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Monolayers of poly(L,D, and DL)alanine and their mixtures with arachidic acid
ColfoHsandSurf~e~ 3(19813147-167 E&e&r Scientitic ~blishing Company. Amrterdun - Rink in Belgium
AND THEIR
MONOLAYERS OF POLY(L, D, AND DL)ALANINE MIXTURES WFFH ARACHIDIC ACID GABRIBLLA
GABRIELLI,
PIER0
InnrZitute of Physical Chemirtxy. Firenze (Italy) (Received August
27th.
BAGLIONI
The Uniwrsity
XS80;rccepted
and ANNA of Florence.
147
FABBRINI Wa 0.
in final form Deamber
Capponi 9. SO 121
lfatb, 1980)
ABSTRAGT Monchyers of poXy(L, D. DL]rlmfne were rtudied on rub&=&8 containing Ua’+, Na+,andK* and in mixtures with arachidic acIdd. Appticatton 05 the Lheoxy of Huggins shows that tte most strbte-metic form,froma thermodynamk otmdpaint,IstheL
fom, while the feut themtodynlmkdly atrble t the DL ape&s. It has ken SIZII that poIy(L znd D)olmhtne, which arc present at the interpha#c aa u helix-, rhow preterentlal inttractbn8 between the polymerfc chalq while poly(DLwanine, whfch Is presentin the8 confomatkn, rhowspreferential interactiona with the support. The presence of the Ca*’ Ions Increases considerably the intemctionr between the polymer and the rub&ate. Mixtures of the three isomers ahaw incompatibility whkh arachidic acid even if interactiona are present between the lipIdi= chain8 and them~eramofeculu micdes in the “bad fit” zoner of the latter.
INTRODUCXION Mixed monolayers of compounds of low and high mokcuIar weights, as we know, have particular importance both from a theoretical and applied Vi@wpOillL
In earlier wclrks [l] bidimensional systems were studied having two macromokcu~ compounds ILLconstituents or a compound of low molecular weight and a poIymeric compound, and the possibility of arriving at reciprocal compatibility of the components, as well as the principal parameters that determine it, was shown, In this context, the study of mixed monolayers of polypeptides and lipidic compounds, which are considered usefuI models of biological membranes, has shown that the reciprocal compatibility of the components in the monobyer can be modified by vztqing the composition of the support [2]. The aim of the present work is to study the modifications that ions present in the support can induce in the configuration and the distribution at the interphase of the Wee isomers (L, D, DL) of the same poIyaminoacid, and in the soXubilityof these isomers in the bidimensionsl state with a lipidic compound selected ss a model, viz., a fatty carboxylic acid. 0166-662213~~066~660/$62.S0 ~1981 Elsetier ScIentificPubthMngtimpany
148
For this purpose a particularly simple poIyaminoacid was selected which can give stable bidimensional films at the waterjair intcerphase and, for the support, were select4?dCay*, Na* and K*, which are particularly interesting from the point of view of monolayers considered as models of biologic membranes. Finally, the lipidic compound selected was arachidic acid, whose bidimensional mixtuxcs with other polypepticies had been studied previously IS], and which gives monolayers at the,wtter/air intcrphsse with phases and orientation that are well known and defilked. EXPERIMENTAL
Po,ly(DL)alanine MW = 3900 lot number AL 69 and poly(L)alsnine MW = 3000 lot number AL 44 were furnished by Miles Ltd. Israel: poly(D)alanine MW = 3000 was furnished bs_Sigma Chemie GmbH, Munich, West Germany, The average molecular weight of all three polymers was deterlnined by titration of the end group in non-a
preparation of the solutions bidistilled water was used, which was then cleared of any colloidal impuri:bs WE h activated carbon. As spreading solvent, both for the pure constitu~:ata as well as for their mixtures, we used chloraform with the addition ui 6% by volume of dichIoroac&ic acid. AU the reagents used in the preparation tif the suppa& and the solvents were fumished by Carlo Elba, Chemical Industrial Division, Milan, Italy. The surface pressure measurements of the mixtures were obtained by spreading the sofutions containing the two components in the various rnoti ratios, The SOIUtions used were freshly pp2pured. renewed every two days and stored at a temperature of about 4°C between one experiment and another. The compressions were carried out in a discontinuous fpshion with in&rruptions every 0.06 m2 mg-’ for about 5 min to ensure that Eurface equilibrium had been reitched before each new compression. That the equilibrium had been reached for each area, before the collapse, was confiied by *he fact that the surface pressure was constant aa a f&ction of time. A variation in the surface pressure at a constant area, during a five-minute interval time, and not less than 1 dyn cm-’ was taken ss the collapse criterion. The apparatus used for the surface pressure measurements has been preViousIy described [21. Tire measurementa were carried out, for the gure components at temperatures of 15,20,26, and 30°C on the three supports, and at 25°C for the mixtures on supports containing CaCl%or KCI.
m
(c~68tlo’)
(cello’)
I---~-----z
; K’ CA(eqdo’) Iw; suppwt NaCl 9
SupportCdl,
A-l;v(.D,Utnhe
4
CA
6
SupportKC1
4
CA
: 6
k r CA(@f#=lo’) K; SupportNaCl
Pdy(L)alanine SopportWI,
Hug&u’ pammstcn
TABLE1
High
-LOW
-16.6
-2.8
11695.1 -9,9 - 16,O
-2.8
_.----.
