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The flow and deformation of synthetic polypeptide monolayers during compression
The Flow and Deformation of Synthetic Polypeptide Monolayers during Compression BENJAMIN R. MALCOLM Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom Received June 30, 1984; accepted September 10, 1984 Monolayers of poly-3'-ethyl-L-glutamate and poly-E-benzyloxycarbonyl-L-lysine have been studied at the air-water interface. Application of lines of sulfur to the monolayer shows that the collapse of the monolayer to bilayer starts in the region of the moving barrier and that this transition is then propagated along the surface. The flow patterns recorded photographically show regions of compression and elongation of the film, while expansion occurs to the rear of a Wilhelmy plate. It is possible in some cases to relate the flow patterns to the orientation of the molecules. Any obstruction to flow can seriously perturb the monolayer and produce flow and orientation effects which depend on the geometry o f the system. These effects are important in relation to measurements with a Wilhelmy plate and deposition of Langmuir-Blodgett films. The results require a revision of conclusions drawn from some recent studies of nucleation and collapse in polypeptide monolayers. © 1985 Academic Press,Inc.
INTRODUCTION
Molecular monolayers of many hydrophobic synthetic polypeptides are believed to exist as a-helices at the air-water interface (1, 2). The area of a monolayer, deduced from the initial rise in the surface pressure, shows that the molecules pack as closely together as in the solid, so that there must be considerable intermolecular cohesion and order in the monolayer state. In many cases a transition to a bilayer takes place associated with a plateau in the force-area curve (1-8). The pressure then increases as the monolayer is further compressed, where either collapse of the bilayer starts or, in certain cases, further layers of molecules form (4, 7). The transition to a bilayer may start in one of two ways. One possibility is that it is initiated from a large number of centers in the monolayer, perhaps associated with dust particles or discontinuities in the order at the molecular level, so that as observed with the Langmuir trough the collapse appears to take place uniformly over the surface. The second
possibility is that it is caused by viscous drag as the monolayer is compressed, which will cause the transition first to appear where the drag is greatest, that is in the region of the moving barrier. Studies described here of the flow of the monolayer under pressure show clearly that this second possibility is the correct one. Furthermore, by introducing a glass plate into the interface, the effect so produced on the flow can be studied. This shows the influence of a Wilhelmy plate on the system and is valuable in relation to the study of deposition and orientation phenomena in Langmuir-Blodgett films. Observations have been made on monolayers of poly-3'-ethyl-L-glutamate (PEG), chosen as a typical a-helical synthetic polypeptide with a long fiat plateau in the pressure-area curve, and on poly-~-benzyloxycarbonyl-L-lysine (PBCL). This not only shows a number of transitions in the pressure-area curve associated with the consecutive formation of several layers of molecules, but also interesting orientation phenomena during deposition of Langmuir-Blodgett films (7).
520 0021-9797/85 $3.00 Copyright© 1985by AcademicPress,Inc. All rightsof reproductionin any form reserved.
Journalof Colloidand InterfaceScience,Vol. 104,No. 2, April 1985
521
POLYPEPTIDE MONOLAYER FLOW EXPERIMENTAL
The Langmuir trough used for this work was 15 cm wide, 60 cm long, and both it and the barriers were made of polytetrafluorethylene. Surface pressures were recorded automatically in two ways, either with a film balance made from a lightly waxed phosphorbronze strip (9) mounted at one end of the trough, or with a Wilhelmy plate that could be mounted anywhere over the trough surface. The sensor for both systems was a DC miniature displacement transducer (Sangamo Schlumberger DF/2.5, Sangamo Transducers, Bognor Regis, P022 9BR, U. K.). The armature was mounted on a phosphor-bronze flexure strip (7.5 m m wide, 0.56 m m thick) 8.5 cm from the clamped end, as in the arrangement for the Wilhelmy system described by Fromherz (10). The Wilhelmy plate was a piece of filter paper 3 cm wide freely suspended by a wire 6 cm long from the sensor. To use the sensor with the film balance it was mounted with the flexure strip vertical, with an arm projecting down below the armature making contact with the back of the phosphor-bronze strip of the film balance. This provided a sensitive and stable balance, which was calibrated by comparison with the Wilhelmy system. The same configuration was used to measure the horizontal force exerted by the monolayer on a glass plate during compression. The glass was fixed vertically directly below the flexure strip. The movement of the monolayer was followed by forming lines of flowers of sulfur powder on the monolayer. For this a mask was made from transparent plastic 6 m m thick, with a series of slots in it 1.5 m m wide, 1.5 cm apart. This was mounted so that it could be placed over and close to the water surface with the slots parallel to the barrier. After application of the sulfur through the slots, the mask was removed from the surface. A sheet of black glass in the bottom of the trough facilitated photographing the surface, using a camera mounted centrally 70 cm above the trough. Direct measurements
of the movement of sulfur were made with a scale mounted close above the monolayer, along the center line of the trough. The monolayer was compressed in 1 cm increments and measurements recorded at 19 positions. Parallax errors were avoided by using the reflection of the eye in the trough. PEG (molecular weight 300,000) was supplied by New England Nuclear, Pilot Chemicals Division, Boston, Mass. and PBCL (molecular weight 400,000) was obtained from Sigma Chemical Company, Poole, Dorset BH17 7NH, U. K. About 8 mg of polymer was dissolved in 0.5 cm 3 of dichloroacetic acid and made up to 10 cm 3 with chloroform. Solvents were reagent grade redistilled. Monolayers were spread by applying a series of small drops evenly over the surface of twice distilled water at an area 5 /~2/residue higher than the first rise in the force-area curve. The amount applied was such that 40 cm of trough length corresponded to 25 A2/ residue. Compression of the monolayer was normally continuous and at a barrier speed of 4 m m per minute. All observations were made at 20 _+ 1°C. RESULTS
Figure 1 shows measurements on PEG monolayers using the film balance and Wil-
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FIG. I. Surface pressure-area curves for PEG, (a) measured with a film balance at the end of the trough, (b) measured with a Wilhelm), plate I0 cm from the end of the trough parallel to the bmTier; (¢) shows the horizontal pressure on a glass plate 3 cm'wide in the same position as the Wilhelmy plate. Journal of Colloid and Interface Science, Vol.
