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Use of VO2+ as a spin probe for dynamics of polar headgroups in phosphatidylcholine bilayers
Chemistry and Physics of Lipids 85 (1997) 45 – 51
Use of VO2 + as a spin probe for dynamics of polar headgroups in phosphatidylcholine bilayers Hisao Tanaka*, Harumi Saito, Hiroshi Kawazura Faculty of Pharmaceutical Sciences, Josai Uni6ersity, Sakado, Saitama 350 -02, Japan Received 20 August 1996; revised 9 October 1996; accepted 9 October 1996
Abstract VO2 + was used as a probe of polar headgroup dynamics in fully hydrated dipalmitoylphosphatidylcholine membranes. The ESR spectrum exhibited an anisotropic pattern consisting of gzz and gxx ( =gyy ) components, which is characteristic of VO2 + in a slow motion regime. The VO2 + probe was capable of detecting the main transition from gel to liquid crystalline phase through a change in spectral characteristics. Liouville lineshape analysis revealed that the rotational diffusion of VO2 + in the liquid crystalline phase is two orders slower than that of the choline terminal methyls, indicating that VO2 + experiences the motion of the headgroup back-bone due to a tight binding to the phosphate group. The probe also detected the lack of phase transition in the membrane containing 40 mol% cholesterol, which agrees with the depiction of recent phase diagrams. As viewed using the probe, incorporation of cholesterol enhanced the rotational diffusion of DPPC headgroups in the gel phase by a factor of approximately 6, but produced no significant effect in the liquid crystalline phase. The VO2 + -probing method is applicable for investigating motion with diffusion rates larger than 1×106 s − 1 in phosphatidylcholine membranes. Copyright © 1997 Elsevier Science Ireland Ltd. Keywords: VO2 + ; Spin probe; Dynamics; Polar head; Phosphatidylcholine; Cholesterol
1. Introduction Oxovanadium (IV) and copper (II) are divalent paramagnetic cations with doublet spin states (S =1/2) and ESR spectra that are easily detectable in a solution state due to comparatively * Corresponding author. Fax: +81 492 71 7984; E-mail: [email protected].
long relaxation times. These cations were the subjects of early ESR studies on spin relaxation (Freed and Fraenkel, 1963; Wilson and Kivelson, 1966; Hudson and Luckhurst, 1969), and in recent years have attracted considerable interest for application to catalysts and polymer gels (Bassetti et al., 1979; Chachaty et al., 1991; Lee et al., 1991). Nitroxides have been used in ESR spin labeling and probing studies on lipid membranes, particu-
0009-3084/97/$17.00 Copyright © 1997 Elsevier Science Ireland Ltd. All rights reserved PII S 0 0 0 9 - 3 0 8 4 ( 9 6 ) 0 2 6 3 9 - 4
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H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
larly in the hydrophobic region (Berliner, 1976). In order to investigate the applicability of VO2 + and Cu2 + in membrane research, we previously studied the interaction between these cations and the headgroups in a phosphatidylcholine – water binary system using the NMR paramagnetic relaxation method (Tanaka et al., 1995). Both cations were found to be preferentially transferred from the aqueous phase to the membrane phase, and the VO2 + of the cations distributed to the surface region was found to undergo strong motional restriction due to binding to the anionic phosphate, suggesting the potential use of VO2 + as a spin probe for headgroup motion. Thus, in the present study, we examined the ESR spectra of VO2 + in a dipalmitoylphosphatidylcholine (DPPC) multilamellar dispersion with excess water and in a dispersion containing a high concentration of cholesterol (CHOL). Cholesterol added at high concentrations to a DPPC membrane is known to eliminate the gel (Lb%)-to-liquid crystalline (La ) phase transition, producing an alternative ‘liquid-gel’ phase(s) (Ipsen et al., 1987; Vist and Davis, 1990; Huang et al., 1993). The conformation of DPPC headgroups in this phase is considered to be similar to that in the La phase in which they are aligned nearly parallel to the membrane surface (Huang et al., 1993), whereas the quantitative headgroup motion is not as clearly understood. Generally, high concentrations of cholesterol are believed to increase headgroup mobility due to a spacing effect that induces a weakened interaction between the headgroups (Shepherd and Bu¨ldt, 1979; Henze, 1980; Browning, 1981). On the other hand, NMR studies indicate that cholesterol at the same concentrations shows no significant effect on the motion and the conformation of the headgroups (Cullis et al., 1976; Brown and Seelig, 1978; Han and Gross, 1991). The effect of cholesterol on the dynamics appears to be at least bifacial, requiring further examination with alternative approaches. In the present study, VO2 + is shown to probe the headgroup backbone over a wide temperature range including the Lb% and La ranges. As a result of applying VO2 + , the effect of cholesterol on headgroup motion is different in the Lb% and La
temperature ranges. Combining this VO2 + -probing method with Liouville lineshape analysis allows the acquisition of quantitative information on the headgroup dynamics.
