Chemistry and Physics of Lipids, 52 (1990) 11--27 Elsevier Scientific Publishers Ireland Ltd.
11
Interaction of 7,7,8,8-tetracyanoquinodimethane with diacylphosphatidylcholines and -phosphatidylglycerols. A photoacoustic Fourier transform infrared study Timo I. Lotta*, Antti Pekka Tulkki, Jorma A. Virtanen and Paavo K.J. Kinnunen** K S V Chemical Corporation, P.O. Box 128, SF-00381 Helsinki (Finland) (Received December 19th, 1988; accepted March 15th, 1989)
7,7,8,8-Tetracyanoquinodimethane (TCNQ) was incorporated in fully hydrated liposomes of the following pyrene-containing as well as non-labelled phospholipids: l-palmitoyl-2-ll0-(pyren-l-yl)decanoyl]-sn-glycero-3-phosphatidylcholine (PPDPC), l-palmitoyl-2-[lO-(pyren-l-yl)decanoyl]-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-PPDPG), l-palmitoyl-2-[l 0(pyren-l°yl)decanoyll-sn-glycero-3-phosphatidyl-sn-3'-glycerol (3'-PPDPG), l-[10-(pyren-l-yl)decanoyl]-2-palmitoyl-sn-glycero-3phosphatidyl-sn-3'-glycerol (3'-PDPPG), 1-[lO-pyren-l-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-sn-l'-~lycerol (l'PDPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-DPPG). Lyophilized charge-transfer (CT) complexes of TCNQ with phospholipids were examined by Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS). Due to the spectral changes observed in the vibrational bands originating from the CH 2 and C = O stretching vibrations, and the bands associated with the polar headgroup of the phospholipids it is evident that TCNQ has only a minor perturbing effect on the hydrocarbon chains. However, the molecular interaction between TCNQ and phospholipids is seen in the polar headgroup region. The donated electrons are most likely located on the oxygens of the phosphate group in the polar head. As judged from the present infrared data interations of TCNQ with phosphatidylcholines (PC) and phosphatidylglycerols (PG) differ. For PG the complex formation produces a second strong C = O stretching band at approx. 1710 cm -~ in addition to the band at approx. 1735 cm -t indicating a specific molecular interaction in the interfacial region.
Keywords: Fourier transform infrared spectroscopy; phospholipids; photoacoustic; complex formation.
Introduction
In order to understand the characteristics of the large cooperative assemblies such as biomembranes and the molecular forces which govern lipid-lipid, lipid-protein, and lipid-drug interactions it is necessary to consider organization and packing properties of lipids and *To whom correspondence should be sent at present address: Orion Corporation Ltd., Orion Pharmaceutica, Research Center, P.O. Box 65, SF-02101 Espoo, Finland. **Present address: Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10, SF-00170 Helsinki, Finland.
their molecular interactions with guest molecules and ions [lml0] (and Refs. cited therein). Some of the membrane processes can be explained on the basis of charge-transfer interactions of the membrane lipid molecules with guest agents. For instance the formation of a very stable complex between Adriamycin® (doxorubicin hydrochloride) and cardiolipin, a phospholipid specific to the inner mitochondrial membrane, has been shown to inhibit several mitochondrial enzymes whose activities depend on the presence of cardiolipin [11]. Another important interaction mode of lipids is their ability to interact via intermolecular hydrogen
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
12 bonds [12--17]. The hydrogen bonded and charge-transfer complexes can be considered as restricted cases of the broad class of electron donor-acceptor complexes where transfer of the negative charge occurs from an electron donor to an electron acceptor [18]. Recently the properties of CT-complexes of organic materials as electron donors with different electron acceptors have been extensively studied [10,19--31]. Nandy and Bhowmik [10] have studied electron donor-acceptor interaction of phospholipids with iodine. They have shown that the electron donor strengths of phospholipids depend on the polar head in the order sphingomyelin ) phosphatidylcholine lysophosphatidylcholine ) phosphatidylethanolamine [10]. In general phospholipids are strong electron donors and they have been shown to interact