31
Advances in Colloid and Interface Science, 34 (1991) 31-72 Elsevier Science Publishers B.V., Amsterdam
MONOLAYERS
AND PLANAR
OR CURVED BILAYERS
G. GABRIELLI Department
of Chemistry,
University of Florence, Via Gino Capponi 9, Florence (Italy)
CONTENTS Abstract
31
........................
32
1.
Introduction
2.
Experimental Methods Used ................ 2.1. Monolayers .................... 2.2. Planar bilayers .................. 2.3. Curvedbilayers ..................
33 33 33 34
3.
Results and Discussion .................. ....... 3.1. Two-dimensional systems made up of polypeptides ......... 3.1.1. Monolayers with only one component ............ 3.1.2. Two-component monolayers 3.2. Two-dimensional systems made of lipids and polypeptides ............. 3.2.1. Single component systems 3.2.2. Systems made of two compounds, one lipidic and one polypeptidic ................... ....... 3.3. Two-dimensional systems with lipidic components .......... 3.3.1. Systems with a lipidic component ......... 3.3.2. Systems with two lipidic components
34 34 35 41 45 45
4.
.....................
Conclusions
References
......................
.
.
,
.
51 57 57 62 69 70
.......................
ABSTRACT Monolayers and planar or curved bilayers can be considered useful models of biologic membranes. With this in perspective we report on studies performed on monolayers and bilayers of polypeptides and lipids. The results obtained show that the study of monolayers allows either the deduction of the interphasal orientation in a one-component system or the reasons of mutual solubility when mixtures are considered. In the case of bilayers and particularly of BLM, LB films and vesicles, it is possible to gain information about stability and thickness of single component bilayers as well as about the influence of the addition of a second component on these properties. The comparison between the various systems taken into consideration allows us to draw a first conclusion: the condition to form BLM and vesicles is to start from monolayers in expanded phases while the condition to form plurilayers and LB films is to start from monolayers in condensed phases. OcKll-8686/9l/$5.60
0 1991-
Elsevier Science Publishers B.V.
32 1. INTRODUCTION
It is well known that many amphiphilic substances are able to give orientated systems, among them mono- and bi-layers. All these systems may be considered useful models of natural membranes, especially if the amphiphilic compounds are constituents or models of compounds actually present in the membranes. Membranes are, in fact, formed, as may be seen from the most commonly accepted model [ll, of a bilayer of lipidic compounds in which the other components such as proteins and enzymes are immersed or bound to the two interfaces. Moreover, comparison between the various above mentioned orientated systems is seen to be particularly useful not only for establishing their representativity, but also to gain complementary information from the study of each one of them, and finally it is possible with mimetic chemistry to construct membranes possessing at least some of the extraordinary properties of natural membranes. In view of this, studies were carried out on the following systems: (1) Monolayers of lipidic compounds and polypeptidic compounds having one or more components of the same or of a different type. Both these classes of compounds may in fact be considered simple and efficient models of the lipid and protein substances which make up each monomolecular layer of the biological membranes. Thus, the study of monolayers both of one component and of mixtures 121may constitute a useful means of establishing both the interfacial distribution of the components and the possible interactions among them in the two-dimensional state. (2) Planar bilayers which can be of two fundamental types, i.e., black lipid membranes (BLM) 131,which are among the most used and studied models of biological membranes, and Langmuir-Blodgett bilayers (LB), which are monolayers transferred from the liquid to the solid support and built up [4]; these are generally used in multilayers for the preparation of special materials, but may also constitute useful and easily studied models of biological membranes. (3) Curved bilayers, these are called liposomes if they consist of actual membrane constituents, or vesicles if made up of synthetic compounds 151, which are often models of natural amphiphilic systems, but are generally more stable and easily characterized. The studies carried out concern both different substances in different systems, and the comparison of the same amphiphiles in different systems in order to deduce the formation conditions of the various mono- and
33
