J. Phys. Chem. 1983, 87. 5354-5357
5354
TABLE VI: Infrared Bands for C=N Stretch in Metal Cyanide Complexes Bound by [(BPQZ+),],,,~ band band complex max, cm-' complex max, cm-' Fe(CN)632104 Ru(CN),~2036 CO(CN),~- 2114 Mo(CN),~- 2097 Fe( CN),42027
C1- or with Fe(CN):-l4-. Other metal cyanides can be detected as well (Table VI) and the infrared measurements can be used to independently order the binding of the various complexes. The procedure involves determining the relative absorbance for a given complex after equilibration of the n-Si/ [ (BPQ2+),],,,f with a particular anion-containing solution. The same ordering as determined electrochemically is found by using the infrared technique. The value of the infrared technique is that the species to be detected need not be electroactive, e.g., C O ( C N ) ~ ~ - .
Summary Species I is an easily prepared surface-derivatizing reagent t h a t can yield a surface-bound polymer, [(BPQ2+)nlsurf, that is durable and redox active. The one-electron reduced state is particularly durable but the two-electron reduced state does not persist in aqueous electrolytes. The [(BPQ+)n]sdis blue (A- = 610 nm), not purple as for the polymer from I1 (A, = 545 nm), consistent with smaller interactions of the BPQ+ units than for the units of the polymer from 11. The smaller interactions of the BPQ+ units and the need for a break-in period for polymers derived from I (and not from 11) are
both consistent with the conclusion that the polymer from I is more rigid than the polymer from 11. However, the order of binding of a series of transition-metal complexes is the same for the polymers from I and 11. Further, the polymers, r > 3 X mol/cm2, derived from reagents I and I1 are reduced with approximately the same rate upon a potential step to a potential negative of Eo' for [ (BPQ2+/+),lsurf.Apparently, the additional rigidity imposed by the benzyl group of I compared to the propyl group of I1 is offset by the fact that the benzyl group is unsaturated and may promote charge transport. It is especially noteworthy that the [(BPQ2+),],,f can be >50% reduced to [(BPQ+),],,,, in C50 ms for I' 6 1 X mol/cm2, corresponding to an optical density change of >1.0. Achnowledgment. We thank the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, for support of this research. Support from the M.I.T. Laboratory for Computer Science, IBM Fund, is also gratefully acknowledged. Use of the Central Facilities of the Center for Materials Science and Engineering is appreciated. Registry NO.I, 87698-68-8;Mo(CN)a", 17845-99-7;Mo(CN)a", 17923-49-8;RU(CN)e3-,54692-27-2;RU(CN)e", 21029-33-4; C O (CN):-, 14897-04-2;Fe(CN)6", 13408-62-3;Fe(CN)t-, 13408-63-4; IrCl:-, 16918-91-5;IrCl:-, 14648-50-1;Mo(CN),~-,38141-22-9; Mo(CN)t-, 87698-69-9; Pt, 7440-06-4; W, 7440-33-7;Si, 7440-21-3; SnOz,18282-10-5;Cl-, 16887-00-6;KCI, 7447-40-7; LiC1,7447-41-8; K2HP04,7758-11-4; 4,4'-bipyridine, 553-26-4; p-(trimethoxysily1)benzyl chloride, 24413-04-5.
I c e Melting Induced Phase Transition in Diacyl Phosphatidylcholinest Hector L. Casal;
Davld G. Cameron, and Henry H. Mantsch
Division of Chemistry, National Research Council of Canada, Ottawa, Ontario, Canada K I A OR6 (Received: March 30, 1983)
Infrared studies were made of the gel phases of 1,2-dipalmitoyl-and 1,2-dibehenoyl-sn-glycero-3-phosphatidylcholine hydrated with HzO and DzO. It is shown that ice melting induces a solid-solid phase change in the lipid.
