- Email: [email protected]
Nuclear magnetic resonance studies of amphiphile hydration
ARCHIVES
OF
BIOCHEMISTRY
Nuclear
Magnetic Effects
RONALD
Department
AND
183,
83-89
Resonance
of Cholesterol
P. TAYLOR,’
of Biochemistry,
BIOPHYSICS
(1977)
Studies
of Amphiphile
on Phosphatidyl
CHING-HSIEN HUANG, LUTHER LEAKE Uniuerslty
of Virginia Received
School March
Choline
Hydration’
ANTHONY
of Medicine,
Hydration
V. BROCCOLI,
Charlottesville,
Virginia
AND
22901
14, 1977
Water which remains unfrozen at -25°C in the presence of phosphatidyl choline (PC) gives rise to a proton magnetic resonance signal which can be used to measure the hydration of single-walled vesicles and multilamellar liposomes of PC. The proton magnetic resonance signal of the unfrozen water in these systems is strongly dependent upon the nature of the molecular domain in which the water is situated. For example, at cholesterol to PC molar ratios below 35 mol%, the vesicle hydration signal consists of a relatively narrow symmetric peak (line width, -150 Hz). At higher molar ratios, however, rather broad asymmetric signals appear (line widths, -300-1000 Hz) which indicate that when significant quantities of cholesterol are packed in the bilayer there must be regions in which there is a preferred direction for motion of the unfrozen water. It is possible to solubilize significant quantities of cholesterol by sonicating it in concentrated solutions of sodium dodecyl sulfate. Addition of cholesterol to PC vesicles via these sodium dodecyl sulfate-cholesterol complexes caused hydration changes in the PC, which, at high cholesterol to PC molar ratios, paralleled the effects of cholesterol on PC hydration in homogeneous vesicles in which the cholesterol and PC were simply cosonicated.
Cholesterol is the major sterol constituent in the plasma membrane of a variety of mammalian cells (l-3) and, in addition, it is known to be one of the critical components in the development of atherosclerotic plaques (4). For these reasons, a host of physicochemical techniques have been employed to determine how specific interactions between water, phospholipids, and cholesterol can affect the structure and function of biomembranes. A great deal of this work has focused on model membranes and, for example, has led to the conclusion that at moderate concentrations cholesterol has a significant effect on reducing the permeability of
phospholipid bilayers (5). Recently evidence has been accumulating that a structure transition occurs in the bilayer if the ratio of cholesterol to phosphatidyl choline exceeds about 0.33 mol fraction of cholesterol (6-g). This structural transition has been inferred from a number of diverse experiments on phospholipid-cholesterol systems such as X-ray diffraction (6), calorimetry (71, hydrodynamic studies (81, and complementmediated attack on haptens in bilayers (9). It is believed that part of this structural reorganization may involve the generation of separate molecular domains which can be specifically enriched in cholesterol. We have examined this apparent structural change by measuring the hydration of phospholipid vesicles as a function of the degree of incorporation of cholesterol. The measurement involves using proton magnetic resonance spectroscopy to determine quantitatively the amount of water which
’ This work was supported by a Grant-in-Aid from the American Heart Association and with funds contributed in part by the Virginia Heart Association. This is paper No. 2; see Ref. 11 for the first paper in this series. 2 Research Career Development Awardee of the National Institutes of Health, No. AI-000062-03. 83 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
84
TAYLOR
does not freeze at -25°C in the presence of phosphatidyl choline vesicles containing varying proportions of cholesterol. Our hydration studies also indicate that a transition does occur as the concentration of cholesterol increases; above about 33 mol% cholesterol we have detected complicated hydration signals which we believe represent water interacting with distinctly different molecular domains in the bilayer. MATERIALS
AND
ET
AL.
water/g of protein at ~25°C) or pure PC vesicles (with a hydration of 6.5 mol of water/m01 of PC at -25°C). The hydration signal of SC was at most 0.03 g of water/g of total lipid (i.e., SDS plus cholesterol), which corresponds to about 0.5 mol of water/m01 of lipid. For this reason, all hydration values were calculated on the basis of moles of water per mole of PC only. In all cases reported, hydrations are based on two or more independent determinations on each of two or more independently prepared samples. RESULTS
METHODS
Materials. Phosphatidyl choline (PC):’ was isolated from egg yolk and purified by two steps of column chromatography (10). Other materials used were discussed in the first paper in this series (11). Vesicles. Purified samples of PC and cholesterol were colyophilized from benzene solutions and then sonicated in distilled water following published procedures (8). Samples were sized to obtain singlewalled vesicles by centrifuging them for 1 h at 40,000 rpm at 4°C in a Beckman Model L5-65 centrifuge. The sizing procedure is not critical for the hydration experiment, however. Sonicated but unsized samples and the solubilized precipitates obtained from the sizing procedures all had hydration signals very similar to the single-walled vesicles, for a given cholesterol to PC ratio. Independent assays for PC and cholesterol (12) indicated that the molar ratio of cholesterol to PC in the sized vesicles was identical to the ratio at which they were first colyophilized. Solubilized cholesterol (SC). In a typical preparation, 250 mg of solid cholesterol was added to 5 ml of a 10% solution of SDS and then the dispersion was sonicated for a total of 12 to 15 min on a Bronson sonifier. The clarified solution was centrifuged briefly to remove particles of titanium. Assays of cholesterol and dry weight measurements on the solution indicated that the final SDS concentration was 100 mg/ml and that the final cholesterol concentration was 40 mg/ml. nmr. Proton magnetic resonance studies (at 100 MHz) were accomplished following published methods (111, which were based on the initial experiments of Kuntz on protein hydration (13a, b). Because some of the signals we detect are asymmetric, when necessary hydrations were calculated by xeroxing traces of the signals and weighing the cutout traces. All signals were then referenced to stock solutions of either BSA (with a hydration of 0.4 g of 3 Abbreviations used: PC, phosphatidyl choline; SDS, sodium dodecyl sulfate; SC, solubilized cholesterol in aqueous solution containing 100 mg/ml of SDS and 40 mgiml of cholesterol; BSA, bovine serum albumin; nmr, nuclear magnetic resonance.
