1032
J. Phys. Chem. 1981, 85,1032-1037
Studies of the Phase Transition in the Single-Lamellar Liposomes. 2. Liposome-Size Effect on the Phase Transition Hiroyuki Takernoto,+ Shoshi Inoue,t Tatsuya Yasunaga,t Mltsunori Sukigara,§ and Yoshinorl Toyoshimat" Faculty of Inregrated Arts and Sciences, and Faculty of Science, Hiroshima University, 1- 1-89, Higashisenda-machl,Naka-ku, Hiroshima 730 Japan; and Institute of Industrial Sciences, University of Tokyo, Roppongi, Minato-ku, Tokyo 106 Japan (Received: February 5, 1980; In Final Form: September 22, 1980)
Uniformly sized dipalmitoylphosphatidylcholine(DPPC) liposome dispersions were prepared in different molecular weights ranging from 3.1 X lo6 to 3.6 X lo7 by means of molecular-sieve column chromatography on a Sepharose 2B gel to investigate the size effect on fluid-solid phase transition in the single-lamellar liposome bilayer membranes. The fluorescence intensity of N,N'-distearyloxacarbocyanine iodide incorporated into the liposomes was used as an indication of the phase transition. Bangham-type multilamellar DPPC liposomes exhibited a completely reversible abrupt large change in the fluorescence intensity associated with the main transition at 41.5 "C. A much smaller change was accompanied by the pretransition around 30 "C which had a real hysteresis. On the other hand, single-lamellar liposomes showed only a single transition. The temperature profiles of the phase transition in the single-lamellar liposomes determined as a function of the liposome size clearly demonstrated that the midpoint temperature and the van't Hoff enthalpy of the transition decrease with the size of liposomes. Systematic data of the size effects on the transition parameters are presented arid discussed.
Introduction Because of the possible importance of the lipid phase transition in the structure and function of biological membranes, there have been a number of theoretical and experimental works on the thermotropic phase transition in lipid bilayer systems. Two types of liposome dispersions have been usually utilized in the studies of the phase transition. The first type, introduced by Bangham et al.,l is multilamellar liposomes composed of many concentric bilayer sheets of various,sizes intersphered with water. The second type of liposome, which consists of a sphere bounded by a single bilayer shell, was introduced by Huang2 using ultrasonic irradiation. The phase-transition behavior of the former has been characterized by a highly cooperative main transition and an additional, somewhat broader transition which occurs at 5-10 "C below the main transition with small latent heat. The main transition was determined to be a transition with regard to the lipid hydrocarbon chains between an all-trans configuration and a more disordered ne,^-^ while the pretransition was recently found to be associated with a structure transformation from the lamellar Lptphase to the two-dimensional P phase which probably arises from periodic distortions o!hte lamellar.6,7 The change of a phospholipid from the multilamellar to single-lamellar structure has been qualitatively known to result in the alternation of the transition behavior, such as the decrease in the latent heat by a factor of -0.5s and the drop of the transition temperature for several degrees accompanied by the decrease in the co~perativity.~ These differences between the multi- and single-lamellar structures were usually interpreted in terms of the disruption of the regular packing in the single-lamellar liposomes due to their strong curvature.1° Consequently, particular interest was focused on the phase transition of the multilamellar liposomes rather than the single-lamellar liposomes. However, because of the lack of interactions between the bilayers within each liposome, the single-lamellar liposomes
might have their own particular character for the phase transition which is essentially different from that of the multilamellar liposomes. Actually, we have found the pretransition to disappear in the single-lamellar liposomes. It has also been suggested that the transition behavior and the related properties of the single-lamellar liposomes depend on their size.11J2 In spite of these findings, the quantitative investigation of the size effects has never been carried out, mainly because the preparation of liposomes well-defined in size was not established over a wide size range. In part 1 of this series,13we reported the preparation of homogeneously sized single-lamellar liposomes of egg phosphatidylcholine and dipalmitoylphosphatidylcholine in the molecular-weight range 2 X 106-3 x 107. In this paper, we present the phase-transition behavior of the dipalmitoylphosphatidylcholine single-lamellar liposomes in comparison with that of the multilamellar liposomes. Particular attention is given to the liposomesize effect on the phase-transition parameters. (1)A. D. Bangham and R. W. Horne, J. Mol. Biol., 8, 660 (1964). (2) C. Huang, Biochemistry, 8, 344 (1969). (3) D. Chapman, R. M. Williams, and B. D. Ladbrooke, Chem. Phys. Lipids, 1, 445 (1967). (4) V. Luzzati, "X-ray Diffraction Studies of Lipid-Water Systems in
Biological Membranes", D. Chapman, Ed., Academic Press, New York, 1968.