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BENJAMIN R. MALCOLM
helmy methods. It is evident that while the monolayer area measured by both methods, by extrapolating the linear rise in pressure, is the same (19.7 ~2/residue), the plateau extends to 9.5 A2/residue measured at the end of the trough with the film balance, but to only 12.5 /~2/residue, measured 10 cm from the end with the Wilhelmy plate. A similar effect was observed also in PBCL monolayers (Fig. 2) where additionally the shape of the transitions associated with the formation of three and four layers of molecules differs between the two methods. The measurements using Wilhelmy plates were made with the plates parallel to the barriers: if the plates were perpendicular to the barriers, the plateau was slightly curved and usually sloped upward at lower areas. It was noted that in all cases the plates were deflected in the direction of compression so that the suspension wire was about 10 ° from the vertical at the end of an experiment. To measure the horizontal force causing deflection of the plate, recordings were made of the force on a fixed plate of glass in the surface; with plates 2 to 5 cm wide the force appeared approximately proportional to their width, when the plates were mounted parallel to the barriers. Figure l c shows that there are two regions where the force is appreciable, in the region of the start of the plateau and at its completion. The length of the plateau
=~10
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25
Area (~/Residue) FIG. 2. Surface pressure-area curves for PBCL: ]ower curve, measured with a film balance at the end of the trough; upper curve (displaced l0 raN/m), measured with a Wilhe]my plate l0 cm from the end of the trough. Journal of Colloid and Interface Science, Yol. ]04, No. 2, April ]985
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FIG. 3. Length of the plateau observed in pressurearea curves of PEG measured with a Wilhelmy plate parallel to the barrier, at various distances from the end of the trough.
at different positions along the trough corresponded to the observations with the Wilhelmy plate. The relation between the position of the Wilhelmy plate and the length of plateau was pursued further for PEG by recording the length of the plateau measured with the Wilhelmy plate at different distances from the end of the trough. The length was defined by extrapolation of the flat plateau to intersect straight lines extrapolated from the curve on each side. Figure 3 shows that the length of the plateau decreases almost linearly as the plate is moved further away from the end of the trough.
Observations on the Flow and Deformation of the Monolayer It was evident from the behavior of the monolayer already described, that deformation and flow in the monolayer required further understanding and the following observations were therefore carried out. Spread monolayers were compressed to the observed monolayer area and lines of sulfur powder applied to the surface. Direct measurements of the movement of the sulfur were made with a scale mounted close above the monolayer, along the center line of the trough. The movement so recorded (Fig. 4) shows four well-defined regions to the graph: (1) a fairly uniform decrease in the monolayer area to the line A with the slopes of the
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POLYPEPTIDE MONOLAYER FLOW Distance 5
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tions; if compression of the monolayer were stopped, or if the plate were withdrawn from the surface, there was little change in the overall pattern.
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DISCUSSION
The Effect of Compression on a Monolayer
The measurements of the line spacings in Fig. 4 provide a clear picture of the way the monolayer behaves, when considered in reFIG. 4. Graphs showing the movement of sulfur lines lation to the pressure-area curves. The initial on a monolayer of PEG, measured from the end of the rise in the surface pressure, as far as line A, trough along the center line, as the monolayer is comat the area where bilayer formation starts, is pressed. Line A corresponds to the area at which the plateau starts to form; at B the transition to bilayer is a stage where decrease in area is fairly uniform taking place. Individual points have been omitted for over the surface. The area between A and B clarity. The monolayer was spread at 25 ,~2/residue, then represents monolayer which shows very little compressed to 20/~2/residue and lines of sulfur applied. movement or compression until it is converted to bilayer in the region of B. The almost parallel sloping lines below B show lines extrapolating (approximately) to zero that the bilayer is not compressed further area; and does not undergo further collapse but (2) a region of very slight movement of moves as a uniform plate until, below 9.5 the monolayer between lines A and B; A2/residue, all the monolayer has formed (3) a region below the line B where the bilayer. The slope of the lines then alters and lines are parallel and spaced at half the initial a more even collapse takes place over the spacing (approximately); and whole surface. This last stage is a large-scale (4) a part where the slopes of the lines process, detectable by eye on a well-illumichange from almost parallel to inclinations nated film, and associated with irregularities which extrapolate to zero area, indicative of developing in the pressure-area curve which a region of uniform collapse. show clearly when continuous automatic reThe movement of the monolayer was also cording is used (1, 11). The separation befollowed by taking a series of photographs tween lines A and B correlates well with the while the monolayer was being compressed length of the plateau measured with the (Fig. 5). To simulate the effect of a Wilhelmy Wilhelmy plate (Fig. 3). The slope of the plate in the surface a glass slide was lowered linear region of line B (with the ordinate into the interface immediately after applica- converted to centimeters) is close to one-half, tion of the sulfur. Figures 6 and 7 show as is to be expected for bilayer formation. examples of patterns recorded with the plates This work shows that bilayer is formed parallel and perpendicular to the barriers. initially in the region of the barrier and that Normally sulfur was not on the monolayer a monolayer-bilayer transition region is when pressure-area curves were being re- propagated along the surface at twice the corded; however a recording made with the speed of the barrier. This can in fact be film balance with lines of sulfur on the visualized by watching the relative movement monolayer showed that the sulfur did not of lines of sulfur when the barrier is rapidly significantly affect the results. These photo- moved. The process of bilayer formation is graphs represent essentially static observa- not therefore initiated uniformly over the 7.5
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BENJAMIN R. MALCOLM
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FIG. 5. Upper photograph, monolayer of PEG spread at 25 /~2/residue, compressed to 20 A2/residue and sulfur lines applied. Lower photograph, the monolayer compressed to 12.5/X2/residue.