Effects with
of PC
Cholesterol:Colyophilization
Up to about 30 mol% cholesterol, the PC hydration signal at -25°C is rather sharp (line width at half height of ca. 120-150 Hz) and symmetric (11). However, as the cholesterol concentration is increased further, the signals become rather broad and asymmetric (Fig. 1). These signals must be due to the water protons, as no signal was detectable (even up to 50 mol% cholesterol) when D,O was substituted for H,O by extensive dialysis of the vesicles. In addition, we have found that by adding small amounts of acid to the samples (generating a final pH of ca. 4.7 or lower) the lines became rather sharp (line width at half height of ca. 250 Hz for 50 mol% cholesterol) and symmetric, but there was no change in the hydration of the samples. Presumably the small amount of acid facilitated proton exchange in the system and, thus, the mechanisms which lead to the increased line widths were eliminated due to rapid exchange. This also provides further evidence that the signals we observe are due to unfrozen water only. We are currently investigating the pH dependence for this exchange phenomenon. PC-cholesterol samples which were not cosonicated but simply colyophilized exhibited very complex behavior (Fig. 2) at high cholesterol concentrations. While the sonicated samples equilibrated rapidly at -25°C (in about 10 min or less), there was a slow continuous change in the nature of the hydration signal for the 50-mol% cholesterol-PC dispersion. Two distinct hydration signals could be detected (one rather sharp signal and a broad one) and, with time, the intensity of the narrow sig-
CHOLESTEROL
AND
PHOSPHATIDYL
CHOLINE
85
HYDRATION
FIG. 1. Typical traces for the proton magnetic resonance signal (100 MHz1 ofunfrozen water at -25°C in the presence of cholesterol-PC vesicles as a function of the mol% of incorporated cholesterol. The arrow represents 100 Hz for 30 and 35 molR cholesterol (A), and it represents 300 Hz for cholesterol concentrations of 40 or 50 mol8 (B). The trace at 50 mol9? cholesterol was obtained on an instrument of considerably higher sensitivity. .
I
I
ia /
Li r
/A
FIG. 2. Apparent
line width of the hydration signal at 100 MHz (see Fig. 1 for typical traces) versus the mol% cholesterol. Closed circles represent data from cholesterol-PC vesicles. Open triangles represent the observed line width for vesicles containing 30 mol% cholesterol to which SC was added. The abscissa is calculated for these points based on the total amount of cholesterol (relative to PC) in solution.
nal decreased while the intensity of the broad signal increased. We also noted that the hydration signal for this sample is considerably larger than that which is measured for unsonicated PC alone (Table I). Acidification of this sample (final pH, ca. 3.8) also gave rise to one sharp (line width of ca. 100 Hz) symmetric peak.
TABLE nmr
I
HYDRATION PARAMETERS FOR VARIOUS PC PREPARATIONS (100 MHz)” Hyty;Line width Sample (Hz)
PC vesicles Cholesterol vesicles Cholesterol vesicles
(30 mol%;i-PC
6.5 9.0
(50 molB)-PC
15.0
PC dispersion Cholesterol (30 dispersion Cholesterol (50 dispersion Epicholesterol PC dispersion Epicholesterol PC dispersion Thiocholesterol PC dispersion Thiocholesterol PC dispersion
150 120 700-900’
mol%)-PC
6.5 9.0
180 100
mol%;)-PC
14.0
100”
(30 mol%;l-
9.0
102
(50 mol%)-
9.0
103
(30 mol%)-
8.0
106
(50 mol%)-
7.0
128 -
” The reproducibility in hydration values was generally 215% or better for vesicle samples. Dispersions gave more erratic results, presumably because of variations due to settling out of material before it was frozen. The reproducibility for a dispersion was about 1-20s or better in hydration value. ’ In all cases hydrations are calculated as moles of water per mole of PC. It is assumed that the cholesterol does not directly contribute to the hydration values. All data refer to -25°C. ’ Considerable variation in the apparent line width (defined as width at half height) was observed for this sample for different preparations. ” The line width of this signal was time dependent (see Fig. 3).
86
TAYLOR
Effects of Epicholesterol and Thiocholesterol We were not able to prepare PC vesicles containing as much as 30 mol% epicholesterol or thiocholesterol; other investigators have also reported that modifications to the P-OH group of cholesterol cause a decrease in solubilization of the sterol by egg PC (14, 15). However, unsonicated dispersions of PC with either cholesterol, epicholesterol, or thiocholesterol (in each case the PC and sterol were colyophilized) gave rise to a very similar signal (Table I) for a final sterol concentration of 30 mol%. However, for samples with 50 mol% sterol, only in the case of cholesterol did we observe two apparent hydration signals (Fig. 31, which exhibited the unusual kinetic behavior described above. This clearly indicates that the presence of the hydroxyl group in the p configuration at C-3 of cholesterol (16) is essential for the specific cholesterol-PC interactions we detect at high cholesterol concentrations. The SDS-Induced Structural tion
Reorganiza-
The addition of moderate amounts of SDS to PC vesicles causes a pronounced
ET
AL.