(5) B. D. Ladbrooke and D. Chapman, Chem. Phys. Lipids, 3, 304 (1969). (6) M. J. Janiak, D. M. Small, and G. G. Shipley, Biochemistry, 15, 4575 (1976). (7) M. J. Janiak, D. M. Small, and G. G. Shipley, J.Biol. Chem., 254, 6068 (1979). (8) J. Suurkuusk, B. R. Lentz, Y. Barenholz, R. L. Biltonen, and T. E. Thompson, Biochemistry, 15, 1393 (1976). (9) M. P. Sheetz and S. I. Chan, Biochemistry, 11, 4573 (1972). (10) J. F. Faucon and C. Lussan, Biochem. Biophys. Acta, 307, 459 (1973). (11) M. I. Kanehisa and T. Y. Tsong, J. Am. Chem. SOC.,100, 424 11978). (12) B. R. Lentz, Y. Barenholz, and T. E. Thompson, Biochemistry, 16,4521 (1976). (13) S. Inoue, H. Takemoto, T. Yasunaga, and Y. Toroshima, Mol. ~~
'Faculty of Integrated Arts and Sciences, Hiroshima University. Faculty of Science, Hiroshima University. University of Tokyo.
*
0022-3654/81/2085-1032$01.25/0
Cryst. Liq. Cryst., in press.
0 1981 American Chemical Society
Phase Transition in the Single-Lamellar Liposomes
Experimental Section Materials. l-a-Dipalmitoylphosphatidylcholine(DPPC) purchased from Sigma Chemical Co. or synthesized in our laboratory by modifying the method reported by Robles and Berg14 was used. Before the preparation of the liposomes, each DPPC was purified by thin-layer chromatography on silica gel (Merck 60 F254) using a mixture of CH3C1, CH30H, and H 2 0 at a volume ratio of 65:25:4 as the developing solvent. N,N'-Distearyloxacarbocyanine iodide (DSOCC) in a crystallized form was purchased from the Japan Research Institute for Photosensitizing Co., Ltd., and was used without further purification. KC1 and tris-(hydroxymethy1)aminomethane of analytical grade were used as received. Preparation of DPPC Liposomes Containing DSOCC as a Fluoroscent Probe and Molecular- Weight Determination. Preparation and size determination of single-lamellar liposomes were carried out in accordance with part 1of this series.13 Briefly, 3 mL of the liposome dispersion prepared by the sonication2or alcohol15 method was subjected to gel-permeation chromatography (GPC) on Sepharose 2B column (1.5 X 40 cm) at 40 "C. The effluent was collected in 3-mL fractions and stored at 45 "C before rechromatography. Several fractions from the first GPC were rechromatographed on the same column, and each peak fraction of the effluent was submitted to the experiments of the phase transition and of the sedimentation equilibrium as soon as it came out from the column. The rechromatographic profiles of the dispersions monitored by the optical density at 300 nm were used to determine the partition coefficient, Kav,of the liposomes at the peak fractions on Sepharose 2B gel. DPPC concentrations in the dispersions were determined by the Fiske Subbarow method,16 while DSOCC concentrations were estimated from the absorption at 492 nm attributed to the DSOCC band by using the molar extinction coefficient determined in ethanol, 6 = 1.20 X lo5 M-l cm-l. The DSOCC-to-DPPC molar ratio thus obtained agreed with that at the formation of the liposome within an experimental error. The molecular weights of typical liposome samples were determined by the sedimentation equilibrium with freshly obtained peak fractions of the second GPC in a Hitachi ultracentrifuge (Model UCA-1A type) using a multichannel cell at 35 "C. The rotor (RAGOH type) was operated at 1500-9500 rpm for 12-30 h to get the equilibrium state, depending on the molecular weight of the sample. Phase- Transition Measurements. The thermotropic