6452
Langmuir 1996, 12, 6452-6458
Compression/Expansion Hysteresis of Poly(L-glutamic acid) Monolayers Spread at the Air/Water Interface Torsten Reda,† Horst Hermel,*,† and Hans-Dieter Ho¨ltje‡ Max-Planck-Institut fu¨ r Kolloid- und Grenzfla¨ chenforschung, Rudower Chaussee 5, D-12489 Berlin, Germany, and Pharmazeutisches Institut der Freien Universita¨ t Berlin, Ko¨ nigin-Luise Strasse 2-4, D-14195 Berlin, Germany Received February 22, 1996. In Final Form: August 12, 1996X In order to study the conformational behavior of poly(L-glutamic acid) (PGA) at the air/water interface under the influence of compression and expansion forces, PGA was spread on an aqueous acidic subphase and studied by the Langmuir technique. Several distinct regions of the first compression surface pressure/ area (π/A) isotherm could be identified by the starting and inflection point of the isotherm and by the beginning and the center of the pseudoplateau. The interpretation of the characteristic shape of the π/A isotherms in the sense of a side-by-side and an interdigitated organization of helical rods is strongly supported by molecular modeling calculations. But the helical surface layer is sensitive to repeated expansion and compression. A well-defined and reaction-kinetic demonstrable change occurs. The reasons for this transformation are discussed. Solidified regions are forming in the layer. Finally, after several compression/ expansion cycles a more rigid monolayer results than that formed by helical rods exclusively. These monolayer ruptures on expansion and clods of PGA molecules were observed using Brewster angle microscopy and ellipsometry.
Introduction Molecular orientation of polypeptides at interfaces is of fundamental significance for biological and technological processes. Therefore, studies of model polypeptides at well-defined interfaces have been of considerable interest. The monolayer technique is an useful means for model studies because surface pressure/area (π/A) isotherms reflect the intermolecular forces operating in the 2D array of molecules and at the same time provide information on the molecular packing.1 For investigations of polypeptides poly(L-glutamic acid) (PGA) is a well-suited model. In the bulk phase it can exist as R-helix, β-sheet, or random coil structures which most likely exist at the air/water interface also.2 Numerous studies of PGA and their γ-esters spread on water have been carried out.2,3-9 The conformation of PGA monolayers depends on the pH of the subphase. Shibata et al.6 presumed from their findings that PGA molecules in monolayers are in the R-helix form at pH 2.0, in the β-sheet form at pH 3.5, and in a heterogeneous form with random coil segments at pH 4.0. Takeda et al.4 concluded from studies of poly(γ-methyl L-glutamate) (PMG) that the molecules in the monolayer were in the helical conformation and that the helical rods lie flat on the water surface. Higashi et al.9 arrived at the same conclusions for PGA at pH 3.0. The plateau region in the π/A isotherm was ascribed to a layer transition from the 2D into the 3D state. * To whom correspondence should be addressed. † Max-Planck-Institut fu ¨ r Kolloid- und Grenzfla¨chenforschung. ‡ Pharmazeutisches Institut der Freien Universita ¨ t Berlin. X Abstract published in Advance ACS Abstracts, December 1, 1996. (1) Lavigne, P.; Tancrede, P.; Lamarche, F.; Max, J. J. Langmuir 1992, 8, 1988. (2) Loeb, G. I.; Baier, R. E. J. Colloid Interface Sci. 1968, 27, 38. (3) Malcolm, B. R. Proc. R. Soc. London, Ser. A 1968, 305, 363. (4) Takeda, F.; Matsumoto, M.; Takenaka, T.; Fujiyoshi, Y. J. Colloid Interface Sci. 1981, 84, 220. (5) Baglioni, P.; Gallori, E.; Gabrielli, G.; Ferroni, E. J. Colloid Interface Sci. 1982, 88, 221. (6) Shibata, A.; Kai, T.; Yamashita, S.; Itoh, Y.; Yamashita, T. Biochim. Biophys. Acta 1985, 812, 587. (7) Baglioni, P.; Dei, L.; Gabrielli, G.; Innocenti, F. M.; Niccolai, A. Colloid Polym. Sci. 1988, 266, 783. (8) Pugelli M.; Gabrielli, G.; Domini, C. Prog. Colloid Polym. Sci. 1989, 79, 52. (9) Higashi, N.; Sunada, M.; Niwa, M. Langmuir 1995, 11, 1864.