structural reorganization in the bilayer which is manifested by an increase in the line width of the PC hydration signal from about 150 Hz to greater than 1000 Hz (11). We have found that vesicles which contain 30 mol% cholesterol show almost no change in the concentration range (about 0.3-0.4 g of SDS/g of PC) in which SDS is most effective on pure PC vesicles. This has led us to attempt to determine the mechanism by which cholesterol prevents this transition. A first step in attacking the problem has been to solubilize significant quantities of cholesterol directly into SDS through prolonged sonication and then to add the solution (designated SC) to pure PC vesicles. Effects of SC on PC Vesicle Hydration The addition of SC to PC vesicles causes considerably different changes in the PC hydration signal than are observed when SDS alone is added. For example, if enough SC is added to give a final SDS to PC ratio of about 0.3 g/g, the line width of the signal is only about 200 Hz, as compared to about 1000 Hz if SDS alone is added (11). Thus, it appears that the cholesterol which is solubilized into the SDS solution is also capable of inhibiting the
FIG. 3. The hydration signal (at 100 MHz) for a cosonicated dispersion of cholesterol (50 mol% cholesterol) as a function of time at -25°C. The arrow represents 100 Hz.
and PC
CHOLESTEROL
AND
PHOSPHATIDYL
SDS-induced structural reorganization of PC vesicles. We emphasize again that if the vesicles already contain 30 mol% cholesterol, then the SDS transition is also inhibited. A more interesting result is obtained if enough SC is added to PC vesicles which already contain 30 mol% cholesterol such that the final total amount of cholesterol in solution relative to PC corresponds to about 50 mol% cholesterol. The signal we obtain (Fig. 4, trace B) is considerably sharper than that observed when SDS alone is added to PC vesicles (11). In fact, the hydration signal for the PC-SC system in trace B of Fig. 4 more nearly resembles the signals obtained for PC vesicles containing 50 mol% cholesterol to which SDS has been added (Fig. 4, trace A). Though the rather high ratio of SDS to PC in the system under consideration obviously will affect the line widths and resolution in the experiment, the results do suggest that much of the cholesterol in the SC must enter the PC vesicles to generate a final structure (which probably also includes some SDS) that resembles a 50 mol% cholesterol sample with respect to the hydration experiment. Thus, in this last experiment in which the final concentration of the three lipid components is about the same, very similar results are obtained for two distinctly different methods of preparation. On the Refreezability
of Samples
We have found that samples which were defrosted after the nmr experiment and stored on ice had essentially identical signals when they were refrozen. For example, a PC vesicle preparation containing 50 mol% cholesterol had the same characteristic broad, asymmetric line shape (Fig. 1B) when it was refrozen 6 h after it was first examined via the nmr experiment. If it had undergone a structural reorganization to that of the dispersion upon defrosting, we would have expected to see the time-dependent hydration signals depicted in Fig. 3 for the dispersions. Thus, although the defrosted samples are polydisperse and “look” as if they had not been sonicated, upon refreezing they show the
CHOLINE
HYDRATION
87
FIG. 4. (Al Hydration signal (at -25°C) for a cholesterol (50 mol%)-PC vesicle preparation to which SDS had been added to give a final SDS to PC ratio of ca. 0.67. (B) Hydration signal for a cholesterol (30 mol%‘o)-PC vesicle preparation to which enough SC had been added such that the final concentrations of SDS, PC, and cholesterol were identical to those in A. The absolute hydrations of these samples corresponded to about 20 water molecules per molecule of PC. The arrow represents 300 Hz in each case.
characteristic samples.
signals
of freshly
sonicated
DISCUSSION
Our results are in agreement with numerous other studies which indicate that a structural change occurs in the cholesterol-PC bilayer if the cholesterol concentration exceeds about 33 mol% (6-9). We believe that at higher cholesterol concentrations the nmr measurement detects a significant amount of unfrozen water which must be located in environments which are distinctly different from those in which the unfrozen water is situated at lower cholesterol concentrations. This follows from the marked changes in both line width and amplitude of the hydration signal. In addition, our studies suggest that, at least over a certain concentration range (e.g., cf. 40 mol% cholesterol, Fig. lB), exchange of water between the different environments may be slow, as rather asymmetric signals are observed. It is most reasonable to assume that the new environments for unfrozen water are generated by significant changes in the packing of the bilayer phospholipids at high cholesterol concentrations. One possibility is that there are local regions within the system which contain different ratios of cholesterol to phospholipid and that both the relative motion and chemical shift of water within these re-
88
TAYLOR
gions may be very different. The exact molecular mechanism for the rather broad lines we observe (e.g., dipolar broadening due to anisotropic motion of unfrozen bound water, differences in diamagnetic susceptibility in different domains) remains to be determined. However, we note that other workers (17, 18) who have examined the hydration of solid materials by proton or deuterium magnetic resonance at room temperature have also observed rather broad, complex signals for water adsorbed to specific surfaces. It is generally believed that these signals contain contributions from dipolar splitting (for protons) and quadrupolar splitting (for deuterium) due to the relatively slow, anisotropic motion of the “bound” water. There is some precedent for the clustering of molecular species of different stoichiometries in PC-cholesterol bilayers. For example, Gershfeld (19) has recently presented evidence based on calorimetric and density measurements of PC-cholesterol mixtures which indicates that a variety of complexes of distinctly different stoichiometries are generated at cholesterol concentrations greater than 33 mol%. Based on the results of proton magnetic resonance spectroscopy (the resonance signals of the methylene protons of the PC acyl chains were monitored), Phillips and Finer (20) have also suggested there can be nonrandom packing of cholesterol in PC bilayers to give regions composed of either 1:l complexes, “boundary” PC, or “free” PC. They also noted that other workers have reached qualitatively somewhat similar conclusions, but there is still some question about the exact stoichiometry of the cholesterol-PC complexes. It is unlikely that the hydration signal at high cholesterol concentrations contains a significant contribution due to direct hydration of the cholesterol hydroxyl group. First, the majority of the cholesterol hydroxyl groups should be hydrogen bonded either with the acyl carbonyls of PC or with themselves (16); this would of course decrease their ability to interact with water. In addition, the net increase in water of hydration at high cholesterol concentrations would appear to be too large for sol-
ET AL.
