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Association of Blood Clotting Factors I and VII with Phospholipid Monolayers at the Air–Water Interface
Journal of Colloid and Interface Science 215, 356 –363 (1999) Article ID jcis.1999.6280, available online at http://www.idealibrary.com on
Association of Blood Clotting Factors I and VII with Phospholipid Monolayers at the Air–Water Interface Alexa Barnoski Serfis, Rebecca Katzenberger, Kristi Williams, and Ngoc Tran Department of Chemistry, Saint Louis University, 221 North Grand Boulevard, St. Louis, Missouri 63103 Received December 7, 1998; accepted April 14, 1999
mation of fibrin from factor I with eventual hard clot formation occurring. Factor I is a soluble plasma protein which does not require cell membranes for blood clot formation. Although the many steps of the blood clotting process have been identified, a molecular level understanding of the protein and lipid interactions has not been achieved. Many of the steps and components involved have been identified by performing blood clotting tests via in vitro assays. We have focused our research on studies of selected blood clotting proteins in an effort to better understand their associations with phospholipids in natural systems. Our model membrane system consists of a phospholipid monolayer on a buffered water subphase. Such lipid membranes are suitable model membranes for probing specific lipid–protein interactions, and other researchers have reported the use of similar methods for studying lipid interactions with drugs and viral agents (6, 7). A protein solution is injected into the subphase beneath the lipid monolayer and then given time to interact with the lipid. By monitoring changes in surface pressure and surface potential, the nature of the protein–lipid interactions can be deduced. We have studied two coagulating proteins with the following goals in mind:
Phospholipid monolayers adsorbed at the air–water interface are useful model membranes and have been employed to study the interactions between phospholipids and blood clotting factors I and VII. Factor I is a non-membrane-binding protein and was found to penetrate both distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylcholine (DPPC) monolayers at low lipid pressures. At high lipid pressures, the protein was crowded out of the interface. Factor I penetration of phospholipid monolayers was independent of hydrocarbon chain length, while penetration was maximized with electrolytes in the subphase. Factor VII is a membrane binding protein and was found to penetrate a DPPC monolayer only when electrolytes were added to the subphase. Factor VII penetrated DSPC monolayers regardless of electrolyte addition in the subphase, and its interactions with DSPC films are attributed to protein–lipid hydrophobic interactions. © 1999 Academic Press Key Words: blood clotting factors; monolayers; surface potential; phospholipids.
INTRODUCTION
Interactions between proteins and cell membranes occur in numerous biochemical processes. The incorporation of proteins into phospholipid Langmuir monolayers is an interesting model for biomembranes which can be used to study specific protein–lipid interactions. Such mixed protein–lipid systems have been the focus of some recent investigations (1– 4), and we have used this model to study the specific interactions between phospholipid membrane components and blood clotting proteins. The activation of some coagulating proteins occurs on the surface of the cell membrane, requiring participation of phospholipid components. The blood clotting cascade consists of a number of steps, but can be divided roughly into two pathways, the intrinsic and extrinsic, which are interrelated (5). In the extrinsic pathway, factor VII becomes activated by binding to a cell membrane in the presence of calcium ion. In the presence of activated factor VII and factor III, factor X becomes activated on a membrane surface, also in the presence of calcium ion. Activated factor X in turn interacts with activated factor V to promote thrombin activation from prothrombin. Thrombin then promotes the for0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
(i) Determine the extent of protein adsorption at the air– water interface, both with and without a lipid monolayer present. (ii) Determine the effect of the phospholipid chain length on the nature of protein adsorption. (iii) Determine the effect of calcium addition on the extent of protein adsorption, since calcium is known to be important for some protein activation processes. (iv) Compare the behavior of a membrane binding protein (factor VII) to that of a non-membrane-binding protein (factor I). MATERIALS AND METHODS
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Synthetic dipalmitoyl- and distearoylphosphatidylcholine (DPPC and DSPC, respectively) were obtained from SigmaAldrich and used as received. The purity given by the manufacturer was 991% and verified with thin layer chromatography. Phospholipid spreading solutions were made in HPLC
INTERACTIONS OF PROTEINS WITH LIPID MONOLAYERS
grade chloroform (Fisher Scientific) in concentrations of approximately 1 mg/mL. A monolayer was formed by dropping the spreading solution onto the buffered water surface with a syringe. Fifteen minutes was allowed for solvent evaporation before monolayer compression. The aqueous subphase consisted of pure water obtained from a Barnstead Nanopure system with a specific resistivity of 18 MV-cm, to which Tris buffer (Fisher) was added in a concentration of 0.10 M. The pH was then adjusted with hydrochloric acid to 7.4 (physiological pH). In some experiments calcium chloride (Fisher, 981%) was added to the buffer in a concentration of 10 mM. Blood coagulation factors I and VII were obtained from Sigma– Aldrich and used as received. Factor I consists of six polypeptide chains, has a molecular weight of 340,000, and was isolated from bovine blood. Factor VII is a single chain globular protein having a molecular weight of 50,000 and was obtained from human blood samples. Each protein was dissolved in a small amount of the buffer solution and then 500 mL of the solution was injected beneath the compressed lipid monolayer. The final concentration of factor I was 3 mg/mL in the subphase, and factor VII had a final concentration of 0.02 mg/mL. These concentrations are smaller than physiological concentrations, but significant changes in surface properties were observed. A NIMA Model 611D Langmuir–Blodgett trough equipped with a KP2 surface potential sensor was employed in these studies. Changes in surface pressure and surface potential were monitored for 75 min after protein injection beneath the compressed monolayer. At the end of this time period, the barrier was returned to its starting position, and a mixed lipid–protein monolayer isotherm was measured. We also attempted to form pure protein monolayers by dropping 500 mL of the protein solutions directly onto the aqueous
FIG. 1. Phospholipid monolayer isotherms (DPPC monolayer on (a) plain subphase and on (b) calcium buffer subphase; DSPC monolayer on (c) plain subphase and on (d) calcium buffer subphase).