2. Materials and methods Multilamellar membrane dispersions of pure DPPC and CHOL-DPPC (2:3, mol/mol) containing 1 mol% VOSO4 were prepared by freeze–drying (Tanaka and Freed, 1984) and subsequent addition to the dry lipids of an equal weight of water. As a result of hydration, almost half the water is held between the bilayers and the remaining water forms an isolated water phase (Scherer, 1989). DPPC and CHOL were obtained from Sigma Co. and used without further purification. Deionized water of 18 MV quality was used for dispersion. The ESR spectra of VO2 + in the membrane dispersions were recorded on a JEOL FE-3XG spectrometer on the X-band with 100 kHz field modulation equipped with an NM-PVT temperature control unit. The spectra observed at a rigid limit (− 196°C) were analyzed in order to determine the principal values of the g and A tensors, using the QCPE SIM14 powder pattern simulation program written by G. Lozos et al. of Northwestern University. In a motional regime, analysis of the spectra was performed at several typical temperatures in order to determine the rotational diffusion rate of VO2 + , using a stochastic Liouville lineshape analysis program prepared by J.H. Freed’s group from Cornell University. The LF version of this program was capable of fully solving the spin Hamiltonian matrix (Schneider and Freed, 1989). The powder pattern and Liouville lineshape programs were modified by one of the authors (H. Tanaka) to run on Microsoft DOS and FORTRAN.
3. Results and discussion Fig. 1 shows the temperature dependence of the ESR spectrum of VO2 + in a DPPC membrane hydrated to 50 w/w%. The corresponding depen-
H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
47
dence in a DPPC membrane containing cholesterol at 40 mol% is shown in Fig. 2. Fig. 3 depicts the temperature dependence of the separation S between the outermost hyperfine (hf) peaks of gzz components from the two spectra. Fig. 4 shows examples of spectral simulation at a rigid limit ( −196°C) and in a motional regime (15°C) obtained using powder pattern and stochastic Liouville lineshape analyses, respectively.
Fig. 2. ESR spectra of VO2 + in fully hydrated DPPC multibilayers containing 40 mol% CHOL. Arrows denote hf lines of MI = 97/2 in the gzz component.
Fig. 1. ESR spectra of VO2 + in fully hydrated DPPC multibilayers. Arrows denote hf lines of MI = 9 7/2 in the gzz component.
First, let us examine the state of VO2 + ions in the membrane dispersions with excess water. The ESR spectra shown in Figs. 1 and 2 exhibit anisotropic patterns characteristic of VO2 + in incomplete motional averaging of the g and A tensors. The spectral characteristics obtained using the Liouville lineshape calculation with a single component of VO2 + in a slow motion regime
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H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
are in good agreement (Fig. 4). Thus, almost all of the VO2 + ions are uniformly distributed to a given site in the membrane polar region and exist in a motionally restricted state, and no substantial population of free VO2 + exists in the water phase squeezed out of the bilayers. This finding confirms our previous assumption (Tanaka et al., 1995) that VO2 + ions in a phosphatidylcholine – water binary solution are preferentially transferred to the membrane surface region, where strong interaction with the phosphate groups occurs. As observed in the spectra, the dynamic characteristics of VO2 + in pure DPPC are as follows. In the range up to −10°C where the rigid limit spectrum is observed, the rotational diffusion is in
Fig. 4. Experimental (——) and best-fitted calculated ( · · · ) spectra of VO2 + at − 196°C in DPPC membrane containing 40 mol% CHOL (A) and at 15°C in pure DPPC (B) and in DPPC membrane containing 40 mol% CHOL (C). g and A tensors determined at rigid limit were used for simulations in motional regime, together with residual inhomogeneous broadening fixed at 0.5 mT.
Fig. 3. Separation S between MI = 9 7/2 hf lines plotted against temperature. , in pure DPPC; , in DPPC membrane containing 40 mol% CHOL. Error in S is within 9 0.3 mT.
