J. Phys. Chem. 1992, 96, 2749-2754 the additional water incoherent scattering which was not subtracted in the upper spectrum of Figure 8. The shape of the curve is not significantly changed, indicating that preferential solvation by water molecules is not probable. As a conclusion it can be stated that thermodynamic and small-angle scattering data combined with solvent-averaged calculations can yield structural information for 2-propanol solutions inasmuch as the 'primitive model" H N C is reliable for solutions of low permittivity. Improvement may be achieved by properly modeling the cation structure and using simulation
2749
techniques such as Brownian dynamics. From the experimental point of view, the extension of SANS to higher q values would be useful to obtain more information concerning distances smaller than 1.2 nm.
Acknowledgment. W.K. is grateful to the Commission of the European Community for a grant. We thank Dr. J. Hodges for helpful comments. Registry No. n-Pe,NBr, 866-97-7; 2-propanol, 67-63-0.
CH, Wagging Progressions as IR Probes of Slightly Disordered Phospholipid Acyl Chain States Laurence Senak, David Moore, and Richard Mendelsobn* Department of Chemistry, Newark College of Arts and Sciences, Rutgers University, 73 Warren Street, Newark, New Jersey 07102 (Received: October 7, 1991)
CH2 wagging progressions have been measured in the IR spectra for a series of saturated gel phase phosphatidylcholines (PC's) over the range of chain lengths diC13PCto diC20PC. Band assignments have been confirmed from a dispersion curve which closely parallels that constructed by Snyder for solid alkanes. The intensity of the progression has been used to monitor the all-trans conformational state in phospholipid aqueous phases and in various regions of the 1,2-dipalmitoylphosphatidylcholine (DPPC)/cholesterol phase diagram. With the assumption that the intensity of the progression arises only from all-trans chains and is thus a highly nonlinear function of the number of gauche rotamers, the data suggest that about 1 gauche bond/chain exists in the "liquid-ordered" phase (33 mol % cholesterol) as compared with 3.6-4.2gauche rotamers for the L, phase of DPPC alone. The strong ordering effect of cholesterol on the DPPC acyl chains is thus evident. At 20 mol % cholesterol, a phase separation region is evident from 39 to 45 OC. The conversion of the 'liquid-ordered" to the "liquid-disordered" phase is accompanied by the formation of 0.15 double gauche conformers/chain, as monitored from localized CH2wagging vibrations. The role of the eight-carbon side chain of cholesterol in the ordering process at 33 mol % is revealed through studies of DPPC with 5-androsten-3-&01 (androsten). Substantially more disorder is observed (- 2-2.5 gauche rotamers/chain at 50 "C). The various IR measurements of acyl chain conformational order are compared.
Introduction It is well-known that the methylene wagging vibrations in the IR spectra of ordered saturated hydrocarbons may be described as coupled oscillators which produce band progressions diagnostic of the all-trans chain conformation. The observed frequency patterns in the region 1175-1380 cm-I which characterize particular chain lengths for alkanes and related molecules have been analy~ed.l-~A detailed summary is a ~ a i l a b l e . ~Structural applications involving these progressions have been reported. For example, Umemura and co-workers5 demonstrated the existence of cis and trans isomers in the spectra of fatty acid dimers while Naselli et a1.6 used these modes to monitor two-dimensional melting in Langmuir-Blodgett monolayer films of cadmium arachidate. Fourier transform infrared (FT-IR) spectroscopy has, in recent years, been widely used to investigate acyl chain conformations in phospholipid bilayers (for reviews see refs 7 and 8), the major structural component of biological membranes. Toward this end, several spectral parameters have been utilized. The most common among these, the frequency of the symmetric CH2 stretching ( I ) Snyder, R. G. J. Mol. Spectrosc. 1960, 4, 41 I . (2) Snyder, R. G.; Schachtschneider, J. H. Spectrochim. Acta 1963, 19, 85. (3) Susi, H.; Panzer, S. Spectrochim. Acta 1962, 18, 499. (4) Painter, P. C.; Coleman. M. M.; Koenig, J. L. The Theory of Vibra-
tional Spectroscopy and its Application to Polymeric Materials; Wiley: New York, 1982. (5) Hayashi, S.; Umemura, J. J. Chem. Phys. 1975, 63, 1732. (6) Naselli, C.; Rabolt, J. F.; Swalen, J. D. J. Chem. Phys. 1985,82, 2136. ( 7 ) Mendelsohn, R.; Mantsch, H. H. In Progress in Prorein-Lipid Interacrions; Watts, A., DePont J. J. H. H. M., Eds.; Elsevier: Amsterdam, 1986; Vol. 2, p 103. (8) Mantsch, H. H.; McElhaney, R. N. Chem. Phys. Lipids 1991,57,213.