phase-transition behavior of DPPC liposomes was studied by measuring the change in the fluorescence intensity of DSOCC incorporated into the liposome bilayers. Fluorescence measurements were performed on a spectrofluorophotometer (Hitachi MPF-4) with a temperature-controlled cell holder. In order to get the temperature profile of the fluoroescence intensity at 505 nm attributed to the DSOCC emission band, the samples were cooled at first from 50 to 25 "C and heated to 50 "C again at the rate of 0.5 "C/min. by using a circulating water bath with a temperature programmer (Neslab Instruments Inc. RT8). The temperature was monitored with a thermocouple dipped in the sample cuvette. The concentration of DSOCC in each sample solution was kept constant at 5.0 X lo-' M irrespective of the DSOCC/DPPC molar ratio. Under these conditions, the temperature profile of the (14) E. C. Robles and D. V. D. Berg, Biochim.Biophys. Acta, 187,520 (1969). (15) S. Batzri and E. D. Korn, Biochim. Biophys. A c t a , 298, 1015 (1973). (16) C. H. Fiske and Y. Subbarow, J. Biol. Ghem., 66, 375 (1925).
The Journal of Physical ChemMry, Vol. 85, No. 8, 1981 1033
.-s 0 E O.' 0
1 A > -.:,/;.,
,
,,,,
2
+'.,3
/ ; ) '
40
20
60
80
Elution Volume, ml
Flgure 1. Elution profiles of DPPC liposomes on the first Sepharose 2 B column chromatograph (A) and rechromatograph (B) at 40 'C: prepared (-) by the sonication method and by the alcohol method. The arrows in A designate the fractlons submitted to the rechromatograph. (-e-)
o'B
0.6 2
u"
0.4
:\
1
01
1
-\
-\
'
'
I
2
'
" " " "
5
IO
20
, . . / I 50
Flw x166 Flgure 2. Relationship between K,, of the single-lamellar DPPC liposomes on Sepharose 28 gel and their molecular weight, Mw: (0) prepared by the sonication method; (0) prepared by the alcohol method. Soli llne represents the corresponding rehtionship previously reported for the egg-PC single-lamellar liposome^.'^
fluoroescence intensity was confirmed to be independent of the molar ratio up to 1/250; thus the main experiments were carried out on the liposomes with a molar ratio of 1/500.
Results and Discussion Size Determination of the Single-Lamellar Liposomes. As illustrated in Figure 1, the elution profiles of the second GPC of which peak fractions were used for the sedimentation-equilibrium and/or phase-transition experiments are highly symmetric, indicating that each parent fraction from the first GPC comprises liposomes which are homogeneous in size. Considering that we used only the peak fractions of the second GPC, we may expect the samples submitted to the phase-transition experiments to have a narrow distribution in size. The partition coefficient, K,,, defined by1' eq 1,was determined for every sample used Kav = ( Ve - VJ / ( Vt - VO) ( 1) in the phase-transition experiments by utilizing the elution profile of the second GPC. Here V , and Voare the total volume and the void volume of the column and V , is the elution volume at the peak position. The sedimentation equilibrium data obtained for the several peak fractions of the second GPC were analyzed by the equation applicable to multicomponent systernsl8 to determine their molecular weights, Mw. In the calculation of A&,, the effective partial specific volume of DPPC in the single-lamellar liposome bilayers was assumed to be 0.9560 mL/g at 35.0 "C, which had been obtained for the (17) T. C. Laurent and J. Killander, J . Chromatogr., 14, 317 (1964). (18) H. Fujita, Phys. Chem. ( N . Y.), 11, 271 (1962).