Introduction Of late, there has been considerable interest in transitions in the gel phases of fully saturated synthetic lipids. In the phosphatidylcholines1 and sulfocholines2it has been found that incubation near 0 "C results in dehydration of the head group, and a change in the acyl chain packing2*, effects which are almost completely reversed when the temperature is elevatede6Two phase transitions have been reported for dilaurylphosphatidylethanolamine;7it has since been showns that the dimyristoyl- and dipalmitoylphosphatidylethanolamines also exhibit this behavior and that the transitions result from molecules in different states of hydration.s-10 Many such studies encompass the solid-liquid transitions of HzO (0 "C) and D 2 0 (3.8 "C). Generally, no anomalous behavior of lipid properties has been reported, with two exceptions.11J2 In this paper we briefly report that the melting of the water in a hydrated diacyl phos'Issued as NRCC No. 21055. 0022-3654/83/2087-5354$0 1.50/0
phatidylcholine gel induces changes in its infrared spectra which are interpreted as a solid-solid phase transition in the lipid. (1) Chen, S.C.; Sturtevant, J. M.; Gaffney, B. J. R o c . Natl. Acad. Sci. U.S.A. 1980, 77, 5060-3. (2) Mantsch, H. H.: Cameron. D. G.: Tremblav, P. A.: Kates, M. Biochin. Biophys. Acta 1982, 689, 63-72. (3) Fuldner, H. H. Biochemistry 1981, 20, 5707-10. (4) Ruocco, M. J.; Shipley, G. G. Biochim. Biophys. Acta 1982, 684, 59-66. (5) Ruocco, M. J.; Shipley, G. G. Biochim.Biophys Acta 1982, 691, 309-20. (6) Cameron, D. G.; Mantsch, H. H. Biophys. J. 1982, 38, 175-84. (7) Wilkinson, D. A.; Nagle, J. F. Biochemistry 1981,20, 187-92. (8) Mantach, H. H.; Hsi, S.C.; Butler, K. W.; Cameron, D. G. Bzochim. Biophys. Acta 1983, 728, 325-30. (9) Chang, H.; Epand, R. M. Biochim. Biophys. Acta 1983, 728, 319-24. (10) Seddon, J. M.; Harlos, K.; Marsh, D. J . Biol. Chem. 1983, 258, 1R.50-A
_.
I---
(11)Davis, J. H. Biophys. J. 1979, 27, 339-58.
(12) Tardieu, A. Ph.D. Thesis, Universit6 de Paris-Sud, 1972. Cited in Janiak, M. J.; Small, D. M.; Shipley, G. G. J. Bid. Chem. 1979, 254, 6068-78. Published 1983 American Chemical Society
The Journal of Physical Chemistty, Vol. 87, No. 26, 1983 5355
Phase Transition in Diacyi Phosphatidylcholines
TEMPERATURE,
OC
TEMPERflTURE,
%
Figure 1. Temperature dependence of the frequency of the asymmetric N+(CH,), stretching band in the spectra of hydrated (A) DPPC and (B) DBPC.
