel to liquid crystalline transition and gives a higher degree of membrane rigidity in general, even when the enzyme was simply added to the dispersion of vesicles. From Figure 6, it can be seen that the DPPC-casein vesicles and DPPC vesicles gave nearly the same polarization value in the gel state, which implies that casein incorporation into the vesicle structure did not significantly perturb the structure of the bilayer in the gel state. The sample with casein added to vesicles
gave a slightly lower value than the other two systems, the differences being small but reproducible. However, in the liquid crystalline state, the DPPC-casein vesicles gave a higher polarization value, that is to say a higher rigidity of the membrane, while the data of DPPC vesicles and DPPC vesicles plus casein coincided at lower values. The above results are in good agreement with an X-ray diffraction study3l on a liposome system containing serum albumin, in which the diffraction pattern revealed that the protein was inserted into the bilayer but the structure of the hydrocarbon chain was preserved. These results on the polarization of fluorescence show that some degree of immobilization of the hydrocarbon chains occurs when casein is present in the bilayer. However, it is not possible to determine whether this is because of retention of local crystallinity in the vicinity of the protein or because of a general decrease in mobility. Because the probe molecule is believed to bind equally in the gel and liquid crystal phases of the DPPC, it is not possible to distinguish between these possibilities.
Conclusion From the results of dynamic light scattering and labeled fluorescence polarization measurements, we can draw the following conclusions. The surfaces of egg-PC and DPPC vesicles are hydrophilic in nature, and casein molecules have little affinity for this surface. However, complexes are formed when casein is homogenized together with the phospholipid during vesicle preparation. This complexed casein behaves very differently from casein adsorbed on the triglyceride-water interfaces in droplets of oil-in-water emulsions. The hydrodynamic thickness of the casein layer on the vesicles is nearly double that of the casein layer adsorbed on oil droplets, suggesting that the molecules of the caseins have different conformationsin the vesicle systems. The proteolytic breakdown of casein in the combined vesicles caused aggregation and fusion, again demonstrating the different conformation of caseins on oil-water interfaces and associated with phospholipid. The fluorescence polarization measurements revealed that the association of casein with DPPC vesicles made the bilayer more rigid than a pure DPPC bilayer in the liquid crystalline state, but in the gel state, both DPPC vesicles and DPPCkasein combined vesicles showed a similar polarization value, so that only a small portion of the casein was inserted into the bilayer, leaving a ordered bilayer fragmented by casein. This casein insertion into the bilayer also modifies significantly the topological response of the vesicles to temperature change.
Acknowledgment. The authors wish to thank Dr. Alejandro Marangoni of the University of Guelph for help in performing the fluorescence measurements. This research was funded jointly by the Ontario Dairy Council and the Natural Sciences and Engineering Research Council of Canada. LA940467L
(29)Evans, E.;Rawicz, W. Phys. Rev.Lett. 1990,64, 2094-2097. (30)Berndl, K.;Kas, J.; Lipowsky, R.; Sackmann, E.; Seifert, U. Europhys. Lett. 1990,13,659-664.
~
(31)Sogor, B.V.;Zull, J. E. Biochim.Biophys.Acta 1976,375,363380.