with different electron acceptors such as 2,4-dinitrophenol, picric acid, trinitrobenzene, etc. [10]. In addition a great deal of interest has been focused on the electron transport properties of quasi-onedimensional chains formed by TCNQ in several different charge-transfer complexes [24-31]. Because the orientation of donors and acceptors has a significant influence on the conductivity of a CT-complex the LangmuirBlodgett (LB) technique has become an effective means of molecular arrangement [24--31]. In the present study we have examined complexes of the pyrene-containing and nonlabelled phospholipid analogues with TCNQ. In general TCNQ, an electron acceptor, is a useful component of many interesting chargetransfer systems. To characterize the structures of the solid complexes Fourier transform infrared spectroscopy was performed using photoacoustic detection (FTIR-PAS). This technique provides a non-destructive way to study the formed CT-complexes of phospholipids with TCNQ without the need to disperse them into alkali halide matrices [32--34]. Samples can be readily analyzed without further preparation, i.e. structural alterations during the pelleting process can be avoided. FTIR-PAS thus represents a major advance which has not been extensively utilized so far. The UV-VIS
spectra of liquid samples of the complexes were also recorded. The aims of the present study are to characterize the localization of TCNQ molecules in phospholipid membranes and the influence of the lipid headgroup on the complexation. Materials and methods
The pyrene-labelled phospholipids, 1-palmitoyl-2- [ 10-(pyren- 1-yl)decanoyl] -sn-glycero-3phosphatidylcholine (PPDPC), 1-palmitoyl-2[ 10-(pyren- 1-yl)decanoyl]-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-PPDPG), 1-palmitoyl-2[ 10-(pyren- 1-yl)decanoyl]-sn-glycero-3-phosphatidyl-sn-3'-glycerol (3'-PPDPG), 1-[10-(pyren-1yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-sn-l'-glycerol (I'-PDPPG) and 1-[10-pyren1-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-sn-3'-glycerol (3'-PDPPG) were products of KSV Chemical Corporation (Helsinki, Finland). 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero3-phosphatidyl-rac'-glycerol (rac'-DPPG) and the cholesterol (CHOL) were purchased from Sigma. Phosphatidylglycerols were used as ammonium salts. 7,7,8,8,-Tetracyanoquinodimethane (TCNQ) was from Eastman Kodak Company. For the chemical structures of the phospholipid analogues, see Refs. 35 and 36. The water used in experiments was freshly deionized in a Milli-RO/Milli-Q (Millipore, U.S.A.) filtering system. Infrared spectra were recorded with a Nicolet 60SX FTIR spectrophotometer equipped with a standard Gilford/Nicolet photoacoustic cell fitted with KRS-5 windows. For each spectrum, 500 scans were coadded and apodized with the Happ-Genzel function. The spectra were recorded at 4 and 8 cm -~ resolutions. Smoothing procedures were not applied. The peak positions were determined by the peakpicking routine supplied with the FTIR software by Nicolet. In the photoacoustic measurements each single-beam spectrum was rationed to a standard carbon black spectrum used as a background and the sample chamber was flushed with dry nitrogen to remove
13 atmospheric CO 2 and water vapour before measurements. Samples for infrared measurements were prepared using the following procedure. (a) Pure phospholipids. Stock solutions of the lipids were made in chloroform. The solvent was removed under a stream of nitrogen whereafter the sample was maintained under reduced pressure overnight with a vacuum dryer (Edwards, U.K.). The dry lipid was hydrated to yield a final concentration of 2 mg/ml in water. Multilamellar liposomes of the hydrated lipids were prepared by vortexing (0.5--1 min) and sonicating (1--2 min) on a Bransonic 221 bath type irradiator at temperatures above the transition temperature ( T ) of the lipid in question. To remove the water solvent samples of fully hydrated liposomes were maintained under reduced pressure (lyophilized) for a few days whereafter infrared spectra were recorded. As discussed below the quantity of crystallization water was equal or less than two molecules of H 2 0 per lipid molecule.
(b) Phospholipid
and
TCNQ complexes.