Experimental Section High-purity samples of 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) and 1,2-dibehenoyl-snglycero-3-phosphatidylcholine(DBPC) were purchased from Sigma Chemical Co. (St. Louis, MO) and Avanti Polar Lipids (Birmingham, AL).Samples were lyophilized and hydrated to 80% (by weight)* with double distilled H 2 0 or 99.7% deuterated D 2 0 (Merck Sharp and Dohme, Montreal, PQ). The samples were assembled into 25-pm pathlength cells with BaF, windows. The cells were left a t room temperature for 1h, placed in a thermostated mount13 a t -15 "C, and left for 1 h before commencing the temperature study.14 Over a period of 4 h infrared spectra were collected a t increasing temperatures from -15 to 15 "C with a Digilab FTS-15 Fourier transform infrared spectrometer equipped with a wide range mercury cadmium telluride detector (Infrared Associates, New Brunswick, NJ). A maximum optical retardation of 0.5 cm was employed; 300 interferograms were co-added, triangularly apodized, zero filled once, and Fourier transformed to yield 2-cm-I resolution spectra with data encoded every 1cm-l. Frequencies were determined by calculating the center of gravity15of the topmost 6 cm-' of the N+(CH& stretching band and the topmost 2 cm-l of the CH2 wagging band. The fullwidth at half-height (fwhh) of the C=O stretching band was determined after reduction of the resolution to 8 cm-l, and subtraction of a linear baseline extending from 1850 to 1705 cmM1.15 Results The infrared spectra of various diacyl phosphatidylcholines in water and heavy water have been reported and assigned e l ~ e w h e r e . ~ ~ J 'In J ~this paper we present data from three bands resulting from functional groups located in various portions of the bilayer: the asymmetric +N(CH,), stretching band from the choline group a t -970 cm-l, the C=O stretching band from the ester linkages at 1740 cm-l, and the first component of the CH2 wagging N
(13)Cameron, D. G.;Jones, R. N. Appl. Spectrosc. 1981,35, 488. (14)Cameron, D.G.;Charette, G. M. Appl. Spectrosc. 1981,35,224-5. (15)Cameron, D.G.;Kauppinen, J. K.; Moffatt, D. J.; Mantsch, H. H.Appl. Spectrosc. 1982,36, 245-50. (16)Chapman, D.;Wallach, D. F. H. "Biological Membranes", Chapman, D.,Ed.; Academic Press: London, 1968;Vol. 1, pp 125-99. (17)Fringeli, U. P.2.Nuturforsch. C 1977,32, 20-45. Biophys. J. 1981,34,173-87. (18)Cameron, D.G.;Casal, H. L.; Mantsch, H. H. J. Biochem. Biop h y ~Methods . 1979,1,21-36.
band progression of the acyl chains a t 1190 cm-'. Figure 1 shows the temperature dependence of the choline band in the spectra of DPPC (Figure 1A) and DBPC (Figure 1B). In both cases the results are shown for samples hydrated with H 2 0 (top) and D 2 0 (bottom). All four plots show abrupt discontinuities at about 0 "C when the sample was hydrated with water and near 4 "C when heavy water was used. Also evident in the plots is the fact that the shift to higher frequency is much larger when H 2 0 is the hydrating agent. While the choline groups can interact with relatively free interbilayer water, the carbonyl groups lie deeper in the bilayer and water in this region should be hydrogen bound. In Figure 2A we show plots of temperature vs. the fullwidth at half-height of the C=O stretching band contour of DPPC dispersed in H 2 0 (top) and D20 (bottom); Figure 2B shows the corresponding data from the spectra of DBPC. As with the choline band, these plots show an abrupt change when ice melts. There are also evident differences in the absolute bandwidth values. However, while the C=O stretching band is isolated in the spectrum when D20 is employed,6in the presence of H 2 0 it overlaps the strong bending band of water near 1640 cm-l.18 While not affecting changes in the fwhh, this leads to uncertainty in the absolute values and we are unable to ascertain if these are real differences. The CH2 wagging band progression is found in the region 1380-1180 cm-l, and is a chain-length-dependent series of bands characteristic of all-trans acyl chains.19 Figure 3A shows the temperature dependence of the first component of this band progression in the spectrum of DPPC in the presence of H 2 0 and D20, while Figure 3B shows the frequencies in the spectra of DBPC. As with the choline and carbonyl stretching bands, the CH2 wagging band progression changes when the water undergoes a transition from solid to liquid. N
Discussion The data presented herein demonstrate that the melting of ice induces a solid-solid phase change in the lipid component of DPPC and DBPC gels. The correlation of the transition temperature with the different melting points of H20 and D 2 0 eliminates the possibility that there is a coincidental agreement between the transition and melting temperatures, and the observation of the transition in DPPC and DBPC gels illustrates that it occurs with widely (19)Snyder, R.G.;Schachtschneider, J. H. Spectrochim. Acta 1963, 19,85-116.
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The Journal of Physical Chemistry, Vol. 87, No. 26, 1983
Casal et al.