vation of a single hydroxyl group, based on the model studies of Kuntz (13b). Also, as previously reported (ll), cholesterol alone has no hydration signal.4 The situation is apparently more complicated for unsonicated dispersions of PCcholesterol mixtures at 50 mol% cholesterol. This system does not rapidly equilibrate at -25”C, but the results again suggest that different types of water are present for a considerable period of time (Fig. 3). That the hydration signals are so significantly different for the unsonicated mixture (liposomes) versus the sonicated system (vesicles) at 50 mol% cholesterol clearly demonstrates that the unfrozen water serves as a sensitive probe of the different structures in these systems. Though the ideas proposed here are at present speculative, experiments are underway to try to obtain more definitive information regarding the nature of the environment of the unfrozen water we detect. We emphasize, however, that the appearance of the “second” hydration signal (as manifested by the first increase in line width, Fig. 2) occurs at a cholesterol to PC molar ratio at which a structure transition has been postulated to occur. Thus, regardless of the detailed explanation for our observations, it is clear that the hydration experiment is capable of detecting specific structure changes in the bilayer. It will be most interesting to determine if biological membranes which have high cholesterol contents also exhibit more complex hydration signals, and such studies are in progress. Our studies with SC suggest that the cholesterol (and presumably some SDS as well) is freely accessible to the bilayer and in fact can enter the vesicles and interact with PC. We draw this conclusion because we have noted that the addition of SC to PC vesicles causes changes in the hydration signal of these vesicles which are com1 Part of the hydration signal we detect at high cholesterol concentrations may be associated with water in the hydrocarbon region of the bilayer, but the present data contain no direct information on this question. Studies are in progress to try and resolve this point.
CHOLESTEROL
AND
PHOSPHATIDYL
pletely consistent with incorporation of considerable amounts of cholesterol into the vesicles. The fact that 30 mol% cholesterol could inhibit the SDS-induced transition in the vesicles suggests that the partition of SDS into the bilayer must decrease considerably on incorporation of cholesterol The fact that so much cholesterol can in fact be solubilized in SDS has significant implications for model studies which have focused on examining the interaction of cholesterol in water with detergents in order to understand the thermodynamics which govern the interactions of cholesterol with the bilayer (21). Biological membranes can contain as much as 1 mol of cholesterol per mole of phospholipid (l31, and it is possible to incorporate large amounts of cholesterol in aqueous dispersions of phospholipids if the materials are colyophilized. In fact, cholesterol can be “solubilized” in the bilayer (i.e., relatively clear solutions can be prepared) if the phospholipid and cholesterol are cosonicated (8). An interesting question of course is whether these systems are in true thermodynamic equilibrium or, instead, represent structures that are trapped kinetically in local free energy minima. For example, it is unlikely that the SC samples are thermodynamically stable, as we have noted that they slowly settle out of solution if stored at room temperature for more than a few days. However, the SC system represents a new method to solubilize large quantities of cholesterol in aqueous solution in structures which apparently maintain their integrity for sufficiently long periods of time that they can be used in meaningful experiments. It is possible that a use for the SC will be found in other
HYDRATION
89
investigations in which it is necessary alter the cholesterol content in a system.
to
CHOLINE
ACKNOWLEDGMENT We thank Dr. Herman Yeh of the National Institutes of Health for generously providing us with considerable periods of working time on his HA-100 nmr spectrophotometer. REFERENCES 1. NES, W. R. (1974) Lipids 9, 596. 2. JAIN, M. (1975) Current Topics Membr. 6, 1. 3. DEMEL, R. A., AND DE KRUYFF, B. (1976) Biochum Biophys. Actu 157. 109. 4. SMALL, D. M., AND SHIPLEY, G. G. (1974) Science 185, 222. H. (19741 Lipids 9, 645. 5. BROCKERHOFF, 6. ENGLEMAN, D. M., AND ROTHMAN, J. E. (1972) J. Biol. Chem. 247, 3694. 7. HINZ, H. J., AND STURTEVANT, J. M. (1972) J. Bid. Chem. 247, 3697. 8. NEWMAN, G. C., AND HUANG, C. (1975) Biochemistry 14, 3363. 9. HUMPHRIES, G. M. K., AND MCCONNELL, H. M. (1975) Proc. Nat. Acad. Sci. USA 72, 2483. 10. HUANG, C. (1969) Biochemistry 8, 344. 11. TAYLOR, R. P. (1976) Arch. B&hem. Biophys. 173, 596. 12. BAUER, J. D., ACKERMAN, P. G., AND TORO, G. (1960) Bray’s Clinical Laboratory Methods, p. 372, C. V. Mosby, St. Louis. 13a.KuNT2, I. D., BRASSFIELD, T. S., LAW, G. D., AND PURCELL, A. V. (1969) Science 163, 1329. 13b.Kuiwz, I. D. (1971) J. Amer. Chem. Sot. 93, 514. 14. HUANG, C., CHARLTON, J. P., SHYR, C. I., AND THOMPSON, T. E. (1970) Biochemistry 9, 3422. 15. OLDFIELD, E., AND CHAPMAN, D. (1971) Biochem. Biophys. Res. Commun. 43, 610. 16. HUANG, C. (1976) Nature iLondon 259, 242. 17. WOESSNER, D. E. (197415. Mugn. RFS. 16,483. 18. FUNG, B. M. (1974) Science 190, 800. 19. GERSHFELD, W. L. (1977)Biophys. J. 17, 94a. 20. PHILLIPS, M. C., AND FINER, E. G. (1974) Biochim. Biophys. Acta 356, 199. 21. GILBERT, D. B., AND REYNOLDS. J. A. (1976) Biochemistry 1.5. 71.