surface at a large n u m b e r of points, but occurs first where the viscous drag is greatest, that is, in the region of the moving barrier. While m o v e m e n t along the centre line of the trough is represented by Fig. 4, Fig. 5 shows that the flow profile is very curved particularly near the edges. There is an increase in the length of the lines of up to about 20% (for this particular trough geometry), most of this increase taking place near Journal of Colloid and Interface Science, Vol. 104,No. 2, April 1985
the edges. Because bilayer, but not monolayer at the same pressure, undergoes bulk flow, it is bilayer that experiences most viscous drag at the sides of the trough and collapses further in consequence. In some areas the film appears is if it were attached to the side of the trough. This is probably not a direct interaction but a consequence of the drag caused by the stationary boundary layer of the water.
POLYPEPTIDE MONOLAYER FLOW
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FIG. 6. The effect on a monolayer produced by a glass plate 3 cm wide inserted into the interface after applying lines of sulfur. Upper photograph, PEG compressed to 10/~2]residue. Lower photograph PBCL compressed to 12/~2]residue. Note the expanded area behind the plates in both photographs.
The pressure required to initiate collapse of the bilayer is only marginally greater than is required for its formation, as shown by the flat plateau in the pressure-area curve. The bilayer appears rather incompressible and not very fluid (an exception is probably poly"y-n-decly-L-glutamate) (11). Consequently when the bilayer has reached an obstruction
such as a Wilhelmy plate, further collapse occurs in front of the plate, while monolayer at a lower pressure remains behind it. The length of the plateau recorded with the Wilhelmy system therefore depends linearly on its position along the trough (Fig. 3). The pressure difference across the plate is considerable at the start of formation of the bilayer Journal of Colloid and Interface Science, Vol. 104, No. 2, April 1985
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BENJAMIN R. MALCOLM
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FIG. 7. The effect on a monolayer of PEG of a glass plate 3 em wide inserted perpendicular to the barrier. Upper photograph, at 15 A2/residue, bilayer is starting to form at the right. Lower photograph, at 10 ~,2/residue, shows very little change to the left of the plate, though as shown by Fig. 4, an unperturbed rnonolayer is then almost entirely converted to bilayer.
and when it reaches the plate. During propagation o f the bilayer however, it drops to a small value (Fig. l c). Figure 6 is consistent with the above, but reveals some further features. For P E G at 10 A2/residue the first three or four lines on the left o f the trough are at spacings characteristic o f compressed monolayer. To the right o f Journal of Colloid and Interface Science, VoL 104, No. 2, April 1985
the plate the spacings are closest in the region o f the plate, where bilayer has further collapsed, while closer to the barrier the spacings correspond to bilayer which extends r o u n d the sides o f the plate. This produces a curved profile to the flow and extension o f the bilayer in the direction o f the lines. A b o u t 3 crn to the left o f the plate there is a region
POLYPEPTIDE MONOLAYER FLOW of film expansion, as shown by the triangular region with a large line spacing at the center, and a broadening of the lines of sulfur. The overall picture for PBCL is similar, but now bilayer extends in curved lines 6 cm to the rear of the plate. The spacings in front of the plate are not even and show that (taken with the results of previous studies (4, 7)), further layers of molecules first form in the region of the plate. Even if a plate is mounted parallel to the direction of compression, similar perturbations to the flow are produced (Fig. 7). For PEG at 10 A2/residue, bilayer to the right of the plate collapses while compressed monolayer remains to the left. At 15 A2/residue, lines of sulfur on the monolayer make an angle of about 45 ° to the line of the plate, so that even before bilayer has formed near the plate, the flow of the monolayer is affected by it. Orientation Phenomena in Films For a close-packed monolayer of a-helices to exist at areas similar to those observed in the fibrous state, it must be supposed that they form ordered two-dimensional micelles of aligned molecules. It is possible that these are elongated in the direction of the molecular axes (as in the nematic state), though there is no direct evidence on this point. The rigidity of the a-helix and the weaker intermolecular interactions will, however, favor such elongation. Conversion of the monolayer to bilayer, and viscous drag effects operating on an initially randomly oriented array of micelles will in any case favor the development of oriented structures, aligned with their mean direction perpendicular to the direction of compression and in the direction of tension, as occurs generally during fiber formation in three dimensions. Where the photographic observations show shrinkage associated with bilayer formation or further collapse, or elongation of lines of polymer, it is reasonable to suppose that the lines of sulfur indicate the overall direction of orientation of the polymer. This assumption is
527
consistent with experimental studies of collapsed films and shows the origin of orientation effects reported by various observers. There is general agreement that when a bartier or plate is mounted across the trough, parallel to the moving barrier, the polymer is deposited with a mean orientation parallel to the interface and the barrier (2, 6, 12). However, when a plate is perpendicular to the barrier, in line with the direction of compression, Takeda et al. (6) showed that in monolayers of poly-~-methyl-L-glutamate, the polymer was deposited at an angle of 30 ° to the direction of withdrawal. Further, they deduced that the molecules on the water surface formed a certain angle on both sides of the plate. Similar conclusions were reached for poly-7-benzyl-L-glutamate (5) and PBCL (7). This is clearly consistent with the inclination of the lines close to the plate in Fig. 7. However, Cornell in similar experiments found the polymer was aligned vertically (12). This is not necessarily