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FIG. 2. Factor I penetration of DPPC monolayers (plain buffer subphase with initial lipid pressures of 3 (E), 4 (h), and 5.6 (‚) mN/m).
subphase and by injecting the solution in the absence of a monolayer. The former method gave the most reproducible results. All experiments were carried out at room temperature, 20°C. RESULTS AND DISCUSSION
I. Factor I Penetration of Lipid Monolayers Pressure-area isotherms of the phospholipid monolayers are shown in Fig. 1. The DPPC monolayer shows a transition region between the liquid expanded and condensed regions, and the isotherm agrees well with those found in the literature (8, 9). Repeated measurements of reported isotherms resulted in a standard deviation no larger than 0.5 mN/m. The monolayer is more condensed on the calcium chloride subphase, with the transition region observed at lower pressures. The addition of electrolytes to the subphase precompresses the monolayer; subphase electrolytes cause screening of lipid head group charges, resulting in closer head group approach. The DSPC monolayer is much more condensed than the DPPC monolayer, primarily due to the longer lipid chain length. This isotherm is also in good agreement with that found in the literature (4). With the addition of calcium chloride to the subphase, a condensing effect is again observed, with the isotherm being shifted to smaller areas. For the protein penetration studies, a variety of initial lipid pressures were chosen encompassing all regions of both the DPPC and DSPC isotherms. For penetration studies with DPPC monolayers, initial lipid pressures of 2.3, 3, 4, 5.6, 10, 15, and 30 mN/m were chosen. The highest pressure was chosen since the pressure of a cell membrane is estimated to be near 30 mN/m. These pressures cover all of the phases of the isotherm, from ex-
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FIG. 3. Mixed factor I–lipid monolayers ((a) DPPC monolayer; mixed films measured after penetration of DPPC monolayers initially at 5.6 mN/m (b), 4 mN/m (c), and 3 mN/m (d)).
panded to very condensed. The pressures chosen for DSPC monolayers were 5, 10, 18, and 30 mN/m. The results for the penetration of factor I into a DPPC monolayer on a plain buffer subphase are shown in Fig. 2. The highest changes in film pressure were observed for the lowest initial lipid pressures, with the 11 mN/m increase occurring for a lipid pressure of 3 mN/m. As the lipid was initially compressed to higher pressures, the amount of protein incorporated into the film decreased. No changes in film pressure were observed when the DPPC monolayer was compressed to 10 mN/m and higher. The tight packing of lipid molecules on the water surface prevents protein incorporation in the monolayer. Typical mixed lipid–protein monolayer isotherms are shown in Fig. 3. Each mixed monolayer is shifted to the right of the plain DPPC isotherm, indicating that significant amounts of protein have been incorporated. The amount of protein incorporated into the film was largest for the lowest initial lipid pressures, and the corresponding mixed monolayer isotherms show increasingly large shifts to larger areas as the amount of adsorbed protein increases. When the same experiments were performed with calcium TABLE 1 Maximum Film Pressure Changes for Factor I Penetration of DPPC Monolayers Initial lipid pressure (mN/m) DP Plain buffer (mN/m) DP Buffer with calcium (mN/m)
3.0 10.9 11.5
4.0 7.8 8.5
5.6 7.2 7.9
10.0 0.0 7.2
15.0 0.0 3.5
30.0 0.0 0.0
FIG. 4. (a) Surface potential of DPPC monolayers. (b) Surface potential of factor I–lipid monolayers (—, plain buffer subphase, - - - - - -, buffer with calcium subphase).
chloride in the subphase, larger changes in surface pressure were observed and are tabulated with the data from Fig. 2 in Table 1. Average surface pressure values are reported with a standard deviation of 0.5 mN/m. Most notably, significant increases at initial lipid pressures of 10 and 15 mN/m were observed. It is likely that the addition of electrolytes thins out the electrical double layer around the charged groups on both the protein and the lipid molecules, allowing for closer approach and more protein penetration in the interface. The addition of electrolytes may also cause a decrease in protein solubility in the bulk subphase, leading to a larger surface concentration. No significant increases in surface pressure were noted at 30 mN/m, indicating that the monolayer is too
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INTERACTIONS OF PROTEINS WITH LIPID MONOLAYERS
TABLE 2 Maximum Surface Potential Changes for Factor I Penetration of DPPC and DSPC Monolayers Initial DPPC lipid pressure (mN/m)
2.3
DPPC monolayer (plain buffer) DPPC monolayer (buffer with calcium) Factor I/DPPC films (plain buffer) Factor I/DPPC films (buffer with calcium) Initial DSPC lipid pressure (mN/m) DSPC monolayer (plain buffer) DSPC monolayer (buffer with calcium) Factor I/DSPC films (plain buffer) Factor I/DSPC films (buffer with calcium)
3.0
4.0
5.6
10.0
390 407 5.0
396 412 10.0
425 420 18.0
451 465
500 520
506 523
516 537
530 554
500 525
540 580
Note. All surface potentials are in DmV.
closely packed for protein penetration. The mixed monolayer isotherms for the systems containing calcium chloride (not shown) were shifted to larger areas than those isotherms on the plain buffer, again indicating more protein incorporation in the monolayer. The surface potentials of DPPC monolayers and mixed factor I–DPPC monolayers on the two subphases are shown in Figs. 4a and 4b, respectively. The surface potential of the DPPC monolayer on a plain buffer subphase is about 175 mV upon spreading of the lipid and rises to nearly 500 mV upon compression. The surface potential on the calcium chloride subphase is slightly higher and reaches a value of 525 mV upon compression. Additional electrolytes in the subphase cause condensation of the monolayer, likely resulting in more vertical alignment of lipid dipoles and higher surface potentials. An additional contribution to the surface potential may arise from modification of the surface charge density by both proteins and electrolytes. This contribution, however, cannot be determined from these measurements, so it must be noted that the overall changes in surface potential may be due to both effects. During protein adsorption, the surface potential decreased from the initial lipid values, and a steady decline in surface potential throughout protein adsorption was observed. All mixed monolayers also indicate surface potentials which are lower than those of a plain DPPC monolayer. For clarity, the results on only one mixed film formed from an initial lipid pressure of 5.6 mN/m is shown, while the complete set of maximum surface potentials for factor I penetration into DPPC and DSPC monolayers is found in Table 2. (The standard deviation for replicate surface potential measurements is 10 TABLE 3 Maximum Film Pressure Changes for Factor I Penetration of DSPC Monolayers Initial lipid pressure (mN/m) DP Plain buffer (mN/m) DP Buffer and calcium (mN/m)
5.0 9.2 11.6
10.0 5.9 8.2
18.0 3.2 3.9
30.0 0.0 0.0
mV.) The mixed film shows a maximum surface potential of only 425 mV upon compression, with similar values obtained on both subphases. This is about 75 mV less than the plain DPPC monolayer. The lowest surface potentials were exhibited by those systems with the most protein incorporated, i.e., the lowest initial lipid pressures. This decrease may indicate interactions between protein and lipid dipole moments. A lipid monolayer exhibiting positive surface potentials has a positive perpendicular dipole moment component, with the positive end of the dipole oriented upward (toward the vapor phase). The negative portion is near the head group. As protein molecules adsorb and interact with lipid head groups, the separation of the lipid’s dipolar ends decreases, leading to a decrease in surface dipole moment. There may also be a significant contribution to the surface potential decrease due to modification of the surface charge density by the protein and subphase ions. It is possible to estimate the total contribution of the protein (electrolyte contributions inclusive) to the surface potential with the data collected. Using the methods outlined in the literature (9), the surface potential of a mixed lipid–protein monolayer is given by V lipid–protein 5 V lipid~ A lipid! 1 V protein~ A protein!,
[1]
where V lipid and V protein are the surface potentials of the plain lipid film and the protein, respectively. The estimated area fraction of the lipid is given by A lipid, and that of the protein is A protein. These area fractions are found by noting the area increase of the mixed monolayer isotherm over that of a plain lipid isotherm. The estimated factor I contribution to the surface potential on the plain buffered subphase (initial lipid pressure 5.6 mN/m) is about 243 mV. When the same calculations are performed to estimate the protein contribution to the surface potential on the calcium chloride subphase, it is estimated that the protein contribution is 330 mV. The increase in protein contribution to the surface potential stems in part from the
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increased interfacial protein incorporation with calcium in the subphase and may be due in part to subphase ions. For the factor I penetration studies involving DSPC monolayers, similar trends were observed. The change in surface pressure was largest for the lowest initial lipid pressures (Table 3). The overall changes in surface pressure were slightly larger than those observed for the shorter chain DPPC lipid at similar pressures. The addition of electrolyte to the subphase leads to higher changes in surface pressure, but no significant increases occurred at an initial lipid pressure of 30 mN/m. The surface potentials also exhibited a similar trend (Table 2), with values lower than for the plain DSPC monolayer and continually decreasing for the lowest initial lipid pressures. The maximum changes in surface potential for DSPC monolayers were 540 mV on a plain buffer subphase and 580 mV with calcium in the
FIG. 6. Factor VII penetration of DPPC monolayers (buffer with calcium subphase; initial lipid pressures of 2.3 (h), 3 (E), 4 (Œ), 5.6 ({), and 10 (‚) mN/m).