S0743-7463(96)00161-8 CCC: $12.00
The aim of this contribution is to investigate the changes in the PGA molecular organization in the monolayer under the influence of mechanic forces. Therefore PGA monolayers (at aqueous acidic subphase) were compressed and expanded repeatedly. During these compression and expansion cycles the monolayer was observed by Brewster angle microscopy and ellipsometry. Brewster angle microscopy is a good tool to observe directly ordering processes in monolayers at the air/water interface.10,11 Changes of the layer thickness during the compression/ expansion cycles were detected by ellipsometry. During compression, the layer becomes optically more dense leading to an increase of the absolute value of the ellipticity. Motschmann et al.12 have calculated that this increasing in ellipticity is among other factors extremely sensitive to small changes of the layer thickness. The working hypothesis of molecular restructuring at the air/water interface derived from the experimental data is supported by the results from molecular modeling calculations. Because it is known that the behavior of polypeptides at interfaces depends on their degree of polymerization (DP),13 we also used PGA with different DP in the course of our investigations. Materials and Methods Poly(L-glutamic acid) (PGA). Poly(L-glutamic acid) sodium salts were purchased from Sigma Chemical Co. (St. Louis, MO). The samples used are shown in Table 1 (data from Sigma). Langmuir Film Formation and Characterization. PGA was dissolved in 0.01 M NaOH (double-distilled water, Milli-RQ plus 10; NaOH, Fixanal, Riedle-de Haen, Seelze, Germany) at a concentration of 1 mg/mL. 2-Propanol (5 vol%) (Sigma Chem. Co., 99+% purity, used without further purification) was added to this solution. PGA monolayers were formed by spreading this solution on the subphase by using a microsyringe (Hamilton, Bonaduz, Switzerland) until residual areas of 35 Å2/residue were attained. Double-distilled water of different pH ranging from 1.5 to 4.5 (adjustment by 1 M HCl, p.a., Riedle-de Haen) was (10) Overbeck, G. A.; Ho¨nig, D.; Mo¨bius, D. Langmuir 1993, 9, 555. (11) Weidemann, G.; Gehlert, U.; Vollhardt, D. Langmuir 1995, 11, 864. (12) Motschmann, H.; Reiter, R.; Lawall, R.; Duda, G.; Stamm, M.; Wegener, G.; Knoll, W. Langmuir 1991, 7, 2743. (13) Enser, M.; Bloomberg, G. B.; Brock, C.; Clark, D. C. Int. J. Biol. Macromol.1990, 12, 118.
© 1996 American Chemical Society
Compression and Expansion of PGA
Langmuir, Vol. 12, No. 26, 1996 6453
Table 1 degree of polymerization DP
av mol wt Mw
polydispersityc Mw/Mn
4a 7a 95b 115b 405b
600 1000 14300b 17500b 61200b
1.20 1.20 1.33
a From capillary electrophoresis. b From viscosity. c From LALLS.
