Adsorption of Glucose Oxidase into Lipid Monolayers. Effect of Lipid

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Adsorption of Glucose Oxidase into Lipid Monolayers. Effect of Lipid Chain Lengths on the Stability and Structure of Mixed Enzyme/Phospholipid Films J. Zhang,† V. Rosilio,*,‡ M. Goldmann,§ M.-M. Boissonnade,‡ and A. Baszkin‡ Laboratory of Disperse Systems and Interfacial Phenomena, Institute of Photographic Chemistry, Academia Sinica, Beijing 100101, People’s Republic of China, Laboratoire de Physico-Chimie des Surfaces, UMR CNRS 8612, 5 rue J. B. Cle´ ment, 92296 Chaˆ tenay-Malabry Cedex, France, and Laboratoire de Physico-Chimie Curie, UMR CNRS 168, Institut Curie, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France Received April 23, 1999 The effect of lipid hydrophobic chain lengths on adsorption of glucose oxidase (GOx) into phospholipid monolayers has been investigated. The results show that at low initial surface pressures, penetration of GOx depended both on hydrophobic interactions and on the orientation of lipid chains with respect to the interface plane. Compared with dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoyl-phosphatidylcholine (DPPC), dibehenoyl-phosphatidylcholine (DBPC) favored enzyme adsorption at low surface pressures. This was attributed to its long hydrophobic chains and to their vertical orientation. At higher initial surface pressures, the fluidity and packing of these monolayers appeared to play a key role in the penetration process. The presence of a double bond in the hydrocarbon chains enhanced enzyme adsorption, and this effect was independent of the initial surface pressure of lipid monolayers.

Introduction The structure of biological membranes and the physical properties of the lipids and proteins that make up these membranes have been a subject of a great number of biophysical and computational investigations using as models Langmuir-Blodgett (LB) films and monolayers.1,2 The incorporation of biomolecules such as enzymes in these films provides them with both biological activity and biospecific recognition properties which can be used in the design of artificial systems such as biosensors.3-5 Proteins adsorb spontaneously on a wide range of surfaces. When a protein adsorbs at the air-water interface its conformation often changes.6-8 It has been shown that molecular rearrangements that occur as the hydrophobic side-chains move inward to the air phase are accompanied by the loss of the protein tertiary structure. The main thermodynamic driving force for protein adsorption at an interface is thus the removal of its nonpolar amino acid side-chains from their unfavorable environment in the bulk aqueous solution. The free energy gain on adsorption strongly depends on dehydration of hydrophobic regions at the surface, protein unfolding, charge redistribution due to overlapping of the electric fields of * To whom correspondence should be addressed: Ve´ronique Rosilio, Ph.D., Tel.: 33 1 46 83 56 45. Fax: 33 1 46 83 53 12. E-mail: [email protected]. † Laboratory of Disperse Systems and Interfacial Phenomena. ‡ Laboratoire de Physico-Chimie des Surfaces. § Laboratoire de Physico-Chimie Curie. (1) Birdi, K. S. J. Colloid Interface Sci. 1976, 57, 228-232. (2) Haas, H.; Mo¨hwald, H. Colloids Surf. B. 1993, 1, 139-148. (3) Dubreuil, N.; Alexandre, S.; Lair, D.; Valleton, J. M. Langmuir 1996, 12, 6721-6723. (4) Dai, G.; Li, J.; Jiang, L. Acta Physico-Chimica Sinica 1997, 13, 200-203. (5) Hodak, J.; Etchenique, R.; Calvo, E. J.; Singhal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708-2716. (6) Norde, W.; Favier, J. P. Colloids Surf. 1992, 64, 87-93. (7) Clark, S. R.; Billsten, P.; Elwing, H. Colloids Surf. B. 1994, 2, 457-461. (8) Kondo, A.; Oku, S.; Higashitani, K. J. Colloid Interface Sci. 1991, 143, 214-221.