FIG. 5. (a) Factor I isotherms. (b) Surface potential of factor I monolayers (—, plain buffer subphase; - - - - - -, buffer with calcium subphase).
buffer subphase. This trend is interpreted the same way as discussed above. As more protein is incorporated into the film, the surface potential continues to decrease, and the mixed monolayer isotherms show shifts to larger areas. The factor I contribution to the surface potential in a DSPC mixed monolayer (DSPC initial pressure 10 mN/m) is slightly smaller, with values of 143 mV. The values may be smaller than those calculated for the DPPC monolayer because the protein may reduce the dipole moment of the DSPC lipid film more than it does for the DPPC monolayer. Since the charge separation and dipole moment are greater for the longer chained DSPC lipid, interaction with protein can cause a larger change in the surface potential. When calcium chloride is added to the subphase, the same trends are observed as with the DPPC monolayers. The changes in surface pressure are higher, but again decrease as the initial lipid pressure becomes larger. The surface potentials show similar trends, with each lying below the plain DSPC monolayer. The calculated protein contribution to the surface potential in this case is 239 mV. The surface potential contribution is larger with calcium in the subphase, and this is the same trend observed with a DPPC monolayer. It was possible to form a pure factor I monolayer by dropping the protein solution directly onto the subphase. Isotherms were obtained on both the plain buffer and electrolyte subphases, shown in Fig. 5a. The protein occupies larger areas on the electrolyte subphase, due to the decreased protein solubility in the bulk. Dziri et al. observed a similar salting out effect for an acetylcholinesterase film on KCl subphases (1). Systematic studies of bull serum albumin monolayers on water and electrolyte subphases revealed increases in protein molecular areas on the surface of salt solutions (12). We were not able to find a collapse pressure for this monolayer, because it was pushed
INTERACTIONS OF PROTEINS WITH LIPID MONOLAYERS
361
lates in the plasma and does not require cell membranes for hard clot formation. It is reasonable that there should not be cooperative interactions between factor I and the phospholipid monolayers. II. Factor VII Penetration of Lipid Monolayers
FIG. 7. Mixed factor VII–lipid monolayers ((a) DPPC monolayer; mixed films measured after penetration of DPPC monolayers initially at 10 mN/m (b), 4 mN/m (c), and 2.3 mN/m (d)).
off the trough surface before collapse. Factor I does form a relatively stable liquid phase monolayer, and we found that the monolayer could be repeatedly compressed and expanded, as long as it was not allowed to overflow the trough. The surface potentials steadily increase through compression (Fig. 5b), with higher values exhibited by the film on an electrolyte subphase. The maximum surface potentials observed were 170 mV on the plain buffer and 220 mV on the buffer subphase which contained calcium. The protein contribution to the surface potential in the mixed lipid–protein monolayer was only slightly larger than that for the protein monolayer alone. It appears that factor I exhibits nonspecific interactions with both lipid monolayers and adsorbs only at the interface if there is room. In the absence of a lipid monolayer, factor I adsorbs at the air–water interface, forming a stable monolayer. This protein monolayer forms on a plain buffer subphase and with additional subphase electrolytes. The lipid monolayer provides a physical barrier to protein adsorption from the bulk phase. At the lowest initial lipid pressures, the molecules are far apart, and there is sufficient room in the interface to accommodate the protein. However, as the lipid molecules become closer together and the film becomes more condensed, there is less room for the protein, and it is crowded out of the interface. There is little dependence on the phospholipid chain length; if there is not space in the interface, it simply cannot adsorb. The DSPC monolayer is a condensed film and likely has similar packing as the DPPC monolayer in the condensed phase region. The effect of electrolyte addition on protein penetration is mainly electrostatic. Screening of electrical charges allows for closer approach of protein and lipid molecules, allowing for more protein incorporation in the interface. This behavior is reasonable, considering that factor I is a protein which circu-
Penetration of factor VII into DPPC monolayers exhibits behavior somewhat different from factor I (Fig. 6). When factor VII was injected below a DPPC monolayer on a plain buffer subphase, no significant changes in film pressure were observed for any initial lipid pressures. However, when calcium chloride was added to the subphase, changes in film pressure occurred. The overall changes in surface pressure are smaller than those for factor I, likely due to the smaller size and amounts of protein in the subphase. For lipid pressures up to 10 mN/m, the largest changes in film pressure were also observed for the highest initial lipid pressures. This trend is opposite to that observed for factor I. However, significant changes in film pressure were not observed for lipid pressures near 30 mN/m. At this pressure, the lipid molecules are too closely packed, preventing protein penetration of the monolayer. The shapes of the adsorption isotherms are also different than those observed for factor I. There is biphasic adsorption, indicated by a change in slope in the adsorption isotherm. There are also lag times observed in some studies, where the pressure does not change until a few minutes have passed. It is likely that the protein penetration and subsequent increase in film pressure causes the lipid monolayer to undergo a transition from the liquid to more condensed phase. Since these initial lipid pressures are in the region where the phase transition occurs, the adsorbed protein can cause the lipid transition to occur in the mixed monolayer.
FIG. 8. Surface potential of factor VII–lipid films (- - - - - -, mixed film; —, DPPC monolayer; both were measured on a buffer with calcium subphase after penetration of a DPPC monolayer at 10 mN/m).
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TABLE 4 Maximum Surface Potential Changes for Factor VII Penetration of DPPC and DSPC Monolayers Initial DPPC lipid pressure (mN/m) DPPC monolayer (buffer with calcium) Factor VII/DPPC films (buffer with calcium) Initial DSPC lipid pressure (mN/m) DSPC monolayer (plain buffer) DSPC monolayer (buffer with calcium) Factor VII/DSPC films (plain buffer) Factor VII/DSPC films (buffer with calcium)
2.3
3.0
4.0
5.6
10.0
607 5.0
505 10.0
485 18.0
431
315
508 518
515 542
533 564
525
540 580
Note. All surface potentials are in Dmv.