bimolecular aggregates and also the possible different distributions and interactions in the various systems. In this study the experimental techniques used will be briefly described, a more detailed description of the working procedures can be found in a previous study; a wider description will, on the other hand, be given of the most important results obtained, above all the observations and the comparisons which may be made from them. 2. EXPERIMENTAL
METHODS USED
2.1. Monolayers For the study of monolayers consisting of one or more components the spreading isotherms were determined by the Langmuir method, using a modified Lauda Filmwaage Balance interfaced to a Digital PDPl1/23 computer which has been described previously 161, or with the Wilhelmy method using a Mettler AE163 balance, also previously described [71. The isotherms were obtained by discontinuous compression, so that an equilibrium state was ensured for each area value and thus for each surface concentration. For monolayers on liquid supports the surface potential was also measured with the ionizing electrode method, using apparatus which essentially consisted of two Am (221Am)electrodes [7,81. Ellipsometric measurements were taken on the transferred monolayers with a D2112 ellipsometer (Rudolph Research), where a 3L= 6320 laser was the monochromatic source 191,and MIR spectra were recorded using a germanium plate and a Perkin Elmer Spectrophotometer model 580 E 1101. The transfer of the monolayers was performed using the LB method (111 using a Joyce Loebl Langmuir Trough Model 4 instrument which allows different extraction speeds and the constancy of surface pressure during the transfer. Finally, electron micrographs using a JSM-13 electron microscope were taken of the monolayers which had been collapsed and transferred onto special supports according to a previously described procedure [121. 2.2. Planar bilayers For BLM preparation, the Mueller et al. method was used [13l. This consists essentially of depositing a solution in suitable solvents of the amphiphile or mixture whose membrane we wish to construct into a hole of
34
-1 mm diameter. The hole is placed in a Teflon partition separating two 0.1 MNaCl solutions or other solutions of electrolytes. After having checked the formation of this, the capacitance and conductance through the membrane are measured by means of special circuits. For the preparation of LB multilayers, the previously-reported technique was used; the transfer was onto different solid supports, mainly quartz or glass plates, the latter being covered with a layer of Cr which was considered thick and which was deposited under vacuum. 2.3. Curved bilayers For liposome or vesicle preparation sonication at suitable temperatures was generally used, the instrument being a Sonifier B-2 Cell Disruptor (Branson Sonic Power Co., Danbury, U.S.A.); for their separation an ultracentrifuge was used [141. For vesicle characterization the following procedure was adopted: electronic micrographs with an SEM-515 Philips scanning microscope; adsorption measurements at set wavelengths; light-scattering measurements for the control of the mean diameter and size distribution of the aggregates with a Bookhaven apparatus. Moreover, for the determination of some interesting properties of the vesicles, various methods were used, the main ones being: Osmotic shock to control the osmometric properties of the curved bilayers by means of turbidity measurements with a Perkin Elmer Lambda 5UV/VI5 Spectrometer 1141; EPR probe insertions and recording of the spectra with a Bruker model 200D spectrometer operating in the X-band, about 9.7 GHz in order to get information on the mobility of the hydrophobic chains making up the lipid double layer. 3. RESULTS AND DISCUSSION
We shall examine the different results obtained separately. 3.1. Two-dimensional systems made up ofpolypeptides The two-dimensional systems studied in this case are only monolayers, because of the evident unlikelihood of obtaining bilayers like BLM or vesicles with polypeptidic components alone and also because of the lack of representativity that these systems would have in relation to natural membranes, which are considered to consist fundamentally of a double
35