- 12.1 0,42 6.4 2418 -3,2
39%ia3 19,9
*63:.* 2242
_
-X6,7
-2,s
---.
-12.8 0.42 6+6 3196 0.2 -2.6 0,57 3,o 62% -0.1
776t4 15,4
- 208 0.38 -362 092 3.6 4824 24,O
-20.0 0.39
-
a I”
.. -
- ,..
- 2.9
-2.9 0.66 5.2 871 0.8
-3.3 0.51 3.8 5176 25,4
35f
- _
- 16.7
-13.7 0,41 6,8 $328 0.7
-20.9 0.38 6A 7296 11.9
-17,3 0.39 6.2 4630 1.6 -3.1 0.64
245? -6.4
-14.6 0.41 6.8 2570 -8.4
meh
pr#rIUe
-3,2 0,52 3.6 1680 4.6
-2,8 0.66
-2.6 0.67 $60 704 0,e
-11.6 OA2
60:: 7426 17.4
-19.2
2oc2 a,2
Low
prmure
403’K
-13.6 0.41
-17.4 0639 6.2 4923 306
-2,6 0.67 3.0 1257 3.5
Mgh
prwrurc
-1710 -3,l O&40 0.54 6.0 3.4 4213 3313 0.46 15,l
-11.7 0642 6.6 1541 -8,O
Low gr=m
298’ K
-3.0 0.5;
73Y 0.9
-2,4 0.59
High
pWSU*
293OX
-2,% 0.67 3,O 674 -0.3
-2,6 0.67 3.0 2368 10.8
- 16.6 0.40
-2.6 0.67 3,O 990 2.3 *79Y -7.3
-l&6 0.43 5.4 816 - 10.7
-2.1 0,61 2.6 670 -0,4
pHli&Llr0 pFeMuET P-m
LOW
2880K
ii IT
Support
4
KCI
@At-10’)
ii
JI
Support N&I
K;
KC1
&I@@)
dppott
“a,
K K’ 3.0
0.67
-3.1 0.53 3,6 281 -2,8 -9*1 0,192 5.4 235 - 11.5
-9.2 OA3 5.4 235 -11*7
-9.6 0.42 S.4 171 -12.7
-2.4 0.57 3.0 184 -2+3
-2,Ii 0.56 3.2 223 -2.4
2SQY -9.7
- 17.1 0.4
2160 - 10*2
ii;:
-2.8 0.57 3.0 1600 6.1
1.0
826
TASLE 1 (continued)
-3,l 0.53 3.6 362 -2,3
-2.6 0.55 3.2 266 -2.2
-2.4 0.57 3,o 193 -2.3
-2.8 0.55 31 191.0 7.4
3.0 1023 2.1
0.57 O,5S 3,2 1258 3.4
249Y 10,3
-3,l 0.66
-3.3 0.51 3.8 526 -1.7
34t4 -2.0
515BQ -9.9 -9.1 0.42 5.4 541 -9.8
-2.8 0.64
3*t2 -1.6
-9,2 0.42
22t4 - 12.1
-9.6 2,6 0.42 0.66
-18.6 0.40 6.0 4073 -2.6
848?O -3.3
0.40
-9.2 0642 564 1280 -ii?
92f4 -7.9
-9.3 0.42
-9.7 0.42 5.4 466 -11.2
t”o 6829’ 7.4
-18,6
3972’ -0.9
it
-
Xl 3:e 770 -0.3
-2.8 0.54 3.4 666 -0,8
-2.6 0,6S 3.2 486 -0.9
26F 11.4
-3.4 O*Sl
0.66 8.2 1288 3.3
-10.6 0.42 5.6 1847 -4.6
-11.5 0.42 5.6 1069 -10.3
-12.0 0.42 5.6 655 -13.4
620t2 8.9
91866 0.39
0.4 600 4191 3.6
152
RESULW
AND ~&CUSSION
We will consider separateIy the resuIts obtained for the pure components and those obtained for their mixtures,
In order to obtain characteristic parameters for the three forms of polyalanine the experimental isotherms were compared with the equation of )fuggins [4]. The applicabiiity to monoIayers of non-polypeptidic polymers and later to polypeptide polymers was recently shown I5J. As 811example, the agreement between experimental and computed data, obtained by applying Huggins’s theory, for the three isomeric forms on KC1 substrate is shown in Fig-l. For the other isotherms there is similar or better agreement, The parameters obtained are shown in Tabb 1, and from these parameters the following cottcIusions can be drawn: (1) The parameter $ l which represents the free surface energy of Gibbs in the theory of Huggins, is negative for alS the isomers and support studied; therefore all the monvtiyers can be considered thermodynamically stable. So far as the absolute value is concerned it can be obse& that: (a) for the same support the isomer to which the highest fibsolute value of G corresponds, and therefore, the greatest thermodynamic stability is poly(L) alanine; (b) for the same isomer, the higher value of 9 considered as an absofute value corresponds to the support containing KC!, and decreases in order in going from the supports contuining N&l to CaCll. (2) The K’ values for all the monolayets studied are different from zero, that is, the values of K, which in the Huggins Theory represent an equilibrium constant among the three types of con:acts, (i.e, K = (r&(4 umL*a~@ where a = polymer, 4 = support), are smaller than one. Thus, the distribution of the contacts between the segments (and of the segments) cannot be considered random. In particular the values of K < 1 indkak a higher stability of the polymer-polymer contacts with respect to the polymemuppoti contacts- Furthermore, as was expected, this probability is greater in the condensed than in the expanded phase. It can be observed, finally, that all the values of K are of the same order of magnitude, independent of the isomer and of the support considered. (3 j The values of Ed (ergs X 10’) are positive for & the mono!ayers considered. Since up is proportional to AE (energy change when two unit lengths of contacts between like segments are rep&cd by two unit lengths of contacts between unlike segments) defined by be = ~E~~-E~~-E~B where ~~3, e,, and EBB are the average energies per unit of Iength of contact between pairs of segments of the type indicated (i.e. polymer-support, polymerpolymer, and supportsupport), we can see that the positive vahles of EA are related to the energies of interaction which are prevalently attractive between the polymer segments. So far as the vaIue of t-he parameter is con-
153