the protein and the surface, difference in ionic hydration between bulk and surface, change in pK values of sidechains on adsorption, and on an overall change in van der Waals interactions of the system. The process of protein adsorption is considered to be a biphasic process highly dependent on the temperature. It is characterized by timedependence in protein conformational structure, proteinprotein interactions within the layer, and lateral molecular mobility within the layer.8-12 The properties of mixed protein-lipid monolayers may be investigated from surface pressure changes (∆π) generated by the injection of a protein under a lipid film at a well-defined initial surface pressure (πi). The magnitude of ∆π is then related to the degree of penetration of a protein molecule into a lipid film and largely depends on the initial surface pressure of the film, lipid organization, protein concentration at the surface, protein charge, and salt concentration of the subphase. The ∆π ) f(π) relationship gives the so-called exclusion pressure which is the initial surface pressure of a monolayer above which the protein is no longer able to penetrate the lipid monolayer and produce an increment in surface pressure. This parameter, which is related to the packing of lipid molecules in a monolayer, is a quantitative measure of the penetration capacity of the protein and after penetration, of the affinity between the protein and the lipids.1,13 Usually, ∆π decreases linearly over the entire range of the lipid π. Since the predominant forces in the stability of a lipid monolayer are the van der Waals forces, when πi is the limiting π, these forces are supposed to be of an such intensity that no more penetration of the protein is possible.1 Glucose oxidase spontaneously adsorbs at the air-water interface.14 Circular dichroism (CD) spectra show that its (9) Dickinson, E.; Matsumura, Y. Colloids Surf. B. 1994, 3, 1-17. (10) Roth, C. M.; Lenhoff, A. M. Langmuir 1995, 11, 3500-3509. (11) Nicholov, R.; Veregin, R. P. N.; DiCosmo, F. Colloids Surf. B. 1995, 4, 45-54. (12) Shirahama, H.; Suzawa, T. J. Colloid Interface Sci. 1988, 126, 269-277. (13) Maget-Dana, R.; Ptak, M. Biophysical J. 1995, 68, 1937-1943.

10.1021/la990490c CCC: $19.00 © 2000 American Chemical Society Published on Web 12/01/1999

