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attenuated total reflection study on subtransition of hydrated dipalmitoylphosphatidylcholine multibilayers
95
t&at&& Spectroscopy, 2 (1991) 95-100 Elsevier Science Publishers B.V., Amsterdam
Fourier transform infrared/ attenuated total reflection study on subtransition of hydrated dipalmitoylphosphatidylcholine multibilayers Emiko Okamura, Junzo Umemura and Tohru Takenaka * Institute for Chemical Research, Kyoto University, Uji, Kyoto-fi 611 (Japan)
(Received 3rd May 1991)
Abstract Changes of the infrared bands of water occurring at the subtransition of a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-water system were studied by Fourier transform infrared/ attenuated total reflection spectroscopy. When hydrated DPPC multibilayers (with 85 wt% of water) were annealed at 2°C for 115 h, the intensities of the water bands were substantially decreased below the subtransition temperature (T,) around 2o”C, whereas those of the hydrocarbon and polar group bands of DPPC were increased. These results suggest that the annealed DPPC multibilayers are in a poorly-hydrated state; water is squeezed out of the bilayer, the phase separation between bulk water and the multibilayer occurs and consequently only bound (unfreezable) water remains in the bilayer region. On heating the sample above T,, the intensity of the water bands was increased and that of the DPPC bands was decreased. This means that the sample acquires a more hydrated state above T, owing to the penetration of bulk water into the interbilayer region. The dichroic ratios of the polar group and water bands also changed at T,. It is concluded that the subtransition is a process in which poorly hydrated DPPC is fully hydrated, being accompanied by the reorientation of the polar groups and water. Keywords: Infrared spectrometry; Dipalmitoylphosphatidylcholine
During the last decade, much attention has been focused on the “subtransition” of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) water systems. Using differential scanning calorimetry, Chen et al. [l] demonstrated that ‘when hydrated DPPC multibilayers are annealed at ca. 0°C for several days, subtransition appears at ca. WC, which is below the pretransition temperature (ca. 34°C). In addition to subsequent calorimetric studies [2-51, several attempts have been made to elucidate the subtransition. From an x-ray diffraction study, Ruocco and Shipley [6] have pointed out that the subtransition corresponds to a structural rearrangement from a bilayer “crystal” to a bilayer gel. They also suggested that the subtransition involves the hydra0924-2031/91/$03.50
multibilayers; Multibilayers; Subtransition
tion of DPPC and an increase in the conformational disorder [7]. Fourier transform infrared (FT-IR) transmission spectrometry by Mantsch and coworkers [8,9] suggested that the two-dimensional acyl chain packing changes at the subtransition from a triclinic-like rigid packing to an orthorhombic subcell packing. A recent FT-IR transmission study by Lewis and McElhaney [lo] indicated that the transition involves reorientation of the chains and changes in hydration and/or hydrogen-bonding interactions at the polar/apolar interfacial region of the bilayer. From infrared spectroscopic changes on prolonged annealing (ca. 2 weeks) at 0-4°C they also demonstrated the appearance of a new phase which is structurally different from that formed after an-
0 1991 - Elsevier Science Publishers B.V. All rights reserved
96
nealing for a few days. As is known, however, nothing has been discussed about changes of infrared bands of water in DPPC multibilayers, although it has been suggested that the transition involves a change in hydration. In previous work [ill, the temperature dependence of polarized FT-IR/attenuated total reflection (ATR) spectra of hydrated DPPC multibilayers was studied, and it was found that the main transition of DPPC can be ascribed to the increase in orientational disorder of the hydrocarbon chains, while the pretransition is due to the reorientation of the polar groups of DPPC and bound water. In this work, the same technique was applied to both annealed and non-annealed DPPC multibilayers. Special attention was paid to the water bands and a characteristic change in the degree of hydration occurring at the subtransition was found. The molecular orientation of DPPC and water in the annealed multibilayers was also studied below and above the subtransition, and compared with that in the non-annealed samples, which do not have the subtransition.