1034
The Journal of Physical Chemistry, Vol. 85, No. 8, 1981
Takemoto et al. I
I
25 20
36
28
T,
44
52
*C
Flgure 3. Temperature dependence of fluorescence intensity in the multilamellar liposome (A) and in methanol (B). Solid line and broken line in A represent the heating scan and the cooling scan, respectively.
suspension of DPPC in the same buffered solution as that in the sedimentation-equilibrium experiments.13 Figure 2 shows plots of K, vs. log Mwwith the data obtained in the present work in comparison with those of egg-yolk phosphatidylcholine (egg PC) which we previously reported.13 Although the values of Mwfor the DPPC liposomes involve larger experimental error than those for egg-PC liposomes, presumably due to the aggregation of the liposomes during the sedimentation-equilibrium experiments, the plots of Kavvs. log ATw satisfy practically the same linear relationship as that obtained in the egg-PC liposomes. Therefore the molecular weights of the samples for the phase-transition experiments were estimated from the values of K,, by using the linear relationship previously reported13except for the samples for which ATw values were obtained from the sedimentation-equilibrium experiments directly. Temperature Profiles of the Fluorescence Intensity of DSOCC i n the Multi- and Single-Lamellar Liposomes. Recently we examined the temperature dependence of the fluorescence intensity of some amphiphilic cyanine dyes incorporated into the liposomes of different kinds of phospholipid and found that only the dye molecules which contain two long acyl chains exhibit the abrupt change in the fluorescence intensity associated with the phase transition of the host-lipid bi1a~ers.l~For instance, when DSOCC or N,N'-distearylthiacarbocyanine (DSSCC) was incorporated into D P P C and l-a-dimyristoylphosphatidylcholine (DMPC) multilamellar liposomes, the abrupt changes in the fluorescence intensity were observed at ca. 42 and 24 "C, respectively. On the other hand, when N-stearyl-N'-methylthiacarbocyanine was used for DSOCC or DSSCC, plots of If vs. T fell on monotonous smooth lines, irrespective of the phase transition of the host lipids over the temperature range between 20 and 50 "C. From these results, together with the results reported by Calzaferri et al.,2O we concluded that the dominant dissipative path of the excitation energy in the cyanine dyes was the twisting motion of the chromophore around the methene bond in the excited singlet state and, consequently, the suppression of this motion by the membrane lipid moiety through the two long acyl chains of the dyes, but not one (19) K. Onugi, K. Kurihara, Y . Toyoshima, and M. Sukigara, Bull. Chern. SOC.Jpn., 53, 669 (1980). (20) G. Calzaferri, H. Gugger, and S. Leutwyler, Helu. Chirn. Acta, 69, 1969 (1976).
30
35 T,
40
45
50
OC
Flgure 4. Temperature profiles of the fluorescence Intensity for the single-lamellar liposomes wkh various slzes: (-) average number of the lipid molecules composing one liposome, N = 0.42 X lo4; (---) N = 0.98 x 104; (-.-) N = 2.5 x 104; (-..-) N = 4.8 x 104.
will increase the fluorescence intensity of the dyes incorporated into the membrane. Thus, the fluorescence intensity of the cyanine dyes with two long acyl chains, such as DSOCC, will reflect the physical state of the lipid bilayer. Figure 3 shows the plots of the fluorescence intensity of DSOCC against temperature in methanol and in the multilamellar DPPC liposomes. In the methanol solution, the plots fall on a monotonous smooth curve, while in the liposomes an abrupt change in the plots appears around the calorimetrically observed main-transition temperature and a much smaller anomalous change takes place at several degrees below the main transition. It should be mentioned here that, although the intensity change associated with the main transition was completely reversible, the small anomalous change at the lower temperature had a real hysteresis as shown in Figure 3. Suurkuusk et alas examined the temperature dependence of the microviscosity in the multilamellar liposomes by the fluorescence depolarization method using 1,6-diphenyl-1,3,5-hexatriene. The DPPC liposomes showed an abrupt 10-fold change in the microviscosity at -42 "C and a small change in a range of 25.2-33.9 "C during a cooling scan. They also mentioned that the calorimetric data taken during a heating scan exhibited pretransition in a range of 33.2-36.7 "C and that instrumental uncertainties could not account for the difference in the results of the low-temperature transition. In the present works, the anomalous change associated with the low-temperature transition was observed in a range of 24.0-31.5 and 31.7-34.5 "C during cooling and heating scans, respectively. These agreements of the present results with those of Suurkuusk strongly suggest that the pretransition in the DPPC multilamellar liposomes has a real hysteresis. Figure 4 illustrates the temperature profiles of I f obtained in the homogeneous DPPC single-lamellar liposomes with different molecular weights. Each profile exhibits an abrupt change at the characteristic temperature which depends on the molecular weight of the sample as increases with the molecular weight up to 42 "C. The thermal change of I f was completely reversible and reproducible, provided that the effluent from the second GPC was immediately submitted to the fluorescence measurements. The temperature profile, however, showed two-step variations, which indicated the aggregation of the liposomes, when the sample had been stored for more than several hours before the fluorescence measurement, par-
Phase Transition in the Single-Lamellar Liposomes h I
31
30
The Journal of Physical Chemistry, Vol. 85,No. 8, 1981 1035 I
35
40 T,
45
O C
Figure 5. Temperature profile of the fluorescence intensity as a function of incubation time at 25 "C: (-) initiil state; (- - -) 5-, (- -) lo-, and (--.-) 20-h incubation.