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R-helix percentage decreases. However, when surface pressure increases, the ratio R/β increases.4 GOx is able to interact with various lipids.15-18 In a previous work we have shown that the presence of phospholipid (DBPC) molecules at the interface strongly enhances adsorption of GOx even at enzyme bulk concentrations (0.001 mg/ mL) at which no increase in surface pressure could be observed in the absence of phospholipid molecules. We have also shown that ∆π decreased with increasing monolayer densities of DBPC (increased πi). The difference in enzyme adsorption in the presence and in the absence of phospholipid molecules was attributed to the existence of hydrophobic interactions at the level of hydrocarbon chains of the lipid and adsorbed enzyme molecules.17 In this work, the interaction of glucose oxidase with various phospholipids differing in the length of their hydrophobic chains was investigated under dynamic conditions of compression of lipid monolayers. Materials and Methods Chemicals. Glucose oxidase (GOx, 180 000 units per gram solid, isoelectric point ) 4.44),18 dimyristoyl-phosphatidylcholine (DMPC, 2 saturated C14 alkyl chains, Mw 677.94), dipalmitoylphosphatidylcholine (DPPC, 2 saturated C16 alkyl chains, Mw 734.0), dibehenoyl-phosphatidylcholine (DBPC, 2 saturated C22 alkyl chains, Mw 902.4), and dioleyl-phosphatidylcholine (DOPC, 2 unsaturated C18 alkyl chains, Mw 786.1) were purchased from Sigma Chemicals (Saint Louis, MO) and were used as received. Ultrapure water was obtained by osmosis from a Milli-RO 6 Plus Millipore apparatus and then doubly distilled from permanganate solution in an all-Pyrex container. Its pH was 5.5 and its surface tension was 71.8 mN/m at 22 °C. Methanol and chloroform used for spreading of lipid monolayers were analytical grade reagents (99+% pure) from Merck (Darmstadt, Germany). Formation of Mixed Enzyme-Phospholipid Monolayers and Surface Pressure-Surface Area Measurements. GOx was dissolved at pH 7 in triply distilled water and the obtained solution was used as the subphase. The π-A isotherms were recorded on a film balance (Lauda, Germany) having the dimensions 34 × 14 × 1 cm and enclosed in a Plexiglas box. Phospholipid molecules were spread from a chloroform/methanol solution (9:1, v/v) onto the aqueous GOx solution after sweeping the substrate with a capillary pipette connected to a vacuum pump. The organic solvents were allowed to evaporate for at least 15 min and the phospholipid monolayer was compressed until a desired initial surface pressure (πi) was reached. The compression barrier moved automatically at the rate of 1.6 or 2 cm/min. The enzyme was then allowed to adsorb from the unstirred subphase to the air-solution interface. The increase in the surface pressure due to enzyme adsorption was recorded as a function of time at 22 °C. After 6 or 20 h of adsorption, the mixed monolayers were compressed until collapse was reached. Grazing Incidence X-ray Diffraction. Grazing incidence X-ray diffraction (GID) experiments were carried out at the D41B beam line on the LURE synchrotron source (Orsay, France). A general description of the experiment has been reported in ref 19. A wavelength of 1.488 Å is selected by a focusing Ge (111) monochromator and deflected with a silica mirror at an incidence of 1.9 mrad, below the water surface critical angle. Scattered intensity is recorded with a vertical focusing position sensitive detector (PSD). The in-plane diffusion is determined by Sollers (14) Sun, S.; Ho-Si, P. H.; Harrison, D. J. Langmuir 1991, 7, 727737. (15) Du, Y.-K.; An, J.-Y.; Tang, J.; Li, Y.; Jiang, L. Colloids Surf. B. 1996, 7, 129-133. (16) Dubreuil, N.; Alexandre, S.; Fiol, C.; Valleton, J. M. J. Colloid Interface Sci. 1996, 181, 393-398. (17) Rosilio, V.; Boissonnade, M. M.; Zhang, J.; Jiang, L.; Baszkin, A. Langmuir 1997, 13, 4669-4675. (18) Baszkin, A.; Boissonnade, M. M.; Rosilio, V.; Kamyshny, A.; Magdassi, S. J. Colloid Interface Sci. 1997, 190, 313-317. (19) Fradin, C.; Daillant, J.; Brasleau, A.; Luzet, D.; Alba, M.; Goldmann, M. Eur. J. Phys., B 1998, 1, 57-69.

Figure 1. π-A isotherms of (1) DMPC, (2) DPPC, (3) DBPC, and (4) DOPC spread at the air-water interface. Table 1. Characteristic Collapse Pressure (πc), Mean Molecular Area (Am), Molecular Area at Collapse (Ac) Deduced from Figure 1, and Phase Transition Temperature (Tc) of the Four Studied Phospholipids phospholipid

πc (mN/m)

Am (Å2)

Ac (Å2)

DMPC DPPC DBPC

41.8 49 52.5

72 56a 57

43 40 38

23 41 74

DOPC

40.5

78

57

-21

Tc (°C)

a

The mean molecular area of DPPC has been considered above the LE-LC transition.

slit with an aperture of 0.9°, leading to a resolution of about 0.008 Å-1 or Qxy ) 1.5 Å-1. The observable Qz window, limited by the height of these Sollers slits, ranges from 0 to 0.8 Å-1. A vertical motion of the slits-PSD device allowed us to explore higher values if necessary.