EXPERIMENTAL
L-DPPC was purchased from Sigma and used without further purification. Chloroform, used as a solvent, was a specially prepared reagent from Nacalai Tesque (Kyoto). Water was purified as described previously [ 121. Dry DPPC multibilayers (3-4) km thick were prepared by uniformly spreading a 12 mg ml - ’ chloroform solution (300-400 ~1) on one face of a germanium ATR plate (52 X 18 X 2 mm) followed by evaporation of the solvent. Then the germanium plate was placed in a thermostated Teflon cell as reported previously [ll]. Temperature control and readings were accurate to within * O.l”C. The temperature dependence of the infrared spectra of annealed and non-annealed samples was studied as follows. After 1.5 ml of water had been gently filled into the sample compartment of the cell, the multibilayer was heated at 50°C for 0.5-l h, then the sample was cooled to 2°C
E. OKAMURA
ET AL.
and maintained at this temperature for 115 h (annealed sample). Subsequently, the sample was cooled to - 10°C and subjected to polarized FTIR/ATR measurements at various temperatures from - 10 to 50°C. Prior to the infrared measurements, the sample was left at each temperature for 10 min to attain thermal equilibrium. On the other hand, the hydrated sample heated at 50°C was rapidly cooled to - 10°C for less than 80 min (non-annealed sample) and subjected to the spectral measurements. The temperature range of the measurements and the waiting time were the same as those for the annealed sample. Infrared spectra were recorded on a Nicolet 6000C FT-IR spectrophotometer equipped with an MCI detector. The contribution of the amorphous ice band due to the MCT detector was 4 X 1Ce3 absorbance and almost negligible. One thousand interferograms, collected with an optical velocity of 1.2 cm s- ’ and a maximum optical retardation of 0.25 cm, were coadded, apodized with the Happ-Genzel function and Fourier transformed with one level of zero filling to yield spectra of high signal-to-noise ratio with a resolution slightly better than 4 cm-‘. The accuracy of the frequency was better than +O.l cm-‘. To improve the polarization purity, two wire-grid polarizers were placed in parallel with each other just after a Perkin-Elmer multiple ATR attachment. The angle of incidence was 45” and the number of total reflections was twelve on the sample side. The absorbance was obtained as the peak height of each absorption band.
RESULTS AND DISCUSSION
FT-IR/ATR spectra of non-annealed and annealed DPPC multibilayers at a lower temperature
Figure la shows the polarized ATR spectra of non-annealed DPPC multibilayers (85 wt% water) at - 10°C. Assignments of the absorption bands were summarized in Table I in ref. 11. The intense and broad band in the OH stretching region (3700-3000 cm-‘) has an asymmetric shape with the peak maximum at 3240 cm-’ and a shoulder at 3400 cm-‘, which are characteristic of amorphous ice [13,14].
FT-IR
STUDY
ON SUBTRANSITION
OF HYDRATED
DlPALMITOYLPHOSPHATIDYLCHOLINE
MULTIBILAYERS
97
A
‘,
00
se00
9600
* 3900
3200
sboo
2600 ” 1600
1600
I%00
1too
lb00
601
Wavenumber / cm-’ Fig. 1. Polarized FT-IR/ATR spectra of non-annealed DPPC multibilayers at (a) - 10, (b) 8 and (c) 31°C. Solid and broken lines refer to the electric vector parallel and perpendicular to the plane of incidence, respectively.
(vOH) band of water is much weaker than that of the non-annealed sample (Fig. la). The same feature is also found for the HOH bending
The polarized ATR spectra of annealed DPPC multibilayers at - 10°C are shown in Fig. 2a. Apparently, the intensity of the OH stretching
c
00
se00
3800
.A
3coo ’
Fig. 2. Polarized FT-IR/ATR Fig. 1.
se00
3boo
2600 ” 1.400
,600
lYO0 ’
1LOIl
1000
30,
Wavenumber / cm-’ spectra of annealed DPPC multibilayers at (a) - 10, (b) 8 and (c) 30°C. Solid and broken lines as in
E. OKAMURA
(SHOH) band of water at 1640 cm-i. In contrast, all of the DPPC bands, that is, the CH stretching (3000-2800 cm-‘), C=O stretching (1740 cm-‘), CH, scissoring (1470 cm-‘), CH, wagging (1200 cm-‘), C-O-C stretching (1170 and 1060 cm-‘), PO; stretching (1230 and 1090 cm-‘) and N+(CH,), stretching (970 cm-‘) bands, exhibit a much stronger intensity in Fig. 2a than in Fig. la. These results indicate that the annealed multibilayer is in a poorly hydrated state with the densely packed DPPC molecules as a consequence of the interbilayer water having been squeezed out. In addition, the vOH band of the annealed DPPC shows a characteristic feature of bound water with the broad peak centre around 3350 cm-’ [15]. These facts suggest that water in the annealed multibilayer is strongly bound to DPPC and is free from freezing even at - 10°C. This may correspond to “unfreezable water” reported by Ladbrooke and Chapman [16] from a differential scanning calorimetric study of phospholipidwater systems. The molecular structure and packing in the annealed DPPC multibilayer will be discussed later.