Stock solutions of lipid in chloroform and TCNQ in chloroform were combined in 1:1 molar ratios. The solvent was removed under a stream of nitrogen after which the samples were maintained under reduced pressure overnight and the dried mixtures were dispersed in excess water as described above. Mixtures of phospholipids and TCNQ appeared as yellowish solutions which were incubated (approx. 2 --3 days) at temperatures above the lipid phase transition until the solution turned blue. The complexes were maintained under reduced pressure for a few days to remove water. The infrared spectra were measured from solid lipid/TCNQ complexes. For the preparation of phospholipid (DPPC or rac'-DPPG) and cholesterol mixtures (1:1) the same procedure was followed. UV-VIS spectra of phospholipid and TCNQ mixtures were measured with a Hitachi U-3200 spectrophotometer using quartz cuvet[es with an optical pathlength of 1.00 cm. The samples were prepared as follows: 30 nmol of
PPDPC, rac'-PPDPG or rac'-DPPG were mixed with 30 nmol of TCNQ in chloroform. The solutions were dried under nitrogen flow and thereafter 2 ml of pure degassed water was added. The sample preparation was continued as described above and the formation of the complexes was observed due to the change of the colour of the samples. All spectroscopic measurements were done at room temperature (=23°C). Results and discussion
The entire infrared spectra of pure phospholipids, pure TCNQ and lipid/TCNQ complexes were studied in detail in order to obtain information on the complexation and the localization of TCNQ in the phospholipid membrane. Previously infrared spectroscopy has been used to discern the molecular characteristics of phospholipid phase states [37]. The changes in the spectra of phospholipids undergoing phase transition are marked and provide information about chain packing, lattice characteristics, headgroup hydration as well as the interfacial region of bilayer, i.e. infrared spectroscopy can be used as a submolecular probe [37]. The C--H stretching region (approx. 3000-2800 cm -1) provides information mainly on the packing of the hydrocarbon chains. The C--H stretching wavenumbers are significantly affected by the conformation of the adjoining C--C bonds. An increased number of gauche C--C bonds (a more disordered lattice) is consistent with the shifts of the C--H stretching bands to higher wavenumbers observed upon melting of n-alkanes and liposomes upon the main phase transition into the liquid crystalline state [37]. Thus wavenumbers of the C H 2 stretching bands depend upon the order of the lipid acyl chains and bandwidths upon the librational and torsional motions of the chains. Raman and infrared spectra of C = O stretch of phospholipids provide a sensitive probe of molecular interactions in the interfacial region [16,37]. Using Fourier selfdeconvolution technique the broad carbonyl
14
stretching mode has been resolved into two bands at approx. 1741 and 1726 cm -~ corresponding to the sn-I and sn-2 ester carbonyl groups, respectively [17,38]. We, as well as others, have previously shown that the ratio of the relative intensities of these two bands depends on the phase state of the phospholipid membrane [39]. The vibrational modes originating from the hydrophilic polar headgroup of the lipids appears in the approx. 1300--900 cm -~ region. However, the bands are overlapped and the interpretation of the spectra is not straightforward.
Spectral changes in the IR and UV- VIS energy regions o f TCNQ due to the complex formation Figure 1 displays the infrared spectrum of solid TCNQ in its neutral form. There are four strong vibrational bands in the IR spectrum assigned to the CH stretch (3050 cm-~), CN stretch (2225 cm-~), C = C stretch (1543 c m -m) and CH out of plane bending (860 cm-~). In addition the following medium intensity bands are found: 1354 (CH bend) and
El) oq
1124 (1114) (C,--CN stretch) cm -~ [40--43]. Mixing of phospholipids and TCNQ yields a yellowish solution, and the IR spectrum resembles one characteristic for neutral TCNQ molecules and pure lipid. Similar behaviour was found for all the lipids in our study. The progression and perfection of the complexation process can be conveniently followed in the 2300--2000 cm -~ region because neutral TCNQ has an intensive nitrile stretching mode in this energy region whereas no fundamental modes are observed for phospholipids in this region. During incubation of lipid/TCNQ mixtures the C - N stretching band originating from the neutral TCNQ molecules decreased. In the 2300--2000 cm -l region there appears new lower-energy vibrational bands due to the complex. Upon continued incubation (depending on the phospholipd in question; average time 2--3 days) the band at 2225 cm -~ originating from the neutral TCNQ disappeared and the solution tu~'ns dark blue. In the case of DPPC/TCNQ mixture for instance there appeared two lower-wavenumber peaks at 2180 and 2125 cm -1. FTIR-PAS spectra of the complexes of phospholipids and TCNQ in the
c
© c/C\c
C~J L~I~ (.3 Z
CO (I= 0o LO 0
C3
C3
~zo
36zo
z>zo
z~zo
21zo
l~zo
WF~VENUMBER
J6zO l~zo
cm. 1
~zo
~zo
Fig. 1. Infrared spectrum of solid T C N Q in a KBr matrix, and the chemical structure of T C N Q molecule.