Results Interfacial Behavior of the Phospholipids under Dynamic Compression. The π-A isotherms of DMPC, DPPC, DBPC, and DOPC monolayers spread at the airwater interface are illustrated in Figure 1. Their main characteristics are summarized in Table 1. The results reveal a simultaneous increase in surface pressure values (πc) and decrease in areas occupied by a phospholipid molecule at collapse (Ac), with an increase in the length of saturated hydrophobic chains. Adsorption of Glucose Oxidase at the Air-Water Interface. GOx adsorption was studied using a Langmuir trough in which the compression barrier was moved after 15 min, 6 h, or 20 h following the beginning of the experiment. The possible formation of an adsorbed enzyme monolayer even at the low GOx bulk concentration (0.001 mg/mL) may be inferred from the results illustrated in Figure 2. The recorded π-A isotherms demonstrated that a slow and spontaneous adsorption of the protein at the interface occurred. Increasing adsorption times led to expanded π-A isotherm profiles. However, from the small increments in molecular areas it was clear that GOx adsorption at the air-water interface although spontaneous was very weak. GOx Adsorption into Phospholipid Monolayers. The effect of GOx adsorption on the interfacial behavior of the studied phospholipids is presented in Figures 3-6 as a function of initial surface pressures (πi) of the monolayers.

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Figure 2. π-A isotherms of adsorbed GOx monolayers in the absence of a lipid film. Film compressed after (1) 15 min, (2) 6 h, and (3) 20 h following the beginning of enzyme adsorption. Enzyme concentration in the subphase was 0.001 mg/mL.

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GOx adsorption was investigated at the three initial surface pressures of the phospholipid monolayers equal to 3.3, 13, and 20.8 mN/m. From the comparison of the results illustrated in Figures 3-6, it was clear that the interaction of the enzyme with phospholipid monolayers depended to a large extent on the nature of the spread lipid. Indeed it may be noted that (i) For DPPC, both at πi ) 13 and 21 mN/m (above the LE-LC transition pressure of the phospholipid), the π-A profiles remained virtually unchanged in the presence of the enzyme (Figure 4). However, at πi ) 3.3 mN/m, a modified LE-LC transition was observed in the presence of the enzyme. (ii) For DBPC (Figure 5), the presence of the enzyme appeared to have a significant effect on the collapse pressure of the monolayer. (iii) Finally, in the presence of the enzyme, the isotherms for DMPC and DOPC exhibited more expanded profiles than those obtained for DPPC and DBPC. The expansion of DMPC monolayers after GOx adsorption was quantified and the results showed that at πi ) 3, 13, and 20 mN/m, it corresponded to the increase in molecular area of +29, +21, and +14%, respectively. The monolayer expansion was accompanied by a decrease in the lipid collapse surface pressure. Kinetics of Adsorption of GOx into Phospholipid Monolayers. In Figure 7, the change in the surface

Figure 3. π-A isotherms of (1) a GOx monolayer compressed after 6 h following the beginning of enzyme adsorption at the free air-water interface, (2) a pure DMPC monolayer spread at the air-water interface, and (3) a mixed DMPC-GOx monolayer compressed after 6 h following the beginning of enzyme adsorption into a DMPC monolayer at πi equal to (a) 3.3 mN/m, (b) 13 mN/m, and (c) 20 mN/m.

Figure 4. π-A isotherms of (1) a GOx monolayer compressed after 6 h following the beginning of enzyme adsorption at the free air-water interface, (2) a pure DPPC monolayer spread at the air-water interface, and (3) a mixed DPPC-GOx monolayer compressed after 6 h following the beginning of enzyme adsorption into a DPPC monolayer at πi equal to (a) 3.3 mN/m, (b) 13 mN/m, and (c) 20 mN/m.

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Figure 5. π-A isotherms of (1) a GOx monolayer compressed after 6 h following the beginning of enzyme adsorption at the free air-water interface, (2) a pure DBPC monolayer spread at the air-water interface, and (3) a mixed DBPC-GOx monolayer compressed after 6 h following the beginning of enzyme adsorption into a DBPC monolayer at πi equal to (a) 3.3 mN/m, (b) 13 mN/m, and (c) 20 mN/m.