0022-3654/92/2096-2149$03.00/0
modes near 2850 cm-l, increases by 2-5 cm-I at the gel-liquid crystal phase transition temperature, T,. This observation has been exploited in studies of lipid m i s ~ i b i l i t yand ~ ~ ' ~lipid-protein interaction.IIJ2 Recent studies have extended the domain of utility of IR spectroscopy into the realm of quantitative determination of conformational disorder in the biologically relevant La and HII phases of phospholipids. The CD2 rocking modes in a series of specifically deuterated derivatives have been used to determine fractional disorder (trans-gauche population ratios) as a function of depth in DPPC bilayers,13 DPPC/cholesterol mixtures,I4 and DPPC/gramicidinI5 mixtures. A related utilizes the (localized) wagging modes of disordered acyl chains to quantitatively determine specific conformer types (e.g. double gauche (gg) or kinks (g'tg')). The current study describes an additional IR probe of acyl chain conformational order, namely the intensity of the aforementioned (9) Dluhy, R. H.; Moffatt, D.; Cameron, D. G.;Mendelsohn, R.; Mantsch,
H.H . Can. J . Chem. 1985,63,
1925.
(10) Mantsch, H. H.; Madec, C.; Lewis, R. N. A. H.; McElhaney, R. N. Biochemistry 1985, 24, 2440. (1 1 ) Anderle, G.; Mendelsohn, R. Biochemisrry 1986, 25, 2174. (12) Babin, Y.; D'Amour, J.; Pigeon, M.; Ptzolet, M. Biochim. Biophys. Acta 1987, 903, 78. (13) Mendelsohn, R.; Davies, M. A,; Brauner, J. W.; Schuster, H. F.; Dluhy, R. A. Biochemistry 1989, 28, 8934. (14) Davies, M. A.; Schuster, H. F.; Brauner, J. W.; Mendelsohn. R. Biochemistry 1990, 29, 4368. (15) Davies, M. A.; Brauner, J. W.; Schuster, H. F.; Mendelsohn, R. Biochem. Biophys. Res. Commun. 1990, 168, 85. (16) Casal, H. L.; McElhaney, R. N. Biochemistry 1990, 29, 5423. (17) Senak, R.; Davies, M. A.; Mendelsohn, R. J. Phys. Chem. 1991, 95, 2565.
0 1992 American Chemical Society
2750 The Journal of Physical Chemistry, Vol. 96,No. 6, 1992
CH2 wagging progression in relatively ordered phases. Fringeli and GiinthardI6 have identified these modes in DPPC, while Cameron et a1.I9 have reported qualitative intensity changes for the progression upon the formation of various phases of the molecule. In the current work, spectral assignments for the wagging modes in the C13-C20 series of saturated PC's are verified with a dispersion curve. The advantage of this measure of conformational order is demonstrated through semiquantitative characterization of the loss of the all-trans form in the biologically important 'liquidordered" state that exists over substantial regions of the DPPC/cholesterol phase diagram.20*21The role of the cholesterol eight-carbon side chain in the ordering process is evaluated through comparative studies of the wagging progression in DPPC/ cholesterol and DPPC/5-androsten-3/3-01 (androsten). Androsten differs from cholesterol only in the absence of this chain at C( 17). The sterol structures are shown below.