15 2300--2000 cm -1 region are shown in Fig. 2 (A--D). Table 1 summarizes the observed infrared absorption bands. Ruaudel-Teixier et al. [24] found a lowerwavenumber shift in the C - N stretching mode of TCNQ when present as a CT-complex with N-docosylpyridinium. They assigned the lowerenergy band at approx. 2190 cm -~ to the nitrile stretching mode of TCNQ-. Richard et al. [31] have recently examined LangmuirBlodgett assemblies of N-docosylpyridinium/ TCNQ CT-complexes and they have assigned the following bands to the C-=N stretch in the charge-transfer complex: 2185, 2178, 2173, 2158 and 2150 cm-L In addition for LiTCNQ CT-complex the bands at 2195, 2184 and 2168 cm -~ are assigned to the C---N stretching bands in water solution [40]. Bozio et al. [40] have suggested that the formation of the self-dimer of TCNQ- is accompanied by the appearance of some additional absorptions in the infrared spectrum. The phenomenon is interpreted in terms of the Ferguson-Person vibronic model, which embodies the basic concepts of the charge transfer interaction, i.e., the additional bands correspond to strong vibronic activation in infrared of the out-of-phase components of the totally symmetric molecular modes such as the totally symmetric C---N stretching mode. Also the other IR spectral regions of the blue lipid/TCNQ solutions reveal no sign of the presence of neutral TCNQ molecules. The diagnostic region is 1550--1500 cm -1, where one observes the band at 1507 cm-' for rac'D P PG/TC NQ and at 1496 cm -t for D P P C / TCNQ attributed to the C = C stretching mode of TCNQ- species in the dimeric state [31], whereas that of the neutral species would appear at 1543 cm -~ as found immediately after the mixing of phospholipid and TCNQ. The bands observed at 1601 and 1599 cm -~ for rac'-DPPG/TCNQ and DPPC/TCNQ, respectively, are also assigned to the C = C stretching modes of TCNQ- anion [31]. In K÷ TCNQ- two bands at 1579 and 1509 cm-' associated with the C = C stretch are observed [41]. Thus the wavenumbers found for TCNQ in a blue solution are typical of the TCNQ-
anion in either its monomeric or dimeric form. Also the electronic spectra for lipid/TCNQ showed changes during complexation. The UV-VIS spectrum of neutral TCNQ in ethyl acetate is shown in Fig. 3A, and reveals an intensive absorption band at approx. 396 nm. The UV-VIS spectral data are consistent with the picture emerging from our IR spectroscopic studies. Immediately after the mixing of phospholipids and TCNQ a band at approx. 396 nm assigned to neutral TCNQ is evident. In the blue solution obtained after incubation broad bands at approx. 630 and 830 nm assigned to the [TCNQ-] 2 and TCNQ-, respectively, appear (Fig. 3B) [31,44]. In addition there is a band at approx. 310 nm which overlaps with pyrene absorption in the pyrenephospholipd/TCNQ complexes. This band is also assigned to the [TCNQ-]2 [31]. The bands at 630 and 830 nm are characteristic for CT-complexes [45] and can be attributed to charge transfer from the phospholipid transfer from the phospholipid to TCNQ. A charge-transfer band at 1050 nm is also evidnet in the UV-VIS spectrum of N-docosylpyridinium [25].
The acyl chain region This spectral region contains primarily the vibrational modes of acyl chains of phospholipids. Unfortunately, there is some overlapping with the methylene modes of the headgroup. Compared for instance to the spectra of pure rac'-DPPG TCNQ causes only minor changes in the observed spectral features. The two strong bands at approx. 2918 and 2850 cm -~ are assigned to the asymmetric and symmetric CH 2 stretching modes of the methylene segment, respectively [37]. The exact position (wavenumber) of the C--H stretching provides a sensitive measure of the degree of conformational disorder of the membrane lipids. Neither the wavenumbers nor the half-bandwidths of the asymmetric and symmetric CH 2 stretching modes are significantly altered upon TCNQ/phospholipid complex formation. This is expected if no
16 penetration of TCNQ into the hydrocarbon chain region occurs. The same kind of tendency is also observed for the other phospholipids studied. However, in the case of pyrene-labelled analogues the C H 2 stretch-
c (D W-0 • Z ~E
Q
O) N 0
a 0 (:3 0
B
(D bJ~ ~_~ Z
m
O
Q (:3 Q
NAVENUMBER
(cm.1)