A
28 1 I
i
B
OPPC-H20
.--?---.-.-.-l-
E
v
"1 33 -
A
A
31 -
29
I
I
I
-5
5
15
TEMPERRTURE,
A-
OBPC-020
-15
A-A-A-A-
I
I
I
'T
Flgure 2. Temperature dependence of the full-width at half-helght of the C=O
stretching band in the spectra of hydrated (A) DPPC and (B) DBPC.
A
B
DPPC-H20
OBPC-H20
119oj
1
1 -15
9 -5
TEMPERRTURE,
8
5
1
15
'T
1189
I
I
I
I
-15
-5
5
15
TEMPERATURE,
T
Figure 3. Temperature dependence of the frequency of the first component of the CH, wagging band progression, in the spectra of hydrated (A) DPPC and (B) DBPC.
differing chain lengths. With regard to the latter point, we have also observed the transition in 1,2-distearoyl-snglycero-3-phosphatidylcholinegels and, at a minimum, believe it to be a general property of the even-numbered diacyl phosphatidylcholines that are gels a t the melting point of water. The exact nature of the changes induced in the bilayer structure by the ice melting require more detailed studies. However, these data indicate that the changes are minor compared to those resulting from incubation near 0 OC,14 the gel to liquid crystal phase transition: or large variations in the temperature. Spectral changes are observed in bands originating from all regions of the lipid molecules: the choline group representing the polar head group, the ester carbonyl stretching band representing the interfacial region of intermediate polarity, and the CH2wagging band progression representing the hydrophobic acyl chains. The changes in the frequency of the asymmetric 'N(CH,), stretching band are probably due to differences in the strength of the hydrogen bonds between this group and solid and liquid HzO (or DzO) molecules. Similarly, the differences in the frequency shifts between H 2 0 or D20 can be rationalized in terms of different H-bond strengths in these two solvents.
The changes observed in the ester carbonyl stretching bands are indicative of rearrangements of the glycerol moiety of the molecule. Since water molecules have been shown to be in contact with the C=O groups in phosphatidylcholinesZ0the melting of the solvent can influence directly the equilibrium between different conformations of the glycerol backbone. The increase in frequency of the CH2 wagging bands indicates an increase in the acyl chain mobility.21 In this respect, two relevant observations have been reported. Tardie@ has calculated that the surface areas of hydrated DMPC and DPPC increase by 25 % and 45 % ,respectively, on melting of ice, while Davis" has reported that the first and second moments of the 2H NMR spectrum of 1,2diperdeuteriopalmitoyl-sn-glycero-3-phosphatidylcholine (in HzO) decrease abruptly as the temperature is increased through 0 "C. This indicates an increase in the acyl chain mobility which is compatible with increased lipid surface areas and the increase in frequency of the CHz wagging band.21 (20) Zaccai, G.; Blasie, J. K.; Schoenborn, B. P. Proc. Natl. Acad. Sei. U.S.A. 1975, 72, 376-80. (21) Casal, H. L.; Cameron, D. G.; Mantsch, H.H.Can. J. Chern. 1983, 61, 1736-42.
J. Phys. Chem. 1983, 87, 5357-5361
The other phenomenon which occurs in this temperature range is a transition to a poorly hydrated, rigidly packed gel phase.14 This transition occurs over several days when the sample is incubated near 0 'C.' We believe the transition reported herein is a separate event for three reasons. Firstly, the time scale of our experiments only encompasses part of the first, rapid ~ t a g eof~the , ~ incubation. Secondly, the rate of change on incubation decreases as the chain length increases;l we have incubated a DBPC gel at 3 "C for 10 days and found no evidence of any change in the system. Finally, incubation of DPPC results in decreased mobility of the acyl chains,24 an effect opposite to that observed in the transition reported in this study. Apart from the details of the structural changes resulting from the transition, there are a variety of other points to be addressed. It will be of interest to learn if the transition
5357
is a general property of phospholipids or, indeed, surfactants. The behavior of the choline band suggests that there may be subtle differences in, at least, the head group region between samples hydrated with H20 and DzO. There is also the point as to what occurs when the amount of water is exactly, or less than, that required to fully hydrate the lipid, these studies having been performed in the presence of excess water. We are currently addressing these questions and expect to report on them in the near future.