inconsistent with the results of Takeda et al. since Cornell's work also showed a pressure-area curve with a longer and flatter plateau, so that the conditions for deposition of film were probably not the same for both groups. With plates parallel to the barriers, low deposition ratios on the rear surface were reported, as is to be expected from Fig. 6. The methods described here are clearly of value in optimizing conditions for the deposition and orientation of polymer to form Langmuir-Blodgett films. Experimental and Theoretical Considerations These observations are important in relation both to the meaningful measurement of monolayer properties, in the understanding of orientation effects in monolayers and in the theoretical analysis of collapse phenomena. While the results presented here are limited to two polymers, it is probable that the conclusions apply equally to other highmolecular-weight polypeptides with similar Journal of Colloid and Interface Science, Vol. 104, No. 2, April 1985
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BENJAMIN
monolayer properties, and possibly to other polymer systems. In particular it shows the limitations of the simple Wilhelmy system when applied to the measurement of the properties of monolayers that show viscous effects and transitions at constant pressure in the monolayer state. These might largely be avoided by the use of a Wilhelmy plate centrally mounted with symmetrical compression from both ends of the trough, or a film balance that measures the horizontal force at one end of the trough. To minimize edge effects the trough should be reasonably wide. The position over a film of an electrode for surface potential measurements also needs to be carefully considered. The photographs show very clearly that wherever the monolayer is moving near to a stationary boundary, changes can be produced in the state of the film which are not reversible; if the glass plates are withdrawn during compression there is little change to the pattern, the film can be regarded as undergoing irreversible changes analogous to "cold work" in metals. Caution is particularly necessary therefore in the interpretation of surface viscosity measurements where, in the region of the measuring device, shear effects will occur. So-called denaturation effects, deduced from viscometric measurements (13), may well therefore arise from the method of observation rather than be an intrinsic property of the polypeptide. . . . . The presence of strong in,termolecular~orientation effects are a serious limitation to the application of polymer solution theory to monolayers. Gabrielli et al. (14) have noted that as the limiting area for a polymer is approached, any possible tendencies of adjacent chains to become parallel, or to assume any preferential orientations, will lead to departures from assumed equations. The application of this theory (15) to studies of poly-~/-methyl-L-glutamate (MW 100,000), which behaves very similarly to PEG, is therefore questionable. Additionally, studies of the kinetics of collapse of monolayers of this polymer have been interpreted as a n u Journal of Colloid and Interface Science, Vol. 104 No. 2 April 1985
R. MALCOLM
cleation phenomenon (8). It is clear that this is not homogeneous and if a simple Wilhdmy balance is used (without symmetrical compression), the observations are complicated further by the perturbations in flow and state of the film in the region of the plate, which produces an ill-defined system. Such effects are serious at pressures close to and in excess o f those required for bilayer formation. The values of activation energies for the collapse of bilayer deduced from such studies, and their physical significance, therefore, require further consideration. CONCLUSIONS
In measuring and understanding the properties of these monolayers it is clearly valuable and important to have a clear physical picture of the processes involved. The studies described on the flow of the monolayer using lines of sulfur show, in a very simple and direct way, a number of features which have not previously been recorded. The formation of the bilayer is not a homogeneous nucleation phenomenon, but starts first in the region of the moving barrier. This transition is then propagated along the surface of the trough and is affected by obstructions to the monolayer flow such as a Wilhelmy plate. In consequence this method can give results which depend directly on the position of the plate with respect to the moving barrier, unless symmetrical compression is used. From a study of the flow, it is possible to infer the direction of mean orientation of the molecules, which is of value in studying orientation effects during deposition of Langmuir-Blodgett films. In all such work the precise geometry of the trough and the position of measuring devices and obstructions to flow, such as plates in the interface, need to be carefully considered and reported. The results require further examination of current theories of polypeptide monolayer behavior. It is possible that the methods described may be of value in other condensed systems where flow conditions are nonideal.
POLYPEPTIDE MONOLAYER FLOW REFERENCES 1. Malcolm, B. R., Prog. Surface Membr. Sci. 7, 183 (1973). 2. Malcolm, B. R., Proc. R. Soc. London Ser. A 305, 363 (1968). 3. Malcolm, B. R., Polymer 7, 595 (1966). 4. Malcolm, B. R., Biochem. J. 110, 733 (1968). 5. Takenaka, T., Harada, K., and Matsumoto, M., J. Colloid Interface ScL 73, 569 (1980). 6. Takeda, F., Matsumoto, M., Takenaka, T., and Fujiyoshi, Y., J. Colloid Interface Sci. 84, 220 (1981). 7. Takeda, F., Matsumoto, M., Takenaka, T., Fujiyoshi, Y., and Uyeda, N., J. Colloid Interface Sci. 91, 267 (1983).
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8. Baglioni, P., Dei, L., and Gabrielli, G., J. Colloid Interface Sci. 93, 402 (1983). 9. Malcolm, B. R., and Davies, S. R., J. Sci. Instrum. 42, 359 (1965). 10. Fromherz, D., Rev. Sci. Instrum. 46, 1380 (1975). 11. Malcolm, B. R., Adv. Chem. Ser. 145, 338 (1975). 12. Cornell, D. G., J. Colloid Interface Sci. 70, 175 (1979). 13. Goupil, D. W., and Goodrich, F. C., J. Colloid Interface Sci. 62, 142 (1977). 14. Gabrielli, G., Ferroni, E., and Huggins, M. L., Prog. Colloid Polymer Sci. 58, 201 (1975). 15. Baglioni, P., Gallori, E., Gabrielli, G., and Ferroni, E., J. Colloid Interface Sci. 88, 221 (1982).