used as subphase. The π/A isotherms were measured on a Teflon trough, 23 × 40 cm, equipped with a Wilhelmy plate system (filter paper 3 × 0.3 mm) and two movable motor-driven barriers (R+K, Mainz, Germany). The Wilhelmy plate was positioned exactly in the middle between the two barriers. The π/A isotherms measured for this paper have a reproducibility of ∆A ) (0.15Å2/ residue at π ) 5 mN/m and of ∆A ) (0.20 Å2/residue in the π range 10-20 mN/m. These were checked with π/A isotherms of three independent sample preparations. Variation of the time space (15 min, 16 h) between the spreading process and the beginning of the compression did not yield differences to these data. Therefore we have started the compression 15 min after spreading by all our π/A isotherm measurements for this paper. The compression and the expansion velocity, checked within a range from 0.05 to 5.00 Å2/(residue min), did not have any effect on the π/A isotherms either, and therefore we used a velocity of 1 Å2/(residue min) for all the following compression and expansion cycles. Each π/A isotherm showed in this paper was confirmed by two repeated measurements. Brewster-Angle Microscopy. A Brewster-angle microscope (BAM 1, Nanofilm Technologie GmbH, Go¨ttingen, Germany) was mounted on the trough in a perpendicular arrangement of the incidence plane with respect to the direction of barrier motion. For viewing and image storage, the microscope was combined with a CCD camera and a video system (recorder, printer, monitor). An area of 1 mm2 was observed at a distance of 5 cm from the trough boundary. The lateral resolution of the BAM was about 4 µm. Other experimental details of the Brewsterangle microscopy at the air/water interface are described by Ho¨nig and Mo¨bius.14 Ellipsometry. A Teflon trough, 14.3 × 24 cm, otherwise described above, was used for these studies. A conventional ellipsometer (Beaglehole Instr. Ltd., Wellington, New Zealand) was mounted in a custom-made vertical arrangement. Trough and ellipsometer were fixed on a heavy vibration-isolated table to provide a smooth subphase surface. A 5-mW HeNe laser, wavelength 633 nm, served as light source. The area of the incident beam on the surface was approximately 1 mm2. The ellipsometer setup and the method have been described in detail by Beaglehole.15 Additional Experimental Performance. The temperature was regulated by thermostats, in the subphase at 20 ( 0.1 °C and in the vapor phase above the trough at 20 ( 1 °C. Molecular Modeling. Molecular models of polypeptides containing 14 glutamic acid residues each in a standard R-helical conformation were constructed using the BIOPOLYMER module of the SYBYL software running on a SGI Indigo2 workstation. In order to allow for the monitoring of interhelical distances, the helices at a time were put together manually in four different geometrical organizations: planar side-by-side; planar interdigitated; folded side-by-side, and compressed folded interdigitated. Standard bond lengths and bond angles of the TRIPOS force field were employed for model building.
Results Formation of PGA Monolayers. The solubility of PGA in water increases with increasing pH. Therefore stock solutions of PGA were prepared by dissolving the polypeptide in 0.01 M NaOH. Aliquots of these stock solutions were then spread on acidic subphases of varying pH. The observed π/A isotherms gradually shift to higher (14) Ho¨nig, D.; Mo¨bius, D. J. Phys. Chem. 1991, 95, 4590. (15) Beaglehole, D. Physica 1980, 100B, 163.
Figure 1. Loss rate of PGA during the spreading process at different pH of the subphase: 0, DP405; 4, DP115; 3, DP95; O, DP7; ], DP4. T ) 20 °C.
areas/molecule, compared at a pressure π of 5 mN/m, with decreasing pH of the subphase until remaining almost identical between pH 1.5 and 3.0. During the spreading process of the basic stock solution on the acidic subphase, some of the PGA molecules diffuse into the bulk phase and are therefore not accessible to monolayer measurements. The number of PGA molecules diffusing into the subphase at pH < 2 is negligible, at pH ) 3.3 it is less than 8%, but it increases significantly at pH > 3.5 independent of the DP of the studied PGAs (Figure 1). On the basis of these findings the experiments were performed by spreading PGA on subphases in the pH range from 1.5 to 3.0. Compression/Expansion Hysteresis. During compression and expansion of the PGA layer, hysteresis was observed. Figure 2 shows for the PGA with DP 405 the very interesting behavior as revealed in the π/A isotherms upon repeated compression and expansion cycles. All compression curves and all expansion curves, respectively, have a common intersection point. We call this point iPπ/A following the example of the isosbestic point observed in absorption spectra. While the compression isotherms shift to smaller area values below iPπ/A and to higher values above iPπ/A in the course of the hysteresis experiments, the exact reciprocal behavior is observed for the expansion curves (Figure 2). The isobaric area values change in a complicated mode with increasing cycle number; different regions could be fitted by the kinetic law of the zero, the first, and the second order term, respectively (Figure 3). The pseudoplateau in the compression/expansion isotherms disappears gradually. The hysteresis occurs independently of the surface pressure as the change from compression to expansion was performed (Figure 2, inset) and is independent also of the compression and expansion velocity. Likewise the π/A isotherms of short chain PGA, for example PGA with DP7, show compression/expansion hysteresis (Figure 4). But these hysteresis curves are strongly shifted to smaller areas/residue, and a pseudoplateau does not exist in the isotherm.