lipidic layer. However, the interest of polypeptidic monolayers lies in the fact that they can be considered as simple and representative models of proteins, and thus their inter-facial distribution and the reciprocal interactions may be considered comparable to the corresponding ones in systems with protein components. 3.1.1. Monolayer-s with only one component It has been demonstrated for monolayers made up of only one polypeptide component that it is possible to obtain stable two-dimensional layers made up of different macromolecular forms of the same.polypeptide, in particular a-helix, F and random-coil forms. The study of the conditions required for obtaining different macromolecular forms and of the corresponding phase transitions takes on a particular importance also for a comparison between two-dimensional and three-dimensional phases and thus for the determination of the role that a surface (in our case generally the liquid/air interface) has in maintaining or modifying the macromolecular conformation present in the bulk phase. The presence at this interface of the CI-and p-forms of the same polypeptide depends on the spreading solvent used for the preparation of the monolayer, i.e., in this case the interphase does not modify the macromolecular form which is present in the bulk solution. To be more precise, if the spreading solvent does not contain a basic substance like pyridine, and is, for example, made up only of chloroform, spreading isotherms are obtained which are seen to correspond to monolayers formed of a-helixes, as has been demonstrated in various cases [10,15-171. Figure 1 shows the spreading isotherms obtained in this way at 25°C for three poly(amino acids), poly-y-methylglutamate (PyMG), poly-y-benzylglutamate (PyBG) and poly-L-alanine (PLA). All three isotherms clearly show an arrest or inflection in surface pressure which we and other authors I191 have attributed, not to a first-order two-dimensional phase transition, but to a particular type of collapse, i.e., a passage from monoto bilayer, which is typical or rigid forms like the a-helix. The MIR spectra shown for the three polypeptides in Fig. 2, which were identical for monolayers transferred at lower and higher ressures than -Y the arrest pressure, present bands at 1650 and 1550 cm which are attributable to a-helix forms and rule out the ascribability of the arrest, for example, to an a-(3 transition, since the latter form presents different adsorption bands which are clearly distinguishable from the above bands. Finally, the ellipsometric measurement of the thickness at lower and higher pressures than at arrest conformed beyond all doubt the formation
36
n(mN/m)
A (m2/mg) Fig. 1. Spreading isotherms of monolayers of PTBG, PyMG and PLA at 25’C obtainedfrom spreading solvent without pyridine.
r-
PrMG
1700
cm1
PLA
1500
1700
cm1
1500
PrBG
1700
cm’
1500
Fig. 2. MIR spectra of transferred monolayers obtained from spreading solvent without pyridine for PrMG, PLA and P-yBG.
37
of a bilayer corresponding to the point of arrest; in fact, in the case of the PLA, the ellipsometric thickness of about 5 A in the monolayer area is seen to double in the collapsed film area 1181,for PyMG the corresponding thicknesses are about 9 and 19 A [201. The fact that the formation of a bilayer may consist, in equilibrium state conditions, both of an arrest or an inflection, i.e., part of the linear trace which is not a plateau, may be attributed to the different flexibility and length of the side chain, as reported by other authors 1211. In the case of PLA the section of the linear trace which is not parallel to the z axis corresponds to -9 mN m-l, which when multiplied by the limit residue area, -13.8 A2/residue, gives an energy of -180 cal [181. This value is comparable to that obtained by Malcolm [211 and attributed by the author to a difference in the interaction energy between parallel and antiparallel disposition in the monolayer. The @-forms are obtained when the spreading solvent is rich in pyridine t10,15,221. Figure 3 shows the isotherms at 20°C for the monolayers obtained by this solvent in the case of two polypeptides, PyMG and poly-P_benzil aspartate
n (mN/m)
25 _
0.5
1.0 A(m2/mg)
Fig. 3. Spreading isotherms (2O’C) of monolayers of P(3BA and PyMG obtained from solvent containing pyridine.
38
(PPBA). In both cases it can be seen that the isotherms show no arrest which would be characteristic of a bilayer formation and the MIR spectra shown in Fig. 4 present adsorption bands at 1625 and 1520 cm-l, which are characteristic of the @forms. A further confirmation of the differences between the forms obtained with the two spreading solvents may be derived from the parameters obtained with the comparison of the experimental isotherms with Huggins’ theory applied to two-dimensional states [231. It has in fact been demonstrated several times [9,16,24,2!51 that theoretical isotherms may be calculated by means of the equation of two-dimensional state proposed by Huggins [231, which is deduced by statistical thermodynamics, considering the monomolecular layer as a two-dimensional solution. In the case of the polymers in question, the agreement between the experimental and the calculated findings is very close L5,16,221, as in the case of other polypeptides [26]. It is thus possible to establish some characteristics of interfacial
1700
1600
1500
-PyMG - -- PpBA Fig. 4. MIR spectra of transferred monolayers of PyMG and PflBA obtained from spreading solvent. containing pyridine.