Temperature dependence of the FT-IR /ATR spectra of non-annealed and annealed DPPC multibilayers
The temperature dependence of the FTIR/ATR spectra of the non-annealed and annealed DPPC multibilayers was studied. On heating the non-annealed sample to 8 and 31°C the peak maximum of the vOH band shifts to near 3400 cm-‘, which is characteristic of free liquid water [15,17] (Fig. lb and c>. This spectral change occurs suddenly at O”C, indicating that frozen water at the interbilayer melts at this temperature just like bulk water. For the annealed DPPC, on the other hand, the intensities of the water bands (YOH and aHOH) are still weak at 8°C (Fig. 2b), making a remarkable contrast with the non-annealed sample shown in Fig. lb. However, these bands are sustantially enhanced at 30°C accompanied by a higher frequency shift to the free-water position near 3400 cm-’ (Fig. 2~). Details of the intensity change will be described in the following section.
ET AL.
1.0 -
b 60.5 -
4 I
-20
0
20
40
60
Temperature 1°C
Fig. 3. Temperature dependence of (a) the absorbance of the symmetric CH, stretching (v,CH,) band of DPPC and (b) of the OH stretching (vOH) band of water in the (0) annealed and (0) non-annealed DPPC multibilayers. (0) Absorbance of the vOH band of ice at 3240 cm-‘.
The temperature dependence of the absorbance of the symmetric CH, stretching (v,CH,) band of DPPC at 2850 cm-’ is shown in Fig. 3a. The intensity is considerably decreased around 20°C for the annealed sample, but only slightly decreased around 0°C for the non-annealed sample. Correspondingly, as seen in Fig. 3b, the intensity of the OH stretching band is substantially increased around 20°C for the annealed sample, but only moderately increased around 0°C for the non-annealed sample. The frequency shift of the vOH band from 3350 to 3400 cm-’ induces a decrease in the molar absorption coefficient of water by ca. 10% [18]. Considering this, it is suggested that the decrease in the intensity of the DPPC band and the concomitant increase in that of the water band observed for the annealed sample are due to the swelling of the multibilayers by hydration [19]. It is therefore apparent that the annealed DPPC multibilayers are much more hydrated at the subtransition temperature (T, = 20°C). For the non-annealed DPPC multibilayers, hydration does not occur at 2O”C, corresponding to the absence of the subtransition. Instead, the multibilayers are hydrated to a certain extent at 0°C. This is valid, as the molar absorption coefficient of water is smaller than that of ice. These
FT-IR STUDY ON SUBTRANSITION
OF HYDRATED
DIPALMITOYLPHOSPHATIDYLCHOLINE
facts indicate that water penetrates into the interbilayer after the ice melting. The intensity decrease of the v,CH, band at 0°C can be explained as the ice-melting-induced phase transition of DPPC, which was previously reported by Casal and co-workers [20,21]. The advantage of the ATR technique for the present study should be pointed out. The difference in degrees of hydration shown in Figs. 1 and 2 cannot be observed by the conventional transmission technique. As the total amount of DPPC and that of water do not vary in each case, the intensities of the DPPC and water bands are always identical in the transmission spectra. Using the ATR technique, however, the degrees of hydration of DPPC and the state of the surrounding water could be successfully determined. This is due to the reduced penetration depth (0.2-0.8 pm> of the evanescent wave of the ATR measurements in the spectral range 4000-800 cm-‘, as compared with the film thickness (3-4 pm>.
a \ l
T 6
2.1
1.9 G ,o 1.7 t
99
MULTIBILAYERS
b,_. . _oet.
l
TSl
.
.=
,o
2 n
2.2
C
Ts
TP Tm 0
2.0 e
l
1.8
lf2~
l
20
Temperature
40
60
/C
Fig. 4. Temperature dependence of the dichroic ratios of (a) the symmetric CH, stretching and (b) the symmetric PO; stretching bands of DPPC and k) the OH stretching band of water in the (0) annealed and (0) non-annealed DPPC multibilayers.