Acknowledgment. The authors thank R. M. Epand and D. Marsh for providing preprints of the reports on their studies of phosphatidylethanolamines. Registry No. DPPC, 63-89-8; DBPC, 37070-48-7; water,
7732-18-5.
Resonance Raman and Molecular Orbital Studies of the Effects of Deuteration on the Vibrational Structure of the p -6enzosemiquinone Radical Anion' Robert H. Schuler," 0. N. R. Tripathi, Michael F. Prebenda,2 and Daniel M. Chlpman Radiation Laboratory and Department of Chemistry, Universiv of Notre Dame, Notre Dame, Indiana 46556 (Received: May 12, 1983)
Deuterium substitution of the p-benzosemiquinone radical anion results in decreases of 23 and 317 cm-l in the vibrational frequencies of its totally symmetric ring stretching (Wilson 8a) and C-H bending (9a) modes as observed by resonance-enhanced Raman methods. Ab initio molecular orbital calculations corroborate the vibrational assignments and predict decreases of a similar magnitude. Both the experimental and theoretical studies show that the bands associated with the CO stretching and C-C-C bending vibrations are affected to only a very minor extent by deuteration, i.e., that there is very little contribution from local modes involving hydrogen motion. The symmetrical ring breathing (1)and C-H stretching (2) modes are not resonance enhanced and not observed in the Raman experiments. Of particular note is the Raman band at the ring stretching frequency of the deuterated radical which is considerably broader than the other lines in H20 and partially resolved into two lines in D20. This feature is tentatively interpreted as being due to an accidental near degeneracy with the combination of the 9a and 1modes. Deuteration of the radical somewhat simplifies its optical absorption spectrum in the region of 400-410 nm indicating that the frequency of the 9a vibration in the excited state is decreased in much the same way as in ground state.
Radicals related to the p-benzosemiquinone anion are important in electron transport processes so that it is of considerable interest to obtain detailed information on their electronic and molecular properties. The unsubstituted p-benzosemiquinone radical anion is relatively stable and can be readily prepared in aqueous solution under well-controlled conditions by radiation chemical oxidation of hydroquinone. The frequencies of the totally symmetric vibrations of this radical have been previously determined by resonance Raman methods3r4and assignments made from symmetry and structural considerations. Recent5 ab initio molecular orbital studies of the vibrational structure of this radical provide information on the 30 possible vibrational modes and completely confirm the assignments made in the Raman experiments. Of interest, particularly (1)The research described herein was supported by the Office of Basic Energy Sciences of the Department of Energy. This is Document No. NDRL-2468 from the Notre Dame Radiation Laboratory. (2) Kalamazoo College, Kalamazoo, MI. (3) G. N. R. Tripathi, J. Chen. Phys., 74, 6044 (1981). (4)G. N. R. Tripathi and R. H. Schuler, J . C h e n . Phys., 76, 2139 (1982). ( 5 ) D. M. Chipman and M. F. Prebenda, manuscript in preparation. 0022-365418312087-5357$01.50/0
with respect to the comparisons between theory and experiment, are the effects of deuterium substitution on the vibrational frequencies since the isotope effects provide information on the contributions from local modes involving hydrogen motion. We report here the results of experimental and theoretical studies in which effects of deuteration on the Raman spectra of this important radical are examined. Also reported are the effects of deuterium substitution on the absorption and ESR spectra of this radical.
Experimental Section The Raman studies were carried out by OH oxidation of 2 mM hydroquinone directly in the Raman cell (0.2 cm3) by pulse radiolytic method^.^ Spectra were examined at microsecond times after production of radical concentraM. This approach allows one to avoid tions of complications from product buildup which are present when chemical oxidation methods are used. Data were recorded a t the rate of 7.5 experiments/s. Studies were at pH -11 where the radical does not decay on the time scale of these experiments. Flow rates of -2 cm3/s were used to replenish the sample between pulses. 0 1983 American Chemical Society