Figure 6. π-A isotherms of (1) a GOx monolayer compressed after 6 h following the beginning of enzyme adsorption at the free air-water interface, (2) a pure DOPC monolayer spread at the air-water interface, and (3) a mixed DOPC-GOx monolayer compressed after 6 h following the beginning of enzyme adsorption into a DOPC monolayer at πi equal to (a) 3.3 mN/m, (b) 13 mN/m, and (c) 20 mN/m.

Figure 7. Surface pressure change (∆π) versus time on enzyme adsorption in (1) DMPC, (2) DPPC, and (3) DBPC monolayers from a 0.001 mg/mL GOx solution.

pressure (∆π) due to GOx adsorption during the first 6 h following its initial compression has been monitored for DMPC, DPPC, and DBPC (the lipids that differed only in their hydrophobic chain lengths). (i) At πi ) 3 mN/m, for DBPC the increase in ∆π was more pronounced than for DMPC and DPPC monolayers. (ii) At πi ) 13 mN/m, whereas DMPC displayed the highest ∆π value, DPPC showed the lowest ∆π. It should

also be noted that at this πi, DPPC and DBPC monolayers exhibited the same molecular area (54 Å2), while the DMPC monolayer was in a more expanded state (67 Å2). (iii) Finally, at πi ) 20 mN/m, no penetration of the enzyme into any of the three studied monolayers was observed. Negative ∆π values recorded for DPPC and DBPC were attributed to the desorption of lipid molecules from the interface as a result of their interaction with

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Figure 8. Diffraction spectra of (a) a DBPC monolayer compressed to 14 mN/m and (b) a DBPC/GOx monolayer after 16 h of enzyme adsorption at πi ) 14 mN/m.

adsorbing GOx. Conversely, for DMPC due to its high packing at this surface pressure such a desorption was not observed. Effect of GOx Penetration on the Organization of a DBPC Monolayer. If it was clear that the interaction of GOx with phospholipid monolayers shifted their π-A relationships to the right (Figures 3-6), the observed phenomenon was not indicative of the changes in the organization of these monolayers. Therefore, the nature of the lattice and the thickness of the monolayer were evaluated by means of the grazing incidence X-ray diffraction method. A DBPC monolayer was spread onto a pure water subphase and then compressed until the desired initial surface pressure (14 mN/m) was reached. The monolayer was then scanned by X-rays and its diffraction spectrum showed a single peak at qxy ) 1.428 Å-1 (Figure 8a) and corresponded to a hexagonal lattice. The correlation length

was longer than 500 Å, and was representative of an ordered system. When a DBPC monolayer was compressed up to πi ) 15.8 mN/m, the qxy value and the correlation length did not change. The thickness of the monolayer deduced from the rodscan was about 27 Å. The same DBPC monolayer spread over a GOx solution (0.001 mg/mL), compressed up to 14 mN/m and then scanned by X-rays for 2.5 h, displayed a different diffraction spectrum. An enlargement and small displacement of the position peak were observed. Also, the correlation length dropped to 50 Å indicating that the monolayer was in a disorganized state. After 16 h of GOx adsorption the surface pressure increased to 16.5 mN/m, and the diffraction peak was significantly displaced (qxy ) 1.433 Å-1). Moreover a second peak appeared at qxy ) 1.34 Å-1 (Figure 8b). For such a system the observed correlation length was longer than 500 Å and the monolayer thickness increased up to 31 Å as a result of