Molecular orientation in non-annealed and annealed DPPC multibilayers
Figure 4a shows the temperature dependence of the dichroic ratio (A,,/A I1 of the v,CH, band of annealed and non-annealed DPPC multibilayers. It is seen that, no matter whether the sample was annealed or not, the dichroic ratio increased from 1.1 to 1.4 at the main transition temperature (T, = 41S”C), but changed little at T, and Tp. The same results are also observed for the antisymmetric CH, stretching (v,CH,) band at 2918 band at cm-’ and the CH, scissoring (XH,) 1470 cm-’ (not shown). It is apparent, therefore, that the orientation of the hydrocarbon chain of DPPC is not changed by either annealing or hydration. The dichroic ratio of about 1.1 observed for the v$H, band below T, means an orientational order parameter of the hydrocarbon chain of 0.71 [ll], which is in good agreement with that obtained for the dry (solid) DPPC film (0.72) in a previous study [12]. It is concluded that the hydrocarbon chains are in a highly oriented state with an all-trans conformation as in the solid state, and that the subtransition does not involve the reorientation of the hydrocarbon chain. This is also supported by the
observed frequencies of the V&H, and v,CH, bands (2850 and 2918 cm-‘, respectively). On the other hand, the temperature dependence of the orientation of the polar groups of DPPC and the surrounding water is different from that of the hydrocarbon chains described above. As shown in Fig. 4b, the dichroic ratio of the symmetric PO; stretching band at 1090 cm-’ is decreased at T, for the annealed DPPC, but increased at Tp for the non-annealed sample. A similar temperature dependence of the dichroism is also found for the OH stretching band of water, as seen in Fig. 4c. These results suggest strong cooperation between the polar groups of DPPC and water in both annealed and non-annealed DPPC multibilayers [ll]. Considering that the annealed DPPC is poorly hydrated below T,, it is concluded that the reorientation of the polar groups of DPPC and water is induced by the hydration of the DPPC multibilayers at T,. For the non-annealed DPPC multibilayers, the reorientation occurs at T, (not at T,) [ll], also corresponding to the absence of the subtransition in this sample.
E. OKAMURA
100
___
-_-> -==--s-_
_-
_ _
-bulk
bound
swollen and finally acquire a fully hydrated state. As the dichroic ratios of the polar group and water bands are changed at T, (Fig. 4), it can be concluded that the subtransition involves the reorientation of the polar groups and bound water. Finally, it should be added that Fig. 5b also represents the structure of the non-annealed DPPC multibilayers above 0°C.
-=
water
water
ET AL.
_-
/
REFERENCES
(a) below
Ts
hea+‘ng\
(b) above
TS
Fig. 5. Schematic illustration of the annealed DPPC multibilayers (a) below and (b) above T,.
Conclusion
This FT-IR/ATR study of hydrated DPPC multibilayers has provided useful information about the changes in the degrees of hydration which occur at the subtransition. The spectra of the annealed multibilayers indicate that DPPC is in a poorly hydrated state below T, (Fig. 2a and b). A plausible structure of this sample below T, is illustrated in Fig. 5a. The interbilayer water is squeezed out of the bilayer, which causes phase separation between bulk water and the multibilayer. Consequently, the multibilayer acquires a less hydrated state and only unfreezable bound water remains in the bilayer region. As the multibilayer is thicker than the penetration depth of the evanescent wave in the ATR measurements (ca. 0.2 pm at 3400 cm-‘), only the weak OH band assignable to the bound water can be detected, as seen in Fig. 2a and b. This may be an advantage of the ATR technique over the transmission technique for studying multibilayers. When the annealed sample is heated above T,, the process (a) + (b) shown in Fig. 5 occurs. Water penetrates into the interbilayer region, and consequently DPPC is more hydrated above T,, as is evident from the intense vOH band of water shown in Figs. 2c and 3b. Here no phase separation shown in Fig. 5a appears; the bilayers are
1 S.C. Chen, J.M. Sturtevant and B.J. Gaffney, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 5060. 2 H.H. Fiildner, Biochemistry, 20 (1981) 5707. 3 L. Ter-Minassian-Saraga and G. Madehuont, J. Colloid Interface Sci., 99 (1984) 420. 4 M. Kodama, H. Hashigami and S. Seki, Biochim. Biophys. Acta, 814 (1985) 300. 5 M. Kodama, H. Hashigami and S. Seki, J. Colloid Interface Sci., 117 (1987) 497. 6 M.J. Ruocco and G.G. Shipley, Biochim. Biophys. Acta, 684 (1982) 59. 7 M.J. Ruocco and G.G. Shipley, Biochim. Biophys. Acta, 691 (1982) 309. 8 D.G. Cameron and H.H. Mantsch, Biophys. J., 38 (1982) 175. 9 H.H. Mantsch and R.N. McElhaney, J. Mol. Struct., 217 (1990) 347. 10 R.N.A.H. Lewis and R.N. McElhaney, Biochemistry, 29 (1990) 7946. 11 E. Okamura, J. Umemura and T. Takenaka, B&him. Biophys. Acta, 1025 (1990) 94. 12 E. Okamura, J. Umemura and T. Takenaka, B&him. Biophys. Acta, 856 (1986) 68. 13 U. Buontempo, Phys. Lett., A42 (1972) 17. 14 E. Whalley, Can. J. Chem., 55 (1977) 3429. 15 P.T.T. Wong and C. Huang, Biochemistry, 28 (1989) 1259. 16 B.D. Ladbrooke and D. Chapman, Chem. Phys. Lipids, 3 (1969) 304. 17 G.E. Walrafen, in F. Franks (Ed.), Water, Vol. 1, Plenum, New York, 1972, p. 151. 18 T. Motojima, S. Ikawa and M. Kimura, J. Quant. Spectrosc. Radiat. Transfer, 26 (1981) 177. 19 L. Ter-Minassian-Saraga, E. Okamura, J. Umemura and T. Takenaka, B&him. Biophys. Acta, 946 (1988) 417. 20 H.L. Casal, D.G. Cameron and H.H. Mantsch, J. Phys. Chem., 87 (1983) 5354. 21 H.L. Casal, H.H. Mantsch and D.G. Cameron, Chem. Phys. Lipids, 35 (1984) 77.