ing bands are slightly shifted in the presence of TCNQ, Table II. This can be compared with the perturbation produced by cholesterol in the structural features of the hydrocarbon chains• For instance in DPPC-cholesterol
17
0
C
0 nn 0 Z
11
Interaction of 7,7,8,8-tetracyanoquinodimethane with diacylphosphatidylcholines and -phosphatidylglycerols. A photoacoustic Fourier transform infrared study Timo I. Lotta*, Antti Pekka Tulkki, Jorma A. Virtanen and Paavo K.J. Kinnunen** K S V Chemical Corporation, P.O. Box 128, SF-00381 Helsinki (Finland) (Received December 19th, 1988; accepted March 15th, 1989)
7,7,8,8-Tetracyanoquinodimethane (TCNQ) was incorporated in fully hydrated liposomes of the following pyrene-containing as well as non-labelled phospholipids: l-palmitoyl-2-ll0-(pyren-l-yl)decanoyl]-sn-glycero-3-phosphatidylcholine (PPDPC), l-palmitoyl-2-[lO-(pyren-l-yl)decanoyl]-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-PPDPG), l-palmitoyl-2-[l 0(pyren-l°yl)decanoyll-sn-glycero-3-phosphatidyl-sn-3'-glycerol (3'-PPDPG), l-[10-(pyren-l-yl)decanoyl]-2-palmitoyl-sn-glycero-3phosphatidyl-sn-3'-glycerol (3'-PDPPG), 1-[lO-pyren-l-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-sn-l'-~lycerol (l'PDPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-DPPG). Lyophilized charge-transfer (CT) complexes of TCNQ with phospholipids were examined by Fourier transform infrared photoacoustic spectroscopy (FTIR-PAS). Due to the spectral changes observed in the vibrational bands originating from the CH 2 and C = O stretching vibrations, and the bands associated with the polar headgroup of the phospholipids it is evident that TCNQ has only a minor perturbing effect on the hydrocarbon chains. However, the molecular interaction between TCNQ and phospholipids is seen in the polar headgroup region. The donated electrons are most likely located on the oxygens of the phosphate group in the polar head. As judged from the present infrared data interations of TCNQ with phosphatidylcholines (PC) and phosphatidylglycerols (PG) differ. For PG the complex formation produces a second strong C = O stretching band at approx. 1710 cm -~ in addition to the band at approx. 1735 cm -t indicating a specific molecular interaction in the interfacial region.
Keywords: Fourier transform infrared spectroscopy; phospholipids; photoacoustic; complex formation.
Introduction
In order to understand the characteristics of the large cooperative assemblies such as biomembranes and the molecular forces which govern lipid-lipid, lipid-protein, and lipid-drug interactions it is necessary to consider organization and packing properties of lipids and *To whom correspondence should be sent at present address: Orion Corporation Ltd., Orion Pharmaceutica, Research Center, P.O. Box 65, SF-02101 Espoo, Finland. **Present address: Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10, SF-00170 Helsinki, Finland.
their molecular interactions with guest molecules and ions [lml0] (and Refs. cited therein). Some of the membrane processes can be explained on the basis of charge-transfer interactions of the membrane lipid molecules with guest agents. For instance the formation of a very stable complex between Adriamycin® (doxorubicin hydrochloride) and cardiolipin, a phospholipid specific to the inner mitochondrial membrane, has been shown to inhibit several mitochondrial enzymes whose activities depend on the presence of cardiolipin [11]. Another important interaction mode of lipids is their ability to interact via intermolecular hydrogen
0009-3084/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
12 bonds [12--17]. The hydrogen bonded and charge-transfer complexes can be considered as restricted cases of the broad class of electron donor-acceptor complexes where transfer of the negative charge occurs from an electron donor to an electron acceptor [18]. Recently the properties of CT-complexes of organic materials as electron donors with different electron acceptors have been extensively studied [10,19--31]. Nandy and Bhowmik [10] have studied electron donor-acceptor interaction of phospholipids with iodine. They have shown that the electron donor strengths of phospholipids depend on the polar head in the order sphingomyelin ) phosphatidylcholine lysophosphatidylcholine ) phosphatidylethanolamine [10]. In general