cerned, it can be obsenred that (a) for the same support the interactions between the segments of the polymers are higher for the L isomer, and decrease on passing to the D and the racemic mixture, This last form is therefore the one which has the greatest interaction with the support; this can be confirmed by the fact that this isomer is found principally in the j3 form ISI at the interphase, as opposed to the opticaMy active isomers, which are stable at the water/air interphase in the form of a helixes. (b) For the same isomer, the polymer-support interactions are greater for the support containing Ca*’ ions, and decrease in pming to Na* and K’. (4) The orientaiion of the polymeric segments is in general dependent on the concentration. There is, therefore, an entropic contribution which may be derived from this dependence. The h: parameter measures especially the average variation of the orientation of a segment when the contacts pass from one type (i.e. a) to another (P). In the present case, the parameter remains negative at all temperatures and on the three supports only for the racemfc polypeptide. This means that only in this case the compression pmcass is not entropically favored, This seems acceptable if we remember that the DL isomer alone is present at the interphase in the B form, which represents a potymetic conPormation that is more distended than the a helix form, with greater support interaction and lower attractive energies between the polymer segments. Far the optically active isomers it can be seen that greater variations of & both with respect to temperature and support are found with the L form.
Following an approach which is well known, from the experimental isotherms, the areas at constant surface pressure as a function of the molar ratios between the monomeric units of the polypeptides rend moles of acid were determined. The behavior, as can be seen from Fig.& shows a negati-: e deviation from the additivity, and therefore the free surface energies of mixing computed A
I.0
a8
06 a4
Fig. 2. Plot of surface areas. A(m’ mg-*), as a function of molar ratios far Arachidic Acid (A-A.) and its mixtures with poIy(L)aIanine (L), poIy(D)alanine (D), and paly(DL)aIanine (DL). Supports containing Cay* and KC; values computed at a surface pressure of LO dyn cm”.
0
9
-I -2 -3
&g-3. &SE surfme euew of mixhg, AC&erg X 10’ )* as a funcaian of mahr ratkas for a*achidk acid and ib mirturre with L,D, and DL paIyahnirte. Supporti containing Ca*’ and K*: eahes camguld up to the prevum OQ2 (a)* 10 (b), and 18 dyn cm’@ (c)-
for the mktwea of the three isomers are different from zero, The free tiace enezgiesof mixing, gee F&3, are computed for the mixtures of the three isomers on the difierent auppor&saccording to the de&Ction of Goodrich [?J, that is: AG,h a{” t#Ia-N,A,-N~A~~dn, The integrations of the isotherms have been computed to the prewures of 2,10, and 18 dyn cm’*, By examining the graph, the following cunckaions can be made: (a) the values of AG,k ahow negative deviations fkom S-XSO and for the same support ate greater in absolute vafue for the L isomer and decrease with passage to the D and DL isomers; (b) the vakes of AG,f, am for some of the isomers lower in absdute vdue for the support containing CaCll with respect to that containing KCI; the values are strongly dependent on the prerraure s to which they have been computQd, end in padicuhr, at tow surface presuumm the &d values are small andincreaw with inaease of the surfkce pressure, becoming maximum s of the three isometic forms. at prewzes that are ne8t the collapse pThe data reported TVthis point are in agreement with Surcacecompatibility among the components, but the colkpse prwsures are not in mment with thb EnTable 2 the pv at 2S’C for the three forms and the three supporfs are shown. It can be swn that the loweat collapwzp-33eurecorre-
185
TABLE
2
Cdhpse
preuurcr (dyn cm-l) at 2693) as a functfoa of mohr ratios on K*
Molu mtbs
L
PoIy~~nine/AA
IlO 4/a 2/l 111
l/2 l/4 O/l -_
20.3 20.4 20.3 20.3 20.2 20.3
D
33.8 33.9 33.2 34.0 33.8 31.8
20.4 20.4 20.5 20.4 20.3 20.5
rupporL
DL
33.5 33.6 33.6 32.8 32.8 31.8
20.9 20.9 20.6 20.8 20.9 20.8
33.0 31.8 32.8 33.4 32.8 31.8
spends to that of pure polyalanfne and is independent of the molar ratioa a simple applicatibn of the phase rule for bidimensional systems, incompatibility among the components. The deviation from additivitq of the areas does therefore not seem to be attributable to a red reciprwal sotubitity and hence the formation of a real mixed film at the waterlair interpha#. An entirely similar behavior was observed by Hookes [S] with bidimentiionalmixtuns of polypeptides with Iipidic compounds. The author found, as we did, a negative deviation of the -as (approximately 5%) that was attributed to a fiiing of the lipid in the eurface micelle of tfrs polymer. The behavior of the mod&s of surface compressibility, even if, as is known, It cannot give direct information about the stiace compatibility, urns to confii the preceding hypothesis. 1n fact, as is shown in Fig.4, they This behavior indicates, tim
IO0
m 60
Fig.4. Plot of the rurf8ce compresional maduli, C, -* (dyn cm-*), as a function of m&u ratios for aracbiddic add-poIy(L,D, ad DL)ahdne_ Supports containing Ca** ami K*r vahes computad
at IO dyn cm-‘.