H/ O
Spectra were obtained at 4-cm-' resolution, under N2 purge, by co-addition of 256 interferograms. These were apodized with a triangular fundon and Fourier-transformed with me lev4 of zcro filling to yield data encoded every 2 cm-I. Data from two or three independent samples were acquired at all temperatures for each sample. For quantitative analysis of the CHI wagging modes in the region 1300-1 150 cm-', subtraction of the underlying PO2-s y m metric stretching band was required. This was acaMlplishcd with spectra of DPPC-& matched for temperature, path length, sterol content, and water content, as reference spectra. Subtraction factors were chosen by maximizing the band heights of the progression and choosing a consistent shape for the baseline of the residual contour of wagging progression components as a function of temperature for a given sample. A flattened baseline was generated for DPPC-containing samples for the k = 1 through k = 4 progression bands by selecting minima at 1280 and 1188 cm-l. Integration was accomplished with the Mattson Instruments data using manufacturer-supplied FIRST software and with the Digilab FTS-40 spectrometer data by transferring spectra to a microcomputer employing software supplied by D. Moffatt of the National Research Council of Canada. The same end points chosen for baseline leveling were used for integration. Progression intensities were estimated by ratioing the intensity of either the sum of the k = 1 to k = 4 components or the k = 1 component alone to that of the underlying phosphate band. Spectral intensities for isolated CH2wagging modes (disordered phases) between 1320 and 1390 cm-' were determined by the methods reported recently from this laboratory."
1 cholostorol
H/ O
Senak et al.
e 5-Androsten-3-pol
Experimental Section Materials. PC's, including acyl-chain perdeuterated DPPC (DPPC-d,,) were obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). These materials, which generally have transition widths for their P r L , interconversions of T,, consistent with the loss of the all-trans conformation. The results of a typical subtraction operation (performed as discussed in the Experimental Section) to eliminate the underlying phosphate band are shown in Figure l(bott0m). Two points are noted. First, for the wagging mode progression of pure alkanes with an even number of oscillators, the koddmodes are symmetry forbidden? However, these bands were observed in the prior study of DPPC19 and in the
The Journal of Physical Chemistry, Vol. 96, No. 6, 1992 2751
CH2 Wagging Progressions as IR Probes
-7
13201305A
E
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p-iase a n g l e
8
I
,
1320
1280
1240
1200
wovenumber
O
1160
(cm-1)
Figure 1. (Top) CH, wagging mode progression for DPPC: evident in the FT-IR spectrum at 15 "C (-) but absent in the spectrum at 65 "C (-). (Bottom) Isolation of the CH2wagging progression. Subtraction was optimized by using the spectrum of DPPC-&, matched for lipid/
water levels, path length, and temperature as a reference.
Figure 2. Dispersion curve for the CHI wagging mode for a series of disaturated PC's in their respective gel phases as follows: diCI3PC (*); diCl4PC (A);diCl5PC (0); diCMPC (0); diC17PC (A);diCl8PC (V); diCI9PC (m); diC2OPC (e). x
.-
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TABLE I: CHI Wagging Band Frequencies for Disaturated PC's
chain length
k = l
k = 2
k = 3
k = 4
13 14 15 16 17 18 19 20
1207.5' 1203.7 1201.7 1199.8 1197.9 1195.9 1194.0 1192.1
1238.4 1228.7 1224.9 1221.0 1219.1 1215.2 1211.4 1211.4
1265.4 1255.7 1250.0 1244.2 1240.3 1236.5 1232.6 1228.7
1288.5 1280.8 1273.1 1265.4 1259.6 1253.8 1249.9 1246.0
k=5
k=6 a
1300.1 1296.3 1288.5 1280.8 1271.2 1267.3 1261.5
2851 . O
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-2850.5
5
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A
A
2850.0 20 25 30 35 40 45 50 55 60 temperature (degrees c )
1284.8 1280.8
Band positions in cm-'.