phospholipids are strong electron donors and they have been shown to interact with different electron acceptors such as 2,4-dinitrophenol, picric acid, trinitrobenzene, etc. [10]. In addition a great deal of interest has been focused on the electron transport properties of quasi-onedimensional chains formed by TCNQ in several different charge-transfer complexes [24-31]. Because the orientation of donors and acceptors has a significant influence on the conductivity of a CT-complex the LangmuirBlodgett (LB) technique has become an effective means of molecular arrangement [24--31]. In the present study we have examined complexes of the pyrene-containing and nonlabelled phospholipid analogues with TCNQ. In general TCNQ, an electron acceptor, is a useful component of many interesting chargetransfer systems. To characterize the structures of the solid complexes Fourier transform infrared spectroscopy was performed using photoacoustic detection (FTIR-PAS). This technique provides a non-destructive way to study the formed CT-complexes of phospholipids with TCNQ without the need to disperse them into alkali halide matrices [32--34]. Samples can be readily analyzed without further preparation, i.e. structural alterations during the pelleting process can be avoided. FTIR-PAS thus represents a major advance which has not been extensively utilized so far. The UV-VIS
spectra of liquid samples of the complexes were also recorded. The aims of the present study are to characterize the localization of TCNQ molecules in phospholipid membranes and the influence of the lipid headgroup on the complexation. Materials and methods
The pyrene-labelled phospholipids, 1-palmitoyl-2- [ 10-(pyren- 1-yl)decanoyl] -sn-glycero-3phosphatidylcholine (PPDPC), 1-palmitoyl-2[ 10-(pyren- 1-yl)decanoyl]-sn-glycero-3-phosphatidyl-rac'-glycerol (rac'-PPDPG), 1-palmitoyl-2[ 10-(pyren- 1-yl)decanoyl]-sn-glycero-3-phosphatidyl-sn-3'-glycerol (3'-PPDPG), 1-[10-(pyren-1yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-sn-l'-glycerol (I'-PDPPG) and 1-[10-pyren1-yl)decanoyl]-2-palmitoyl-sn-glycero-3-phosphatidyl-sn-3'-glycerol (3'-PDPPG) were products of KSV Chemical Corporation (Helsinki, Finland). 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero3-phosphatidyl-rac'-glycerol (rac'-DPPG) and the cholesterol (CHOL) were purchased from Sigma. Phosphatidylglycerols were used as ammonium salts. 7,7,8,8,-Tetracyanoquinodimethane (TCNQ) was from Eastman Kodak Company. For the chemical structures of the phospholipid analogues, see Refs. 35 and 36. The water used in experiments was freshly deionized in a Milli-RO/Milli-Q (Millipore, U.S.A.) filtering system. Infrared spectra were recorded with a Nicolet 60SX FTIR spectrophotometer equipped with a standard Gilford/Nicolet photoacoustic cell fitted with KRS-5 windows. For each spectrum, 500 scans were coadded and apodized with the Happ-Genzel function. The spectra were recorded at 4 and 8 cm -~ resolutions. Smoothing procedures were not applied. The peak positions were determined by the peakpicking routine supplied with the FTIR software by Nicolet. In the photoacoustic measurements each single-beam spectrum was rationed to a standard carbon black spectrum used as a background and the sample chamber was flushed with dry nitrogen to remove
13 atmospheric CO 2 and water vapour before measurements. Samples for infrared measurements were prepared using the following procedure. (a) Pure phospholipids. Stock solutions of the lipids were made in chloroform. The solvent was removed under a stream of nitrogen whereafter the sample was maintained under reduced pressure overnight with a vacuum dryer (Edwards, U.K.). The dry lipid was hydrated to yield a final concentration of 2 mg/ml in water. Multilamellar liposomes of the hydrated lipids were prepared by vortexing (0.5--1 min) and sonicating (1--2 min) on a Bransonic 221 bath type irradiator at temperatures above the transition temperature ( T ) of the lipid in question. To remove the water solvent samples of fully hydrated liposomes were maintained under reduced pressure (lyophilized) for a few days whereafter infrared spectra were recorded. As discussed below the quantity of crystallization water was equal or less than two molecules of H 2 0 per lipid molecule.
(b) Phospholipid
and
TCNQ complexes.