Journal of Colloid and Interface Science. Vol. 104. No. 2. April 1985
INTRODUCTION
Molecular monolayers of many hydrophobic synthetic polypeptides are believed to exist as a-helices at the air-water interface (1, 2). The area of a monolayer, deduced from the initial rise in the surface pressure, shows that the molecules pack as closely together as in the solid, so that there must be considerable intermolecular cohesion and order in the monolayer state. In many cases a transition to a bilayer takes place associated with a plateau in the force-area curve (1-8). The pressure then increases as the monolayer is further compressed, where either collapse of the bilayer starts or, in certain cases, further layers of molecules form (4, 7). The transition to a bilayer may start in one of two ways. One possibility is that it is initiated from a large number of centers in the monolayer, perhaps associated with dust particles or discontinuities in the order at the molecular level, so that as observed with the Langmuir trough the collapse appears to take place uniformly over the surface. The second
possibility is that it is caused by viscous drag as the monolayer is compressed, which will cause the transition first to appear where the drag is greatest, that is in the region of the moving barrier. Studies described here of the flow of the monolayer under pressure show clearly that this second possibility is the correct one. Furthermore, by introducing a glass plate into the interface, the effect so produced on the flow can be studied. This shows the influence of a Wilhelmy plate on the system and is valuable in relation to the study of deposition and orientation phenomena in Langmuir-Blodgett films. Observations have been made on monolayers of poly-3'-ethyl-L-glutamate (PEG), chosen as a typical a-helical synthetic polypeptide with a long fiat plateau in the pressure-area curve, and on poly-~-benzyloxycarbonyl-L-lysine (PBCL). This not only shows a number of transitions in the pressure-area curve associated with the consecutive formation of several layers of molecules, but also interesting orientation phenomena during deposition of Langmuir-Blodgett films (7).
520 0021-9797/85 $3.00 Copyright© 1985by AcademicPress,Inc. All rightsof reproductionin any form reserved.
Journalof Colloidand InterfaceScience,Vol. 104,No. 2, April 1985
521
POLYPEPTIDE MONOLAYER FLOW EXPERIMENTAL
The Langmuir trough used for this work was 15 cm wide, 60 cm long, and both it and the barriers were made of polytetrafluorethylene. Surface pressures were recorded automatically in two ways, either with a film balance made from a lightly waxed phosphorbronze strip (9) mounted at one end of the trough, or with a Wilhelmy plate that could be mounted anywhere over the trough surface. The sensor for both systems was a DC miniature displacement transducer (Sangamo Schlumberger DF/2.5, Sangamo Transducers, Bognor Regis, P022 9BR, U. K.). The armature was mounted on a phosphor-bronze flexure strip (7.5 m m wide, 0.56 m m thick) 8.5 cm from the clamped end, as in the arrangement for the Wilhelmy system described by Fromherz (10). The Wilhelmy plate was a piece of filter paper 3 cm wide freely suspended by a wire 6 cm long from the sensor. To use the sensor with the film balance it was mounted with the flexure strip vertical, with an arm projecting down below the armature making contact with the back of the phosphor-bronze strip of the film balance. This provided a sensitive and stable balance, which was calibrated by comparison with the Wilhelmy system. The same configuration was used to measure the horizontal force exerted by the monolayer on a glass plate during compression. The glass was fixed vertically directly below the flexure strip. The movement of the monolayer was followed by forming lines of flowers of sulfur powder on the monolayer. For this a mask was made from transparent plastic 6 m m thick, with a series of slots in it 1.5 m m wide, 1.5 cm apart. This was mounted so that it could be placed over and close to the water surface with the slots parallel to the barrier. After application of the sulfur through the slots, the mask was removed from the surface. A sheet of black glass in the bottom of the trough facilitated photographing the surface, using a camera mounted centrally 70 cm above the trough. Direct measurements
of the movement of sulfur were made with a scale mounted close above the monolayer, along the center line of the trough. The monolayer was compressed in 1 cm increments and measurements recorded at 19 positions. Parallax errors were avoided by using the reflection of the eye in the trough. PEG (molecular weight 300,000) was supplied by New England Nuclear, Pilot Chemicals Division, Boston, Mass. and PBCL (molecular weight 400,000) was obtained from Sigma Chemical Company, Poole, Dorset BH17 7NH, U. K. About 8 mg of polymer was dissolved in 0.5 cm 3 of dichloroacetic acid and made up to 10 cm 3 with chloroform. Solvents were reagent grade redistilled. Monolayers were spread by applying a series of small drops evenly over the surface of twice distilled water at an area 5 /~2/residue higher than the first rise in the force-area curve. The amount applied was such that 40 cm of trough length corresponded to 25 A2/ residue. Compression of the monolayer was normally continuous and at a barrier speed of 4 m m per minute. All observations were made at 20 _+ 1°C. RESULTS
Figure 1 shows measurements on PEG monolayers using the film balance and Wil-
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FIG. I. Surface pressure-area curves for PEG, (a) measured with a film balance at the end of the trough, (b) measured with a Wilhelm), plate I0 cm from the end of the trough parallel to the bmTier; (¢) shows the horizontal pressure on a glass plate 3 cm'wide in the same position as the Wilhelmy plate. Journal of Colloid and Interface Science, Vol.