current work for all PC chain lengths studied. Second, the k = 0 mode (missing in alkanes) appears weakly as a shoulder near 1182 cm-l (Figure 1A). Both observations emphasize the importance of the ester moiety to the intensity of the progression and to the removal of the center symmetry from the normal modes. The observed wagging bands are collected in Table I and the dispersion curve constructed therefrom for the diCI3to diCzoseries of saturated PC's is shown in Figure 2. The shape of the dispersion curve over the range of phase angles where $ / T varies from 0 to 0.4 is in good agreement with that for alkanes. A slight (3-7 cm-l) shift to higher frequency is apparent for a given phase angle for the phospholipids. The monotonic, smooth nature of the curve is evidence for the correctness of the band assignments. The detailed temperature dependence of the band intensities (measured as described in the Experimental Section) of the sum of the k = 1 through k = 4 components for DPPC is shown in Figure 3. Also included in the figure is the temperature dependence of the CH2 symmetric stretching mode, widely used as an index of acyl chain conformational order. Although there is some scatter in the wagging progression intensity data due to the inherent weakness of the bands, two trends are evident. First, there is a substantial, progressive decrease in the intensity from 1 5 to 40 OC with a possible discontinuity (close to experimental uncertainty) at the pretransition (Lp Pb) near 30-35 OC. The intensity at 40 O C Cjust below T,) is reduced to about 50% of its initial value. Second, the remaining intensity essentially vanishes at T,. Similar behavior is noted for the entire set of saturated PC's. We note that Fringeli and Giinthard'* have suggested that in pure phospholipids, the entire intensity of the progression arises from the C( 1) position of the glycerol backbone. That suggestion is currently being evaluated. To reconcile the rapid diminution in intensity below T,,, with the widely accepted notion that the phospholipid acyl chains are
-
Q1
2851 . 5 2
Figure 3. Temperature variation for the wagging progression intensity ( k = 1 through 4 components) for DPPC (A,left-hand ordinate scale)
and for the CH, symmetric stretching frequency (0, right hand ordinate scale). Note the rapid variation of the wagging progression intensity prior to 35 "C. essentially all-trans in their gel state, the simple model proposed for the relative intensity of the progression is adequate. According to this scheme, the predicted intensities are very sensitive to the introduction of small amounts of conformational disorder. To illustrate, it is assumed that the fraction of trans bonds at the lowest temperature studied is 1. With the introduction of small amounts of disorder in the gel phase at higher temperatures, the probability of a trans bond is p , where p is close to (but less than) 1. The probability of a gauche bond is then 1 - p . The relative intensity of the progression (which is proportional to the remaining fraction of all-trans chains) is
(where n is the number of methylenes). Introduction of 5% gauche forms at each position (p = 0.95) at 40 'C corresponds to 0.6-0.7 gauche bond/chain [determined as (1 - p) X N o r 0.05 X 13 C-C bonds, neglecting the 1-2 and 15-16 bonds]. This slight conformational disorder is sufficient to reduce the intensity of the band progression by a factor of -2, since 0.9513 0.5. Thus, the observed 50% diminution of the progression intensity between 15 and 40 'C in DPPC corresponds to about a 5% probability of gauche rotamer formation. The calculation assumes a uniform distribution along the chain but can easily be modified for a nonuniform case. The result is then consistent with the well-known Occurrence of high conformational order in phospholipid gel phases. Above T,, the band progression is not distinguishable from experimental noise. This too may be understood from prior quantitative measurements of acyl chain conformational order from this laboratory. Mendelsohn et aL20 showed the probability of gauche rotamer formation at 50 OC to be about 20% at most acyl chain positions, with the exception of the 14-15 bond, where it was found to be about 40%. In this case, the intensity of the wagging progression, would be at most 4.1% [(0.8)12(0.6)] of the
-
Senak et al.
2752 The Journal of Physical Chemistry, Vol. 96, No. 6, 1992 x
I
A
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1370
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-2849
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I
I
I
9
I
jJ
0.00
(degrees c )
temperature
Figure 4. Temperature dependence of the k = 1 component for a 4:l
(mo1:mol) DPPC/cholesterol sample (m), left ordinate scale. Included for comparison is the temperature dependence for the CH2 symmetric stretching frequency (0),right ordinate scale. 0 050,
c
f
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i'
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;
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8 45
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4 50
55
temperature (degrees c )
Figare 5. Temperature dependence of the k = 1 component of the wagging mode progression for a 2:l (mokmol) DPFC/cholesterol sample (m, kft ordinate scale). Included for comparison is the temperature dependence for the CH2 symmetric stretching frequency (A,right ordinate scale).