.
ticularly below 30 "C. Figure 5 shows a time variation of the temperature profile of Iffor a given sample incubated at 25 "C. In addition to an abrupt change at 37 "C, another anomalous change due to the liposome aggregation appeared at 41.5 "C after -3-h incubation, and it increased with time at the expense of the change a t 37 "C. The induction period for the aggregation to occur mainly depends on the temperature and the concentration of the lipid in the dispersion. Thus, the Occurence of the liposome aggregation was easily checked by the temperature profile of If;consequently we could exclude the data for the sample which contained the aggregated liposomes, although it was a very rare case. With regard to the pretransition, it is of interest to compare the results shown in Figures 3 and 4. The anomalous change in the fluorescence intensity associated with the pretransition in the multilamellar liposomes (Figure 3) completely disappears in the single-lamellar liposomes, irrespective of their molecular weights (Figure 4). The pretransition was observed by a number of methods other than calorimetry, and various molecular interpretations of the transition were proposed. For instance, Chapman et a1.21reported that the pretransition was associated with conformational rearrangement of the head-group portion of the lipid molecule, while the deuterium and phosphorus NMR data obtained by Gally et a1.22denied the conformational change in the head group at the pretransition, and the fluorescence studies by Jacobson and Papahadojopo~los~~ indicated that the pretransition had a pronounced effect on the arrangement of the acyl chains. Recently Janiak et al.617 demonstrated by differential scanning calorimetry and x-ray diffraction that the pretransition was associated with a structure transformation from the Lo,to the Pp phase upon heating. The L, phase was assigned to the lamellar bilayer organization with the acyl chains fully extended and tilted with respect to the normal to the bilayer plane but packed in a distorted quasihexagonal lattice, while the P, to the structure which consists of bilayer lamellae distorted by a periodic ripple in the plane of the lamellae with the acyl chains tilted but packed in a regular hexagonal lattice. Combining the results of Janiak et al. and the findings in the present work that the pretransition disappears in the single-lamellar liposomes (even very large liposomes), we may suggest that (21) D. Chapman, Biomembranes, 7, 1 (1975). (22) H. U. Gally, W. Niederberger, and J. Seelig, Biochemistry, 14, 3647 (1975). (23) K. Jacobson and D. Papahadjopoulos, Biochemistry, 14, 152 (1975).