a frozen state or in a very slow motional state beyond a limit. Lineshape analysis indicated that this limit of the diffusion rate DR occurred at 1× 106 s − 1, which is in agreement with the DR of 104 to 105 s − 1 expected for DPPC phosphate groups below 0°C based on the 31P-NMR relaxation measurements (Ruocco et al., 1985). As the temperature rises in the range above 0°C, the anisotropic spectrum shows shifting and linebroadening of the hf peaks (Fig. 1). In such a motional regime, a separation S between the outermost gzz peaks, as has been established for nitroxides and for VO2 + (Berliner, 1976; Chachaty et al., 1991), is related to the rotational diffusion rate DR in the empirical form:
H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
DR = a(1 − SN)b
(1)
where SN is the S normalized by the corresponding S0 at the rigid limit and a and b are positive constants. In the present case, the S value for pure DPPC maintained a rigid limit of 142 mT up to − 10°C, and then decreased with further increase in temperature, indicating a profile of the thermal activation process in the rotational diffusion of VO2 + (Fig. 3). A dramatic break in this process is observed between 40 and 50°C due to the main phase transition of Lb% to La occurring at Tc = 42°C in fully hydrated DPPC membranes. Lineshape analysis revealed a jump in the diffusion rate from 6.0( 9 0.5) × 106 s − 1 at 40°C to 7.0(91.0)×107 s − 1 at 50°C due to this phase transition, indicating a large release of motional freedom brought about by the order-disorder change in the headgroups organization. Although no data from the Lb% phase are available for comparison, the rate in the La phase can be compared with some data from previous NMR T1 relaxation studies: the correlation time tR of 2 ns at 50°C, which is found from the relationship tR =1/(6DR) (Berliner, 1976), is two orders longer than the tR of 36 ps at 30°C reported for choline terminal methyls in fully hydrated dioleylphosphatidycholine (DOPC) membranes (Tc = − 22°C) (Ulrich et al., 1990). Furthermore, the tR approaches 1 ns at 4°C for reorientation of DOPC phosphate groups (Seelig et al., 1981), falling within the upper limit of 3 – 4 ns imposed for motion of DPPC phosphate and choline methylene groups (Shepherd and Bu¨ldt, 1979). These comparisons indicate that VO2 + ions experience slow motion on the headgroup backbone due to a tight binding to the phosphate group, rather than rapid internal motion as observed in the terminal methyls, being consistent with the preceding inference (Tanaka et al., 1995). Thus, VO2 + appears to function as a headgroup spin probe, especially for the backbone. This VO2 + -probing method tested for a pure DPPC membrane was applied in order to examine the effect of cholesterol on polar-head motion. The general appearance of the spectrum shown in Fig. 2 is similar to that of pure DPPC. However, in the membrane containing cholesterol, the rigid
49
lattice spectrum only exists below − 40°C, and the phase transition phenomenon observed in pure DPPC is not observed, resulting in a continuous change in the spectral characteristics over the entire temperature range. Notwithstanding such spectral changes in the motional regime, the rigid limit spectra of VO2 + in DPPC and in CHOL/DPPC were very similar, and powder pattern lineshape analysis yielded essentially equivalent g and A tensors, i.e. gzz = 1.935, gxx = gyy = 1.985, Azz = 20.3 mT, and Axx = Ayy = 7.5 mT (Fig. 4). This suggests that the static nature of the VO2 + -phosphate linkage is not significantly altered by the presence of cholesterol and that the spectral change caused by cholesterol should essentially be ascribed to the dynamic characteristics of the polar head attached by VO2 + . Thus, the S plots in Fig. 3 indicate that incorporation of cholesterol noticeably promotes rotational diffusion in the Lb% phase of DPPC, but produces only slight enhancement in the La phase. The diffusion rates of VO2 + determined by lineshape analysis were 2.1(9 0.3)× 106 s − 1 for DPPC and 1.3(90.2)×107 s − 1 for CHOL/ DPPC at 15°C in the Lb% range and 7.0(9 1.0)× 107 s − 1 and 8.5(9 1.0)× 107 s − 1 for the respective dispersions at 50°C in the La range. The approximately 6-fold increase in the rate at 15°C induced by cholesterol was nearly equivalent to the thermal activation observed upon increasing the temperature by 25–35°C in the Lb% phase. Another aspect of the cholesterol effect is observed in the replots of data in Fig. 3, based on the relationship: log(1− SN) = C− Ea/(2.303bRT)
(2)
which is derived from Eq. (1) and the Arrhenius equation, DR = A×exp(−Ea/RT), where Ea is the activation energy of the rotational diffusion of VO2 + , and A and C are constants. A simple linear relationship is observed for log(1-SN) vs. 1/T with a jump at the phase transition in pure DPPC (Fig. 5). As expected, the plots in the CHOL/DPPC exhibit no steep change at Tc, but tend to decrease in slope above Tc. The values of Ea in the gel-phase range were determined from the slopes and parameter b found with DR at 15 and 40°C, resulting in 31(9 3) kJ/mol for both