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BENJAMIN R. MALCOLM
helmy methods. It is evident that while the monolayer area measured by both methods, by extrapolating the linear rise in pressure, is the same (19.7 ~2/residue), the plateau extends to 9.5 A2/residue measured at the end of the trough with the film balance, but to only 12.5 /~2/residue, measured 10 cm from the end with the Wilhelmy plate. A similar effect was observed also in PBCL monolayers (Fig. 2) where additionally the shape of the transitions associated with the formation of three and four layers of molecules differs between the two methods. The measurements using Wilhelmy plates were made with the plates parallel to the barriers: if the plates were perpendicular to the barriers, the plateau was slightly curved and usually sloped upward at lower areas. It was noted that in all cases the plates were deflected in the direction of compression so that the suspension wire was about 10 ° from the vertical at the end of an experiment. To measure the horizontal force causing deflection of the plate, recordings were made of the force on a fixed plate of glass in the surface; with plates 2 to 5 cm wide the force appeared approximately proportional to their width, when the plates were mounted parallel to the barriers. Figure l c shows that there are two regions where the force is appreciable, in the region of the start of the plateau and at its completion. The length of the plateau
=~10
;
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25
Area (~/Residue) FIG. 2. Surface pressure-area curves for PBCL: ]ower curve, measured with a film balance at the end of the trough; upper curve (displaced l0 raN/m), measured with a Wilhe]my plate l0 cm from the end of the trough. Journal of Colloid and Interface Science, Yol. ]04, No. 2, April ]985
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15
20
25
Position of Wilhetmy prate (cm)
FIG. 3. Length of the plateau observed in pressurearea curves of PEG measured with a Wilhelmy plate parallel to the barrier, at various distances from the end of the trough.
at different positions along the trough corresponded to the observations with the Wilhelmy plate. The relation between the position of the Wilhelmy plate and the length of plateau was pursued further for PEG by recording the length of the plateau measured with the Wilhelmy plate at different distances from the end of the trough. The length was defined by extrapolation of the flat plateau to intersect straight lines extrapolated from the curve on each side. Figure 3 shows that the length of the plateau decreases almost linearly as the plate is moved further away from the end of the trough.
Observations on the Flow and Deformation of the Monolayer It was evident from the behavior of the monolayer already described, that deformation and flow in the monolayer required further understanding and the following observations were therefore carried out. Spread monolayers were compressed to the observed monolayer area and lines of sulfur powder applied to the surface. Direct measurements of the movement of the sulfur were made with a scale mounted close above the monolayer, along the center line of the trough. The movement so recorded (Fig. 4) shows four well-defined regions to the graph: (1) a fairly uniform decrease in the monolayer area to the line A with the slopes of the
523
POLYPEPTIDE MONOLAYER FLOW Distance 5
20
10
(tin) ~5
20
25
30
17.5 A--
~
.
____
tions; if compression of the monolayer were stopped, or if the plate were withdrawn from the surface, there was little change in the overall pattern.
~= 15
~12.5 10
DISCUSSION
The Effect of Compression on a Monolayer
The measurements of the line spacings in Fig. 4 provide a clear picture of the way the monolayer behaves, when considered in reFIG. 4. Graphs showing the movement of sulfur lines lation to the pressure-area curves. The initial on a monolayer of PEG, measured from the end of the rise in the surface pressure, as far as line A, trough along the center line, as the monolayer is comat the area where bilayer formation starts, is pressed. Line A corresponds to the area at which the plateau starts to form; at B the transition to bilayer is a stage where decrease in area is fairly uniform taking place. Individual points have been omitted for over the surface. The area between A and B clarity. The monolayer was spread at 25 ,~2/residue, then represents monolayer which shows very little compressed to 20/~2/residue and lines of sulfur applied. movement or compression until it is converted to bilayer in the region of B. The almost parallel sloping lines below B show lines extrapolating (approximately) to zero that the bilayer is not compressed further area; and does not undergo further collapse but (2) a region of very slight movement of moves as a uniform plate until, below 9.5 the monolayer between lines A and B; A2/residue, all the monolayer has formed (3) a region below the line B where the bilayer. The slope of the lines then alters and lines are parallel and spaced at half the initial a more even collapse takes place over the spacing (approximately); and whole surface. This last stage is a large-scale (4) a part where the slopes of the lines process, detectable by eye on a well-illumichange from almost parallel to inclinations nated film, and associated with irregularities which extrapolate to zero area, indicative of developing in the pressure-area curve which a region of uniform collapse. show clearly when continuous automatic reThe movement of the monolayer was also cording is used (1, 11). The separation befollowed by taking a series of photographs tween lines A and B correlates well with the while the monolayer was being compressed length of the plateau measured with the (Fig. 5). To simulate the effect of a Wilhelmy Wilhelmy plate (Fig. 3). The slope of the plate in the surface a glass slide was lowered linear region of line B (with the ordinate into the interface immediately after applica- converted to centimeters) is close to one-half, tion of the sulfur. Figures 6 and 7 show as is to be expected for bilayer formation. examples of patterns recorded with the plates This work shows that bilayer is formed parallel and perpendicular to the barriers. initially in the region of the barrier and that Normally sulfur was not on the monolayer a monolayer-bilayer transition region is when pressure-area curves were being re- propagated along the surface at twice the corded; however a recording made with the speed of the barrier. This can in fact be film balance with lines of sulfur on the visualized by watching the relative movement monolayer showed that the sulfur did not of lines of sulfur when the barrier is rapidly significantly affect the results. These photo- moved. The process of bilayer formation is graphs represent essentially static observa- not therefore initiated uniformly over the 7.5
Journal of Colloid and Interface Science, Vol. 104. No. 2, April 1985
524
BENJAMIN R. MALCOLM
A2/ Residue
10
lo
Cm o
15
I
2o
I
20
3o
I
J I I I
~2/=,=id°o c=o
=
,o lo
i " . j+~
,~
I
2o
I
2o 3o
I
', / /~/'i'/
II
i I
I
}
FIG. 5. Upper photograph, monolayer of PEG spread at 25 /~2/residue, compressed to 20 A2/residue and sulfur lines applied. Lower photograph, the monolayer compressed to 12.5/X2/residue.
surface at a large n u m b e r of points, but occurs first where the viscous drag is greatest, that is, in the region of the moving barrier. While m o v e m e n t along the centre line of the trough is represented by Fig. 4, Fig. 5 shows that the flow profile is very curved particularly near the edges. There is an increase in the length of the lines of up to about 20% (for this particular trough geometry), most of this increase taking place near Journal of Colloid and Interface Science, Vol. 104,No. 2, April 1985
the edges. Because bilayer, but not monolayer at the same pressure, undergoes bulk flow, it is bilayer that experiences most viscous drag at the sides of the trough and collapses further in consequence. In some areas the film appears is if it were attached to the side of the trough. This is probably not a direct interaction but a consequence of the drag caused by the stationary boundary layer of the water.