are ideally additive, or with negative deviations only in the case of the L and D isomers on C&l, support. The additive behavior of the modules can be considered in agreemr!ntwith an insoIubti&y between the two components, since the hypothesis of id4 insolubility is fo be excIuded considering the behavior of the surf&e areas as well as the collapse pressures. The rxqative
156
deviation, on the other hand, does not go along with the exi&nce of attractive interactions, and therefore with the hypothesis of reciprocal solubility among the components, since it signifies the presence at the surface of phases that are more expanded with respect to ideal@, that can instead be justified by a decreased interaction between the poIypeptidic chains on account of the filling of the latter by the carboxylic acid. This hypothesis is supported by the fact that the effect ia found for the Land D isomers on the CaCI, support, for which we have the greatest polymer support interactions and hence the least possible interaction between potymeric chains, so that there is a condition of “bad fit” among the surface macromolecular micelIes, indicated by MakoIm 191as a favorable condition for the entrance of the lipidic substance among the polypeptidic chains. It should be noted that there is no such effect for the GL isomer, for which the modules are additive. However, this is to be attributed to the fact that the macromokcuIar form present at the interphase is different, as we have shown, that is 8, whose interfacial distribution can be unfavorabb to the entrance of the lipidic substances in the surface macromokcular Pim. CONCLUSIONS
The application of the theory of Huggins to the monolayers of poIy(L, D, and DLIaIanine allows us to draw some interesting conclusions. The isomer that is the most thermodynamically stableisthe one that is generallypresent in nature, that is, the L isomer, whae the feast stable is the DL form’. This stability is a btained principalIy &om the interactions between the po!ymeric chains, The polymer-polymer interactions are considenbty higher for the D and L isomers, which are present at the interphase in the u helix form, while they decrease rapidly in favor of the polymersupport interactions for the poIy(DL)-alanine present at the interphase prirrcipally In the 0 conformatian, The present of ions in the support, and in particular the Ca” ion, has a
different effec depending on the macomokcuk form present at thein&rphase (a or #3). n particuIar, for the L and D isomers thert! is a considerabb decrease of ink% ctions between the polymeric chains in favor of interactions with the support and a thermodynamic destabikation cIe=ly indicated by increase of the 9 parameter (from about -21 to about -14 for the L isomer); for the racemic species there is also an increase of the interactions with the support but these stabilize the macromoIe~uIar form present at the interphase (increase in the # parameter). In conclusion, the ar conformation is thermodynamically more stabIe than the 0 form at the air/water interphaw. The presence of Cal* ions, and in a lesser way, of Na* ions acts as a destabilizer of the othelix, This inhibits the interactions between the polymeric chains while favoring the atabillzation of the B forms probably due to a condensation of the fibn, indicated also by the increase of the madulus of the coeffitient of compressibirity
167
to the one conthat is obtained in passing from the support containir ‘. taining CaCl~. The mixtures of the polyaminoacids with arachidic acid show the incompatibility of the two componel a even if it seems possibte for the lipid to insert into the surface micelle of the polyaminoacid. This type of filling is greater for the optically active polyaminoacids which are present in the Q helix form.
REFERENCES 1 2
3 4 6
6 7 8 9
G. Gab&M and A. hfaddii, J. Callord Interface Sci., 64 (1978) 19; G. Gabrielli, P. Baglioni and A. Maddii, J. CaUoid Intprfacc Sci,. 79 (1?81) 268. G. Gabcielti and C. D’Auber\ Colfoid Polym. Sci., 256 (1978) 1165. G. GabrielI& d. Coltotd Interface Sci,, 63 (1975). l-IS_ ML Huggins, 2. Kollotd Z. Polym., 25111973). 449. G. Gabrblli, E. Fenoni and M.L. Huggins~ Prune Coltold Polym. Sci., 58 (1976). 201; G. Gabrialll, E. Ferront, RI, Puggelli and hf.L_ Huggins, Colloid Potym, Sci., 266 (1978), 417. G. Gabcielli, P, Baglioni and E. Fertoni, J. Callaid Interface Sci., 8 I(198 1) 139. P.C. Goodrich, Proceedings of the 2nd lnternattonal Congress of Surface Activity, Vol. 1, bndon, 1957, p_ 8s. D.E_ Haokes, Ph.D, Thesis, Udvcraity of Londan. 1971. B.R. hlalcolm, Pragr. Surf. Membr, Sci., 7 (1973), 183.