all-trans intensity. Thus, the absence of the progression above T,, as monitored in Figure 3, is anticipated. It is apparent from the above that this spectral parameter will be most useful when the phospholipid acyl chains are relatively ordered. B. DPPC/Cbkstd Mixtures. The requirement that a relatively ordered phase be present for the CHI wagging progression intendy to be a useful index for studies of conformtional disorder is satisfied for DPPC/cholesterol mixtures at certain compositions and temperatures. The phasc diagram for this system has been determined by Davis and his co-workers21and discussed in detail by Ipsen et a1.12 Under typical physiological conditions (37"C, cholesterol mole fractions > 0.25-0.3),the system contains a positionally disordered, conformationally ordered state termed the "liquid-ordered" phase. The temperature dependence of the k = 1 component of the methylene wagging mode progressions for 4/1 and 2/ 1 DPPC/ cholesterol (mol/mol) mixtures are shown in Figures 4 and 5 respectively. Included for comparison in each case are the temperature dependencits of the CH, symmetric stretching vibrations. Several features of interest are noted. At 20 mol % cholesterol (Figure 4). a monotonic decrease in the intensity of the progression is noted between 15 and 39 O C . This is followed by a phase separation region characterized by more rapid loss of intensity until 45 "C; additional gradual intensity loss resumes from this point. The thermotropic behavior for the sample containing 33 mol % is strikingly different (Figure 5 ) . The rate of intensity diminution in the k = 1 component is approximately constant over the temperature range 15-60 "C.The observation of substantial residual intensity at temperatures as high as 60 "C reflects the (22) Ipen, J. H.;Mouritsen, 0. G.; Zuckermann, M.J. Biophys. J. 1989, 56, 661.
1360
wavenumber
I
I
1350
1340
I
1
1330
(cm-')
Figure 6. Temperature dependence of the disordered CHI wagging region (1330-1390 cm-l) for a 41 DPPC/cholesterol sample at 60 "C (-) The arrows point to spectral contributions to the and 40 "C (-). contour from end gauche, double gauche, and the sum of kink + gtg forms.
E
8 8
W
b
-7
,
1390
I
A
,
I I
persistence of conformational order in the phospholipid acyl chain. No phase transition or phase separation can be discerned. To explore the validity of the model chosen for the intensity, the current data for the 2:l DPPC/cholesterol mixture may be compared with earlier results from this l a b o r a t ~ r y , I ~in*which ~~ the position dependence of conformational disorder in the DPPC/cholesterol system was monitored at 50 "C. Conformational disorder (3-4%) was noted at acyl chain positions 4 and 6 and an average of 11-12% disorder at acyl chain positions 10, 12, and 13. The wagging modes in the current work show an intensity reduction of 54% (46% all-trans chains remaining, according to the current model) from DPPC at 15 "C (assumed to be in the all-trans form) to DPPC/cholesterol(2:1) at 50 OC. To facilitate comparisons, we assume that conformational order remains constant at 3 4 % from positions 2-9 in 2:l DPPC/cholesterol mixtures, at which point the disorder increases to 11-12%. The residual fraction of trans conformers as deduced from the CD2 rocking experiments is (0.965)7(0.88# = 0.37. This value is in adequate agreement with the observed value of 0.46 from the wagging mode experiment, considering the assumptions involved. Thus the liquid ordered state in DPPC/cholesterol consists of about 1 gauche rotamer/chain compared with the 3.6-4.2 gauche rotamers/chain deduced for the La phase of the pure phospholipid. The thermotropic behavior of the k = 1 component intensity in the sample containing 20 mol % cholesterol (Figure 4) can be readily interpreted. From 15 to 39 "C,the monotonic reduction corresponds to the gradual incorporation of gauche rotamers. Numerically, -0.4 gauche rotamer/chain is introduced over this interval at a rate of -0.016 gauche rotamer/degree. From 39 to 45 "C the more rapid rate of gauche rotamer formation (0.1/deg, 0.6 gauche rotamer/chain) arises from the disordering of the liquid-ordered phase that coexists with the liquid-disordered state over this temperature range. Above 45 "C, the continuing introduction of disorder into the liquid disordered phase is evident. Thus the broad phase transition evident in the FT-IRdata in corresponds well with the region of phase separation that delineatesI2the coexistence of the liquid-ordered and liquid-disordered phases. As the fraction of all-trans state in the acyl chains revealed by the wagging modes is diminished, the increasing disorder may be quantiatively monitored through the intensity of the gg marker band from localized wagging vibrations.I6J7 The band appears in alkanes near 1353 cm-l and is shifted to a slightly higher frequency in the current work. The temperature dependence of this spectral region is shown in Figure 6, and the CHI wagging vibrations from other two- and three-bond conformational states are also indicated. The protocol for converting band intensities obtained from curve-fitting of this spectral region to numbers of
CH2 Wagging Progressions as IR Probes
The Journal of Physical Chemistry, Vol. 96, No. 6 , 1992 2753 x 4 '
0 025T
o nor) 1 1'7
temperature (degrees c )
Figure 7. Temperature dependence of the number of double gauche bonds in the DPPC acyl chains as determined from the contribution of the gg band to the spectral contour in Figure 6. The data were processed as described in ref 17. Data are for 4/ 1 DPPC/cholesterol dispersions.