6454 Langmuir, Vol. 12, No. 26, 1996
Reda et al.
Figure 2. Compression and expansion π/A isotherms of PGA with DP 405: subphase pH ) 3; speed of compression and expansion 1 Å2/(residue min); change from compression to expansion at π ) 22.5 mN/m (6 s of waiting time before expansion). The delay between consecutive cycles was 10 min: dark line, first cycle; light line, eighth cycle. The arrows indicate the direction of the barrier motion (compression or expansion). Inset: Change from compression to expansion at π ) 3.5 mN/m (s), 7 mN/m (- - -), 14.5 mN/m (- ‚ -), and 22.5 mN/m (‚‚‚).
Figure 3. Change of isobaric (0 during compression at 7 mN/ m; O, during expansion at 14 mN/m) areas in dependence of the cycle number: zero (s), first (‚‚‚), and second (---) order.
Chain-Length Dependence of the π/A Isotherm. Figure 5 shows the π/A isotherms of PGA with different DP. It looks as if the curve DP405 does not fit into this scheme. But that is not the case: DP4 and DP7 cannot construct helical molecules (see text p 8) like DP95, DP115, and DP405. DP95 and DP115 have similar π/A isotherms, divergent from DP405. But DP405 is the largest of the molecules and has the most β-turns also and therefore the helical molecule segments are intramolecular orientated to one another in the best way of the three long chain PGAs. On calculation of the compressibility from the data in Figure 5,
Ks ) -A(dπ/dA)T
(1)
and a plot of the result versus A, a clear separation between
short chain PGAs (DP4, DP7) and long chain PGAs (DP95, DP115, DP405) is obtained (Figure 5, inset), and DP405 fits into this scheme. Direct Visualization of Changes in the Monolayer Arrangement. Brewster angle microscopy was used to observe the monolayer structure during the consecutive compression/expansion cycles of PGA DP405. It appears that the monolayer is homogeneous during the compression of the first four cycles until the pseudoplateau region is reached. At this point, the inhomogeneities begin to develop. But the effect is too small, and representative pictures were not obtained. Likewise during the expansion the surface layer appears homogeneous. Already after four compression/expansion cycles solidified regions in the surface layer have formed which rupture apart in the direction of the moving barrier during expansion in the region π e 2 mN/m and A > 15 Å2/residue (Figure 6a,b). This process finally leads to the formation of PGA clods (Figure 6c,d). Figure 7 shows the ellipsometric data of the fifth compression/expansion cycle of the PGA layer. It is known that the ellipticity depends in a complex way on the layer thickness and the refractive indices in the x, y, and z direction of the surface layer. The ellipsometric measurements cannot be easily adapted to independently determine both the refractive indices and the film thickness for surface layers simultaneously. But on the assumption that the refractive indices of the surface layer do not change drastically during the measurement, the shown data are qualitatively proportional to the layer thickness. During the compression the transformations of the layer structure are accompanied by an increase in layer thickness. This thickness is not completely reduced on expansion but remains almost canstant below 10 Å2/ residue. Interestingly, sudden drops of the ellipticity to zero occur in the region greater as 13 Å2/residue. This finding is in excellent agreement with the Brewster angle microscopic pictures. During the expansion the rigid layer ruptures and PGA clods of constant thickness are formed. Ellipticity values of zero correspond to the bare water surface in between.
Compression and Expansion of PGA
Langmuir, Vol. 12, No. 26, 1996 6455
Figure 4. Compression and expansion π/A isotherms of PGA with DP7. Speed of compression and expansion, change from compression to expansion and waiting time between consecutive cycles (see the legend of Figure 2): (s) first cycle, (‚‚‚) third cycle. The arrows indicate the direction of the barrier motion.
Figure 5. π/A Isotherms of PGA with different DP: (s) DP405, (- - -) DP115, (- ‚ -) DP95, (- ‚‚ -) DP7, (‚‚‚) DP4; subphase-pH ) 1.4; speed of compression, 1 Å2/(residue min). Inset: Compressibility modulus (see eq 1 in the text) of the PGA isotherms in this figure.