39
TABLE 1 Parameters of monolayers of PyMG Spreading solvent without pyridine 288K 298K La Hb L” Hb -3.0 4.0 K 0.50 &A10’ ergs loo0
+ K’
-29.6 9.4 0.30 10900
-3.2 4.4 0.48 4000
-24.5 9.8 0.29 16500
Spreading solvent with pyridine 288K 298 K La Hb La Hb -2.9 3.0 0.57 100
-30.8 7.2 0.36 1900
-3.1 3.2 0.56 40
-28.9 7.2 0.36 1600
%ow surface pressure bHigh surface pressure TABLE 2 Parameters of monolayer of PgBA
* K’ K
eAlO’erg
Spreading solvent without pyridine 288K 298K La Hb L” Hb
Spreading solvent with pyridineC 288-298 K
-10.26 9.0 0.31 4411
-26.0 10.2 0.28 -445
-42.72 12.8 0.24 23090
-12.47 10.2 0.28 4428
-49.0 13.8 0.22 23238
*Low surface pressure. bHigh surface presure. ‘The values are valid for all pressures.
polymeric distribution. These are given in Tables 1 and 2 for two polypeptides at two temperatures. To make a comparison of the theoretical data with the experimental data as close as.possible, it was necessary to consider two pressure areas, one lower and one higher than 2 dyn cm-‘, as has often been noted for other polymers P6,27l. The following parameters are shown in the Tables: (1) I), which represents the Gibbs free surface energy and which is negative for all the forms and all the temperatures considered. This indicates the stability of the monolayers in each case, the reproducibility of the isotherms being a further confirmation. (2) K represents an equilibrium constant between the various types of contact: polymer/polymer, polymer/substrate and substrate/substrate.
40
The values below 1 in all cases show that the distribution is not random and that the polymer/polymer contacts are more probable than those between different segments. The relationship of K’ to K is: K’ = 4(1/K - 1) and thus K’ has the same significance. The higher K values for the forms obtained from a spreading solvent with pyridine are a preliminary indication of a more diffuse from with more interactions with the subphase. (3) &Ais an energy parameter proportional to hE: AE = 2~~~ - E, - tzPP are the mean energies per unit of length of contact for where E+., E,, EMU polymer/polymer (a/a), polymer/support (a/(3)and support/support
Advances in Colloid and Interface Science, 34 (1991) 31-72 Elsevier Science Publishers B.V., Amsterdam
MONOLAYERS
AND PLANAR
OR CURVED BILAYERS
G. GABRIELLI Department
of Chemistry,
University of Florence, Via Gino Capponi 9, Florence (Italy)
CONTENTS Abstract
31
........................
32
1.
Introduction
2.
Experimental Methods Used ................ 2.1. Monolayers .................... 2.2. Planar bilayers .................. 2.3. Curvedbilayers ..................
33 33 33 34
3.
Results and Discussion .................. ....... 3.1. Two-dimensional systems made up of polypeptides ......... 3.1.1. Monolayers with only one component ............ 3.1.2. Two-component monolayers 3.2. Two-dimensional systems made of lipids and polypeptides ............. 3.2.1. Single component systems 3.2.2. Systems made of two compounds, one lipidic and one polypeptidic ................... ....... 3.3. Two-dimensional systems with lipidic components .......... 3.3.1. Systems with a lipidic component ......... 3.3.2. Systems with two lipidic components
34 34 35 41 45 45
4.
.....................
Conclusions
References
......................
.
.
,
.
51 57 57 62 69 70
.......................
ABSTRACT Monolayers and planar or curved bilayers can be considered useful models of biologic membranes. With this in perspective we report on studies performed on monolayers and bilayers of polypeptides and lipids. The results obtained show that the study of monolayers allows either the deduction of the interphasal orientation in a one-component system or the reasons of mutual solubility when mixtures are considered. In the case of bilayers and particularly of BLM, LB films and vesicles, it is possible to gain information about stability and thickness of single component bilayers as well as about the influence of the addition of a second component on these properties. The comparison between the various systems taken into consideration allows us to draw a first conclusion: the condition to form BLM and vesicles is to start from monolayers in expanded phases while the condition to form plurilayers and LB films is to start from monolayers in condensed phases. OcKll-8686/9l/$5.60
0 1991-
Elsevier Science Publishers B.V.