gg states has been discussed in our prior work," and the results for the samples containing 20 and 33 mol % cholesterol are shown in Figure 7. As noted above, the loss of the all-trans state in the former between 39 and 45 OC which involves the introduction of about 0.6gauche bond per chain, is accompanied by the formation of about 0.15 gg forms in the chain. This suggests that some of the disordering of the liquid-ordered state in the phase separation region arises from gg forms which presumably are localized in those regions of the acyl chains adjacent to the side chain at C( 17) of cholesterol, i.e. from acyl chain position 10 to the bilayer center. The IR approaches described here and in our previous studies lend strong support to the general features of the DPPC/cholesterol phase diagram. C. DPPC/Androsten Mixtures. The sensitivity of the current approach to slight changes in all-trans conformer population is illustrated in studies of DPPC/androsten interaction. This molecule differs from cholesterol only in the absence of the eight-carbon side chain at position 17. The thermodynamics of this interaction have been evaluated.23 The effect of androsten on the transition enthalpy of the DPPC gel-liquid crystal transition is similar to that of cholesterol. This is a perhaps unanticipated result, since the absence of the sterol aliphatic side chain is expected to remove the packing constraints to phospholipid disordering from acyl chain position 9 or 10 toward the bilayer center. Thus, the observed fractional disorder (10%13% gauche forms) observed for DPPC/cholester~l'~~~~ toward the bilayer center might be expected to substantially increase in the case of DPPC/ androsten. The intensity of the k = 1 wagging mode for a 2:l (mo1:mol) DPPC/androsten complex is compared with DPPC/ cholesterol at the same composition in Figure 8. The diminished intensity at all studied temperatures for the DPPC/androsten system is striking. At 40 OC the intensity of the progression is that in DPPC/cholesterol. This corresponds to an average additional gauche population of 0.1 gauche rotamers/C atom compared with cholesterol, or an additional 1-1.5 gauch bonds/chain. Corroborating this observation is the presence (data not shown) of gg conformers as deduced from the localized CH2 wagging modes. These increase approximately linearly from 0.45/chain at 45 OC to 0.65/chain at 70 OC. This suggests that most of the additional disorder in DPPC/androsten arises from gg states near the bilayer center. If these gauche bonds form cooperatively (probably unlikely in a poorly packed system), they will contribute a maximal additional 500-750 cal/mol to the calorimetric enthalpy (assuming 500 cal/gauche bond24)compared with that observed for DPPC/cholesterol. This small energy difference is probably within the uncertainty of the DSC meas u r e m e n t ~ .The ~ ~ CH2 wagging intensity data confirm the sensitivity of this parameter to the introduction of small numbers of gauche forms. (23) Singer, M. A.; Finegold, L. Chem. Phys. Lipids 1990, 56, 217. (24) Scherer, J. R.; Snyder, R. G. J . Chem. Phys. 1980, 72, 5798.
i
0
70
7'1
50
35
:emprroture
40
45
50
55
(oegrees c )
Figure 8. Intensity of the k = 1 component of the C H 2 wagging progression for 2:l DPPC/cholesterol (V),and for 2:l DPPC androsten ).( Note the persistence of the band at elevated temperatures for the former.