OF
BIOCHEMISTRY
Nuclear
Magnetic Effects
RONALD
Department
AND
183,
83-89
Resonance
of Cholesterol
P. TAYLOR,’
of Biochemistry,
BIOPHYSICS
(1977)
Studies
of Amphiphile
on Phosphatidyl
CHING-HSIEN HUANG, LUTHER LEAKE Uniuerslty
of Virginia Received
School March
Choline
Hydration’
ANTHONY
of Medicine,
Hydration
V. BROCCOLI,
Charlottesville,
Virginia
AND
22901
14, 1977
Water which remains unfrozen at -25°C in the presence of phosphatidyl choline (PC) gives rise to a proton magnetic resonance signal which can be used to measure the hydration of single-walled vesicles and multilamellar liposomes of PC. The proton magnetic resonance signal of the unfrozen water in these systems is strongly dependent upon the nature of the molecular domain in which the water is situated. For example, at cholesterol to PC molar ratios below 35 mol%, the vesicle hydration signal consists of a relatively narrow symmetric peak (line width, -150 Hz). At higher molar ratios, however, rather broad asymmetric signals appear (line widths, -300-1000 Hz) which indicate that when significant quantities of cholesterol are packed in the bilayer there must be regions in which there is a preferred direction for motion of the unfrozen water. It is possible to solubilize significant quantities of cholesterol by sonicating it in concentrated solutions of sodium dodecyl sulfate. Addition of cholesterol to PC vesicles via these sodium dodecyl sulfate-cholesterol complexes caused hydration changes in the PC, which, at high cholesterol to PC molar ratios, paralleled the effects of cholesterol on PC hydration in homogeneous vesicles in which the cholesterol and PC were simply cosonicated.
Cholesterol is the major sterol constituent in the plasma membrane of a variety of mammalian cells (l-3) and, in addition, it is known to be one of the critical components in the development of atherosclerotic plaques (4). For these reasons, a host of physicochemical techniques have been employed to determine how specific interactions between water, phospholipids, and cholesterol can affect the structure and function of biomembranes. A great deal of this work has focused on model membranes and, for example, has led to the conclusion that at moderate concentrations cholesterol has a significant effect on reducing the permeability of
phospholipid bilayers (5). Recently evidence has been accumulating that a structure transition occurs in the bilayer if the ratio of cholesterol to phosphatidyl choline exceeds about 0.33 mol fraction of cholesterol (6-g). This structural transition has been inferred from a number of diverse experiments on phospholipid-cholesterol systems such as X-ray diffraction (6), calorimetry (71, hydrodynamic studies (81, and complementmediated attack on haptens in bilayers (9). It is believed that part of this structural reorganization may involve the generation of separate molecular domains which can be specifically enriched in cholesterol. We have examined this apparent structural change by measuring the hydration of phospholipid vesicles as a function of the degree of incorporation of cholesterol. The measurement involves using proton magnetic resonance spectroscopy to determine quantitatively the amount of water which
’ This work was supported by a Grant-in-Aid from the American Heart Association and with funds contributed in part by the Virginia Heart Association. This is paper No. 2; see Ref. 11 for the first paper in this series. 2 Research Career Development Awardee of the National Institutes of Health, No. AI-000062-03. 83 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
84
TAYLOR
does not freeze at -25°C in the presence of phosphatidyl choline vesicles containing varying proportions of cholesterol. Our hydration studies also indicate that a transition does occur as the concentration of cholesterol increases; above about 33 mol% cholesterol we have detected complicated hydration signals which we believe represent water interacting with distinctly different molecular domains in the bilayer. MATERIALS
AND
ET
AL.
water/g of protein at ~25°C) or pure PC vesicles (with a hydration of 6.5 mol of water/m01 of PC at -25°C). The hydration signal of SC was at most 0.03 g of water/g of total lipid (i.e., SDS plus cholesterol), which corresponds to about 0.5 mol of water/m01 of lipid. For this reason, all hydration values were calculated on the basis of moles of water per mole of PC only. In all cases reported, hydrations are based on two or more independent determinations on each of two or more independently prepared samples. RESULTS
METHODS
Materials. Phosphatidyl choline (PC):’ was isolated from egg yolk and purified by two steps of column chromatography (10). Other materials used were discussed in the first paper in this series (11). Vesicles. Purified samples of PC and cholesterol were colyophilized from benzene solutions and then sonicated in distilled water following published procedures (8). Samples were sized to obtain singlewalled vesicles by centrifuging them for 1 h at 40,000 rpm at 4°C in a Beckman Model L5-65 centrifuge. The sizing procedure is not critical for the hydration experiment, however. Sonicated but unsized samples and the solubilized precipitates obtained from the sizing procedures all had hydration signals very similar to the single-walled vesicles, for a given cholesterol to PC ratio. Independent assays for PC and cholesterol (12) indicated that the molar ratio of cholesterol to PC in the sized vesicles was identical to the ratio at which they were first colyophilized. Solubilized cholesterol (SC). In a typical preparation, 250 mg of solid cholesterol was added to 5 ml of a 10% solution of SDS and then the dispersion was sonicated for a total of 12 to 15 min on a Bronson sonifier. The clarified solution was centrifuged briefly to remove particles of titanium. Assays of cholesterol and dry weight measurements on the solution indicated that the final SDS concentration was 100 mg/ml and that the final cholesterol concentration was 40 mg/ml. nmr. Proton magnetic resonance studies (at 100 MHz) were accomplished following published methods (111, which were based on the initial experiments of Kuntz on protein hydration (13a, b). Because some of the signals we detect are asymmetric, when necessary hydrations were calculated by xeroxing traces of the signals and weighing the cutout traces. All signals were then referenced to stock solutions of either BSA (with a hydration of 0.4 g of 3 Abbreviations used: PC, phosphatidyl choline; SDS, sodium dodecyl sulfate; SC, solubilized cholesterol in aqueous solution containing 100 mg/ml of SDS and 40 mgiml of cholesterol; BSA, bovine serum albumin; nmr, nuclear magnetic resonance.