Stock solutions of lipid in chloroform and TCNQ in chloroform were combined in 1:1 molar ratios. The solvent was removed under a stream of nitrogen after which the samples were maintained under reduced pressure overnight and the dried mixtures were dispersed in excess water as described above. Mixtures of phospholipids and TCNQ appeared as yellowish solutions which were incubated (approx. 2 --3 days) at temperatures above the lipid phase transition until the solution turned blue. The complexes were maintained under reduced pressure for a few days to remove water. The infrared spectra were measured from solid lipid/TCNQ complexes. For the preparation of phospholipid (DPPC or rac'-DPPG) and cholesterol mixtures (1:1) the same procedure was followed. UV-VIS spectra of phospholipid and TCNQ mixtures were measured with a Hitachi U-3200 spectrophotometer using quartz cuvet[es with an optical pathlength of 1.00 cm. The samples were prepared as follows: 30 nmol of
PPDPC, rac'-PPDPG or rac'-DPPG were mixed with 30 nmol of TCNQ in chloroform. The solutions were dried under nitrogen flow and thereafter 2 ml of pure degassed water was added. The sample preparation was continued as described above and the formation of the complexes was observed due to the change of the colour of the samples. All spectroscopic measurements were done at room temperature (=23°C). Results and discussion
The entire infrared spectra of pure phospholipids, pure TCNQ and lipid/TCNQ complexes were studied in detail in order to obtain information on the complexation and the localization of TCNQ in the phospholipid membrane. Previously infrared spectroscopy has been used to discern the molecular characteristics of phospholipid phase states [37]. The changes in the spectra of phospholipids undergoing phase transition are marked and provide information about chain packing, lattice characteristics, headgroup hydration as well as the interfacial region of bilayer, i.e. infrared spectroscopy can be used as a submolecular probe [37]. The C--H stretching region (approx. 3000-2800 cm -1) provides information mainly on the packing of the hydrocarbon chains. The C--H stretching wavenumbers are significantly affected by the conformation of the adjoining C--C bonds. An increased number of gauche C--C bonds (a more disordered lattice) is consistent with the shifts of the C--H stretching bands to higher wavenumbers observed upon melting of n-alkanes and liposomes upon the main phase transition into the liquid crystalline state [37]. Thus wavenumbers of the C H 2 stretching bands depend upon the order of the lipid acyl chains and bandwidths upon the librational and torsional motions of the chains. Raman and infrared spectra of C = O stretch of phospholipids provide a sensitive probe of molecular interactions in the interfacial region [16,37]. Using Fourier selfdeconvolution technique the broad carbonyl
14
stretching mode has been resolved into two bands at approx. 1741 and 1726 cm -~ corresponding to the sn-I and sn-2 ester carbonyl groups, respectively [17,38]. We, as well as others, have previously shown that the ratio of the relative intensities of these two bands depends on the phase state of the phospholipid membrane [39]. The vibrational modes originating from the hydrophilic polar headgroup of the lipids appears in the approx. 1300--900 cm -~ region. However, the bands are overlapped and the interpretation of the spectra is not straightforward.
Spectral changes in the IR and UV- VIS energy regions o f TCNQ due to the complex formation Figure 1 displays the infrared spectrum of solid TCNQ in its neutral form. There are four strong vibrational bands in the IR spectrum assigned to the CH stretch (3050 cm-~), CN stretch (2225 cm-~), C = C stretch (1543 c m -m) and CH out of plane bending (860 cm-~). In addition the following medium intensity bands are found: 1354 (CH bend) and
El) oq
1124 (1114) (C,--CN stretch) cm -~ [40--43]. Mixing of phospholipids and TCNQ yields a yellowish solution, and the IR spectrum resembles one characteristic for neutral TCNQ molecules and pure lipid. Similar behaviour was found for all the lipids in our study. The progression and perfection of the complexation process can be conveniently followed in the 2300--2000 cm -~ region because neutral TCNQ has an intensive nitrile stretching mode in this energy region whereas no fundamental modes are observed for phospholipids in this region. During incubation of lipid/TCNQ mixtures the C - N stretching band originating from the neutral TCNQ molecules decreased. In the 2300--2000 cm -l region there appears new lower-energy vibrational bands due to the complex. Upon continued incubation (depending on the phospholipd in question; average time 2--3 days) the band at 2225 cm -~ originating from the neutral TCNQ disappeared and the solution tu~'ns dark blue. In the case of DPPC/TCNQ mixture for instance there appeared two lower-wavenumber peaks at 2180 and 2125 cm -1. FTIR-PAS spectra of the complexes of phospholipids and TCNQ in the
c
© c/C\c
C~J L~I~ (.3 Z
CO (I= 0o LO 0
C3
C3
~zo
36zo
z>zo
z~zo
21zo
l~zo
WF~VENUMBER
J6zO l~zo
cm. 1
~zo
~zo
Fig. 1. Infrared spectrum of solid T C N Q in a KBr matrix, and the chemical structure of T C N Q molecule.