Discussion Four IR spectral parameters are now available for evaluation of conformational states in disordered phospholipid acyl chains. These are (i) the frequency of the CH2stretching modes, (ii) the relative intensity of the CH2 wagging progression (current experiments), (iii) the intensity of the CD2 rocking modes in specifically deuterated acyl chains, and (iv) the positions and intensities of isolated (uncoupled) CH2wagging modes in disordered phases. As this set of spectral parameters provides the best available experimental means for determination of phospholipid conformation, it is appropriate to define their optimal domains of utility. The most intense features in acyl chain spectra arise from the CH2 stretching vibrations. The symmetric and antisymmetric frequencies increase mostly as the result of conformational disorder?$ but quantitative correlations between frequency and extent of disorder are not available. The magnitude of the frequency shift is so small (2-5 cm-' during a transition) that other factors, as noted many years ago for small molecules,26 may contribute to changed band positions. However, melting curves constructed from the measured positions (and their CDz counterparts in acyl-chain perdeuterated derivatives) have been used as an empirical parameter for the construction of phase diagram^.^ A highly nonlinear relationship has been found between the frequency change and the fraction of lipid melted. In contrast to the intensities of the CH2wagging progression components studied in the current work, the CHI symmetric stretching frequency is not sensitive to the introduction of small amounts of conformational disorder. As seen in Figure 3 there is only a slight change in this parameter prior to the Lp,-P, transition at 35-36 O C , unlike the progression which loses 50% of intensity between 15 and 35 OC. Thus, the CH2 frequency measurement is best for qualitative studies of rather disordered phases, while the current measurement of wagging progression intensity is best for monitoring the introduction of the first 1-2 gauche bends into an ordered system. The quantitative aspects of the current work invoke the assumption that the intensity of the progression is derived solely from all-trans chains. Three factors suggest that this approach is a reasonable starting point. First, the lack of substantial frequency shifts as a function of temperature in any of the observed progressions implies that there is no intensity arising from chains with effectively shortened lengths that might occur upon gauche rotamer formation, had the progression appeared in spectra of disordered phases. Second, in the two situations (e.g. DPPC/ cholesterol mixtures and DPPC in the gel phase) where conformational order has been estimated from both the CD2 rocking modes and the CH2 wagging bands, there is adequate agreement between the methods. Third, as can be seen from the simple (25) Snyder, R. G.; Straws, H.; Elliger, C. A. J . Phys. Chem. 1982, 86, 5145. (26) Bellamy, L. J. The Infra-red Spectra of Complex Molecules, 2nd ed.; Methuen: London, 1958; p 380.
J. Phys. Chem. 1992, 96, 2154-2761
2754
sample calculations above, the fractional disorder required at each position to produce a particular loss of intensity is not a very strong function of the power dependence assumed in eq 1, for values of p close to 1 that occur in slightly disordered phases. From a biological perspective, the current results suggest that membranes with high levels of cholesterol, such as mammalian cells, may contain more conformational order than might be anticipated from the original fluid mosaic model of membrane str~cture.~’ We are currently examining native membranes enriched in a single acyl chain length to quantitatively explore this hypothesis. (27) Singer, S. J.; Nicolson,
G.L. Science
1972, 175, 720.
Finally, previous experiments from this l a b o r a t ~ r y I ~have -‘~ introduced spectral parameters for the study of conformational disorder in phospholipid liquid crystalline phases. The CD2rocking modes of specifically deuterated acyl chains provide a depth-dependent probe of trans-gauche isomerization, especially in the L, phase. However, in ordered phases, the bands that reveal gauche rotamer formation are extremely weak and yield band intensities of low precision. The approach presented in the current investigation thus complements the earlier studies and permits a more detailed evaluation of the whole range of conformational order available to hydrocarbon chains.
Acknowledgment. This work was supported by PHS Grant (GM 29864) to R.M.