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H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
DPPC and CHOL/DPPC (a =5.8 ×108 s − 1 and b= 1.27 in Eq. (1)). The lack of substantial alteration in Ea suggests that cholesterol does not change the mode of rotational diffusion in the headgroups but only affects its magnitude. Disappearance of the phase transition phenomenon induced by cholesterol, which was also observed in the previous studies on DPPC headgroups (Cullis et al., 1976; Brown and Seelig, 1978), agrees with recent phase diagrams of DPPC/CHOL showing that cholesterol at concentrations greater than 22 mol% produces ‘liquid-gel’ phase(s) called LG, b, or lo (Ipsen et al., 1987; Vist and Davis, 1990; Huang et al.,
1993). NMR studies on the headgroup motion in phosphatidylcholine membranes with 40–50 mol% cholesterol have been performed based on the 31P chemical shielding anisotropy and line width of the phosphates (Cullis et al., 1976), 2H quadrupole splitting and relaxation time T1 of the deuterated choline methylenes (Brown and Seelig, 1978; Browning, 1981), and 1H T1 of the choline methylene (Han and Gross, 1991). The NMR results can be summarized as indicating no significant effect of cholesterol on headgroup dynamics, which is consistent with the present results found in the La range. With respect to dynamics in the gel-phase range, no detailed NMR information has been reported because the parameters cited above are nearly independent of temperature due to their insensitivity to motion in that range (Cullis et al., 1976; Brown and Seelig, 1978; Browning, 1981; Vist and Davis, 1990). Distinct promotion of motion by cholesterol in the gel-phase range is reflected by an apparent increase in the relaxation frequency, as indicated from dielectric measurements on DPPC headgroup dipoles (Shepherd and Bu¨ldt, 1979; Henze, 1980). The development of the cholesterol effect in this range can be interpreted in terms of its spacing effect on the headgroups, which supplies adequate space for their motion (Shepherd and Bu¨ldt, 1979; Yeagle, 1985). In conclusion, the VO2 + -probing method allows not only observation of the headgroup motion in a wide temperature range including both the Lb% and La ranges, but also direct determination of the rotational diffusion rate in combination with lineshape analysis. We plan to apply this probing method to lipid membranes consisting of polar heads other than phosphocholine.
Acknowledgements Fig. 5. Relationship of log(1-SN) vs. 1/T in motional regime, made from data of S in Fig. 3. , in pure DPPC; , in DPPC membrane containing 40 mol% CHOL. Solid lines represent those obtained by least-squares fit.
The authors would like to thank Prof. J.H. Freed of Cornell University for his gift of the FORTRAN source programs (LF version) of Liouville lineshape analysis.
H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
References Bassetti, V., Burlamacchi, L. and Martini, G. (1979) Use of paramagnetic probes for the study of liquid adsorbed on porous supports. J. Am. Chem. Soc. 101, 5471–5477. Berliner, L.J. (Ed.) (1976) Spin Labeling-Theory and Applications, Academic Press, New York. Brown, M.F. and Seelig, J. (1978) Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers. Biochemistry 17, 381–384. Browning, J.L. (1981) Motions and interactions of phospholipid head groups at the membrane surface. 3. Dynamic properties of amine-containing head groups. Biochemistry, 20, 7144 – 7151. Chachaty, C., Soulie, E. and Wolf, C. (1991) Some recent applications of computer simulations of ESR spectra to the dynamics of spin probes in gels and membranes. J. Chim. Phys. 88, 153 – 172. Cullis, P.R., De Kruyff, B. and Richards, R.E. (1976) Factors affecting the motion of the polar headgroup on phopholipid bilayers. Biochim. Biophys. Acta 426, 433 – 446. Freed, J.H. and Fraenkel, G.K. (1963) Theory of linewidths in electron spin resonance spectra. J. Chem. Phys. 39, 326 – 348. Han, X. and Gross, R.W. (1991) Proton magnetic resonance studies on the molecular dynamics of plasmenylcholine/ cholesterol and phosphatidylcholine/cholesterol bilayers. Biochim. Biophys. Acta. 1063, 129–136. Henze, R. (1980) Dielectric relaxation in lecithin/cholesterol/ water mixtures. Chem. Phys. Lipids 27, 165–175. Huang, T.-H., Lee, C.W.B., Das Gupta, S.K., Blume, A. and Griffin, R.G. (1993) A 13C and 2H nuclear magnetic resonance study of phosphatidylcholine/cholesterol interactions: characterization of liquid-gel phases. Biochemistry 32, 13277 – 13287. Hudson, A. and Luckhurst, G.R. (1969) The electron resonance lineshapes of radicals in solution. Chem. Rev. 69, 191 – 225. Ipsen, J.H., Karlstro¨m, G., Mouritsen, O.G., Wennerstrom, H. and Zuckermann, M.J. (1987) Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim. Biophys. Acta 905, 162 – 172.