AND THEIR
MONOLAYERS OF POLY(L, D, AND DL)ALANINE MIXTURES WFFH ARACHIDIC ACID GABRIBLLA
GABRIELLI,
PIER0
InnrZitute of Physical Chemirtxy. Firenze (Italy) (Received August
27th.
BAGLIONI
The Uniwrsity
XS80;rccepted
and ANNA of Florence.
147
FABBRINI Wa 0.
in final form Deamber
Capponi 9. SO 121
lfatb, 1980)
ABSTRAGT Monchyers of poXy(L, D. DL]rlmfne were rtudied on rub&=&8 containing Ua’+, Na+,andK* and in mixtures with arachidic acIdd. Appticatton 05 the Lheoxy of Huggins shows that tte most strbte-metic form,froma thermodynamk otmdpaint,IstheL
fom, while the feut themtodynlmkdly atrble t the DL ape&s. It has ken SIZII that poIy(L znd D)olmhtne, which arc present at the interpha#c aa u helix-, rhow preterentlal inttractbn8 between the polymerfc chalq while poly(DLwanine, whfch Is presentin the8 confomatkn, rhowspreferential interactiona with the support. The presence of the Ca*’ Ions Increases considerably the intemctionr between the polymer and the rub&ate. Mixtures of the three isomers ahaw incompatibility whkh arachidic acid even if interactiona are present between the lipIdi= chain8 and them~eramofeculu micdes in the “bad fit” zoner of the latter.
INTRODUCXION Mixed monolayers of compounds of low and high mokcuIar weights, as we know, have particular importance both from a theoretical and applied Vi@wpOillL
In earlier wclrks [l] bidimensional systems were studied having two macromokcu~ compounds ILLconstituents or a compound of low molecular weight and a poIymeric compound, and the possibility of arriving at reciprocal compatibility of the components, as well as the principal parameters that determine it, was shown, In this context, the study of mixed monolayers of polypeptides and lipidic compounds, which are considered usefuI models of biological membranes, has shown that the reciprocal compatibility of the components in the monobyer can be modified by vztqing the composition of the support [2]. The aim of the present work is to study the modifications that ions present in the support can induce in the configuration and the distribution at the interphase of the Wee isomers (L, D, DL) of the same poIyaminoacid, and in the soXubilityof these isomers in the bidimensionsl state with a lipidic compound selected ss a model, viz., a fatty carboxylic acid. 0166-662213~~066~660/$62.S0 ~1981 Elsetier ScIentificPubthMngtimpany
148
For this purpose a particularly simple poIyaminoacid was selected which can give stable bidimensional films at the waterjair intcerphase and, for the support, were select4?dCay*, Na* and K*, which are particularly interesting from the point of view of monolayers considered as models of biologic membranes. Finally, the lipidic compound selected was arachidic acid, whose bidimensional mixtuxcs with other polypepticies had been studied previously IS], and which gives monolayers at the,wtter/air intcrphsse with phases and orientation that are well known and defilked. EXPERIMENTAL
Po,ly(DL)alanine MW = 3900 lot number AL 69 and poly(L)alsnine MW = 3000 lot number AL 44 were furnished by Miles Ltd. Israel: poly(D)alanine MW = 3000 was furnished bs_Sigma Chemie GmbH, Munich, West Germany, The average molecular weight of all three polymers was deterlnined by titration of the end group in non-a
preparation of the solutions bidistilled water was used, which was then cleared of any colloidal impuri:bs WE h activated carbon. As spreading solvent, both for the pure constitu~:ata as well as for their mixtures, we used chloraform with the addition ui 6% by volume of dichIoroac&ic acid. AU the reagents used in the preparation tif the suppa& and the solvents were fumished by Carlo Elba, Chemical Industrial Division, Milan, Italy. The surface pressure measurements of the mixtures were obtained by spreading the sofutions containing the two components in the various rnoti ratios, The SOIUtions used were freshly pp2pured. renewed every two days and stored at a temperature of about 4°C between one experiment and another. The compressions were carried out in a discontinuous fpshion with in&rruptions every 0.06 m2 mg-’ for about 5 min to ensure that Eurface equilibrium had been reitched before each new compression. That the equilibrium had been reached for each area, before the collapse, was confiied by *he fact that the surface pressure was constant aa a f&ction of time. A variation in the surface pressure at a constant area, during a five-minute interval time, and not less than 1 dyn cm-’ was taken ss the collapse criterion. The apparatus used for the surface pressure measurements has been preViousIy described [21. Tire measurementa were carried out, for the gure components at temperatures of 15,20,26, and 30°C on the three supports, and at 25°C for the mixtures on supports containing CaCl%or KCI.
m
(c~68tlo’)
(cello’)
I---~-----z
; K’ CA(eqdo’) Iw; suppwt NaCl 9
SupportCdl,
A-l;v(.D,Utnhe
4
CA
6
SupportKC1
4
CA
: 6
k r CA(@f#=lo’) K; SupportNaCl
Pdy(L)alanine SopportWI,
Hug&u’ pammstcn
TABLE1
High
-LOW
-16.6
-2.8
11695.1 -9,9 - 16,O
-2.8
_.----.