Isotherms of the mixed lipid–protein monolayer are shown in Fig. 7. These were measured after penetration into a DPPC monolayer initially at 2.3, 4, and 10 mN/m. The mixed monolayers are shifted to higher areas than the DPPC monolayer, indicating significant protein incorporation. It is interesting to note, however, that the largest shifts are observed for the lowest initial lipid pressures. These isotherms indicate that the most protein incorporation in the film occurs when the initial lipid pressure is lowest. This further supports the idea that the protein causes the phase transition in the lipid monolayer upon adsorption. Some of the lipid–protein mixed films exhibit surface potentials much lower than that of a plain DPPC monolayer (Fig. 8 and Table 4). However, the largest surface potential was found for the lowest initial lipid pressure. The mixed monolayer pressure-area isotherms indicate the highest factor VII incorporation at this pressure, so we may expect this mixed monolayer film to have the smallest surface potential. If there is a cooperative interaction or protein–lipid binding which occurs, it is possible that there are specific interactions which would lead to changes in surface potential which are greater than for the DPPC film alone. If the protein dipoles align with the lipid dipoles in the interface, an overall surface potential greater than the plain lipid would be observed. For the other films exhibiting surface potentials lower than that of a plain DPPC monolayer, the protein may bind to the lipid during the protein adsorption. Subsequently, the lipid undergoes a transition to the more condensed phase, with the result that the protein orientation changes in a way to cancel lipid dipoles and reduce the surface potential. The protein contribution to the surface potential in the mixed films were small, and in one case only about 15 mV. This is significantly smaller than the factor I contribution, and the electrolyte contributions still remain unclear. Penetration of factor VII into a DSPC monolayer occurs with and without calcium in the subphase, but the trend differs from that observed with the DPPC monolayer (Table 5). The highest observed film pressure changes are at the lowest initial film pressures. This is the same behavior observed with factor I. The same crowding out effect is observed, with less protein
penetration occurring at higher initial lipid pressures. The surface potential measurements for the mixed protein–lipid monolayers exhibit the same trend as factor I. All mixed monolayers show surface potentials which are lower than for the pure lipid monolayer. As factor VII is incorporated into the interface, the protein may align in such a way that its dipoles are aligned opposite to the lipids’, thus canceling surface dipole contributions. The more protein that is incorporated, the lower is the surface potential of the mixed monolayer films. The hydrocarbon chain length clearly affects the adsorption of factor VII. The protein penetrated the DSPC monolayer from both subphases, but only penetrated the DPPC monolayer when calcium was present. Hydrophobic interactions between lipid hydrocarbon chains and the protein are more important for DSPC monolayers. The hydrophobic regions on the protein are attracted to the hydrophobic DSPC film, even in the absence of subphase calcium ions. These hydrophobic interactions promote factor VII incorporation in the interface. Bos et al. observed a similar hydrophobic effect for the penetration of b-lactoglobulin into DSPA and DPPA (PA, phosphatidic acid) monolayers, with greater adsorption rates observed for the longer chained compound (4). For both films, subphase calcium addition facilitates the incorporation of factor VII. This indicates that electrostatic repulsion between DSPC and DPPC films with the protein occurs, but the repulsions are hampered when electrolytes are added to the subphase. It is interesting to note that this reduction in electrostatic interactions occurred for both factor I and factor VII. It has been proposed (13, 5) that the calcium ion serves as a bridge between the protein and the phospholipid components in the coagulation process. Our studies with plain DPPC monolayers indicate that calcium serves as a facilitator TABLE 5 Maximum Film Pressure Changes for Factor VII Penetration of DSPC Monolayers Initial lipid pressure (mN/m) DP Plain buffer (mN/m) DP Buffer and calcium (mN/m)
5.0 3.6 4.4
10.0 2.0 3.5
18.0 0.4 0.5
30.0 0.0 0.0
INTERACTIONS OF PROTEINS WITH LIPID MONOLAYERS
for the lipid–protein interactions since both the membranebinding and the non-membrane-binding proteins behaved similarly with added electrolytes. It is possible, however, that the incorporation of negatively charged phospholipids such as phosphatidylserines may show more specific interaction with the calcium ions. Studies of factor VII with mixed phosphatidylcholine and -serine components have already been initiated. It has recently been shown (14) that hydrophobic domains of factor X contact hydrophobic phospholipid membrane components. Insertion of protein domains in the bilayer results. It was proposed that the presence of calcium ions contributed to membrane attachment through electrostatic interactions and did not serve as a bridge between the protein domains and the membrane phospholipids. The hydrophobic residue on factor X is identical to factor VII, and our studies indicate similar hydrophobic interactions between proteins and phospholipids. Hydrophobic interactions between the protein and lipid account for the increased factor VII penetration of DSPC monolayers compared to that seen with DPPC films. It is interesting to note that penetration of lipid monolayers near 30 mN/m did not occur for factor VII under any conditions. Even though the cell membrane pressure has been estimated, a natural membrane is in a dynamic state of fluid motion in which the actual pressure is likely variable. These experiments indicate that very tight lipid packing precludes protein penetration, and lower pressures are necessary for protein incorporation. Further studies of factor VII penetration of DPPC monolayers are currently being investigated with fluorescence imaging techniques. It is hoped that more detailed studies will further elucidate the nature of these lipid–protein interactions. SUMMARY
Factor I is a non-membrane-binding protein and interacts similarly with DSPC and DPPC monolayer films on water. There are nonspecific interactions between the protein and the lipid molecules, resulting in crowding out of the protein at high
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lipid pressures. Factor VII also interacts with lipid monolayers, but the lipid–protein interactions are more specific. The hydrocarbon chain length affects the adsorption, with factor VII penetrating a DSPC film to greater extents. These studies indicate that hydrophobic interactions are important for factor VII penetration of both types of films. The effect of calcium addition was the same in all studies. Reduction of electrostatic interactions allowed further incorporation of both types of proteins into both DPPC and DSPC films. ACKNOWLEDGMENTS This research was supported by an award from Research Corporation. This research was also sponsored in part by Saint Louis University (Summer Research Award).