POLYPEPTIDE MONOLAYER FLOW
~2/ Residue
10
5
l 10 I,,,,,,,,,I,,,,,
Cm 0
/~2/ Residue Cmo I I I I I I I
525
2o I I I i l I i
5
10
20
10 I
I
IIJ
1 I I
IIIII
FIG. 6. The effect on a monolayer produced by a glass plate 3 cm wide inserted into the interface after applying lines of sulfur. Upper photograph, PEG compressed to 10/~2]residue. Lower photograph PBCL compressed to 12/~2]residue. Note the expanded area behind the plates in both photographs.
The pressure required to initiate collapse of the bilayer is only marginally greater than is required for its formation, as shown by the flat plateau in the pressure-area curve. The bilayer appears rather incompressible and not very fluid (an exception is probably poly"y-n-decly-L-glutamate) (11). Consequently when the bilayer has reached an obstruction
such as a Wilhelmy plate, further collapse occurs in front of the plate, while monolayer at a lower pressure remains behind it. The length of the plateau recorded with the Wilhelmy system therefore depends linearly on its position along the trough (Fig. 3). The pressure difference across the plate is considerable at the start of formation of the bilayer Journal of Colloid and Interface Science, Vol. 104, No. 2, April 1985
526
BENJAMIN R. MALCOLM
~2/ Residue Cm o I
I
I
i ,o I I I
TOII
10
~2/ Residue Cm o I I 1 I I I I
'I
'I °
I I I I I I I I I I I
10
I
I I
15 20
I I I L I
i
I I I
I t L 1 t
FIG. 7. The effect on a monolayer of PEG of a glass plate 3 em wide inserted perpendicular to the barrier. Upper photograph, at 15 A2/residue, bilayer is starting to form at the right. Lower photograph, at 10 ~,2/residue, shows very little change to the left of the plate, though as shown by Fig. 4, an unperturbed rnonolayer is then almost entirely converted to bilayer.
and when it reaches the plate. During propagation o f the bilayer however, it drops to a small value (Fig. l c). Figure 6 is consistent with the above, but reveals some further features. For P E G at 10 A2/residue the first three or four lines on the left o f the trough are at spacings characteristic o f compressed monolayer. To the right o f Journal of Colloid and Interface Science, VoL 104, No. 2, April 1985
the plate the spacings are closest in the region o f the plate, where bilayer has further collapsed, while closer to the barrier the spacings correspond to bilayer which extends r o u n d the sides o f the plate. This produces a curved profile to the flow and extension o f the bilayer in the direction o f the lines. A b o u t 3 crn to the left o f the plate there is a region
POLYPEPTIDE MONOLAYER FLOW of film expansion, as shown by the triangular region with a large line spacing at the center, and a broadening of the lines of sulfur. The overall picture for PBCL is similar, but now bilayer extends in curved lines 6 cm to the rear of the plate. The spacings in front of the plate are not even and show that (taken with the results of previous studies (4, 7)), further layers of molecules first form in the region of the plate. Even if a plate is mounted parallel to the direction of compression, similar perturbations to the flow are produced (Fig. 7). For PEG at 10 A2/residue, bilayer to the right of the plate collapses while compressed monolayer remains to the left. At 15 A2/residue, lines of sulfur on the monolayer make an angle of about 45 ° to the line of the plate, so that even before bilayer has formed near the plate, the flow of the monolayer is affected by it. Orientation Phenomena in Films For a close-packed monolayer of a-helices to exist at areas similar to those observed in the fibrous state, it must be supposed that they form ordered two-dimensional micelles of aligned molecules. It is possible that these are elongated in the direction of the molecular axes (as in the nematic state), though there is no direct evidence on this point. The rigidity of the a-helix and the weaker intermolecular interactions will, however, favor such elongation. Conversion of the monolayer to bilayer, and viscous drag effects operating on an initially randomly oriented array of micelles will in any case favor the development of oriented structures, aligned with their mean direction perpendicular to the direction of compression and in the direction of tension, as occurs generally during fiber formation in three dimensions. Where the photographic observations show shrinkage associated with bilayer formation or further collapse, or elongation of lines of polymer, it is reasonable to suppose that the lines of sulfur indicate the overall direction of orientation of the polymer. This assumption is
527
consistent with experimental studies of collapsed films and shows the origin of orientation effects reported by various observers. There is general agreement that when a bartier or plate is mounted across the trough, parallel to the moving barrier, the polymer is deposited with a mean orientation parallel to the interface and the barrier (2, 6, 12). However, when a plate is perpendicular to the barrier, in line with the direction of compression, Takeda et al. (6) showed that in monolayers of poly-~-methyl-L-glutamate, the polymer was deposited at an angle of 30 ° to the direction of withdrawal. Further, they deduced that the molecules on the water surface formed a certain angle on both sides of the plate. Similar conclusions were reached for poly-7-benzyl-L-glutamate (5) and PBCL (7). This is clearly consistent with the inclination of the lines close to the plate in Fig. 7. However, Cornell in similar experiments found the polymer was aligned vertically (12). This is not necessarily inconsistent with the results of Takeda et al. since Cornell's work also showed a pressure-area curve with a longer and flatter plateau, so that the conditions for deposition of film were probably not the same for both groups. With plates parallel to the barriers, low deposition ratios on the rear surface were reported, as is to be expected from Fig. 6. The methods described here are clearly of value in optimizing conditions for the deposition and orientation of polymer to form Langmuir-Blodgett films. Experimental and Theoretical Considerations These observations are important in relation both to the meaningful measurement of monolayer properties, in the understanding of orientation effects in monolayers and in the theoretical analysis of collapse phenomena. While the results presented here are limited to two polymers, it is probable that the conclusions apply equally to other highmolecular-weight polypeptides with similar Journal of Colloid and Interface Science, Vol. 104, No. 2, April 1985