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Lee, C.W., Chen, X. and Kevan, L. (1991) Electron spin resonance and electron spin echo modulation studies of cupricion. Location and adsorbate interactions in Cu2 + exchanged H-SAPO-11 molecular sieve. J. Phys. Chem. 95, 8626 – 8632. Ruocco, M.J., Simonovitch, D.J. and Griffin, R.G. (1985) Comparative study of the gel phase of ether- and esterlinked phosphatidylcholines. Biochemistry 24, 2406 – 2411. Scherer, J.R. (1989) On the position of the hydrophobic/hydrophilic boundary in lipid bilayers. Biophys. J. 55, 957 – 964. Schneider, D.J. and Freed, J.H. (1989) Calculating slow motional magnetic resonance spectra: a user’s guide, in: Berliner, L.J. and Reuben, J. (Eds.), Biological Magnetic Resonance, Vol. 8, Plenum Press, New York, pp. 1 – 76. Seelig, J., Tamm, L., Hymel, L. and Fleischer, S. (1981) Deuterium and phosphorous nuclear magnetic resonance and fluorescence depolarization studies of functional reconstituted sarcoplasmic reticulum membrane vesicles. Biochemistry 20, 3922 – 3932. Shepherd, J.C.W. and Bu¨ldt, G. (1979) The influence of cholesterol on the head group mobility in phospholipid membranes. Biochim. Biophys. Acta 558, 41 – 47. Tanaka, H. and Freed, J.H. (1984) Electron spin resonance studies on ordering and rotational diffusion in oriented phosphatidylcholine multilayers: evidence for a new chainordering transition. J. Phys. Chem. 88, 6633 – 6644. Tanaka, H., Saito, H. and Kawazura, H. (1995) Interactions of Cu2 + and VO2 + ions with phosphatidylcholine vesicular membranes as studied by the 1H NMR paramagnetic relaxation method. Bull. Chem. Soc. Jpn. 68, 502 – 505. Ulrich, A.S., Volke, F. and Watts, A. (1990) The dependence of phospholipid head-group mobility on hydration as studied by deuterium-NMR spin-lattice relaxation time measurements. Chem. Phys. Lipids 55, 61 – 66. Vist, M.R. and Davis, J.H. (1990) Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and differential scanning calorimetry. Biochemistry 29, 451 – 464. Wilson, R. and Kivelson, D. (1966) ESR linewidths in solution. 1. Experiments on anisotropic and spin-rotational effect. J. Chem. Phys. 44, 154 – 168. Yeagle, P.L. (1985) Cholesterol and the cell membrane. Biochim. Biophys. Acta 822, 267 – 287.
Use of VO2 + as a spin probe for dynamics of polar headgroups in phosphatidylcholine bilayers Hisao Tanaka*, Harumi Saito, Hiroshi Kawazura Faculty of Pharmaceutical Sciences, Josai Uni6ersity, Sakado, Saitama 350 -02, Japan Received 20 August 1996; revised 9 October 1996; accepted 9 October 1996
Abstract VO2 + was used as a probe of polar headgroup dynamics in fully hydrated dipalmitoylphosphatidylcholine membranes. The ESR spectrum exhibited an anisotropic pattern consisting of gzz and gxx ( =gyy ) components, which is characteristic of VO2 + in a slow motion regime. The VO2 + probe was capable of detecting the main transition from gel to liquid crystalline phase through a change in spectral characteristics. Liouville lineshape analysis revealed that the rotational diffusion of VO2 + in the liquid crystalline phase is two orders slower than that of the choline terminal methyls, indicating that VO2 + experiences the motion of the headgroup back-bone due to a tight binding to the phosphate group. The probe also detected the lack of phase transition in the membrane containing 40 mol% cholesterol, which agrees with the depiction of recent phase diagrams. As viewed using the probe, incorporation of cholesterol enhanced the rotational diffusion of DPPC headgroups in the gel phase by a factor of approximately 6, but produced no significant effect in the liquid crystalline phase. The VO2 + -probing method is applicable for investigating motion with diffusion rates larger than 1×106 s − 1 in phosphatidylcholine membranes. Copyright © 1997 Elsevier Science Ireland Ltd. Keywords: VO2 + ; Spin probe; Dynamics; Polar head; Phosphatidylcholine; Cholesterol
1. Introduction Oxovanadium (IV) and copper (II) are divalent paramagnetic cations with doublet spin states (S =1/2) and ESR spectra that are easily detectable in a solution state due to comparatively * Corresponding author. Fax: +81 492 71 7984; E-mail: [email protected].