Lipid Chain Length Effect on GOx Adsorption in ML Films

a more vertical orientation of the phospholipid hydrocarbon chains with respect to the interfacial plane. The presence of the second peak may be attributed either to a more ordered structure of the lipid phase resulting from the penetration of GOx into the lipid or from diffraction of the enzyme itself. Discussion GOx Interaction with Phospholipid Monolayers. GOx interaction with the studied lipids resulted in their different characteristics depending both on the nature of the phospholipids and on the initial surface pressure of their spread monolayers. At low surface pressures, for all studied phospholipids, the orientation of hydrocarbon chains predominantly influenced the adsorption process. If one compares molecular areas of the three phospholipids at πi ) 3 mN/m (Figure 1), then for DBPC a more condensed state was observed (65 Å2), despite its longer hydrophobic chains, than for DPPC and DMPC (85 Å2). As the polar head of these three phospholipids was the same, the difference in the molecular area, at this πi, should result from the differences in the orientation of their chains with respect to the planes. In the case of DBPC they were probably less inclined to the surface plane than in the case of DMPC or DPPC. For DBPC there was a large space available and the vertical orientation of the hydrophobic chains favored GOx penetration (Figure 7). To explain a higher adsorption of biological molecules into lipids with increased chain lengths, numerous authors quote hydrophobic interaction parameters. Thus, Du and co-workers15 observed that GOx adsorption in glycolipid monolayers increased with the glycolipid chain length increase. Also Bos and Nylander20 referred to hydrophobic interactions between a protein and lipid hydrocarbon chains. They have pointed out that hydrophobic interactions might generate conformation changes during the adsorption process that would facilitate the insertion of a protein into lipid monolayers; Brown et al.21 have found that when the R-helix content of β-lactoglobulin was increased, phosphatidylcholine was able to bind to the protein. They suggested that the hydrocarbon chain of the phospholipid interacted with the hydrophobic interior of the R-helix while the hydrophilic part of the protein interacted with the headgroups. From the comparison of the GOx adsorption data to DMPC, DOPC, and DBPC at low πi it appeared obvious that long DBPC chains favored hydrophobic interaction with the enzyme. However, low adsorption of the enzyme into DPPC monolayers and preferential adsorption of the enzyme to DMPC at higher πi seem to demonstrate that the hydrophobic effect was not the only possible explanation of the observed phenomenon and that other parameters such as fluidity and packing of lipid chains should account for the increase in monolayer surface pressure. At 13 mN/m the adsorbed amounts of GOx into DMPC and DOPC were higher than into DBPC and DPPC. It is reasonable to think that since less space was available to the enzyme penetration, the fluidity of the lipid monolayer and its compressibility played a critical role. For both DMPC with its critical transition temperature Tc ) 23 °C, and DOPC Tc ) -21 °C, lipid molecules were close to or above their phase transition temperatures. At the experimental temperature both lipids were in the liquid crystal state. Conversely, for DPPC (Tc ) 41 °C) and DBPC (Tc ) 74.5 °C) they were in a gel phase (Table 1).22 (20) Bos, M. A.; Nylander, T. Langmuir 1996, 12, 2791-2797. (21) Brown, E. M.; Carroll, R. J.; Pfeffer, P. E.; Sampugna, J. Lipids 1983, 18, 111-118.