32 1. INTRODUCTION
It is well known that many amphiphilic substances are able to give orientated systems, among them mono- and bi-layers. All these systems may be considered useful models of natural membranes, especially if the amphiphilic compounds are constituents or models of compounds actually present in the membranes. Membranes are, in fact, formed, as may be seen from the most commonly accepted model [ll, of a bilayer of lipidic compounds in which the other components such as proteins and enzymes are immersed or bound to the two interfaces. Moreover, comparison between the various above mentioned orientated systems is seen to be particularly useful not only for establishing their representativity, but also to gain complementary information from the study of each one of them, and finally it is possible with mimetic chemistry to construct membranes possessing at least some of the extraordinary properties of natural membranes. In view of this, studies were carried out on the following systems: (1) Monolayers of lipidic compounds and polypeptidic compounds having one or more components of the same or of a different type. Both these classes of compounds may in fact be considered simple and efficient models of the lipid and protein substances which make up each monomolecular layer of the biological membranes. Thus, the study of monolayers both of one component and of mixtures 121may constitute a useful means of establishing both the interfacial distribution of the components and the possible interactions among them in the two-dimensional state. (2) Planar bilayers which can be of two fundamental types, i.e., black lipid membranes (BLM) 131,which are among the most used and studied models of biological membranes, and Langmuir-Blodgett bilayers (LB), which are monolayers transferred from the liquid to the solid support and built up [4]; these are generally used in multilayers for the preparation of special materials, but may also constitute useful and easily studied models of biological membranes. (3) Curved bilayers, these are called liposomes if they consist of actual membrane constituents, or vesicles if made up of synthetic compounds 151, which are often models of natural amphiphilic systems, but are generally more stable and easily characterized. The studies carried out concern both different substances in different systems, and the comparison of the same amphiphiles in different systems in order to deduce the formation conditions of the various mono- and
33
bimolecular aggregates and also the possible different distributions and interactions in the various systems. In this study the experimental techniques used will be briefly described, a more detailed description of the working procedures can be found in a previous study; a wider description will, on the other hand, be given of the most important results obtained, above all the observations and the comparisons which may be made from them. 2. EXPERIMENTAL
METHODS USED
2.1. Monolayers For the study of monolayers consisting of one or more components the spreading isotherms were determined by the Langmuir method, using a modified Lauda Filmwaage Balance interfaced to a Digital PDPl1/23 computer which has been described previously 161, or with the Wilhelmy method using a Mettler AE163 balance, also previously described [71. The isotherms were obtained by discontinuous compression, so that an equilibrium state was ensured for each area value and thus for each surface concentration. For monolayers on liquid supports the surface potential was also measured with the ionizing electrode method, using apparatus which essentially consisted of two Am (221Am)electrodes [7,81. Ellipsometric measurements were taken on the transferred monolayers with a D2112 ellipsometer (Rudolph Research), where a 3L= 6320 laser was the monochromatic source 191,and MIR spectra were recorded using a germanium plate and a Perkin Elmer Spectrophotometer model 580 E 1101. The transfer of the monolayers was performed using the LB method (111 using a Joyce Loebl Langmuir Trough Model 4 instrument which allows different extraction speeds and the constancy of surface pressure during the transfer. Finally, electron micrographs using a JSM-13 electron microscope were taken of the monolayers which had been collapsed and transferred onto special supports according to a previously described procedure [121. 2.2. Planar bilayers For BLM preparation, the Mueller et al. method was used [13l. This consists essentially of depositing a solution in suitable solvents of the amphiphile or mixture whose membrane we wish to construct into a hole of
34