Effects with
of PC
Cholesterol:Colyophilization
Up to about 30 mol% cholesterol, the PC hydration signal at -25°C is rather sharp (line width at half height of ca. 120-150 Hz) and symmetric (11). However, as the cholesterol concentration is increased further, the signals become rather broad and asymmetric (Fig. 1). These signals must be due to the water protons, as no signal was detectable (even up to 50 mol% cholesterol) when D,O was substituted for H,O by extensive dialysis of the vesicles. In addition, we have found that by adding small amounts of acid to the samples (generating a final pH of ca. 4.7 or lower) the lines became rather sharp (line width at half height of ca. 250 Hz for 50 mol% cholesterol) and symmetric, but there was no change in the hydration of the samples. Presumably the small amount of acid facilitated proton exchange in the system and, thus, the mechanisms which lead to the increased line widths were eliminated due to rapid exchange. This also provides further evidence that the signals we observe are due to unfrozen water only. We are currently investigating the pH dependence for this exchange phenomenon. PC-cholesterol samples which were not cosonicated but simply colyophilized exhibited very complex behavior (Fig. 2) at high cholesterol concentrations. While the sonicated samples equilibrated rapidly at -25°C (in about 10 min or less), there was a slow continuous change in the nature of the hydration signal for the 50-mol% cholesterol-PC dispersion. Two distinct hydration signals could be detected (one rather sharp signal and a broad one) and, with time, the intensity of the narrow sig-
CHOLESTEROL
AND
PHOSPHATIDYL
CHOLINE
85
HYDRATION
FIG. 1. Typical traces for the proton magnetic resonance signal (100 MHz1 ofunfrozen water at -25°C in the presence of cholesterol-PC vesicles as a function of the mol% of incorporated cholesterol. The arrow represents 100 Hz for 30 and 35 molR cholesterol (A), and it represents 300 Hz for cholesterol concentrations of 40 or 50 mol8 (B). The trace at 50 mol9? cholesterol was obtained on an instrument of considerably higher sensitivity. .
I
I
ia /
Li r
/A
FIG. 2. Apparent
line width of the hydration signal at 100 MHz (see Fig. 1 for typical traces) versus the mol% cholesterol. Closed circles represent data from cholesterol-PC vesicles. Open triangles represent the observed line width for vesicles containing 30 mol% cholesterol to which SC was added. The abscissa is calculated for these points based on the total amount of cholesterol (relative to PC) in solution.
nal decreased while the intensity of the broad signal increased. We also noted that the hydration signal for this sample is considerably larger than that which is measured for unsonicated PC alone (Table I). Acidification of this sample (final pH, ca. 3.8) also gave rise to one sharp (line width of ca. 100 Hz) symmetric peak.
TABLE nmr
I
HYDRATION PARAMETERS FOR VARIOUS PC PREPARATIONS (100 MHz)” Hyty;Line width Sample (Hz)
PC vesicles Cholesterol vesicles Cholesterol vesicles
(30 mol%;i-PC
6.5 9.0
(50 molB)-PC
15.0
PC dispersion Cholesterol (30 dispersion Cholesterol (50 dispersion Epicholesterol PC dispersion Epicholesterol PC dispersion Thiocholesterol PC dispersion Thiocholesterol PC dispersion
150 120 700-900’
mol%)-PC
6.5 9.0
180 100
mol%;)-PC
14.0
100”
(30 mol%;l-
9.0
102
(50 mol%)-
9.0
103
(30 mol%)-
8.0
106
(50 mol%)-
7.0
128 -
” The reproducibility in hydration values was generally 215% or better for vesicle samples. Dispersions gave more erratic results, presumably because of variations due to settling out of material before it was frozen. The reproducibility for a dispersion was about 1-20s or better in hydration value. ’ In all cases hydrations are calculated as moles of water per mole of PC. It is assumed that the cholesterol does not directly contribute to the hydration values. All data refer to -25°C. ’ Considerable variation in the apparent line width (defined as width at half height) was observed for this sample for different preparations. ” The line width of this signal was time dependent (see Fig. 3).
86
TAYLOR
Effects of Epicholesterol and Thiocholesterol We were not able to prepare PC vesicles containing as much as 30 mol% epicholesterol or thiocholesterol; other investigators have also reported that modifications to the P-OH group of cholesterol cause a decrease in solubilization of the sterol by egg PC (14, 15). However, unsonicated dispersions of PC with either cholesterol, epicholesterol, or thiocholesterol (in each case the PC and sterol were colyophilized) gave rise to a very similar signal (Table I) for a final sterol concentration of 30 mol%. However, for samples with 50 mol% sterol, only in the case of cholesterol did we observe two apparent hydration signals (Fig. 31, which exhibited the unusual kinetic behavior described above. This clearly indicates that the presence of the hydroxyl group in the p configuration at C-3 of cholesterol (16) is essential for the specific cholesterol-PC interactions we detect at high cholesterol concentrations. The SDS-Induced Structural tion
Reorganiza-
The addition of moderate amounts of SDS to PC vesicles causes a pronounced
ET
AL.