- 12.1 0,42 6.4 2418 -3,2
39%ia3 19,9
*63:.* 2242
_
-X6,7
-2,s
---.
-12.8 0.42 6+6 3196 0.2 -2.6 0,57 3,o 62% -0.1
776t4 15,4
- 208 0.38 -362 092 3.6 4824 24,O
-20.0 0.39
-
a I”
.. -
- ,..
- 2.9
-2.9 0.66 5.2 871 0.8
-3.3 0.51 3.8 5176 25,4
35f
- _
- 16.7
-13.7 0,41 6,8 $328 0.7
-20.9 0.38 6A 7296 11.9
-17,3 0.39 6.2 4630 1.6 -3.1 0.64
245? -6.4
-14.6 0.41 6.8 2570 -8.4
meh
pr#rIUe
-3,2 0,52 3.6 1680 4.6
-2,8 0.66
-2.6 0.67 $60 704 0,e
-11.6 OA2
60:: 7426 17.4
-19.2
2oc2 a,2
Low
prmure
403’K
-13.6 0.41
-17.4 0639 6.2 4923 306
-2,6 0.67 3.0 1257 3.5
Mgh
prwrurc
-1710 -3,l O&40 0.54 6.0 3.4 4213 3313 0.46 15,l
-11.7 0642 6.6 1541 -8,O
Low gr=m
298’ K
-3.0 0.5;
73Y 0.9
-2,4 0.59
High
pWSU*
293OX
-2,% 0.67 3,O 674 -0.3
-2,6 0.67 3.0 2368 10.8
- 16.6 0.40
-2.6 0.67 3,O 990 2.3 *79Y -7.3
-l&6 0.43 5.4 816 - 10.7
-2.1 0,61 2.6 670 -0,4
pHli&Llr0 pFeMuET P-m
LOW
2880K
ii IT
Support
4
KCI
@At-10’)
ii
JI
Support N&I
K;
KC1
&I@@)
dppott
“a,
K K’ 3.0
0.67
-3.1 0.53 3,6 281 -2,8 -9*1 0,192 5.4 235 - 11.5
-9.2 OA3 5.4 235 -11*7
-9.6 0.42 S.4 171 -12.7
-2.4 0.57 3.0 184 -2+3
-2,Ii 0.56 3.2 223 -2.4
2SQY -9.7
- 17.1 0.4
2160 - 10*2
ii;:
-2.8 0.57 3.0 1600 6.1
1.0
826
TASLE 1 (continued)
-3,l 0.53 3.6 362 -2,3
-2.6 0.55 3.2 266 -2.2
-2.4 0.57 3,o 193 -2.3
-2.8 0.55 31 191.0 7.4
3.0 1023 2.1
0.57 O,5S 3,2 1258 3.4
249Y 10,3
-3,l 0.66
-3.3 0.51 3.8 526 -1.7
34t4 -2.0
515BQ -9.9 -9.1 0.42 5.4 541 -9.8
-2.8 0.64
3*t2 -1.6
-9,2 0.42
22t4 - 12.1
-9.6 2,6 0.42 0.66
-18.6 0.40 6.0 4073 -2.6
848?O -3.3
0.40
-9.2 0642 564 1280 -ii?
92f4 -7.9
-9.3 0.42
-9.7 0.42 5.4 466 -11.2
t”o 6829’ 7.4
-18,6
3972’ -0.9
it
-
Xl 3:e 770 -0.3
-2.8 0.54 3.4 666 -0,8
-2.6 0,6S 3.2 486 -0.9
26F 11.4
-3.4 O*Sl
0.66 8.2 1288 3.3
-10.6 0.42 5.6 1847 -4.6
-11.5 0.42 5.6 1069 -10.3
-12.0 0.42 5.6 655 -13.4
620t2 8.9
91866 0.39
0.4 600 4191 3.6
152
RESULW
AND ~&CUSSION
We will consider separateIy the resuIts obtained for the pure components and those obtained for their mixtures,
In order to obtain characteristic parameters for the three forms of polyalanine the experimental isotherms were compared with the equation of )fuggins [4]. The applicabiiity to monoIayers of non-polypeptidic polymers and later to polypeptide polymers was recently shown I5J. As 811example, the agreement between experimental and computed data, obtained by applying Huggins’s theory, for the three isomeric forms on KC1 substrate is shown in Fig-l. For the other isotherms there is similar or better agreement, The parameters obtained are shown in Tabb 1, and from these parameters the following cottcIusions can be drawn: (1) The parameter $ l which represents the free surface energy of Gibbs in the theory of Huggins, is negative for alS the isomers and support studied; therefore all the monvtiyers can be considered thermodynamically stable. So far as the absolute value is concerned it can be obse& that: (a) for the same support the isomer to which the highest fibsolute value of G corresponds, and therefore, the greatest thermodynamic stability is poly(L) alanine; (b) for the same isomer, the higher value of 9 considered as an absofute value corresponds to the support containing KC!, and decreases in order in going from the supports contuining N&l to CaCll. (2) The K’ values for all the monolayets studied are different from zero, that is, the values of K, which in the Huggins Theory represent an equilibrium constant among the three types of con:acts, (i.e, K = (r&(4 umL*a~@ where a = polymer, 4 = support), are smaller than one. Thus, the distribution of the contacts between the segments (and of the segments) cannot be considered random. In particular the values of K < 1 indkak a higher stability of the polymer-polymer contacts with respect to the polymemuppoti contacts- Furthermore, as was expected, this probability is greater in the condensed than in the expanded phase. It can be observed, finally, that all the values of K are of the same order of magnitude, independent of the isomer and of the support considered. (3 j The values of Ed (ergs X 10’) are positive for & the mono!ayers considered. Since up is proportional to AE (energy change when two unit lengths of contacts between like segments are rep&cd by two unit lengths of contacts between unlike segments) defined by be = ~E~~-E~~-E~B where ~~3, e,, and EBB are the average energies per unit of Iength of contact between pairs of segments of the type indicated (i.e. polymer-support, polymerpolymer, and supportsupport), we can see that the positive vahles of EA are related to the energies of interaction which are prevalently attractive between the polymer segments. So far as the vaIue of t-he parameter is con-
153