t&at&& Spectroscopy, 2 (1991) 95-100 Elsevier Science Publishers B.V., Amsterdam
Fourier transform infrared/ attenuated total reflection study on subtransition of hydrated dipalmitoylphosphatidylcholine multibilayers Emiko Okamura, Junzo Umemura and Tohru Takenaka * Institute for Chemical Research, Kyoto University, Uji, Kyoto-fi 611 (Japan)
(Received 3rd May 1991)
Abstract Changes of the infrared bands of water occurring at the subtransition of a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)-water system were studied by Fourier transform infrared/ attenuated total reflection spectroscopy. When hydrated DPPC multibilayers (with 85 wt% of water) were annealed at 2°C for 115 h, the intensities of the water bands were substantially decreased below the subtransition temperature (T,) around 2o”C, whereas those of the hydrocarbon and polar group bands of DPPC were increased. These results suggest that the annealed DPPC multibilayers are in a poorly-hydrated state; water is squeezed out of the bilayer, the phase separation between bulk water and the multibilayer occurs and consequently only bound (unfreezable) water remains in the bilayer region. On heating the sample above T,, the intensity of the water bands was increased and that of the DPPC bands was decreased. This means that the sample acquires a more hydrated state above T, owing to the penetration of bulk water into the interbilayer region. The dichroic ratios of the polar group and water bands also changed at T,. It is concluded that the subtransition is a process in which poorly hydrated DPPC is fully hydrated, being accompanied by the reorientation of the polar groups and water. Keywords: Infrared spectrometry; Dipalmitoylphosphatidylcholine
During the last decade, much attention has been focused on the “subtransition” of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) water systems. Using differential scanning calorimetry, Chen et al. [l] demonstrated that ‘when hydrated DPPC multibilayers are annealed at ca. 0°C for several days, subtransition appears at ca. WC, which is below the pretransition temperature (ca. 34°C). In addition to subsequent calorimetric studies [2-51, several attempts have been made to elucidate the subtransition. From an x-ray diffraction study, Ruocco and Shipley [6] have pointed out that the subtransition corresponds to a structural rearrangement from a bilayer “crystal” to a bilayer gel. They also suggested that the subtransition involves the hydra0924-2031/91/$03.50
multibilayers; Multibilayers; Subtransition
tion of DPPC and an increase in the conformational disorder [7]. Fourier transform infrared (FT-IR) transmission spectrometry by Mantsch and coworkers [8,9] suggested that the two-dimensional acyl chain packing changes at the subtransition from a triclinic-like rigid packing to an orthorhombic subcell packing. A recent FT-IR transmission study by Lewis and McElhaney [lo] indicated that the transition involves reorientation of the chains and changes in hydration and/or hydrogen-bonding interactions at the polar/apolar interfacial region of the bilayer. From infrared spectroscopic changes on prolonged annealing (ca. 2 weeks) at 0-4°C they also demonstrated the appearance of a new phase which is structurally different from that formed after an-
0 1991 - Elsevier Science Publishers B.V. All rights reserved
96
nealing for a few days. As is known, however, nothing has been discussed about changes of infrared bands of water in DPPC multibilayers, although it has been suggested that the transition involves a change in hydration. In previous work [ill, the temperature dependence of polarized FT-IR/attenuated total reflection (ATR) spectra of hydrated DPPC multibilayers was studied, and it was found that the main transition of DPPC can be ascribed to the increase in orientational disorder of the hydrocarbon chains, while the pretransition is due to the reorientation of the polar groups of DPPC and bound water. In this work, the same technique was applied to both annealed and non-annealed DPPC multibilayers. Special attention was paid to the water bands and a characteristic change in the degree of hydration occurring at the subtransition was found. The molecular orientation of DPPC and water in the annealed multibilayers was also studied below and above the subtransition, and compared with that in the non-annealed samples, which do not have the subtransition.