528
BENJAMIN
monolayer properties, and possibly to other polymer systems. In particular it shows the limitations of the simple Wilhelmy system when applied to the measurement of the properties of monolayers that show viscous effects and transitions at constant pressure in the monolayer state. These might largely be avoided by the use of a Wilhelmy plate centrally mounted with symmetrical compression from both ends of the trough, or a film balance that measures the horizontal force at one end of the trough. To minimize edge effects the trough should be reasonably wide. The position over a film of an electrode for surface potential measurements also needs to be carefully considered. The photographs show very clearly that wherever the monolayer is moving near to a stationary boundary, changes can be produced in the state of the film which are not reversible; if the glass plates are withdrawn during compression there is little change to the pattern, the film can be regarded as undergoing irreversible changes analogous to "cold work" in metals. Caution is particularly necessary therefore in the interpretation of surface viscosity measurements where, in the region of the measuring device, shear effects will occur. So-called denaturation effects, deduced from viscometric measurements (13), may well therefore arise from the method of observation rather than be an intrinsic property of the polypeptide. . . . . The presence of strong in,termolecular~orientation effects are a serious limitation to the application of polymer solution theory to monolayers. Gabrielli et al. (14) have noted that as the limiting area for a polymer is approached, any possible tendencies of adjacent chains to become parallel, or to assume any preferential orientations, will lead to departures from assumed equations. The application of this theory (15) to studies of poly-~/-methyl-L-glutamate (MW 100,000), which behaves very similarly to PEG, is therefore questionable. Additionally, studies of the kinetics of collapse of monolayers of this polymer have been interpreted as a n u Journal of Colloid and Interface Science, Vol. 104 No. 2 April 1985
R. MALCOLM
cleation phenomenon (8). It is clear that this is not homogeneous and if a simple Wilhdmy balance is used (without symmetrical compression), the observations are complicated further by the perturbations in flow and state of the film in the region of the plate, which produces an ill-defined system. Such effects are serious at pressures close to and in excess o f those required for bilayer formation. The values of activation energies for the collapse of bilayer deduced from such studies, and their physical significance, therefore, require further consideration. CONCLUSIONS
In measuring and understanding the properties of these monolayers it is clearly valuable and important to have a clear physical picture of the processes involved. The studies described on the flow of the monolayer using lines of sulfur show, in a very simple and direct way, a number of features which have not previously been recorded. The formation of the bilayer is not a homogeneous nucleation phenomenon, but starts first in the region of the moving barrier. This transition is then propagated along the surface of the trough and is affected by obstructions to the monolayer flow such as a Wilhelmy plate. In consequence this method can give results which depend directly on the position of the plate with respect to the moving barrier, unless symmetrical compression is used. From a study of the flow, it is possible to infer the direction of mean orientation of the molecules, which is of value in studying orientation effects during deposition of Langmuir-Blodgett films. In all such work the precise geometry of the trough and the position of measuring devices and obstructions to flow, such as plates in the interface, need to be carefully considered and reported. The results require further examination of current theories of polypeptide monolayer behavior. It is possible that the methods described may be of value in other condensed systems where flow conditions are nonideal.
POLYPEPTIDE MONOLAYER FLOW REFERENCES 1. Malcolm, B. R., Prog. Surface Membr. Sci. 7, 183 (1973). 2. Malcolm, B. R., Proc. R. Soc. London Ser. A 305, 363 (1968). 3. Malcolm, B. R., Polymer 7, 595 (1966). 4. Malcolm, B. R., Biochem. J. 110, 733 (1968). 5. Takenaka, T., Harada, K., and Matsumoto, M., J. Colloid Interface ScL 73, 569 (1980). 6. Takeda, F., Matsumoto, M., Takenaka, T., and Fujiyoshi, Y., J. Colloid Interface Sci. 84, 220 (1981). 7. Takeda, F., Matsumoto, M., Takenaka, T., Fujiyoshi, Y., and Uyeda, N., J. Colloid Interface Sci. 91, 267 (1983).
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8. Baglioni, P., Dei, L., and Gabrielli, G., J. Colloid Interface Sci. 93, 402 (1983). 9. Malcolm, B. R., and Davies, S. R., J. Sci. Instrum. 42, 359 (1965). 10. Fromherz, D., Rev. Sci. Instrum. 46, 1380 (1975). 11. Malcolm, B. R., Adv. Chem. Ser. 145, 338 (1975). 12. Cornell, D. G., J. Colloid Interface Sci. 70, 175 (1979). 13. Goupil, D. W., and Goodrich, F. C., J. Colloid Interface Sci. 62, 142 (1977). 14. Gabrielli, G., Ferroni, E., and Huggins, M. L., Prog. Colloid Polymer Sci. 58, 201 (1975). 15. Baglioni, P., Gallori, E., Gabrielli, G., and Ferroni, E., J. Colloid Interface Sci. 88, 221 (1982).
Journal of Colloid and Interface Science. Vol. 104. No. 2. April 1985