-1 mm diameter. The hole is placed in a Teflon partition separating two 0.1 MNaCl solutions or other solutions of electrolytes. After having checked the formation of this, the capacitance and conductance through the membrane are measured by means of special circuits. For the preparation of LB multilayers, the previously-reported technique was used; the transfer was onto different solid supports, mainly quartz or glass plates, the latter being covered with a layer of Cr which was considered thick and which was deposited under vacuum. 2.3. Curved bilayers For liposome or vesicle preparation sonication at suitable temperatures was generally used, the instrument being a Sonifier B-2 Cell Disruptor (Branson Sonic Power Co., Danbury, U.S.A.); for their separation an ultracentrifuge was used [141. For vesicle characterization the following procedure was adopted: electronic micrographs with an SEM-515 Philips scanning microscope; adsorption measurements at set wavelengths; light-scattering measurements for the control of the mean diameter and size distribution of the aggregates with a Bookhaven apparatus. Moreover, for the determination of some interesting properties of the vesicles, various methods were used, the main ones being: Osmotic shock to control the osmometric properties of the curved bilayers by means of turbidity measurements with a Perkin Elmer Lambda 5UV/VI5 Spectrometer 1141; EPR probe insertions and recording of the spectra with a Bruker model 200D spectrometer operating in the X-band, about 9.7 GHz in order to get information on the mobility of the hydrophobic chains making up the lipid double layer. 3. RESULTS AND DISCUSSION
We shall examine the different results obtained separately. 3.1. Two-dimensional systems made up ofpolypeptides The two-dimensional systems studied in this case are only monolayers, because of the evident unlikelihood of obtaining bilayers like BLM or vesicles with polypeptidic components alone and also because of the lack of representativity that these systems would have in relation to natural membranes, which are considered to consist fundamentally of a double
35
lipidic layer. However, the interest of polypeptidic monolayers lies in the fact that they can be considered as simple and representative models of proteins, and thus their inter-facial distribution and the reciprocal interactions may be considered comparable to the corresponding ones in systems with protein components. 3.1.1. Monolayer-s with only one component It has been demonstrated for monolayers made up of only one polypeptide component that it is possible to obtain stable two-dimensional layers made up of different macromolecular forms of the same.polypeptide, in particular a-helix, F and random-coil forms. The study of the conditions required for obtaining different macromolecular forms and of the corresponding phase transitions takes on a particular importance also for a comparison between two-dimensional and three-dimensional phases and thus for the determination of the role that a surface (in our case generally the liquid/air interface) has in maintaining or modifying the macromolecular conformation present in the bulk phase. The presence at this interface of the CI-and p-forms of the same polypeptide depends on the spreading solvent used for the preparation of the monolayer, i.e., in this case the interphase does not modify the macromolecular form which is present in the bulk solution. To be more precise, if the spreading solvent does not contain a basic substance like pyridine, and is, for example, made up only of chloroform, spreading isotherms are obtained which are seen to correspond to monolayers formed of a-helixes, as has been demonstrated in various cases [10,15-171. Figure 1 shows the spreading isotherms obtained in this way at 25°C for three poly(amino acids), poly-y-methylglutamate (PyMG), poly-y-benzylglutamate (PyBG) and poly-L-alanine (PLA). All three isotherms clearly show an arrest or inflection in surface pressure which we and other authors I191 have attributed, not to a first-order two-dimensional phase transition, but to a particular type of collapse, i.e., a passage from monoto bilayer, which is typical or rigid forms like the a-helix. The MIR spectra shown for the three polypeptides in Fig. 2, which were identical for monolayers transferred at lower and higher ressures than -Y the arrest pressure, present bands at 1650 and 1550 cm which are attributable to a-helix forms and rule out the ascribability of the arrest, for example, to an a-(3 transition, since the latter form presents different adsorption bands which are clearly distinguishable from the above bands. Finally, the ellipsometric measurement of the thickness at lower and higher pressures than at arrest conformed beyond all doubt the formation
36
n(mN/m)
A (m2/mg) Fig. 1. Spreading isotherms of monolayers of PTBG, PyMG and PLA at 25’C obtainedfrom spreading solvent without pyridine.
r-
PrMG
1700
cm1
PLA
1500
1700
cm1
1500
PrBG
1700
cm’
1500
Fig. 2. MIR spectra of transferred monolayers obtained from spreading solvent without pyridine for PrMG, PLA and P-yBG.