the P, structure does not exist in the single-bilayer systems and that consequently a combination of some unknown effects arising from the interactions among the concentric bilayer sheets are responsible for the formation of the P, structure. Addition of fatty acids to DPPC multilamellar liposomes was found to cause the disappearance of the pretransition.24 We also found that addition of a lipophilic anion such as tetraphenylborate (TPB-) into DPPC multilamellar liposome dispersions a t a TPB-/DPPC molar ratio of ca. 1/100 results in the disappearance of the pretransition, the main transition remaining unchanged except for a slight decrease in the cooperativity. These results seem to support the above interpretation. Electrostatic repulsive force introduced by the charged groups on the membrane surfaces expands the distance of the adjoining bilayers and consequently leads to reduction of the intrinsic interactions between them. Effect of the Size on the Transition Behavior of the Single-LamellarLiposomes. Hereafter we focus our attention on the phase-transition behavior of the single-lamellar liposomes with different sizes. In contrast with the case of the multilamellar liposome dispersions, plots of If vs. T with the data for the single-lamellar liposomes are clearly divided into three parts with no additional change associated with the pretransition, as shown in Figure 4. This fact may suggest that the lipid molecules are in either of two states, namely, solid or fluid states. If this is the case, the low-temperature part corresponds to the solid state, the high-temperature part to the fluid state, and the intermediate to the state of coexistence of the lipids in the solid and fluid states. Consequently the observed fluorescence intensity in the intermediate region may be given in the form of eq 2. Here I f l ( T )and I&T) are the
+ UT)(1- x )
(2) fluorescence intensities of the dye in the fluid-phase region and the solid-phase region, respectively, at temperature T, and x is the fraction of the dye exisitng in the fluid phase at T. Rearranging eq 2, we get eq 3 as the expression If(T)= Iil(T)x
x =
I f ( T ) - Ism MT) - U T )
(3)
for x . If one assumes the solubility of the dyes in the lipid bilayer to be independent of the physical state of the lipids, the value of x is regarded as the degree of the transition, i.e., the fraction of lipids in the fluid phase at T. Thus, if we know the equation giving the temperature dependence of the fluorescence intensity in each phase, we can estimate the fraction of the phase transition at any temperature by measuring the temperature profile of the fluorescence intensity. The fluorescence quantum yield, 4, will be given in the form of eq 4 , where k f is the rate
4 = k f / h + k1 + k z exp[-E/(RT)IJ
(4)
constant of the fluorescence process and lzl and k2exp[-E/(RT)] represent those of the temperature-independent and -dependent nonradiative process, respectively. A rearrangement of eq 4 yields eq 5. An instrumental ln [$/{I - $0+ kl/kJIl = In ( k d k 2 ) + E / ( R T )
(5)
constant, a , converting the observed If into 4 i.e., aIf = 4, has been determined as 1.31 X under the experimental conditions in the present work. In the calculation of a, a value of 0.053 for the fluorescence quantum yield of DSOCC determined by Roth et al. in methanol and at 25 (24) S. Mabrey and J. M. Sturtevant, Biochim. Biophys. Acta, 486,444 (1977).
1036
The Journal of Physical Chemistty, Vol. 85,No. 8, 1981
Takemoto et al. .
,
.-
42
1
I
I
I -
0
*A
y.
"/"-
40
I-
"
-3
-
3a 3 4 5
3.2
3.3
l/TxlO:deg'
+)I
Flgure 6. Plots of In (4/(1 vs. 1 / T : (-) (-. -) in DMPC liposome; (- -) in methanol.
-
In DPPC liposome;
20
50
N XIO-~
Flgure 8. Effect of the liposome size on the midpoint of transition temperature, T,.
-4
3.1
10
s
5
I
3 00 250 200
9
150 100
3 4 5
10
20
50
N x
Flgure 9, Effect of the liposome size on vant't Hoff enthalpy, AHvH.
34
36
3a
40
42
44
T,'C
Flgure 7. Effect of the liposome size on the phase transition; the value of Ncorresponding to each transition curve is 3.7 X lo', 4.8 X lo4, 1.4 X lo4, 2.5 X lo', 0.79 X lo', 0.98 X lo4, 0.52 X lo', 0.55 X lo4, and 0.50 X lo4 molecules from right to left: (-) prepared by the sonication method; (- -) prepared by the alcohol method.