Mdan)sm of Antioxhiant Reaction ot Vitamin E. Charge Transfer and Tunnew Effect in Proton-Transfer Reaction Shin-ichi Nagaoka,*pt Aya Kuranaka,+ Hideki Tsuboi,+Umpei Nagashima,* and Kazuo Mukai**+ Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790, Japan, and Institute for Molecular Science, Myodaiji, Okazaki 444, Japan (Received: November 1I , 1991)
In order to shed light on the mechanism of proton-transfer reactions, a kinetic and ab initio study of the antioxidant action (intermolecular proton transfer) of vitamin E derivatives has been carried out. The second-order rate constants (k,’s) for the reaction of tocopherols (TocH’s) with variously substituted phenoxy1 radicals (Ph0”s) in ethanol were measured with a stopped-flow spectrophotometer. The half-wave reduction potentials (Ellis) of Ph0”s were obtained by using a cyclic voltammetry technique. The result indicates that k, increases as the total electron-donating capacity of the alkyl substituents at the aromatic ring of TocH or the electron-withdrawing capacity of the substituent of Pho’ increases. k, for the reaction of deuterated tocopherol derivatives (TocDs) with a PhO’ in deuterated ethanol (C2H50D, ethanol-d,) was also measured. A substantial deuterium kinetic isotope effect on k, is observed. In the reactions of each Pho’ with various TocH’s, a plot of log k, vs peak oxidation potential (E,) of TocH is found to be linear. The slope of its plot for TocDs is close to that for TocHs. In the reactions of each TocH with various PhWs, a plot of log k,vs Ell2of Pho’ is found to be linear. The geometries of TocH’s were optimized with the semiempirical modified neglect of diatomic overlap (MNDO)method. The Koopmans’ theorem first ionization energies (IP) for those geometries were calculated with the ab initio method. In the reactions of a PhO’ with various TocH’s, plots of log k, vs IP, the activation energy (Eaa)vs IP, and E, vs IP are also found to be linear. From these results, it is considered that both the charge transfer and the proton tunneling play important roles in the antioxidant reaction of TocH. The transition state has the property of the charge-transfer species. The proton tunneling takes place below the transition state. Tunnefing allows the proton to cut a corner on the potential energy surface. Our explanation will be widely applicable to many proton-transfer reactions.
Introduction
In recent years, proton transfer has been a topic of much interest because of its importance in many chemical and biological pro-
cesses.14 It is a chemically very simple process, which is readily accessible to both accurate measurements and quantitative theoretical analyses. However, the details of the reaction mechanism have not necessarily been elucidated so far. It would be especially interesting to study the tunneling effect. Thus, we have carried out a kinetic and a b initio study to shed light on the mechanism of proton-transfer reactions. As a representative of the protontransfer reaction, we have chosen the antioxidant action of vitamin E derivatives. It is well-known that vitamin E (a-, 8-, y-, and &tocopherols, Figure 1) inhibits the autoxidation of organic molecules, and the reaction has been studied extensively by numerous Furthermore, vitamin E is present in cellular membranes and edible oils and acts as an antioxidant by protecting polyunsaturated lipids or fatty acids from peroxidation. The antioxidant properties of tocopherols (TocH’s) have been ascribed to intermolecular proton transfer (hydrogen transfer) Ehime University. *Institutefor Molecular Science. Current address: Department of Computational Science, Faculty of Science, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 1 1 2, Japan.
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in the ground state as a whole. The proton (hydrogen) transfers from the OH group in TocH’s to a peroxyl radical (LOO’). The proton transfer (hydrogen transfer) produces a tocopheroxyl radical (Toc’), which combines with another peroxyl radical (reactions 1 and 2).9J0
+ TocH 2LOOH + Toc’ LOO’ + Toc’ nonradical products LOO’
-
(1)
(2)
(1) Proton-Transfer Reactions; Caldin, E., Gold, V., Eds.; Chapman and Hall: London, 1975. (2) Spectroscopy and Dynamics of Elementary Proton Transfers in Polyatomic Systems; Barbara, P. F., Trommsdorff, H. P., Eds. Chem. Phys. 1989, 136, 153-360. (3) Nagaoka, S.; Nagashima, U. Chem. Phys. 1989, 136, 153. (4) Nagaoka, S. Kagaku To Kogyo (Tokyo) 1991, 44, 182. ( 5 ) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194, and references cited therein. (6) Niki, E. Yuki Gosei Kagaku Kyokaishi 1989,47,902, and referenccs cited therein. (7) Barclay, L. R. C.; Baskin, K. A.; Locke, S. J.; Vinqvist, M. R. Can. J. Chem. 1989,67, 1366. (8) Pryor, W. A,; Strickland, T.; Church, D. F. J . Am. Chem. SOC.1988, 110, 2224. (9) Burton, G.W.; Ingold, K. U. J. Am. Chem. SOC.1981, 103, 6472. (10) Niki, E.; Kawakami, A.; Saito, M.; Yamamoto, Y.; Tsuchiya, J.; Kamiya, Y. J. Biol. Chem. 1985, 260, 2191.
0 1992 American Chemical Society