EXPERIMENTAL
L-DPPC was purchased from Sigma and used without further purification. Chloroform, used as a solvent, was a specially prepared reagent from Nacalai Tesque (Kyoto). Water was purified as described previously [ 121. Dry DPPC multibilayers (3-4) km thick were prepared by uniformly spreading a 12 mg ml - ’ chloroform solution (300-400 ~1) on one face of a germanium ATR plate (52 X 18 X 2 mm) followed by evaporation of the solvent. Then the germanium plate was placed in a thermostated Teflon cell as reported previously [ll]. Temperature control and readings were accurate to within * O.l”C. The temperature dependence of the infrared spectra of annealed and non-annealed samples was studied as follows. After 1.5 ml of water had been gently filled into the sample compartment of the cell, the multibilayer was heated at 50°C for 0.5-l h, then the sample was cooled to 2°C
E. OKAMURA
ET AL.
and maintained at this temperature for 115 h (annealed sample). Subsequently, the sample was cooled to - 10°C and subjected to polarized FTIR/ATR measurements at various temperatures from - 10 to 50°C. Prior to the infrared measurements, the sample was left at each temperature for 10 min to attain thermal equilibrium. On the other hand, the hydrated sample heated at 50°C was rapidly cooled to - 10°C for less than 80 min (non-annealed sample) and subjected to the spectral measurements. The temperature range of the measurements and the waiting time were the same as those for the annealed sample. Infrared spectra were recorded on a Nicolet 6000C FT-IR spectrophotometer equipped with an MCI detector. The contribution of the amorphous ice band due to the MCT detector was 4 X 1Ce3 absorbance and almost negligible. One thousand interferograms, collected with an optical velocity of 1.2 cm s- ’ and a maximum optical retardation of 0.25 cm, were coadded, apodized with the Happ-Genzel function and Fourier transformed with one level of zero filling to yield spectra of high signal-to-noise ratio with a resolution slightly better than 4 cm-‘. The accuracy of the frequency was better than +O.l cm-‘. To improve the polarization purity, two wire-grid polarizers were placed in parallel with each other just after a Perkin-Elmer multiple ATR attachment. The angle of incidence was 45” and the number of total reflections was twelve on the sample side. The absorbance was obtained as the peak height of each absorption band.
RESULTS AND DISCUSSION
FT-IR/ATR spectra of non-annealed and annealed DPPC multibilayers at a lower temperature
Figure la shows the polarized ATR spectra of non-annealed DPPC multibilayers (85 wt% water) at - 10°C. Assignments of the absorption bands were summarized in Table I in ref. 11. The intense and broad band in the OH stretching region (3700-3000 cm-‘) has an asymmetric shape with the peak maximum at 3240 cm-’ and a shoulder at 3400 cm-‘, which are characteristic of amorphous ice [13,14].
FT-IR
STUDY
ON SUBTRANSITION
OF HYDRATED
DlPALMITOYLPHOSPHATIDYLCHOLINE
MULTIBILAYERS
97
A
‘,
00
se00
9600
* 3900
3200
sboo
2600 ” 1600
1600
I%00
1too
lb00
601
Wavenumber / cm-’ Fig. 1. Polarized FT-IR/ATR spectra of non-annealed DPPC multibilayers at (a) - 10, (b) 8 and (c) 31°C. Solid and broken lines refer to the electric vector parallel and perpendicular to the plane of incidence, respectively.