cerned, it can be obsenred that (a) for the same support the interactions between the segments of the polymers are higher for the L isomer, and decrease on passing to the D and the racemic mixture, This last form is therefore the one which has the greatest interaction with the support; this can be confirmed by the fact that this isomer is found principally in the j3 form ISI at the interphase, as opposed to the opticaMy active isomers, which are stable at the water/air interphase in the form of a helixes. (b) For the same isomer, the polymer-support interactions are greater for the support containing Ca*’ ions, and decrease in pming to Na* and K’. (4) The orientaiion of the polymeric segments is in general dependent on the concentration. There is, therefore, an entropic contribution which may be derived from this dependence. The h: parameter measures especially the average variation of the orientation of a segment when the contacts pass from one type (i.e. a) to another (P). In the present case, the parameter remains negative at all temperatures and on the three supports only for the racemfc polypeptide. This means that only in this case the compression pmcass is not entropically favored, This seems acceptable if we remember that the DL isomer alone is present at the interphase in the B form, which represents a potymetic conPormation that is more distended than the a helix form, with greater support interaction and lower attractive energies between the polymer segments. Far the optically active isomers it can be seen that greater variations of & both with respect to temperature and support are found with the L form.
Following an approach which is well known, from the experimental isotherms, the areas at constant surface pressure as a function of the molar ratios between the monomeric units of the polypeptides rend moles of acid were determined. The behavior, as can be seen from Fig.& shows a negati-: e deviation from the additivity, and therefore the free surface energies of mixing computed A
I.0
a8
06 a4
Fig. 2. Plot of surface areas. A(m’ mg-*), as a function of molar ratios far Arachidic Acid (A-A.) and its mixtures with poIy(L)aIanine (L), poIy(D)alanine (D), and paly(DL)aIanine (DL). Supports containing Cay* and KC; values computed at a surface pressure of LO dyn cm”.
0
9
-I -2 -3
&g-3. &SE surfme euew of mixhg, AC&erg X 10’ )* as a funcaian of mahr ratkas for a*achidk acid and ib mirturre with L,D, and DL paIyahnirte. Supporti containing Ca*’ and K*: eahes camguld up to the prevum OQ2 (a)* 10 (b), and 18 dyn cm’@ (c)-
for the mktwea of the three isomers are different from zero, The free tiace enezgiesof mixing, gee F&3, are computed for the mixtures of the three isomers on the difierent auppor&saccording to the de&Ction of Goodrich [?J, that is: AG,h a{” t#Ia-N,A,-N~A~~dn, The integrations of the isotherms have been computed to the prewures of 2,10, and 18 dyn cm’*, By examining the graph, the following cunckaions can be made: (a) the values of AG,k ahow negative deviations fkom S-XSO and for the same support ate greater in absolute vafue for the L isomer and decrease with passage to the D and DL isomers; (b) the vakes of AG,f, am for some of the isomers lower in absdute vdue for the support containing CaCll with respect to that containing KCI; the values are strongly dependent on the prerraure s to which they have been computQd, end in padicuhr, at tow surface presuumm the &d values are small andincreaw with inaease of the surfkce pressure, becoming maximum s of the three isometic forms. at prewzes that are ne8t the collapse pThe data reported TVthis point are in agreement with Surcacecompatibility among the components, but the colkpse prwsures are not in mment with thb EnTable 2 the pv at 2S’C for the three forms and the three supporfs are shown. It can be swn that the loweat collapwzp-33eurecorre-
185
TABLE
2
Cdhpse
preuurcr (dyn cm-l) at 2693) as a functfoa of mohr ratios on K*
Molu mtbs
L
PoIy~~nine/AA
IlO 4/a 2/l 111
l/2 l/4 O/l -_
20.3 20.4 20.3 20.3 20.2 20.3
D
33.8 33.9 33.2 34.0 33.8 31.8
20.4 20.4 20.5 20.4 20.3 20.5
rupporL
DL
33.5 33.6 33.6 32.8 32.8 31.8
20.9 20.9 20.6 20.8 20.9 20.8
33.0 31.8 32.8 33.4 32.8 31.8
spends to that of pure polyalanfne and is independent of the molar ratioa a simple applicatibn of the phase rule for bidimensional systems, incompatibility among the components. The deviation from additivitq of the areas does therefore not seem to be attributable to a red reciprwal sotubitity and hence the formation of a real mixed film at the waterlair interpha#. An entirely similar behavior was observed by Hookes [S] with bidimentiionalmixtuns of polypeptides with Iipidic compounds. The author found, as we did, a negative deviation of the -as (approximately 5%) that was attributed to a fiiing of the lipid in the eurface micelle of tfrs polymer. The behavior of the mod&s of surface compressibility, even if, as is known, It cannot give direct information about the stiace compatibility, urns to confii the preceding hypothesis. 1n fact, as is shown in Fig.4, they This behavior indicates, tim
IO0
m 60
Fig.4. Plot of the rurf8ce compresional maduli, C, -* (dyn cm-*), as a function of m&u ratios for aracbiddic add-poIy(L,D, ad DL)ahdne_ Supports containing Ca** ami K*r vahes computad
at IO dyn cm-‘.