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Furthermore, if one compares the values of Am with those of Ac, then it appears that the packing of DMPC and DOPC molecules was looser than that of DBPC and DPPC. This was probably due to the increased fluidity of DBPC and DPPC at the experimental temperature. The packing of molecules in a monolayer appeared to strongly influence the penetrant ability of the enzyme. At 13 mN/m, the DPPC monolayer was already in a highly condensed packing state as shown by the low value of its mean molecular area (Am ) 56 Å2). The available space at the interface was too small to allow enzyme penetration. It should be noted that if GOx had no effect on the mean and collapse molecular areas, it also had no effect on the collapse surface pressure of the DPPC monolayer. It may be considered that above πi ) 10 mN/m (LE-LC transition) the formation of mixed DPPC-GOx monolayers could not be accomplished because of the existence of a strong intermolecular interaction between DPPC molecules which acted as a barrier against GOx penetration. The presence of this intermolecular DPPC-DPPC interaction is strongly supported by the poor postcollapse respreading of DPPC in monolayer expansion experiments.23 Since the enzyme could not penetrate into the DPPC monolayers, it was depleted from the interface and, thus, negative values of ∆π were observed. DMPC and DBPC molecules which exhibit an expanded behavior do not develop such a strong intermolecular interaction on compression, and do not hinder enzyme penetration. These results are in good agreement with those reported by Ibdah and Phillips24 who showed that adsorption of proteins into lipid monolayers was generally affected by the physical state of the lipid monolayer. They have studied the influence of lipid packing by spreading the monolayers at various initial surface pressures. They demonstrated that increasing the πi of lipid monolayers generally reduced protein adsorption, and that the degree of adsorption was also influenced by the physical state of lipid monolayers. They have postulated that at a given πi, proteins adsorb to a higher extent to expanded monolayers than to condensed monolayers and that the lateral compressibility was a major determinant of the amount of adsorbed protein. They have also observed the specific behavior of DPPC and they have attributed it to the existence of the LE-LC transition characteristic of this lipid. Adsorption of GOx in DOPC monolayers showed an additional feature. At all initial surface pressures, an expansion of the monolayer was observed. Even at high πi the enzyme penetrated the lipid monolayer (Figure 6). This behavior may be explained by the presence of a double bond in DOPC hydrocarbon chains which generally led to more liquidlike structures. Moreover, the presence of this double bond would enable establishment of strong links of GOx to the phospholipid through the formation of hydrogen bonding. Effect of GOx on the Stability and Structure of Mixed Lipid/Protein Films. Many lipids which form films on water with significant surface pressures may desorb to the bulk subphase at rates which depend on the molecular weight of the lipid, pH, temperature, and surface pressure.16,25 This phenomenon has also been observed with all phospholipids investigated in this study at the initial surface pressure of 13 mN/m and above. When a (22) Small, D. M. Handbook of Lipid Research; Hanaham, D. J., Ed.; Plenum Press: New York, 1986; p 490. (23) Tchoreloff, P.; Denizot, B.; Proust, J. E.; Puisieux, F. In Basic Research on Lung Surfactant; Von Wichert, P., Mu¨ller, B., Eds.; Progress in Respiration Research; Karger: Basel, 1990; Vol. 25, pp 168-175. (24) Ibdah, J. A.; Phillips, M. C. Biochemistry 1988, 27, 7155-7162. (25) Gershfeld, N. J. Colloid Interface Sci. 1982, 85, 28-40.

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lipid monolayer was spread at the air-water interface its surface pressure first decreased for about 4 h and then increased to reach the initial surface pressure. Gershfeld25 suggested that lipid films were stabilized by their surface properties rather than by their insolubility. He attributed this film desorption to two major processes: (a) a dissolution of the film from the surface to a thin region just beneath the film, and (b) the diffusion of the dissolved lipid across an unstirred layer to the bulk of the subphase which is stirred by convection. It seems reasonable to think that the presence of GOx in the subphase even at very low concentrations, and its adsorption at the interface, hinders the decrease in surface pressure. However, the extent of this effect on the two invoked processes is difficult to estimate. Dubreuil et al.16 have also observed that GOx was able to improve the stability of a lipid film. They attributed this phenomenon to the existence of interactions between the enzyme and polar headgroups of fatty acids. The structure of a lipid-protein film strongly depends on the miscibility of lipids and proteins at the interface. Demel et al.26 have shown that the molecules interacting solely with the lipid headgroups without penetration into the monolayer would not affect the surface pressure. In this study as well as in the previous one17 we have shown that adsorption of GOx into lipid monolayers induced a change in the surface pressure (∆π) and that this ∆π decreased when the lipid surface density increased. This would certainly indicate that the enzyme penetrated the monolayer and that its interaction with the lipids was not limited to the sole interaction with lipid polar headgroups. As suggested by Bos and Nylander,20 it is important to determine whether the proteins at the interface exist in separate domains without lipids, that is if the proteins adsorb to form their own monolayers or if they penetrate into the lipid monolayers and disorganize the lipid phase. Cornell and Carroll27 using electron microscopy analysis have shown that phospholipids which form condensed films at the air-water air interface do not mix with protein in monolayers. We have also observed this type of behavior for DPPC (above 3 mN/m) and for DMPC and DBPC at πi of 20 mN/m; that is when the monolayers were in the condensed state. The surface behavior of a lipid in single component monolayers may thus serve to predict its miscibility behavior or at least its penetration behavior with proteins. According to Cornell and Carroll,28 factors such as van der Waals forces which are responsible for the close packing of molecules in condensed films of pure lipid systems would also contribute to the separation of the components when lipids and proteins are spread together in monolayers. They have also reported that a phase separation between a protein such as β-lactoglobulin and DPPC takes place when the latter forms a condensed monolayer.28 The effect of temperature on lipid/protein segregation has been studied by Lookman et al.29 When the temperature of an experiment was higher than the phase transition temperature (Tc) of the phospholipid, a bilayer (26) Demel, R.; London, Y.; Geurts van Kessel, W.; Vossenberg, F.; van Deenen, L. Biochim. Biophys. Acta 1973, 311, 507-519. (27) Cornell, D. G.; Carroll, R. J. J. Colloid Interface Sci. 1985, 108, 226-233. (28) Cornell, D. G.; Carroll, R. J. Colloid Surf. 1983, 6, 385-393.