(vOH) band of water is much weaker than that of the non-annealed sample (Fig. la). The same feature is also found for the HOH bending
The polarized ATR spectra of annealed DPPC multibilayers at - 10°C are shown in Fig. 2a. Apparently, the intensity of the OH stretching
c
00
se00
3800
.A
3coo ’
Fig. 2. Polarized FT-IR/ATR Fig. 1.
se00
3boo
2600 ” 1.400
,600
lYO0 ’
1LOIl
1000
30,
Wavenumber / cm-’ spectra of annealed DPPC multibilayers at (a) - 10, (b) 8 and (c) 30°C. Solid and broken lines as in
E. OKAMURA
(SHOH) band of water at 1640 cm-i. In contrast, all of the DPPC bands, that is, the CH stretching (3000-2800 cm-‘), C=O stretching (1740 cm-‘), CH, scissoring (1470 cm-‘), CH, wagging (1200 cm-‘), C-O-C stretching (1170 and 1060 cm-‘), PO; stretching (1230 and 1090 cm-‘) and N+(CH,), stretching (970 cm-‘) bands, exhibit a much stronger intensity in Fig. 2a than in Fig. la. These results indicate that the annealed multibilayer is in a poorly hydrated state with the densely packed DPPC molecules as a consequence of the interbilayer water having been squeezed out. In addition, the vOH band of the annealed DPPC shows a characteristic feature of bound water with the broad peak centre around 3350 cm-’ [15]. These facts suggest that water in the annealed multibilayer is strongly bound to DPPC and is free from freezing even at - 10°C. This may correspond to “unfreezable water” reported by Ladbrooke and Chapman [16] from a differential scanning calorimetric study of phospholipidwater systems. The molecular structure and packing in the annealed DPPC multibilayer will be discussed later.
Temperature dependence of the FT-IR /ATR spectra of non-annealed and annealed DPPC multibilayers
The temperature dependence of the FTIR/ATR spectra of the non-annealed and annealed DPPC multibilayers was studied. On heating the non-annealed sample to 8 and 31°C the peak maximum of the vOH band shifts to near 3400 cm-‘, which is characteristic of free liquid water [15,17] (Fig. lb and c>. This spectral change occurs suddenly at O”C, indicating that frozen water at the interbilayer melts at this temperature just like bulk water. For the annealed DPPC, on the other hand, the intensities of the water bands (YOH and aHOH) are still weak at 8°C (Fig. 2b), making a remarkable contrast with the non-annealed sample shown in Fig. lb. However, these bands are sustantially enhanced at 30°C accompanied by a higher frequency shift to the free-water position near 3400 cm-’ (Fig. 2~). Details of the intensity change will be described in the following section.
ET AL.
1.0 -
b 60.5 -
4 I
-20
0
20
40
60
Temperature 1°C
Fig. 3. Temperature dependence of (a) the absorbance of the symmetric CH, stretching (v,CH,) band of DPPC and (b) of the OH stretching (vOH) band of water in the (0) annealed and (0) non-annealed DPPC multibilayers. (0) Absorbance of the vOH band of ice at 3240 cm-‘.
The temperature dependence of the absorbance of the symmetric CH, stretching (v,CH,) band of DPPC at 2850 cm-’ is shown in Fig. 3a. The intensity is considerably decreased around 20°C for the annealed sample, but only slightly decreased around 0°C for the non-annealed sample. Correspondingly, as seen in Fig. 3b, the intensity of the OH stretching band is substantially increased around 20°C for the annealed sample, but only moderately increased around 0°C for the non-annealed sample. The frequency shift of the vOH band from 3350 to 3400 cm-’ induces a decrease in the molar absorption coefficient of water by ca. 10% [18]. Considering this, it is suggested that the decrease in the intensity of the DPPC band and the concomitant increase in that of the water band observed for the annealed sample are due to the swelling of the multibilayers by hydration [19]. It is therefore apparent that the annealed DPPC multibilayers are much more hydrated at the subtransition temperature (T, = 20°C). For the non-annealed DPPC multibilayers, hydration does not occur at 2O”C, corresponding to the absence of the subtransition. Instead, the multibilayers are hydrated to a certain extent at 0°C. This is valid, as the molar absorption coefficient of water is smaller than that of ice. These
FT-IR STUDY ON SUBTRANSITION
OF HYDRATED
DIPALMITOYLPHOSPHATIDYLCHOLINE
facts indicate that water penetrates into the interbilayer after the ice melting. The intensity decrease of the v,CH, band at 0°C can be explained as the ice-melting-induced phase transition of DPPC, which was previously reported by Casal and co-workers [20,21]. The advantage of the ATR technique for the present study should be pointed out. The difference in degrees of hydration shown in Figs. 1 and 2 cannot be observed by the conventional transmission technique. As the total amount of DPPC and that of water do not vary in each case, the intensities of the DPPC and water bands are always identical in the transmission spectra. Using the ATR technique, however, the degrees of hydration of DPPC and the state of the surrounding water could be successfully determined. This is due to the reduced penetration depth (0.2-0.8 pm> of the evanescent wave of the ATR measurements in the spectral range 4000-800 cm-‘, as compared with the film thickness (3-4 pm>.
a \ l
T 6
2.1
1.9 G ,o 1.7 t
99
MULTIBILAYERS
b,_. . _oet.
l
TSl
.