REFERENCES 1. Dziri, L., Puppala, K., and Leblanc, R. M., J. Colloid Interface Sci. 194, 37 (1997). 2. Cho, D., Narsimhan, G., and Franses, E. I., Langmuir 13, 4710 (1997). 3. Rosilio, V., Boissonnade, M., Zhang, J., Jiang, L., and Baszkin, A., Langmuir 13, 4669 (1997). 4. Bos, M. A., and Nylander, T., Langmuir 12, 2791 (1996). 5. Voet, D., and Voet, J. G., “Biochemistry.” Wiley, New York, 1990. 6. Bennouna, M., Ferreira-Marques, J., Banerjee, S., Caspers, J., and Ruysschaert, J. M., Langmuir 13, 6533 (1997). 7. Haro, I., Perez, J. A., Reig, F., Mestres, C., Egea, M. A., and Alsina, M. A., Langmuir 12, 5120 (1996). 8. Charron, J. R., and Tilton, R. D., Langmuir 13, 5524 (1997). 9. Heckl, W. M., Thompson, M., and Mohwald, H., Langmuir 5, 390 (1989). 10. Weisel, J. W., Stauffacher, C. V., Bullitt, E., and Cohen, C., Science 230, 1388 (1985). 11. McClellan, A. L., “Tables of Experimental Dipole Moments.” Freeman, San Francisco, 1963. 12. Chudinova, G. K., Pokrovskaya, O. N., and Savitskii, A. P., Russ. Chem. Bull. 44, 1958 (1995). 13. Schwalbe, R. A., Ryans, J., Stern, D. M., Kisiel, W., Dahlback, B., and Nelsestuen, G. L., J. Biol. Chem. 264, 20288 (1989). 14. Sunnerhagen, M., Forsen, A. H., Drakenberg, T., Teleman, O., and Stenflo, J., Nature Struct. Biol. 2, (1995).
Association of Blood Clotting Factors I and VII with Phospholipid Monolayers at the Air–Water Interface Alexa Barnoski Serfis, Rebecca Katzenberger, Kristi Williams, and Ngoc Tran Department of Chemistry, Saint Louis University, 221 North Grand Boulevard, St. Louis, Missouri 63103 Received December 7, 1998; accepted April 14, 1999
mation of fibrin from factor I with eventual hard clot formation occurring. Factor I is a soluble plasma protein which does not require cell membranes for blood clot formation. Although the many steps of the blood clotting process have been identified, a molecular level understanding of the protein and lipid interactions has not been achieved. Many of the steps and components involved have been identified by performing blood clotting tests via in vitro assays. We have focused our research on studies of selected blood clotting proteins in an effort to better understand their associations with phospholipids in natural systems. Our model membrane system consists of a phospholipid monolayer on a buffered water subphase. Such lipid membranes are suitable model membranes for probing specific lipid–protein interactions, and other researchers have reported the use of similar methods for studying lipid interactions with drugs and viral agents (6, 7). A protein solution is injected into the subphase beneath the lipid monolayer and then given time to interact with the lipid. By monitoring changes in surface pressure and surface potential, the nature of the protein–lipid interactions can be deduced. We have studied two coagulating proteins with the following goals in mind:
Phospholipid monolayers adsorbed at the air–water interface are useful model membranes and have been employed to study the interactions between phospholipids and blood clotting factors I and VII. Factor I is a non-membrane-binding protein and was found to penetrate both distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylcholine (DPPC) monolayers at low lipid pressures. At high lipid pressures, the protein was crowded out of the interface. Factor I penetration of phospholipid monolayers was independent of hydrocarbon chain length, while penetration was maximized with electrolytes in the subphase. Factor VII is a membrane binding protein and was found to penetrate a DPPC monolayer only when electrolytes were added to the subphase. Factor VII penetrated DSPC monolayers regardless of electrolyte addition in the subphase, and its interactions with DSPC films are attributed to protein–lipid hydrophobic interactions. © 1999 Academic Press Key Words: blood clotting factors; monolayers; surface potential; phospholipids.
INTRODUCTION
Interactions between proteins and cell membranes occur in numerous biochemical processes. The incorporation of proteins into phospholipid Langmuir monolayers is an interesting model for biomembranes which can be used to study specific protein–lipid interactions. Such mixed protein–lipid systems have been the focus of some recent investigations (1– 4), and we have used this model to study the specific interactions between phospholipid membrane components and blood clotting proteins. The activation of some coagulating proteins occurs on the surface of the cell membrane, requiring participation of phospholipid components. The blood clotting cascade consists of a number of steps, but can be divided roughly into two pathways, the intrinsic and extrinsic, which are interrelated (5). In the extrinsic pathway, factor VII becomes activated by binding to a cell membrane in the presence of calcium ion. In the presence of activated factor VII and factor III, factor X becomes activated on a membrane surface, also in the presence of calcium ion. Activated factor X in turn interacts with activated factor V to promote thrombin activation from prothrombin. Thrombin then promotes the for0021-9797/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
(i) Determine the extent of protein adsorption at the air– water interface, both with and without a lipid monolayer present. (ii) Determine the effect of the phospholipid chain length on the nature of protein adsorption. (iii) Determine the effect of calcium addition on the extent of protein adsorption, since calcium is known to be important for some protein activation processes. (iv) Compare the behavior of a membrane binding protein (factor VII) to that of a non-membrane-binding protein (factor I). MATERIALS AND METHODS
356
Synthetic dipalmitoyl- and distearoylphosphatidylcholine (DPPC and DSPC, respectively) were obtained from SigmaAldrich and used as received. The purity given by the manufacturer was 991% and verified with thin layer chromatography. Phospholipid spreading solutions were made in HPLC
INTERACTIONS OF PROTEINS WITH LIPID MONOLAYERS
grade chloroform (Fisher Scientific) in concentrations of approximately 1 mg/mL. A monolayer was formed by dropping the spreading solution onto the buffered water surface with a syringe. Fifteen minutes was allowed for solvent evaporation before monolayer compression. The aqueous subphase consisted of pure water obtained from a Barnstead Nanopure system with a specific resistivity of 18 MV-cm, to which Tris buffer (Fisher) was added in a concentration of 0.10 M. The pH was then adjusted with hydrochloric acid to 7.4 (physiological pH). In some experiments calcium chloride (Fisher, 981%) was added to the buffer in a concentration of 10 mM. Blood coagulation factors I and VII were obtained from Sigma– Aldrich and used as received. Factor I consists of six polypeptide chains, has a molecular weight of 340,000, and was isolated from bovine blood. Factor VII is a single chain globular protein having a molecular weight of 50,000 and was obtained from human blood samples. Each protein was dissolved in a small amount of the buffer solution and then 500 mL of the solution was injected beneath the compressed lipid monolayer. The final concentration of factor I was 3 mg/mL in the subphase, and factor VII had a final concentration of 0.02 mg/mL. These concentrations are smaller than physiological concentrations, but significant changes in surface properties were observed. A NIMA Model 611D Langmuir–Blodgett trough equipped with a KP2 surface potential sensor was employed in these studies. Changes in surface pressure and surface potential were monitored for 75 min after protein injection beneath the compressed monolayer. At the end of this time period, the barrier was returned to its starting position, and a mixed lipid–protein monolayer isotherm was measured. We also attempted to form pure protein monolayers by dropping 500 mL of the protein solutions directly onto the aqueous
FIG. 1. Phospholipid monolayer isotherms (DPPC monolayer on (a) plain subphase and on (b) calcium buffer subphase; DSPC monolayer on (c) plain subphase and on (d) calcium buffer subphase).