are ideally additive, or with negative deviations only in the case of the L and D isomers on C&l, support. The additive behavior of the modules can be considered in agreemr!ntwith an insoIubti&y between the two components, since the hypothesis of id4 insolubility is fo be excIuded considering the behavior of the surf&e areas as well as the collapse pressures. The rxqative
156
deviation, on the other hand, does not go along with the exi&nce of attractive interactions, and therefore with the hypothesis of reciprocal solubility among the components, since it signifies the presence at the surface of phases that are more expanded with respect to ideal@, that can instead be justified by a decreased interaction between the poIypeptidic chains on account of the filling of the latter by the carboxylic acid. This hypothesis is supported by the fact that the effect ia found for the Land D isomers on the CaCI, support, for which we have the greatest polymer support interactions and hence the least possible interaction between potymeric chains, so that there is a condition of “bad fit” among the surface macromolecular micelIes, indicated by MakoIm 191as a favorable condition for the entrance of the lipidic substance among the polypeptidic chains. It should be noted that there is no such effect for the GL isomer, for which the modules are additive. However, this is to be attributed to the fact that the macromokcuIar form present at the interphase is different, as we have shown, that is 8, whose interfacial distribution can be unfavorabb to the entrance of the lipidic substances in the surface macromokcular Pim. CONCLUSIONS
The application of the theory of Huggins to the monolayers of poIy(L, D, and DLIaIanine allows us to draw some interesting conclusions. The isomer that is the most thermodynamically stableisthe one that is generallypresent in nature, that is, the L isomer, whae the feast stable is the DL form’. This stability is a btained principalIy &om the interactions between the po!ymeric chains, The polymer-polymer interactions are considenbty higher for the D and L isomers, which are present at the interphase in the u helix form, while they decrease rapidly in favor of the polymersupport interactions for the poIy(DL)-alanine present at the interphase prirrcipally In the 0 conformatian, The present of ions in the support, and in particular the Ca” ion, has a
different effec depending on the macomokcuk form present at thein&rphase (a or #3). n particuIar, for the L and D isomers thert! is a considerabb decrease of ink% ctions between the polymeric chains in favor of interactions with the support and a thermodynamic destabikation cIe=ly indicated by increase of the 9 parameter (from about -21 to about -14 for the L isomer); for the racemic species there is also an increase of the interactions with the support but these stabilize the macromoIe~uIar form present at the interphase (increase in the # parameter). In conclusion, the ar conformation is thermodynamically more stabIe than the 0 form at the air/water interphaw. The presence of Cal* ions, and in a lesser way, of Na* ions acts as a destabilizer of the othelix, This inhibits the interactions between the polymeric chains while favoring the atabillzation of the B forms probably due to a condensation of the fibn, indicated also by the increase of the madulus of the coeffitient of compressibirity
167
to the one conthat is obtained in passing from the support containir ‘. taining CaCl~. The mixtures of the polyaminoacids with arachidic acid show the incompatibility of the two componel a even if it seems possibte for the lipid to insert into the surface micelle of the polyaminoacid. This type of filling is greater for the optically active polyaminoacids which are present in the Q helix form.
REFERENCES 1 2
3 4 6
6 7 8 9
G. Gab&M and A. hfaddii, J. Callord Interface Sci., 64 (1978) 19; G. Gabrielli, P. Baglioni and A. Maddii, J. CaUoid Intprfacc Sci,. 79 (1?81) 268. G. Gabcielti and C. D’Auber\ Colfoid Polym. Sci., 256 (1978) 1165. G. GabrielI& d. Coltotd Interface Sci,, 63 (1975). l-IS_ ML Huggins, 2. Kollotd Z. Polym., 25111973). 449. G. Gabrblli, E. Fenoni and M.L. Huggins~ Prune Coltold Polym. Sci., 58 (1976). 201; G. Gabrialll, E. Ferront, RI, Puggelli and hf.L_ Huggins, Colloid Potym, Sci., 266 (1978), 417. G. Gabcielli, P, Baglioni and E. Fertoni, J. Callaid Interface Sci., 8 I(198 1) 139. P.C. Goodrich, Proceedings of the 2nd lnternattonal Congress of Surface Activity, Vol. 1, bndon, 1957, p_ 8s. D.E_ Haokes, Ph.D, Thesis, Udvcraity of Londan. 1971. B.R. hlalcolm, Pragr. Surf. Membr, Sci., 7 (1973), 183.