Different Computer Modeled Helical Organizations. The geometrical features of PGA R-helices are presented in Figure 8. The width of a singular helix was measured to be 12.5 Å (Figure 8a). Depending on the force applied, associated helices can exist in a so-called side-by-side configuration, unfolded (Figure 8a) and folded (Figure 8c). If the external force is larger, helices may form an interdigitated configuration, also unfolded (Figure 8b) and folded (Figure 8d), which is characterized by much lesser volume per residue. The measured helical rod length of 12 residues (without C and N terminal end group) was 18.3 Å. Using these values the following areas/residue were calculated: planar side-by-side, 19.1 Å2; planar inter-
digitated, 15.3 Å2; folded side-by-side, 12.2 Å2; folded interdigitated, 10.2 Å2. Discussion It is well-known that PGA molecules spread on an acidic subphase of pH ) 1.5 form R-helical rods6 which lie flat on the water surface.4 Moreover the pronounced pseudoplateau in the compression π/A isotherm for the first cycle as seen in Figure 2 is characteristic for this organization.3,16 The PGA carboxyl side chain is protonated at pH ) 1.5 (pK ) 4.2517), which means that PGA is an uncharged poly(amino acid) in this state. For the interpretation of (16) Fasman, G. D. Mater Res Soc. Symp. Proc. 1991, 218, 49.
6456 Langmuir, Vol. 12, No. 26, 1996
Reda et al.
Figure 6. Brewster angle microscopy. Images a and b show the tearing of solidified random coil regions of the surface layer and images c and d show the following formation of clods during the expansion of the fifth compression/expansion cycle. Their positions in the expansion isotherm are (a) 2 mN/m, 14 Å2/residue; (b) 1 mN/m, 15 Å2/residue; (c) 0.7 mN/m, 18 Å2/residue; (d) 0.3 mN/m, 20 Å2/residue. Every picture segment is 650 × 650 µm; the darkness in the picture is the water; the arrow indicates the direction of the barrier motion.
our results documented in Figure 2 for the first compression π/A isotherm in the sense of our model calculations, three different steps must be assumed: The First Step. The available surface is gradually reduced and the helical rods are ordered until the surface is covered by helical PGA rods with their side chains just coming into contact. This complete occupation of the surface by the side-by-side molecular organization is attained at 18-21 Å2/residue, in good agreement with the result obtained from the computer model: 19.1 Å2/residue (Figure 8). The Second Step. Further reduction of the available area leads to an interdigitation of the side chains of the helical rods. This behavior is well-known.1 The computer model predicts an area of 15.3 Å2/residue for this organization. This value corresponds with the reversal point (17) Narath, A.; Gundlach, D. Photograph. Korrespondenz 1969, 105, 93.
in the measured π/A isotherm of the first cycle (15.5 Å2/ residue). It is remarkable that the measured maximum compressibility for all the three long chain PGAs (Figure 5, inset) is about 15.3 Å2/residue. The two short chain PGAs have significantly smaller compressibilities. That is in agreement with the fact that poly(amino acids) of DP < 15 units cannot form an R-helical conformation.18 Vice versa, the measured difference in the compressibilities between the long and the short chain PGAs is more evidence for the helical conformation of the spread long chain PGAs in this investigation. The Third Step. The dense 2D arrangement of the interdigitated helical rods evades a further pressure increase by folding of the monolayer into the third dimension. The computer simulation has obtained for this transition from the 2D to the 3D state with side-byside rods an area of 12.2 Å2/residue, corresponding to the (18) Mutter, M. Angew. Chem., Int. Ed. Engl. 1985, 24, 639.
Compression and Expansion of PGA
Langmuir, Vol. 12, No. 26, 1996 6457
Figure 7. Ellipsometric raw F-values during the fifth compression/expansion cycle. Change from compression to expansion at π ) 22.5 mN/m. The arrows indicate the direction of the barrier motion. At // the F value returns to zero.