"C was used. The test of the applicability of eq 5 with the data obtained in methanol and in the DPPC and DMPC single-lamellar liposomes is presented in Figure 6 under the assumption of k4 >> kl. The results show good linear relationships between In [ 4 / ( 1 - 4)] and 1 / T not only in methanol but also in the liposomes except in the region of the phase transition. It should be noted that the contribution of the second term in the ordinate is negligibly small compared with the first term except for the data obtained in the solid phase of the DPPC liposomes, where 4 is no longer negligibly small to unity. Thus, since the plots of In [4/(1- 4)] vs. 1/T were comfirmed to show each straight line when the system was in monophase, irrespective of the solid phase and the fluid phase, the values of Ifl(T) and I&T)in the range of the phase transition were estimated by extraporating each straight line into the phase-transition region. The phase-transition curves thus obtained by using eq 3 with the data for the DPPC single-lamellar liposomes are illustrated in Figure 7. The data were taken for the samples prepared by the two methods, i.e., the sonication and alcohol methods. It is demonstrated that the midpoint and the steepness of the transition increase with increasing liposome size. This behavior of the transition was reporducible and independent of the preparation method of the liposomes, provided that they were determined for the freshly obtained samples from the second GPC. Figure 8 represents a relationship between T, and the average
number of the lipid molecules composing one liposome, N. T , increases with N until N = 1.4 X lo4 and approaches a value of 41.7 "C, in agreement with the value of the multilamellar liposomes within experimental error. If the fluid-solid phase transition is considered as a simple two-state reaction, the van't Hoff equation leads to eq 6, (dx /dT)Tm = M V(4RTm2) ~/
(6)
where AHvH is the effective reaction enthalpy, the so-called van't Hoff enthalpy. The values of AHvHcalculated from eq 6 with the curves in Figure 7 are plotted against N in Figure 9. AHvHexhibits a behavior similar to that of T, with respect to N dependency, i.e., AHvHincreases with N until N = 1.4 X lo4and approaches a value of 280 kcal. The cooperativity of the transition has been usually evaluated by the ratio of AHVHto the molar transition enthalpy, AHcd. The value of AHcdin the multilamellar liposomes was reported as 8.74 kcal/mol for DPPC by Mabrey and Sturtevant using high-sensitivity DSC,%while quantitative investigation of the transition enthalpy of the homogeneous single-lamellar liposomes has never been carried out. Suurkuusk et al.* reported a value of 6.3 kcal/mol for the phase transition in a single-lamellar small DPPC liposome system. The value, however, was not obtained in the liposomes with a homogeneous size, but in the unfractionated sample. Therefore it cannot be directly applied to the estimation of the cooperativity, because AHc, may depend on the liposome size, as mentioned by Gruenwald et a1.26 So at present we have to confine ourselves to showing the liposome-size effect on the van't Hoff enthalpy of the transition. In the multilamellar liposomes, AHvH obtained by the present method was 780 f 50 kcal. This value together with AHc* of 8.74 kcal/mol yields the cooperative unit of 90, which is smaller than the value obtained from high-sensitivity calorimetry (25) S. Mabrey and J. M. Sturtevant, Proc. Natl. Acad. Sci. U S A . , 73, 3862 (1976). (26) B. Gruenwald, S. Stankowski, and A. Blume, FEBS Lett., 102,227 (1979).
J. Phys. Chem. 1981, 85,1037-1042
(260).% The decrease of the transition cooperativity of the multilamellar liposomes in the present work may be attributed to the existence of a trace of DSOCC in DPPC. With regard to the single-lamellar liposomes, if we tentatively use a value of 6.4 kcal/mol for AHcd,the cooperative unit of the largest-size liposomes in the present work is estimated as 44. This value, however, may be an overestimation, because 6.4kcal/mol is an average value for AHd determined in the unfractionated liposomes, and AHcalhas been suggested to increase with increasing liposome size.26 Thus, with regard to the value of 90 obtained in the multilamellar liposomes, we may conclude that the cooperative unit of the single-lamellar liposomes does not exceed a half of that of the corresponding multilamellar liposomes, even if they have a molecular weight as large as 3 X 10’. This decrease of the cooperativity in the single-lamellar liposomes cannot be interpreted in terms of the size effect, such as the packing constraint due to the strong curvature associated with the single-lamellar
1037
liposomes, because the effect was excluded by extraporating the molecular weight to infinity. We suppose that the decrease of the cooperativity in the single-lamellar liposomes is due to the lack of the interactions among the bilayers in each liposomes; conversely, the interactions among many concentric bilayers in each multilamellar liposome are responsible for their high cooperativity. Finally, we just mention that the rate constant of the phase transition of the single-lamellar liposomes has been found to depend on their sizes dramatically, by means of the fluorescence temperature-jump method. The experimental results on the kinetic behavior of the phase transition in the single-lamellar liposome systems and a kinetic model based on a two-dimensional Ising lattice shall be presented in succeeding papers.