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FIG. 2. Factor I penetration of DPPC monolayers (plain buffer subphase with initial lipid pressures of 3 (E), 4 (h), and 5.6 (‚) mN/m).
subphase and by injecting the solution in the absence of a monolayer. The former method gave the most reproducible results. All experiments were carried out at room temperature, 20°C. RESULTS AND DISCUSSION
I. Factor I Penetration of Lipid Monolayers Pressure-area isotherms of the phospholipid monolayers are shown in Fig. 1. The DPPC monolayer shows a transition region between the liquid expanded and condensed regions, and the isotherm agrees well with those found in the literature (8, 9). Repeated measurements of reported isotherms resulted in a standard deviation no larger than 0.5 mN/m. The monolayer is more condensed on the calcium chloride subphase, with the transition region observed at lower pressures. The addition of electrolytes to the subphase precompresses the monolayer; subphase electrolytes cause screening of lipid head group charges, resulting in closer head group approach. The DSPC monolayer is much more condensed than the DPPC monolayer, primarily due to the longer lipid chain length. This isotherm is also in good agreement with that found in the literature (4). With the addition of calcium chloride to the subphase, a condensing effect is again observed, with the isotherm being shifted to smaller areas. For the protein penetration studies, a variety of initial lipid pressures were chosen encompassing all regions of both the DPPC and DSPC isotherms. For penetration studies with DPPC monolayers, initial lipid pressures of 2.3, 3, 4, 5.6, 10, 15, and 30 mN/m were chosen. The highest pressure was chosen since the pressure of a cell membrane is estimated to be near 30 mN/m. These pressures cover all of the phases of the isotherm, from ex-
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FIG. 3. Mixed factor I–lipid monolayers ((a) DPPC monolayer; mixed films measured after penetration of DPPC monolayers initially at 5.6 mN/m (b), 4 mN/m (c), and 3 mN/m (d)).
panded to very condensed. The pressures chosen for DSPC monolayers were 5, 10, 18, and 30 mN/m. The results for the penetration of factor I into a DPPC monolayer on a plain buffer subphase are shown in Fig. 2. The highest changes in film pressure were observed for the lowest initial lipid pressures, with the 11 mN/m increase occurring for a lipid pressure of 3 mN/m. As the lipid was initially compressed to higher pressures, the amount of protein incorporated into the film decreased. No changes in film pressure were observed when the DPPC monolayer was compressed to 10 mN/m and higher. The tight packing of lipid molecules on the water surface prevents protein incorporation in the monolayer. Typical mixed lipid–protein monolayer isotherms are shown in Fig. 3. Each mixed monolayer is shifted to the right of the plain DPPC isotherm, indicating that significant amounts of protein have been incorporated. The amount of protein incorporated into the film was largest for the lowest initial lipid pressures, and the corresponding mixed monolayer isotherms show increasingly large shifts to larger areas as the amount of adsorbed protein increases. When the same experiments were performed with calcium TABLE 1 Maximum Film Pressure Changes for Factor I Penetration of DPPC Monolayers Initial lipid pressure (mN/m) DP Plain buffer (mN/m) DP Buffer with calcium (mN/m)
3.0 10.9 11.5
4.0 7.8 8.5
5.6 7.2 7.9
10.0 0.0 7.2
15.0 0.0 3.5
30.0 0.0 0.0
FIG. 4. (a) Surface potential of DPPC monolayers. (b) Surface potential of factor I–lipid monolayers (—, plain buffer subphase, - - - - - -, buffer with calcium subphase).
chloride in the subphase, larger changes in surface pressure were observed and are tabulated with the data from Fig. 2 in Table 1. Average surface pressure values are reported with a standard deviation of 0.5 mN/m. Most notably, significant increases at initial lipid pressures of 10 and 15 mN/m were observed. It is likely that the addition of electrolytes thins out the electrical double layer around the charged groups on both the protein and the lipid molecules, allowing for closer approach and more protein penetration in the interface. The addition of electrolytes may also cause a decrease in protein solubility in the bulk subphase, leading to a larger surface concentration. No significant increases in surface pressure were noted at 30 mN/m, indicating that the monolayer is too
359
INTERACTIONS OF PROTEINS WITH LIPID MONOLAYERS
TABLE 2 Maximum Surface Potential Changes for Factor I Penetration of DPPC and DSPC Monolayers Initial DPPC lipid pressure (mN/m)
2.3
DPPC monolayer (plain buffer) DPPC monolayer (buffer with calcium) Factor I/DPPC films (plain buffer) Factor I/DPPC films (buffer with calcium) Initial DSPC lipid pressure (mN/m) DSPC monolayer (plain buffer) DSPC monolayer (buffer with calcium) Factor I/DSPC films (plain buffer) Factor I/DSPC films (buffer with calcium)
3.0
4.0
5.6
10.0
390 407 5.0
396 412 10.0
425 420 18.0
451 465
500 520
506 523
516 537
530 554
500 525
540 580
Note. All surface potentials are in DmV.
closely packed for protein penetration. The mixed monolayer isotherms for the systems containing calcium chloride (not shown) were shifted to larger areas than those isotherms on the plain buffer, again indicating more protein incorporation in the monolayer. The surface potentials of DPPC monolayers and mixed factor I–DPPC monolayers on the two subphases are shown in Figs. 4a and 4b, respectively. The surface potential of the DPPC monolayer on a plain buffer subphase is about 175 mV upon spreading of the lipid and rises to nearly 500 mV upon compression. The surface potential on the calcium chloride subphase is slightly higher and reaches a value of 525 mV upon compression. Additional electrolytes in the subphase cause condensation of the monolayer, likely resulting in more vertical alignment of lipid dipoles and higher surface potentials. An additional contribution to the surface potential may arise from modification of the surface charge density by both proteins and electrolytes. This contribution, however, cannot be determined from these measurements, so it must be noted that the overall changes in surface potential may be due to both effects. During protein adsorption, the surface potential decreased from the initial lipid values, and a steady decline in surface potential throughout protein adsorption was observed. All mixed monolayers also indicate surface potentials which are lower than those of a plain DPPC monolayer. For clarity, the results on only one mixed film formed from an initial lipid pressure of 5.6 mN/m is shown, while the complete set of maximum surface potentials for factor I penetration into DPPC and DSPC monolayers is found in Table 2. (The standard deviation for replicate surface potential measurements is 10 TABLE 3 Maximum Film Pressure Changes for Factor I Penetration of DSPC Monolayers Initial lipid pressure (mN/m) DP Plain buffer (mN/m) DP Buffer and calcium (mN/m)
5.0 9.2 11.6
10.0 5.9 8.2
18.0 3.2 3.9
30.0 0.0 0.0
mV.) The mixed film shows a maximum surface potential of only 425 mV upon compression, with similar values obtained on both subphases. This is about 75 mV less than the plain DPPC monolayer. The lowest surface potentials were exhibited by those systems with the most protein incorporated, i.e., the lowest initial lipid pressures. This decrease may indicate interactions between protein and lipid dipole moments. A lipid monolayer exhibiting positive surface potentials has a positive perpendicular dipole moment component, with the positive end of the dipole oriented upward (toward the vapor phase). The negative portion is near the head group. As protein molecules adsorb and interact with lipid head groups, the separation of the lipid’s dipolar ends decreases, leading to a decrease in surface dipole moment. There may also be a significant contribution to the surface potential decrease due to modification of the surface charge density by the protein and subphase ions. It is possible to estimate the total contribution of the protein (electrolyte contributions inclusive) to the surface potential with the data collected. Using the methods outlined in the literature (9), the surface potential of a mixed lipid–protein monolayer is given by V lipid–protein 5 V lipid~ A lipid! 1 V protein~ A protein!,
[1]
where V lipid and V protein are the surface potentials of the plain lipid film and the protein, respectively. The estimated area fraction of the lipid is given by A lipid, and that of the protein is A protein. These area fractions are found by noting the area increase of the mixed monolayer isotherm over that of a plain lipid isotherm. The estimated factor I contribution to the surface potential on the plain buffered subphase (initial lipid pressure 5.6 mN/m) is about 243 mV. When the same calculations are performed to estimate the protein contribution to the surface potential on the calcium chloride subphase, it is estimated that the protein contribution is 330 mV. The increase in protein contribution to the surface potential stems in part from the
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increased interfacial protein incorporation with calcium in the subphase and may be due in part to subphase ions. For the factor I penetration studies involving DSPC monolayers, similar trends were observed. The change in surface pressure was largest for the lowest initial lipid pressures (Table 3). The overall changes in surface pressure were slightly larger than those observed for the shorter chain DPPC lipid at similar pressures. The addition of electrolyte to the subphase leads to higher changes in surface pressure, but no significant increases occurred at an initial lipid pressure of 30 mN/m. The surface potentials also exhibited a similar trend (Table 2), with values lower than for the plain DSPC monolayer and continually decreasing for the lowest initial lipid pressures. The maximum changes in surface potential for DSPC monolayers were 540 mV on a plain buffer subphase and 580 mV with calcium in the
FIG. 6. Factor VII penetration of DPPC monolayers (buffer with calcium subphase; initial lipid pressures of 2.3 (h), 3 (E), 4 (Œ), 5.6 ({), and 10 (‚) mN/m).