long relaxation times. These cations were the subjects of early ESR studies on spin relaxation (Freed and Fraenkel, 1963; Wilson and Kivelson, 1966; Hudson and Luckhurst, 1969), and in recent years have attracted considerable interest for application to catalysts and polymer gels (Bassetti et al., 1979; Chachaty et al., 1991; Lee et al., 1991). Nitroxides have been used in ESR spin labeling and probing studies on lipid membranes, particu-
0009-3084/97/$17.00 Copyright © 1997 Elsevier Science Ireland Ltd. All rights reserved PII S 0 0 0 9 - 3 0 8 4 ( 9 6 ) 0 2 6 3 9 - 4
46
H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
larly in the hydrophobic region (Berliner, 1976). In order to investigate the applicability of VO2 + and Cu2 + in membrane research, we previously studied the interaction between these cations and the headgroups in a phosphatidylcholine – water binary system using the NMR paramagnetic relaxation method (Tanaka et al., 1995). Both cations were found to be preferentially transferred from the aqueous phase to the membrane phase, and the VO2 + of the cations distributed to the surface region was found to undergo strong motional restriction due to binding to the anionic phosphate, suggesting the potential use of VO2 + as a spin probe for headgroup motion. Thus, in the present study, we examined the ESR spectra of VO2 + in a dipalmitoylphosphatidylcholine (DPPC) multilamellar dispersion with excess water and in a dispersion containing a high concentration of cholesterol (CHOL). Cholesterol added at high concentrations to a DPPC membrane is known to eliminate the gel (Lb%)-to-liquid crystalline (La ) phase transition, producing an alternative ‘liquid-gel’ phase(s) (Ipsen et al., 1987; Vist and Davis, 1990; Huang et al., 1993). The conformation of DPPC headgroups in this phase is considered to be similar to that in the La phase in which they are aligned nearly parallel to the membrane surface (Huang et al., 1993), whereas the quantitative headgroup motion is not as clearly understood. Generally, high concentrations of cholesterol are believed to increase headgroup mobility due to a spacing effect that induces a weakened interaction between the headgroups (Shepherd and Bu¨ldt, 1979; Henze, 1980; Browning, 1981). On the other hand, NMR studies indicate that cholesterol at the same concentrations shows no significant effect on the motion and the conformation of the headgroups (Cullis et al., 1976; Brown and Seelig, 1978; Han and Gross, 1991). The effect of cholesterol on the dynamics appears to be at least bifacial, requiring further examination with alternative approaches. In the present study, VO2 + is shown to probe the headgroup backbone over a wide temperature range including the Lb% and La ranges. As a result of applying VO2 + , the effect of cholesterol on headgroup motion is different in the Lb% and La
temperature ranges. Combining this VO2 + -probing method with Liouville lineshape analysis allows the acquisition of quantitative information on the headgroup dynamics.
2. Materials and methods Multilamellar membrane dispersions of pure DPPC and CHOL-DPPC (2:3, mol/mol) containing 1 mol% VOSO4 were prepared by freeze–drying (Tanaka and Freed, 1984) and subsequent addition to the dry lipids of an equal weight of water. As a result of hydration, almost half the water is held between the bilayers and the remaining water forms an isolated water phase (Scherer, 1989). DPPC and CHOL were obtained from Sigma Co. and used without further purification. Deionized water of 18 MV quality was used for dispersion. The ESR spectra of VO2 + in the membrane dispersions were recorded on a JEOL FE-3XG spectrometer on the X-band with 100 kHz field modulation equipped with an NM-PVT temperature control unit. The spectra observed at a rigid limit (− 196°C) were analyzed in order to determine the principal values of the g and A tensors, using the QCPE SIM14 powder pattern simulation program written by G. Lozos et al. of Northwestern University. In a motional regime, analysis of the spectra was performed at several typical temperatures in order to determine the rotational diffusion rate of VO2 + , using a stochastic Liouville lineshape analysis program prepared by J.H. Freed’s group from Cornell University. The LF version of this program was capable of fully solving the spin Hamiltonian matrix (Schneider and Freed, 1989). The powder pattern and Liouville lineshape programs were modified by one of the authors (H. Tanaka) to run on Microsoft DOS and FORTRAN.
3. Results and discussion Fig. 1 shows the temperature dependence of the ESR spectrum of VO2 + in a DPPC membrane hydrated to 50 w/w%. The corresponding depen-
H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
47
dence in a DPPC membrane containing cholesterol at 40 mol% is shown in Fig. 2. Fig. 3 depicts the temperature dependence of the separation S between the outermost hyperfine (hf) peaks of gzz components from the two spectra. Fig. 4 shows examples of spectral simulation at a rigid limit ( −196°C) and in a motional regime (15°C) obtained using powder pattern and stochastic Liouville lineshape analyses, respectively.
Fig. 2. ESR spectra of VO2 + in fully hydrated DPPC multibilayers containing 40 mol% CHOL. Arrows denote hf lines of MI = 97/2 in the gzz component.