Zhang et al.

containing an integral protein existed as a single homogeneous phase. Above Tc, a lipid-protein system separated into an essentially pure lipid phase and a protein-rich phase, provided the lipid-protein interaction parameter was sufficiently weak. According to Heck et al.30 proteins in monolayers are mainly located in the fluid membrane phase which coexists with solid lipid domains without proteins. Using the grazing incidence diffraction method (GID) we have studied the interaction between GOx and DBPC at a temperature below Tc and at a πi at which the monolayer was not yet in the condensed state (between 13 and 16 mN/m). The profile of the π-A isotherm observed at this πi showed almost no alternation with respect to that obtained at πi equal to 20 mN/m (Figure 5b,c). As it may be inferred from the dramatic decrease in the correlation length (Figure 8) the enzyme penetrated the DBPC monolayer and disorganized its structure at the early stage of the adsorption process. Then a reorganization occurred which led to an increase in this correlation length up to its original value. However, when compared with a film of pure DBPC at the same surface pressure, two major changes were observed: a new peak appeared and the thickness of the monolayer increased from 27 to 31 Å. Both of these factors would account for the rearrangement of the protein and the lipid phase from the expanded fluid DBPC monolayer into a phase of a higher intrinsic order. A reorientation of DBPC hydrocarbon chains toward the vertical allows partial penetration of GOx into the monolayer (as inferred from the slight decrease in πc (Figure 5b) and the increase in π (Figure 7). This penetration of glucose oxidase probably had a stronger effect on the molecular area of the mixed monolayer than can be noted from Figure 5b. The expansion of the mixed monolayer appeared to be quite limited. As proposed by Ibdah and Phillips24 it is reasonable to assume that the compressibility of a lipid monolayer changes in the presence of a protein. As the results obtained with GID do not support the hypothesis of an ideal mixing between the enzyme and DBPC, a segregation of constituents gives rise to the existence of patches of GOx surrounded by zones of pure DBPC. The increase in the thickness of the mixed monolayer measured by GID may only be related to the reorientation of the phospholipid hydrophobic chains toward an almost vertical position. However, the increase in surface pressure together with the decrease in the correlation length demonstrate that at least a partial penetration of the enzyme into the monolayer took place. Our work shows that the interaction of a protein with phospholipid monolayers is a complex phenomenon. Even though the studied phospholipids differed only in the length of their hydrophobic chains, the onset of a change in this single parameter on fluidity and packing of lipid monolayers markedly influenced the penetrating ability of glucose oxidase into these lipids. LA990490C (29) Lookman, T.; Pink, D. A.; Grundke, E. W.; Zuckermann, M. J.; deVerteuil, F. Biochemistry 1982, 21, 5593-5601. (30) Heckl, W. M.; Zaba, B. N.; Mo¨hwald, H. Biochim. Biophys. Acta 1987, 903, 166-176.