15 2300--2000 cm -1 region are shown in Fig. 2 (A--D). Table 1 summarizes the observed infrared absorption bands. Ruaudel-Teixier et al. [24] found a lowerwavenumber shift in the C - N stretching mode of TCNQ when present as a CT-complex with N-docosylpyridinium. They assigned the lowerenergy band at approx. 2190 cm -~ to the nitrile stretching mode of TCNQ-. Richard et al. [31] have recently examined LangmuirBlodgett assemblies of N-docosylpyridinium/ TCNQ CT-complexes and they have assigned the following bands to the C-=N stretch in the charge-transfer complex: 2185, 2178, 2173, 2158 and 2150 cm-L In addition for LiTCNQ CT-complex the bands at 2195, 2184 and 2168 cm -~ are assigned to the C---N stretching bands in water solution [40]. Bozio et al. [40] have suggested that the formation of the self-dimer of TCNQ- is accompanied by the appearance of some additional absorptions in the infrared spectrum. The phenomenon is interpreted in terms of the Ferguson-Person vibronic model, which embodies the basic concepts of the charge transfer interaction, i.e., the additional bands correspond to strong vibronic activation in infrared of the out-of-phase components of the totally symmetric molecular modes such as the totally symmetric C---N stretching mode. Also the other IR spectral regions of the blue lipid/TCNQ solutions reveal no sign of the presence of neutral TCNQ molecules. The diagnostic region is 1550--1500 cm -1, where one observes the band at 1507 cm-' for rac'D P PG/TC NQ and at 1496 cm -t for D P P C / TCNQ attributed to the C = C stretching mode of TCNQ- species in the dimeric state [31], whereas that of the neutral species would appear at 1543 cm -~ as found immediately after the mixing of phospholipid and TCNQ. The bands observed at 1601 and 1599 cm -~ for rac'-DPPG/TCNQ and DPPC/TCNQ, respectively, are also assigned to the C = C stretching modes of TCNQ- anion [31]. In K÷ TCNQ- two bands at 1579 and 1509 cm-' associated with the C = C stretch are observed [41]. Thus the wavenumbers found for TCNQ in a blue solution are typical of the TCNQ-
anion in either its monomeric or dimeric form. Also the electronic spectra for lipid/TCNQ showed changes during complexation. The UV-VIS spectrum of neutral TCNQ in ethyl acetate is shown in Fig. 3A, and reveals an intensive absorption band at approx. 396 nm. The UV-VIS spectral data are consistent with the picture emerging from our IR spectroscopic studies. Immediately after the mixing of phospholipids and TCNQ a band at approx. 396 nm assigned to neutral TCNQ is evident. In the blue solution obtained after incubation broad bands at approx. 630 and 830 nm assigned to the [TCNQ-] 2 and TCNQ-, respectively, appear (Fig. 3B) [31,44]. In addition there is a band at approx. 310 nm which overlaps with pyrene absorption in the pyrenephospholipd/TCNQ complexes. This band is also assigned to the [TCNQ-]2 [31]. The bands at 630 and 830 nm are characteristic for CT-complexes [45] and can be attributed to charge transfer from the phospholipid transfer from the phospholipid to TCNQ. A charge-transfer band at 1050 nm is also evidnet in the UV-VIS spectrum of N-docosylpyridinium [25].
The acyl chain region This spectral region contains primarily the vibrational modes of acyl chains of phospholipids. Unfortunately, there is some overlapping with the methylene modes of the headgroup. Compared for instance to the spectra of pure rac'-DPPG TCNQ causes only minor changes in the observed spectral features. The two strong bands at approx. 2918 and 2850 cm -~ are assigned to the asymmetric and symmetric CH 2 stretching modes of the methylene segment, respectively [37]. The exact position (wavenumber) of the C--H stretching provides a sensitive measure of the degree of conformational disorder of the membrane lipids. Neither the wavenumbers nor the half-bandwidths of the asymmetric and symmetric CH 2 stretching modes are significantly altered upon TCNQ/phospholipid complex formation. This is expected if no
16 penetration of TCNQ into the hydrocarbon chain region occurs. The same kind of tendency is also observed for the other phospholipids studied. However, in the case of pyrene-labelled analogues the C H 2 stretch-
c (D W-0 • Z ~E
Q
O) N 0
a 0 (:3 0
B
(D bJ~ ~_~ Z
m
O
Q (:3 Q
NAVENUMBER
(cm.1)
ing bands are slightly shifted in the presence of TCNQ, Table II. This can be compared with the perturbation produced by cholesterol in the structural features of the hydrocarbon chains• For instance in DPPC-cholesterol
17
0
C
0 nn 0 Z