FIG. 5. (a) Factor I isotherms. (b) Surface potential of factor I monolayers (—, plain buffer subphase; - - - - - -, buffer with calcium subphase).
buffer subphase. This trend is interpreted the same way as discussed above. As more protein is incorporated into the film, the surface potential continues to decrease, and the mixed monolayer isotherms show shifts to larger areas. The factor I contribution to the surface potential in a DSPC mixed monolayer (DSPC initial pressure 10 mN/m) is slightly smaller, with values of 143 mV. The values may be smaller than those calculated for the DPPC monolayer because the protein may reduce the dipole moment of the DSPC lipid film more than it does for the DPPC monolayer. Since the charge separation and dipole moment are greater for the longer chained DSPC lipid, interaction with protein can cause a larger change in the surface potential. When calcium chloride is added to the subphase, the same trends are observed as with the DPPC monolayers. The changes in surface pressure are higher, but again decrease as the initial lipid pressure becomes larger. The surface potentials show similar trends, with each lying below the plain DSPC monolayer. The calculated protein contribution to the surface potential in this case is 239 mV. The surface potential contribution is larger with calcium in the subphase, and this is the same trend observed with a DPPC monolayer. It was possible to form a pure factor I monolayer by dropping the protein solution directly onto the subphase. Isotherms were obtained on both the plain buffer and electrolyte subphases, shown in Fig. 5a. The protein occupies larger areas on the electrolyte subphase, due to the decreased protein solubility in the bulk. Dziri et al. observed a similar salting out effect for an acetylcholinesterase film on KCl subphases (1). Systematic studies of bull serum albumin monolayers on water and electrolyte subphases revealed increases in protein molecular areas on the surface of salt solutions (12). We were not able to find a collapse pressure for this monolayer, because it was pushed
INTERACTIONS OF PROTEINS WITH LIPID MONOLAYERS
361
lates in the plasma and does not require cell membranes for hard clot formation. It is reasonable that there should not be cooperative interactions between factor I and the phospholipid monolayers. II. Factor VII Penetration of Lipid Monolayers
FIG. 7. Mixed factor VII–lipid monolayers ((a) DPPC monolayer; mixed films measured after penetration of DPPC monolayers initially at 10 mN/m (b), 4 mN/m (c), and 2.3 mN/m (d)).
off the trough surface before collapse. Factor I does form a relatively stable liquid phase monolayer, and we found that the monolayer could be repeatedly compressed and expanded, as long as it was not allowed to overflow the trough. The surface potentials steadily increase through compression (Fig. 5b), with higher values exhibited by the film on an electrolyte subphase. The maximum surface potentials observed were 170 mV on the plain buffer and 220 mV on the buffer subphase which contained calcium. The protein contribution to the surface potential in the mixed lipid–protein monolayer was only slightly larger than that for the protein monolayer alone. It appears that factor I exhibits nonspecific interactions with both lipid monolayers and adsorbs only at the interface if there is room. In the absence of a lipid monolayer, factor I adsorbs at the air–water interface, forming a stable monolayer. This protein monolayer forms on a plain buffer subphase and with additional subphase electrolytes. The lipid monolayer provides a physical barrier to protein adsorption from the bulk phase. At the lowest initial lipid pressures, the molecules are far apart, and there is sufficient room in the interface to accommodate the protein. However, as the lipid molecules become closer together and the film becomes more condensed, there is less room for the protein, and it is crowded out of the interface. There is little dependence on the phospholipid chain length; if there is not space in the interface, it simply cannot adsorb. The DSPC monolayer is a condensed film and likely has similar packing as the DPPC monolayer in the condensed phase region. The effect of electrolyte addition on protein penetration is mainly electrostatic. Screening of electrical charges allows for closer approach of protein and lipid molecules, allowing for more protein incorporation in the interface. This behavior is reasonable, considering that factor I is a protein which circu-
Penetration of factor VII into DPPC monolayers exhibits behavior somewhat different from factor I (Fig. 6). When factor VII was injected below a DPPC monolayer on a plain buffer subphase, no significant changes in film pressure were observed for any initial lipid pressures. However, when calcium chloride was added to the subphase, changes in film pressure occurred. The overall changes in surface pressure are smaller than those for factor I, likely due to the smaller size and amounts of protein in the subphase. For lipid pressures up to 10 mN/m, the largest changes in film pressure were also observed for the highest initial lipid pressures. This trend is opposite to that observed for factor I. However, significant changes in film pressure were not observed for lipid pressures near 30 mN/m. At this pressure, the lipid molecules are too closely packed, preventing protein penetration of the monolayer. The shapes of the adsorption isotherms are also different than those observed for factor I. There is biphasic adsorption, indicated by a change in slope in the adsorption isotherm. There are also lag times observed in some studies, where the pressure does not change until a few minutes have passed. It is likely that the protein penetration and subsequent increase in film pressure causes the lipid monolayer to undergo a transition from the liquid to more condensed phase. Since these initial lipid pressures are in the region where the phase transition occurs, the adsorbed protein can cause the lipid transition to occur in the mixed monolayer.