Fig. 1. ESR spectra of VO2 + in fully hydrated DPPC multibilayers. Arrows denote hf lines of MI = 9 7/2 in the gzz component.
First, let us examine the state of VO2 + ions in the membrane dispersions with excess water. The ESR spectra shown in Figs. 1 and 2 exhibit anisotropic patterns characteristic of VO2 + in incomplete motional averaging of the g and A tensors. The spectral characteristics obtained using the Liouville lineshape calculation with a single component of VO2 + in a slow motion regime
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H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
are in good agreement (Fig. 4). Thus, almost all of the VO2 + ions are uniformly distributed to a given site in the membrane polar region and exist in a motionally restricted state, and no substantial population of free VO2 + exists in the water phase squeezed out of the bilayers. This finding confirms our previous assumption (Tanaka et al., 1995) that VO2 + ions in a phosphatidylcholine – water binary solution are preferentially transferred to the membrane surface region, where strong interaction with the phosphate groups occurs. As observed in the spectra, the dynamic characteristics of VO2 + in pure DPPC are as follows. In the range up to −10°C where the rigid limit spectrum is observed, the rotational diffusion is in
Fig. 4. Experimental (——) and best-fitted calculated ( · · · ) spectra of VO2 + at − 196°C in DPPC membrane containing 40 mol% CHOL (A) and at 15°C in pure DPPC (B) and in DPPC membrane containing 40 mol% CHOL (C). g and A tensors determined at rigid limit were used for simulations in motional regime, together with residual inhomogeneous broadening fixed at 0.5 mT.
Fig. 3. Separation S between MI = 9 7/2 hf lines plotted against temperature. , in pure DPPC; , in DPPC membrane containing 40 mol% CHOL. Error in S is within 9 0.3 mT.
a frozen state or in a very slow motional state beyond a limit. Lineshape analysis indicated that this limit of the diffusion rate DR occurred at 1× 106 s − 1, which is in agreement with the DR of 104 to 105 s − 1 expected for DPPC phosphate groups below 0°C based on the 31P-NMR relaxation measurements (Ruocco et al., 1985). As the temperature rises in the range above 0°C, the anisotropic spectrum shows shifting and linebroadening of the hf peaks (Fig. 1). In such a motional regime, a separation S between the outermost gzz peaks, as has been established for nitroxides and for VO2 + (Berliner, 1976; Chachaty et al., 1991), is related to the rotational diffusion rate DR in the empirical form:
H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
DR = a(1 − SN)b
(1)
where SN is the S normalized by the corresponding S0 at the rigid limit and a and b are positive constants. In the present case, the S value for pure DPPC maintained a rigid limit of 142 mT up to − 10°C, and then decreased with further increase in temperature, indicating a profile of the thermal activation process in the rotational diffusion of VO2 + (Fig. 3). A dramatic break in this process is observed between 40 and 50°C due to the main phase transition of Lb% to La occurring at Tc = 42°C in fully hydrated DPPC membranes. Lineshape analysis revealed a jump in the diffusion rate from 6.0( 9 0.5) × 106 s − 1 at 40°C to 7.0(91.0)×107 s − 1 at 50°C due to this phase transition, indicating a large release of motional freedom brought about by the order-disorder change in the headgroups organization. Although no data from the Lb% phase are available for comparison, the rate in the La phase can be compared with some data from previous NMR T1 relaxation studies: the correlation time tR of 2 ns at 50°C, which is found from the relationship tR =1/(6DR) (Berliner, 1976), is two orders longer than the tR of 36 ps at 30°C reported for choline terminal methyls in fully hydrated dioleylphosphatidycholine (DOPC) membranes (Tc = − 22°C) (Ulrich et al., 1990). Furthermore, the tR approaches 1 ns at 4°C for reorientation of DOPC phosphate groups (Seelig et al., 1981), falling within the upper limit of 3 – 4 ns imposed for motion of DPPC phosphate and choline methylene groups (Shepherd and Bu¨ldt, 1979). These comparisons indicate that VO2 + ions experience slow motion on the headgroup backbone due to a tight binding to the phosphate group, rather than rapid internal motion as observed in the terminal methyls, being consistent with the preceding inference (Tanaka et al., 1995). Thus, VO2 + appears to function as a headgroup spin probe, especially for the backbone. This VO2 + -probing method tested for a pure DPPC membrane was applied in order to examine the effect of cholesterol on polar-head motion. The general appearance of the spectrum shown in Fig. 2 is similar to that of pure DPPC. However, in the membrane containing cholesterol, the rigid
49