pseudoplateau beginning of the experimental curve, and 10.2 Å2/residue for the organization with interdigitated rods, corresponding to the center of the pseudoplateau region (Figure 8). An increase of the layer thickness in the pseudoplateau region is known for other polypeptides also and has been demonstrated for poly(γ-glutamate esters)3,4 and polyalanine,19 for example. A layer-folding, comparable with that in our experiment, has been shown for polylysine already.1 In the compression/expansion cycles a hysteresis was observed. Their occurrence can be attributed to intermolecular H-bridge interaction between the PGA molecules. The rapid drop at the beginning of the expansion indicates a high elasticity of the surface layer. If the same monolayer is repeatedly compressed and expanded, a conversion of the helical surface layer occurs and is finished after about eight cycles. This conversion process can be characterized by the so-called isosbestic point of the isotherms. The alteration of the isobaric area values (Figure 3) are to describe with the zero order fit from the first up to the fourth cycle (the solid line); and it results in a more densely packed and less compressible layer. What kind of conversion could this be? (1) The helical conformation was preserved, but the helical rods have set up from the planar in the perpendicular direction within the compression cycles, or (2) the transition from the folded helical state toward the random coil state takes place, step by step within the eight cycles, because the hydrogen bonds that stabilize the helical structure were broken by the mechanical forces during the repeated compression/expansion process. There is something to be said for this: In the random coil state the polypeptide molecules have probably a spheroidal shape. The area occupied by these speroidal molecules at the surface is smaller than that of the helical rods lying flat on the surface. Therefore the PGA layer in the random coil conformation must be more densely packed and less compressible than the layer of flat lying helical rods. This could explain the measured smaller area value below and the greater area value above the iPπ/A of the curves in Figure 2 with increasing random coil content compared with the monolayer of exclusively helical molecules. All (19) Gabrielli, G.; Baglioni, P.; Ferroni, E. J. Colloid Interface Sci. 1981, 81, 139.
hysteresis cycles of PGA, DP7, show this more densily packed and less compressible π/A isotherm type only, point to random coils, because the short chain PGA cannot develop the helical secondary structure. The shift of the π/A isotherms to the smaller area/residue region in comparison with the long chain PGAs could have a simple geometric reason: The random coils are spheroidal rather than spherical. Clearly a perpendicular to the surface orientated spheroid of the extremly short chain PGA with DP7 has a smaller occupied area than that possible by PGA with DP405. Futhermore, if the conversion product was more and more dominant (above the fifth cycle) the kinetics of decreasing isobaric molecule areas by compression at repeated cycles can be fitted by the secondorder term in reasonable approximation to the measured values (Figure 3, broken line), directing to a network forming by bimolecular interactions. The kinetics of the increasing isobaric molecule areas at the corresponding expansion can be fitted to conform with the measured values by the first-order term (dotted line). That indicates that molecules interlock together probably more as the helical rods. Stronger solidified regions as the regions of helical rods result in the surface layer. During the expansion these do not react in an isolated way, but the rigid layer ruptures partially with the retention of its thickness and clods can be observed in the region π e 2 mN/m by Brewster angle microscopy. All these facts indicate a helix/ coil transition, and therefore direct measurements in situ at the interface could be very fascinating, by IRRAS for example. But direct measurements are not easy and must be reserved for following investigations. Conclusions The experimental results allow some assertions about the conformation and the conformational changes of PGA helical rods at the air/water interface during compression and expansion of the surface layer. The experimentally measured molecular areas are in good agreement with values derived from computer-modeled side-by-side or interdigitated organizations of helical rods. The helical rods of PGA at the interface form a high elastic layer, which is sensitive to compression and expansion. A more rigid layer results and ruptures during expansion. These results are important for further experiments in situ
6458 Langmuir, Vol. 12, No. 26, 1996
Reda et al.
Figure 8. Computer-modeled PGA R-helical organizations: (a, top left) side view of the standard helix, all side chains are assumed to exist in extended conformations (color code: C, white; H, cyan; O, red; N, blue); (b, top right) side view of two associated helices with an interdigitated configuration; (c, d) top view of a set of three associated helices in side-by-side (c, bottom left) as well as interdigitated (d, bottom right) configuration.
with the PGA model layer at the interface, like penetrations, enzymological studies and interactions with lipids or dyes. Acknowledgment. T.R. thanks Thomas Pfohl and Volkar Melzer for helpful assistance and discussion of the
ellipsometric and Brewster angle measurements, respectively. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) and the Fonds der Chemischen Industrie. LA960161+