FIG. 8. Surface potential of factor VII–lipid films (- - - - - -, mixed film; —, DPPC monolayer; both were measured on a buffer with calcium subphase after penetration of a DPPC monolayer at 10 mN/m).
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SERFIS ET AL.
TABLE 4 Maximum Surface Potential Changes for Factor VII Penetration of DPPC and DSPC Monolayers Initial DPPC lipid pressure (mN/m) DPPC monolayer (buffer with calcium) Factor VII/DPPC films (buffer with calcium) Initial DSPC lipid pressure (mN/m) DSPC monolayer (plain buffer) DSPC monolayer (buffer with calcium) Factor VII/DSPC films (plain buffer) Factor VII/DSPC films (buffer with calcium)
2.3
3.0
4.0
5.6
10.0
607 5.0
505 10.0
485 18.0
431
315
508 518
515 542
533 564
525
540 580
Note. All surface potentials are in Dmv.
Isotherms of the mixed lipid–protein monolayer are shown in Fig. 7. These were measured after penetration into a DPPC monolayer initially at 2.3, 4, and 10 mN/m. The mixed monolayers are shifted to higher areas than the DPPC monolayer, indicating significant protein incorporation. It is interesting to note, however, that the largest shifts are observed for the lowest initial lipid pressures. These isotherms indicate that the most protein incorporation in the film occurs when the initial lipid pressure is lowest. This further supports the idea that the protein causes the phase transition in the lipid monolayer upon adsorption. Some of the lipid–protein mixed films exhibit surface potentials much lower than that of a plain DPPC monolayer (Fig. 8 and Table 4). However, the largest surface potential was found for the lowest initial lipid pressure. The mixed monolayer pressure-area isotherms indicate the highest factor VII incorporation at this pressure, so we may expect this mixed monolayer film to have the smallest surface potential. If there is a cooperative interaction or protein–lipid binding which occurs, it is possible that there are specific interactions which would lead to changes in surface potential which are greater than for the DPPC film alone. If the protein dipoles align with the lipid dipoles in the interface, an overall surface potential greater than the plain lipid would be observed. For the other films exhibiting surface potentials lower than that of a plain DPPC monolayer, the protein may bind to the lipid during the protein adsorption. Subsequently, the lipid undergoes a transition to the more condensed phase, with the result that the protein orientation changes in a way to cancel lipid dipoles and reduce the surface potential. The protein contribution to the surface potential in the mixed films were small, and in one case only about 15 mV. This is significantly smaller than the factor I contribution, and the electrolyte contributions still remain unclear. Penetration of factor VII into a DSPC monolayer occurs with and without calcium in the subphase, but the trend differs from that observed with the DPPC monolayer (Table 5). The highest observed film pressure changes are at the lowest initial film pressures. This is the same behavior observed with factor I. The same crowding out effect is observed, with less protein
penetration occurring at higher initial lipid pressures. The surface potential measurements for the mixed protein–lipid monolayers exhibit the same trend as factor I. All mixed monolayers show surface potentials which are lower than for the pure lipid monolayer. As factor VII is incorporated into the interface, the protein may align in such a way that its dipoles are aligned opposite to the lipids’, thus canceling surface dipole contributions. The more protein that is incorporated, the lower is the surface potential of the mixed monolayer films. The hydrocarbon chain length clearly affects the adsorption of factor VII. The protein penetrated the DSPC monolayer from both subphases, but only penetrated the DPPC monolayer when calcium was present. Hydrophobic interactions between lipid hydrocarbon chains and the protein are more important for DSPC monolayers. The hydrophobic regions on the protein are attracted to the hydrophobic DSPC film, even in the absence of subphase calcium ions. These hydrophobic interactions promote factor VII incorporation in the interface. Bos et al. observed a similar hydrophobic effect for the penetration of b-lactoglobulin into DSPA and DPPA (PA, phosphatidic acid) monolayers, with greater adsorption rates observed for the longer chained compound (4). For both films, subphase calcium addition facilitates the incorporation of factor VII. This indicates that electrostatic repulsion between DSPC and DPPC films with the protein occurs, but the repulsions are hampered when electrolytes are added to the subphase. It is interesting to note that this reduction in electrostatic interactions occurred for both factor I and factor VII. It has been proposed (13, 5) that the calcium ion serves as a bridge between the protein and the phospholipid components in the coagulation process. Our studies with plain DPPC monolayers indicate that calcium serves as a facilitator TABLE 5 Maximum Film Pressure Changes for Factor VII Penetration of DSPC Monolayers Initial lipid pressure (mN/m) DP Plain buffer (mN/m) DP Buffer and calcium (mN/m)
5.0 3.6 4.4
10.0 2.0 3.5
18.0 0.4 0.5
30.0 0.0 0.0
INTERACTIONS OF PROTEINS WITH LIPID MONOLAYERS
for the lipid–protein interactions since both the membranebinding and the non-membrane-binding proteins behaved similarly with added electrolytes. It is possible, however, that the incorporation of negatively charged phospholipids such as phosphatidylserines may show more specific interaction with the calcium ions. Studies of factor VII with mixed phosphatidylcholine and -serine components have already been initiated. It has recently been shown (14) that hydrophobic domains of factor X contact hydrophobic phospholipid membrane components. Insertion of protein domains in the bilayer results. It was proposed that the presence of calcium ions contributed to membrane attachment through electrostatic interactions and did not serve as a bridge between the protein domains and the membrane phospholipids. The hydrophobic residue on factor X is identical to factor VII, and our studies indicate similar hydrophobic interactions between proteins and phospholipids. Hydrophobic interactions between the protein and lipid account for the increased factor VII penetration of DSPC monolayers compared to that seen with DPPC films. It is interesting to note that penetration of lipid monolayers near 30 mN/m did not occur for factor VII under any conditions. Even though the cell membrane pressure has been estimated, a natural membrane is in a dynamic state of fluid motion in which the actual pressure is likely variable. These experiments indicate that very tight lipid packing precludes protein penetration, and lower pressures are necessary for protein incorporation. Further studies of factor VII penetration of DPPC monolayers are currently being investigated with fluorescence imaging techniques. It is hoped that more detailed studies will further elucidate the nature of these lipid–protein interactions. SUMMARY
Factor I is a non-membrane-binding protein and interacts similarly with DSPC and DPPC monolayer films on water. There are nonspecific interactions between the protein and the lipid molecules, resulting in crowding out of the protein at high
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lipid pressures. Factor VII also interacts with lipid monolayers, but the lipid–protein interactions are more specific. The hydrocarbon chain length affects the adsorption, with factor VII penetrating a DSPC film to greater extents. These studies indicate that hydrophobic interactions are important for factor VII penetration of both types of films. The effect of calcium addition was the same in all studies. Reduction of electrostatic interactions allowed further incorporation of both types of proteins into both DPPC and DSPC films. ACKNOWLEDGMENTS This research was supported by an award from Research Corporation. This research was also sponsored in part by Saint Louis University (Summer Research Award).
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