structural reorganization in the bilayer which is manifested by an increase in the line width of the PC hydration signal from about 150 Hz to greater than 1000 Hz (11). We have found that vesicles which contain 30 mol% cholesterol show almost no change in the concentration range (about 0.3-0.4 g of SDS/g of PC) in which SDS is most effective on pure PC vesicles. This has led us to attempt to determine the mechanism by which cholesterol prevents this transition. A first step in attacking the problem has been to solubilize significant quantities of cholesterol directly into SDS through prolonged sonication and then to add the solution (designated SC) to pure PC vesicles. Effects of SC on PC Vesicle Hydration The addition of SC to PC vesicles causes considerably different changes in the PC hydration signal than are observed when SDS alone is added. For example, if enough SC is added to give a final SDS to PC ratio of about 0.3 g/g, the line width of the signal is only about 200 Hz, as compared to about 1000 Hz if SDS alone is added (11). Thus, it appears that the cholesterol which is solubilized into the SDS solution is also capable of inhibiting the
FIG. 3. The hydration signal (at 100 MHz) for a cosonicated dispersion of cholesterol (50 mol% cholesterol) as a function of time at -25°C. The arrow represents 100 Hz.
and PC
CHOLESTEROL
AND
PHOSPHATIDYL
SDS-induced structural reorganization of PC vesicles. We emphasize again that if the vesicles already contain 30 mol% cholesterol, then the SDS transition is also inhibited. A more interesting result is obtained if enough SC is added to PC vesicles which already contain 30 mol% cholesterol such that the final total amount of cholesterol in solution relative to PC corresponds to about 50 mol% cholesterol. The signal we obtain (Fig. 4, trace B) is considerably sharper than that observed when SDS alone is added to PC vesicles (11). In fact, the hydration signal for the PC-SC system in trace B of Fig. 4 more nearly resembles the signals obtained for PC vesicles containing 50 mol% cholesterol to which SDS has been added (Fig. 4, trace A). Though the rather high ratio of SDS to PC in the system under consideration obviously will affect the line widths and resolution in the experiment, the results do suggest that much of the cholesterol in the SC must enter the PC vesicles to generate a final structure (which probably also includes some SDS) that resembles a 50 mol% cholesterol sample with respect to the hydration experiment. Thus, in this last experiment in which the final concentration of the three lipid components is about the same, very similar results are obtained for two distinctly different methods of preparation. On the Refreezability
of Samples
We have found that samples which were defrosted after the nmr experiment and stored on ice had essentially identical signals when they were refrozen. For example, a PC vesicle preparation containing 50 mol% cholesterol had the same characteristic broad, asymmetric line shape (Fig. 1B) when it was refrozen 6 h after it was first examined via the nmr experiment. If it had undergone a structural reorganization to that of the dispersion upon defrosting, we would have expected to see the time-dependent hydration signals depicted in Fig. 3 for the dispersions. Thus, although the defrosted samples are polydisperse and “look” as if they had not been sonicated, upon refreezing they show the
CHOLINE
HYDRATION
87
FIG. 4. (Al Hydration signal (at -25°C) for a cholesterol (50 mol%)-PC vesicle preparation to which SDS had been added to give a final SDS to PC ratio of ca. 0.67. (B) Hydration signal for a cholesterol (30 mol%‘o)-PC vesicle preparation to which enough SC had been added such that the final concentrations of SDS, PC, and cholesterol were identical to those in A. The absolute hydrations of these samples corresponded to about 20 water molecules per molecule of PC. The arrow represents 300 Hz in each case.
characteristic samples.
signals
of freshly
sonicated
DISCUSSION
Our results are in agreement with numerous other studies which indicate that a structural change occurs in the cholesterol-PC bilayer if the cholesterol concentration exceeds about 33 mol% (6-9). We believe that at higher cholesterol concentrations the nmr measurement detects a significant amount of unfrozen water which must be located in environments which are distinctly different from those in which the unfrozen water is situated at lower cholesterol concentrations. This follows from the marked changes in both line width and amplitude of the hydration signal. In addition, our studies suggest that, at least over a certain concentration range (e.g., cf. 40 mol% cholesterol, Fig. lB), exchange of water between the different environments may be slow, as rather asymmetric signals are observed. It is most reasonable to assume that the new environments for unfrozen water are generated by significant changes in the packing of the bilayer phospholipids at high cholesterol concentrations. One possibility is that there are local regions within the system which contain different ratios of cholesterol to phospholipid and that both the relative motion and chemical shift of water within these re-
88
TAYLOR
gions may be very different. The exact molecular mechanism for the rather broad lines we observe (e.g., dipolar broadening due to anisotropic motion of unfrozen bound water, differences in diamagnetic susceptibility in different domains) remains to be determined. However, we note that other workers (17, 18) who have examined the hydration of solid materials by proton or deuterium magnetic resonance at room temperature have also observed rather broad, complex signals for water adsorbed to specific surfaces. It is generally believed that these signals contain contributions from dipolar splitting (for protons) and quadrupolar splitting (for deuterium) due to the relatively slow, anisotropic motion of the “bound” water. There is some precedent for the clustering of molecular species of different stoichiometries in PC-cholesterol bilayers. For example, Gershfeld (19) has recently presented evidence based on calorimetric and density measurements of PC-cholesterol mixtures which indicates that a variety of complexes of distinctly different stoichiometries are generated at cholesterol concentrations greater than 33 mol%. Based on the results of proton magnetic resonance spectroscopy (the resonance signals of the methylene protons of the PC acyl chains were monitored), Phillips and Finer (20) have also suggested there can be nonrandom packing of cholesterol in PC bilayers to give regions composed of either 1:l complexes, “boundary” PC, or “free” PC. They also noted that other workers have reached qualitatively somewhat similar conclusions, but there is still some question about the exact stoichiometry of the cholesterol-PC complexes. It is unlikely that the hydration signal at high cholesterol concentrations contains a significant contribution due to direct hydration of the cholesterol hydroxyl group. First, the majority of the cholesterol hydroxyl groups should be hydrogen bonded either with the acyl carbonyls of PC or with themselves (16); this would of course decrease their ability to interact with water. In addition, the net increase in water of hydration at high cholesterol concentrations would appear to be too large for sol-
ET AL.