37
of a bilayer corresponding to the point of arrest; in fact, in the case of the PLA, the ellipsometric thickness of about 5 A in the monolayer area is seen to double in the collapsed film area 1181,for PyMG the corresponding thicknesses are about 9 and 19 A [201. The fact that the formation of a bilayer may consist, in equilibrium state conditions, both of an arrest or an inflection, i.e., part of the linear trace which is not a plateau, may be attributed to the different flexibility and length of the side chain, as reported by other authors 1211. In the case of PLA the section of the linear trace which is not parallel to the z axis corresponds to -9 mN m-l, which when multiplied by the limit residue area, -13.8 A2/residue, gives an energy of -180 cal [181. This value is comparable to that obtained by Malcolm [211 and attributed by the author to a difference in the interaction energy between parallel and antiparallel disposition in the monolayer. The @-forms are obtained when the spreading solvent is rich in pyridine t10,15,221. Figure 3 shows the isotherms at 20°C for the monolayers obtained by this solvent in the case of two polypeptides, PyMG and poly-P_benzil aspartate
n (mN/m)
25 _
0.5
1.0 A(m2/mg)
Fig. 3. Spreading isotherms (2O’C) of monolayers of P(3BA and PyMG obtained from solvent containing pyridine.
38
(PPBA). In both cases it can be seen that the isotherms show no arrest which would be characteristic of a bilayer formation and the MIR spectra shown in Fig. 4 present adsorption bands at 1625 and 1520 cm-l, which are characteristic of the @forms. A further confirmation of the differences between the forms obtained with the two spreading solvents may be derived from the parameters obtained with the comparison of the experimental isotherms with Huggins’ theory applied to two-dimensional states [231. It has in fact been demonstrated several times [9,16,24,2!51 that theoretical isotherms may be calculated by means of the equation of two-dimensional state proposed by Huggins [231, which is deduced by statistical thermodynamics, considering the monomolecular layer as a two-dimensional solution. In the case of the polymers in question, the agreement between the experimental and the calculated findings is very close L5,16,221, as in the case of other polypeptides [26]. It is thus possible to establish some characteristics of interfacial
1700
1600
1500
-PyMG - -- PpBA Fig. 4. MIR spectra of transferred monolayers of PyMG and PflBA obtained from spreading solvent. containing pyridine.
39
TABLE 1 Parameters of monolayers of PyMG Spreading solvent without pyridine 288K 298K La Hb L” Hb -3.0 4.0 K 0.50 &A10’ ergs loo0
+ K’
-29.6 9.4 0.30 10900
-3.2 4.4 0.48 4000
-24.5 9.8 0.29 16500
Spreading solvent with pyridine 288K 298 K La Hb La Hb -2.9 3.0 0.57 100
-30.8 7.2 0.36 1900
-3.1 3.2 0.56 40
-28.9 7.2 0.36 1600
%ow surface pressure bHigh surface pressure TABLE 2 Parameters of monolayer of PgBA
* K’ K
eAlO’erg
Spreading solvent without pyridine 288K 298K La Hb L” Hb
Spreading solvent with pyridineC 288-298 K
-10.26 9.0 0.31 4411
-26.0 10.2 0.28 -445
-42.72 12.8 0.24 23090
-12.47 10.2 0.28 4428
-49.0 13.8 0.22 23238
*Low surface pressure. bHigh surface presure. ‘The values are valid for all pressures.
polymeric distribution. These are given in Tables 1 and 2 for two polypeptides at two temperatures. To make a comparison of the theoretical data with the experimental data as close as.possible, it was necessary to consider two pressure areas, one lower and one higher than 2 dyn cm-‘, as has often been noted for other polymers P6,27l. The following parameters are shown in the Tables: (1) I), which represents the Gibbs free surface energy and which is negative for all the forms and all the temperatures considered. This indicates the stability of the monolayers in each case, the reproducibility of the isotherms being a further confirmation. (2) K represents an equilibrium constant between the various types of contact: polymer/polymer, polymer/substrate and substrate/substrate.
40
The values below 1 in all cases show that the distribution is not random and that the polymer/polymer contacts are more probable than those between different segments. The relationship of K’ to K is: K’ = 4(1/K - 1) and thus K’ has the same significance. The higher K values for the forms obtained from a spreading solvent with pyridine are a preliminary indication of a more diffuse from with more interactions with the subphase. (3) &Ais an energy parameter proportional to hE: AE = 2~~~ - E, - tzPP are the mean energies per unit of length of contact for where E+., E,, EMU polymer/polymer (a/a), polymer/support (a/(3)and support/support