Acknowledgment. This work was partially supported by Scientific Research Grants (421321 and 447129) from the Ministry of Education of Japan.
Adsorption on Electrodes and Micellization of Some Alkyl Sulfates Ghoiem Naficy, Department of Chemistty, Universlv of Teheran, Teheran, Iran
Pierre Vanel, Daniel Schuhmann, * Reni Bennes, and Emmanuel Tronel-Peyror Groupe de Recherche de Physicochimie des Interfaces, C.N.R.S., E.P. 5051, 34033 Montpelller C a e x , France (Received: April 14, 1980)
The adsorption of alkyl sulfates from aqueous solution at a mercury electrode at concentrationsbelow the cmc has been studied. A comparison with the adsorption of neutral substances and mineral ions shows the probable existence of a coadsorption of the counterions. A very sudden desorption is observed for a charge of the electrode which depends on the surfactant but which in all cases is identical with the superficial charge of the micelle. The results obtained are discussed against the background of the known properties of micelles and vice versa, leading to a description of the probable contribution of the hydrophobic and electrostatic interactions to the formation of micelles.
Introduction The studies on aqueous solutions of sodium dodecyl sulfate (SDS) found in the literature deal essentially14 with the aspect of the formation of micelles which can be described as being due to the competition between the hydrophobic bond between the alkyl chains and the repulsion between the polar groups of the surfactant. The major remaining problem3 area concerns the electrostatic interactions, but at present we are far from being able to give a well-founded description of counterion binding phenomena. It is a natural starting point4 to analyze counterion binding in terms of the general electric double-layer theory of charged interfaces, which has been adapted to the particular case of micelles by Stigter.5-9 (1) G. C. Kresheck, Water: Compr. Treatrise, 4, 95 (1975). (2) 51. Shinoda, T. Nakagawa, B. I. Tamamushi, and T. Isemura in
The superficial part of the micelle is constituted of the polar heads and the interstitial counterions and is termed the Stern layer by analogy with the inner compact layer associated with the diffuse layer in the classical doublelayer model of electrodes.1° However, the structure of the water in this region in micelles is less icelike than at an electrode, the counterions remaining hydrated,11J2which means that they do not form covalent linkages with the polar heads. It would also seem that the discrete charge effects are more important for micelles than in the case of a metal like mercury, while imaging effects must also be different for these two systems. In our group, we have studied the adsorption of different types of surfactants at a mercury electrode,13 and a comparison of the results obtained with those for other in-
“Colloid Surfactants: Some Physicochemical Properties”, Academic Press, New York, 1963. (3) H. Wennerstrom and B. Lindman, Phys. Rep., 52, 1 (1979). (4) B. Ljndman, G. Lindblom, H. Wennerstrom, and G. Gustavsson, Micellizatzon, Solubilization,Microemulsions, [Proc. Int. Symp.],1976,
(8) D. Stigter, J . Phys. Chem., 79, 1008 (1975). (9) D. Stigter, J. Phys. Chem., 79, 1015 (1975). (10) 0. Stern, 2. Elecktrochem, 30, 508 (1924). 97, 3923 (11) H. Gustavsson and B. Lindman, J. Am. Chem. SOC., (1975). (12) M. J. Rosen, J. Colloid Interface Sci., 56, 320 (1976). (13) D. Schuhmann, R. Bennes, M. Privat, E. Tronel-Peyroz, and P.
195 (1977). (5) D. Stigter, J . Phys. Chem., 68, 3603 (1964). (6) D. Stigter, J. Colloid Interface Sci., 47, 473 (1974). (7) D. Stigter, J . Phys. Chem., 78, 2480 (1974).
Vanel, “Adsorption de tensioactifs I l’6lectrode de mercure. L’Blectrode comme modgle d’interfaces”. Communication to “Journ6es d’Etude de la Socidt6 de Chimie Physique: L’Apport de l’6iectrochimie 5. la physicochimie des tensioactifs”, Thiais, Nov 1979.
0022-3654/81/2085-1037$01.25/00 1981 American Chemical Society