vation of a single hydroxyl group, based on the model studies of Kuntz (13b). Also, as previously reported (ll), cholesterol alone has no hydration signal.4 The situation is apparently more complicated for unsonicated dispersions of PCcholesterol mixtures at 50 mol% cholesterol. This system does not rapidly equilibrate at -25”C, but the results again suggest that different types of water are present for a considerable period of time (Fig. 3). That the hydration signals are so significantly different for the unsonicated mixture (liposomes) versus the sonicated system (vesicles) at 50 mol% cholesterol clearly demonstrates that the unfrozen water serves as a sensitive probe of the different structures in these systems. Though the ideas proposed here are at present speculative, experiments are underway to try to obtain more definitive information regarding the nature of the environment of the unfrozen water we detect. We emphasize, however, that the appearance of the “second” hydration signal (as manifested by the first increase in line width, Fig. 2) occurs at a cholesterol to PC molar ratio at which a structure transition has been postulated to occur. Thus, regardless of the detailed explanation for our observations, it is clear that the hydration experiment is capable of detecting specific structure changes in the bilayer. It will be most interesting to determine if biological membranes which have high cholesterol contents also exhibit more complex hydration signals, and such studies are in progress. Our studies with SC suggest that the cholesterol (and presumably some SDS as well) is freely accessible to the bilayer and in fact can enter the vesicles and interact with PC. We draw this conclusion because we have noted that the addition of SC to PC vesicles causes changes in the hydration signal of these vesicles which are com1 Part of the hydration signal we detect at high cholesterol concentrations may be associated with water in the hydrocarbon region of the bilayer, but the present data contain no direct information on this question. Studies are in progress to try and resolve this point.
CHOLESTEROL
AND
PHOSPHATIDYL
pletely consistent with incorporation of considerable amounts of cholesterol into the vesicles. The fact that 30 mol% cholesterol could inhibit the SDS-induced transition in the vesicles suggests that the partition of SDS into the bilayer must decrease considerably on incorporation of cholesterol The fact that so much cholesterol can in fact be solubilized in SDS has significant implications for model studies which have focused on examining the interaction of cholesterol in water with detergents in order to understand the thermodynamics which govern the interactions of cholesterol with the bilayer (21). Biological membranes can contain as much as 1 mol of cholesterol per mole of phospholipid (l31, and it is possible to incorporate large amounts of cholesterol in aqueous dispersions of phospholipids if the materials are colyophilized. In fact, cholesterol can be “solubilized” in the bilayer (i.e., relatively clear solutions can be prepared) if the phospholipid and cholesterol are cosonicated (8). An interesting question of course is whether these systems are in true thermodynamic equilibrium or, instead, represent structures that are trapped kinetically in local free energy minima. For example, it is unlikely that the SC samples are thermodynamically stable, as we have noted that they slowly settle out of solution if stored at room temperature for more than a few days. However, the SC system represents a new method to solubilize large quantities of cholesterol in aqueous solution in structures which apparently maintain their integrity for sufficiently long periods of time that they can be used in meaningful experiments. It is possible that a use for the SC will be found in other
HYDRATION
89
investigations in which it is necessary alter the cholesterol content in a system.
to
CHOLINE
ACKNOWLEDGMENT We thank Dr. Herman Yeh of the National Institutes of Health for generously providing us with considerable periods of working time on his HA-100 nmr spectrophotometer. REFERENCES 1. NES, W. R. (1974) Lipids 9, 596. 2. JAIN, M. (1975) Current Topics Membr. 6, 1. 3. DEMEL, R. A., AND DE KRUYFF, B. (1976) Biochum Biophys. Actu 157. 109. 4. SMALL, D. M., AND SHIPLEY, G. G. (1974) Science 185, 222. H. (19741 Lipids 9, 645. 5. BROCKERHOFF, 6. ENGLEMAN, D. M., AND ROTHMAN, J. E. (1972) J. Biol. Chem. 247, 3694. 7. HINZ, H. J., AND STURTEVANT, J. M. (1972) J. Bid. Chem. 247, 3697. 8. NEWMAN, G. C., AND HUANG, C. (1975) Biochemistry 14, 3363. 9. HUMPHRIES, G. M. K., AND MCCONNELL, H. M. (1975) Proc. Nat. Acad. Sci. USA 72, 2483. 10. HUANG, C. (1969) Biochemistry 8, 344. 11. TAYLOR, R. P. (1976) Arch. B&hem. Biophys. 173, 596. 12. BAUER, J. D., ACKERMAN, P. G., AND TORO, G. (1960) Bray’s Clinical Laboratory Methods, p. 372, C. V. Mosby, St. Louis. 13a.KuNT2, I. D., BRASSFIELD, T. S., LAW, G. D., AND PURCELL, A. V. (1969) Science 163, 1329. 13b.Kuiwz, I. D. (1971) J. Amer. Chem. Sot. 93, 514. 14. HUANG, C., CHARLTON, J. P., SHYR, C. I., AND THOMPSON, T. E. (1970) Biochemistry 9, 3422. 15. OLDFIELD, E., AND CHAPMAN, D. (1971) Biochem. Biophys. Res. Commun. 43, 610. 16. HUANG, C. (1976) Nature iLondon 259, 242. 17. WOESSNER, D. E. (197415. Mugn. RFS. 16,483. 18. FUNG, B. M. (1974) Science 190, 800. 19. GERSHFELD, W. L. (1977)Biophys. J. 17, 94a. 20. PHILLIPS, M. C., AND FINER, E. G. (1974) Biochim. Biophys. Acta 356, 199. 21. GILBERT, D. B., AND REYNOLDS. J. A. (1976) Biochemistry 1.5. 71.