lattice spectrum only exists below − 40°C, and the phase transition phenomenon observed in pure DPPC is not observed, resulting in a continuous change in the spectral characteristics over the entire temperature range. Notwithstanding such spectral changes in the motional regime, the rigid limit spectra of VO2 + in DPPC and in CHOL/DPPC were very similar, and powder pattern lineshape analysis yielded essentially equivalent g and A tensors, i.e. gzz = 1.935, gxx = gyy = 1.985, Azz = 20.3 mT, and Axx = Ayy = 7.5 mT (Fig. 4). This suggests that the static nature of the VO2 + -phosphate linkage is not significantly altered by the presence of cholesterol and that the spectral change caused by cholesterol should essentially be ascribed to the dynamic characteristics of the polar head attached by VO2 + . Thus, the S plots in Fig. 3 indicate that incorporation of cholesterol noticeably promotes rotational diffusion in the Lb% phase of DPPC, but produces only slight enhancement in the La phase. The diffusion rates of VO2 + determined by lineshape analysis were 2.1(9 0.3)× 106 s − 1 for DPPC and 1.3(90.2)×107 s − 1 for CHOL/ DPPC at 15°C in the Lb% range and 7.0(9 1.0)× 107 s − 1 and 8.5(9 1.0)× 107 s − 1 for the respective dispersions at 50°C in the La range. The approximately 6-fold increase in the rate at 15°C induced by cholesterol was nearly equivalent to the thermal activation observed upon increasing the temperature by 25–35°C in the Lb% phase. Another aspect of the cholesterol effect is observed in the replots of data in Fig. 3, based on the relationship: log(1− SN) = C− Ea/(2.303bRT)
(2)
which is derived from Eq. (1) and the Arrhenius equation, DR = A×exp(−Ea/RT), where Ea is the activation energy of the rotational diffusion of VO2 + , and A and C are constants. A simple linear relationship is observed for log(1-SN) vs. 1/T with a jump at the phase transition in pure DPPC (Fig. 5). As expected, the plots in the CHOL/DPPC exhibit no steep change at Tc, but tend to decrease in slope above Tc. The values of Ea in the gel-phase range were determined from the slopes and parameter b found with DR at 15 and 40°C, resulting in 31(9 3) kJ/mol for both
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H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
DPPC and CHOL/DPPC (a =5.8 ×108 s − 1 and b= 1.27 in Eq. (1)). The lack of substantial alteration in Ea suggests that cholesterol does not change the mode of rotational diffusion in the headgroups but only affects its magnitude. Disappearance of the phase transition phenomenon induced by cholesterol, which was also observed in the previous studies on DPPC headgroups (Cullis et al., 1976; Brown and Seelig, 1978), agrees with recent phase diagrams of DPPC/CHOL showing that cholesterol at concentrations greater than 22 mol% produces ‘liquid-gel’ phase(s) called LG, b, or lo (Ipsen et al., 1987; Vist and Davis, 1990; Huang et al.,
1993). NMR studies on the headgroup motion in phosphatidylcholine membranes with 40–50 mol% cholesterol have been performed based on the 31P chemical shielding anisotropy and line width of the phosphates (Cullis et al., 1976), 2H quadrupole splitting and relaxation time T1 of the deuterated choline methylenes (Brown and Seelig, 1978; Browning, 1981), and 1H T1 of the choline methylene (Han and Gross, 1991). The NMR results can be summarized as indicating no significant effect of cholesterol on headgroup dynamics, which is consistent with the present results found in the La range. With respect to dynamics in the gel-phase range, no detailed NMR information has been reported because the parameters cited above are nearly independent of temperature due to their insensitivity to motion in that range (Cullis et al., 1976; Brown and Seelig, 1978; Browning, 1981; Vist and Davis, 1990). Distinct promotion of motion by cholesterol in the gel-phase range is reflected by an apparent increase in the relaxation frequency, as indicated from dielectric measurements on DPPC headgroup dipoles (Shepherd and Bu¨ldt, 1979; Henze, 1980). The development of the cholesterol effect in this range can be interpreted in terms of its spacing effect on the headgroups, which supplies adequate space for their motion (Shepherd and Bu¨ldt, 1979; Yeagle, 1985). In conclusion, the VO2 + -probing method allows not only observation of the headgroup motion in a wide temperature range including both the Lb% and La ranges, but also direct determination of the rotational diffusion rate in combination with lineshape analysis. We plan to apply this probing method to lipid membranes consisting of polar heads other than phosphocholine.
Acknowledgements Fig. 5. Relationship of log(1-SN) vs. 1/T in motional regime, made from data of S in Fig. 3. , in pure DPPC; , in DPPC membrane containing 40 mol% CHOL. Solid lines represent those obtained by least-squares fit.
The authors would like to thank Prof. J.H. Freed of Cornell University for his gift of the FORTRAN source programs (LF version) of Liouville lineshape analysis.
H. Tanaka et al. / Chemistry and Physics of Lipids 85 (1997) 45–51
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