.=
,o
2 n
2.2
C
Ts
TP Tm 0
2.0 e
l
1.8
lf2~
l
20
Temperature
40
60
/C
Fig. 4. Temperature dependence of the dichroic ratios of (a) the symmetric CH, stretching and (b) the symmetric PO; stretching bands of DPPC and k) the OH stretching band of water in the (0) annealed and (0) non-annealed DPPC multibilayers.
Molecular orientation in non-annealed and annealed DPPC multibilayers
Figure 4a shows the temperature dependence of the dichroic ratio (A,,/A I1 of the v,CH, band of annealed and non-annealed DPPC multibilayers. It is seen that, no matter whether the sample was annealed or not, the dichroic ratio increased from 1.1 to 1.4 at the main transition temperature (T, = 41S”C), but changed little at T, and Tp. The same results are also observed for the antisymmetric CH, stretching (v,CH,) band at 2918 band at cm-’ and the CH, scissoring (XH,) 1470 cm-’ (not shown). It is apparent, therefore, that the orientation of the hydrocarbon chain of DPPC is not changed by either annealing or hydration. The dichroic ratio of about 1.1 observed for the v$H, band below T, means an orientational order parameter of the hydrocarbon chain of 0.71 [ll], which is in good agreement with that obtained for the dry (solid) DPPC film (0.72) in a previous study [12]. It is concluded that the hydrocarbon chains are in a highly oriented state with an all-trans conformation as in the solid state, and that the subtransition does not involve the reorientation of the hydrocarbon chain. This is also supported by the
observed frequencies of the V&H, and v,CH, bands (2850 and 2918 cm-‘, respectively). On the other hand, the temperature dependence of the orientation of the polar groups of DPPC and the surrounding water is different from that of the hydrocarbon chains described above. As shown in Fig. 4b, the dichroic ratio of the symmetric PO; stretching band at 1090 cm-’ is decreased at T, for the annealed DPPC, but increased at Tp for the non-annealed sample. A similar temperature dependence of the dichroism is also found for the OH stretching band of water, as seen in Fig. 4c. These results suggest strong cooperation between the polar groups of DPPC and water in both annealed and non-annealed DPPC multibilayers [ll]. Considering that the annealed DPPC is poorly hydrated below T,, it is concluded that the reorientation of the polar groups of DPPC and water is induced by the hydration of the DPPC multibilayers at T,. For the non-annealed DPPC multibilayers, the reorientation occurs at T, (not at T,) [ll], also corresponding to the absence of the subtransition in this sample.
E. OKAMURA
100
___
-_-> -==--s-_
_-
_ _
-bulk
bound
swollen and finally acquire a fully hydrated state. As the dichroic ratios of the polar group and water bands are changed at T, (Fig. 4), it can be concluded that the subtransition involves the reorientation of the polar groups and bound water. Finally, it should be added that Fig. 5b also represents the structure of the non-annealed DPPC multibilayers above 0°C.
-=
water
water
ET AL.
_-
/
REFERENCES
(a) below
Ts
hea+‘ng\
(b) above
TS
Fig. 5. Schematic illustration of the annealed DPPC multibilayers (a) below and (b) above T,.
Conclusion
This FT-IR/ATR study of hydrated DPPC multibilayers has provided useful information about the changes in the degrees of hydration which occur at the subtransition. The spectra of the annealed multibilayers indicate that DPPC is in a poorly hydrated state below T, (Fig. 2a and b). A plausible structure of this sample below T, is illustrated in Fig. 5a. The interbilayer water is squeezed out of the bilayer, which causes phase separation between bulk water and the multibilayer. Consequently, the multibilayer acquires a less hydrated state and only unfreezable bound water remains in the bilayer region. As the multibilayer is thicker than the penetration depth of the evanescent wave in the ATR measurements (ca. 0.2 pm at 3400 cm-‘), only the weak OH band assignable to the bound water can be detected, as seen in Fig. 2a and b. This may be an advantage of the ATR technique over the transmission technique for studying multibilayers. When the annealed sample is heated above T,, the process (a) + (b) shown in Fig. 5 occurs. Water penetrates into the interbilayer region, and consequently DPPC is more hydrated above T,, as is evident from the intense vOH band of water shown in Figs. 2c and 3b. Here no phase separation shown in Fig. 5a appears; the bilayers are
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