Effect of Cholesterol on Location of Organic Molecules in Lipid Bilayers

Chapter 4

Effect of Cholesterol on Location of Organic Molecules in Lipid Bilayers Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on February 24, 2016 | http://pubs.acs.org Publication Date: December 1, 1990 | doi: 10.1021/bk-1990-0447.ch004

An Infrared Spectroscopic Study 1

A. Muga and H. L. Casal

Division of Chemistry, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada

An aliphatic ketone (9-heptadecanone) and two keto derivatives of stearic acid (as potassium salts) containing a ketone functionality either at position 5 or 12 were incorporated into bilayers of the phospholipid 1,2-dihexadecyl-sn-glycero-3-phosphocholine. Infrared spectra of these mixtures were measured as a function of temperature and amount of added cholesterol. It was found that the presence of cholesterol in these bilayers induces changes in the location of the guest ketone and that these changes are dependent on both temperature and cholesterol concentration. It is also demonstrated that, in the gelphase,the presence of cholesterol induces larger intersheadgroup separations and, therefore, water penetrates deeper into the lipid bilayer.

Cholesterol is an important and abundant constituent of most eukaryotic membranes. In cell membranes, cholesterol constitutes up to 50 mol % of the lipid. The physiological significance of cholesterol and its effect on the properties of lipid bilayers have been studied thoroughly. Most studies have dealt with cholesterol-induced changes in the phase behavior of lipids (7). There have also been studies of the effect of cholesterol in the rate of water permeation through membranes (2) and in the effect of this steroid on the insertion of metabolites into membranes (3,4). Recently, it was shown that cholesterol modifies the pressure-induced expulsion of the local anesthetic tetracaine from lipid bilayers (5). It is accepted that cholesterol has a modulating effect on membrane properties; this modulating effect is summarized in the statement that cholesterol "fluidizes the gel phase of lipid bilayers while rigidifying their liquidcrystalline phase". 1

Current address: BP Research, 4440 Warrensville Center Road, Cleveland, O H 44128

0097-6156/91/0447-0056$06.00/0 © 1991 American Chemical Society In Fourier Transform Infrared Spectroscopy in Colloid and Interface Science; Scheuing, David R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on February 24, 2016 | http://pubs.acs.org Publication Date: December 1, 1990 | doi: 10.1021/bk-1990-0447.ch004

4.

MUGA AND CASAL

Effect of Cholesterol on Organic Molecules

In the work reported here, we examine the effect of cholesterol on the extent of hydrogen bonding between water and probe molecules. The probe molecules contain ketone functionalities and the extent of hydrogen bonding between these and water is determined directly from infrared spectroscopy. We have reported before (6-10) that this approach may be used in the characterization of different mesomorphic phases of surfactants and lipid bilayers. We find that the presence of cholesterol in bilayers of DHPC induces changes in the location of organic molecules within the bilayer and that these changes are dependent on both temperature and cholesterol concentration. We also find that, in the gel phase, the presence of cholesterol allows water to penetrate deeper into the lipid bilayer in accord with the recent findings of Hiff and Kevan (77). Materials and Methods l,2-Dihexadecyl-sn-glycero-3-phosphocholine (DHPC) was obtained from Fluka Chemical Corp. (Hauppauge, NY) and 9-heptadecanone (9HP) was from Aldrich (Milwaukee, WI); they were used as received. The preparation of 5-oxo potassium stéarate (5-oxo KSA) and 12-oxo potassium stéarate (12-oxo KSA) has been described before (10). DHPC bilayers containing cholesterol and 9HP were prepared by codissolving in CHCI3 the desired amounts and evaporating the solvent overnight under vacuum; the resulting lipid film was dispersed in excess D 0 and centrifuged. The lipid pellet was resuspended in D 0 to yield samples containing 0.15M DHPC. In all cases, the probe was present at concentrations of 0.01 M with the cholesterol concentration being 8,29 and 45 mol % with respect to DHPC. For all the samples, the supernatant obtained after centrifugation was analyzed, the absence of the probe molecules in the supernatant shows that they are incorporated in DHPC. This is expected since their solubility in aqueous solution is practically nil. DHPC bilayers containing cholesterol and S-oxo KSA or 12-oxo KSA were prepared as follows: DHPC and DHPC/cholesterol mixtures were dispersed in excess H 0 (1 mL) by gentle warming (50 *C); micelles of 5-oxo KSA or 12-oxo KSA were prepared in H 0 at 70 C. These micelles were added to the ready-made DHPC dispersion (containing the desired amount of cholesterol) and this mixture was subjected to three cycles of heating to 70 · C, vortexing while hot and cooling to 4 · C. The diluted dispersion thus obtained was centrifuged at 14000 rpm for 15 min. in an Eppendorf centrifuge. The resulting lipid pellet was removed from the supernatant, dispersed in excess water and lyophilized for 14 hours. The finely divided powder thus obtained was redispersed in D 0 to yield a final DHPC concentration of 0.15 M in samples containing different amounts of cholesterol. For the incorporation of 5-oxo KSA and 12-oxo KSA in DHPC bilayers no alcohols were used as solvents (72) because alcohols may be incorporated in the membrane interior and could interact with the C=0 groups of 5-oxo KSA and 12-oxo KSA. 2

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Infrared spectra at 2 cm" resolution were recorded on a Digilab FTS-60 Fouriertransform spectrometer equipped with a DTGS detector. Typically, 200 interferograms were coadded for each spectrum. The samples were held in cells of 50-/xm path lengthfittedwith C a F windows. For the hydration study (cf. Figure 2), a dry film was deposited on a CaF window and this assembled as one window of a closed chamber, the atmosphere of this chamber may be controlled by circulating either dry nitrogen or nitrogen containing D 0 . 2

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In Fourier Transform Infrared Spectroscopy in Colloid and Interface Science; Scheuing, David R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

58

FTIR SPECTROSCOPY IN COLLOID AND INTERFACE SCIENCE Results and Discussion

In this work, we examine the effect of cholesterol on the infrared spectra of DHPC bilayers containing a long-chain aliphatic ketone (9HP) or stearic acids (as potassium salts) with keto substituents at positions 5 or 12. By measuring the C = Ο stretching bands of these guest molecules it is possible to determine the extent of hydrogen bonding with the solvent (in this case D 0 ) . As shown recently (10) this yields location of the guest molecules in the bilayers. The approach is based on the sensitivity of the C = Ο stretching vibration of ketones to hydrogen bonding. The different species formed may be identified (13) as follows: a ketone C = Ο group not hydrogen bonded gives a C = Ο stretching band between 1718 and 1721 cm" ; the C = 0 band of a monosolvate formed between a C=0 group and water is between 1703 and 1706 cm' ; the corresponding C = 0 band for a disolvate appears between 1695 and 1699 cm" . Therefore, it is possible to detect hydrogen bonding and to distinguish among the different species formed. The lipid studied, DHPC, is an ether-linked phospholipid; it was chosen because it does not give ester C = 0 stretching bands which would interfere with the bands of the guest molecules. We recall here the thermal phase behavior of DHPC since we study the temperature-induced changes in the C = Ο stretching bands as a function of cholesterol concentration. This phospholipid has been shown to form subgel, gel, ripple, and liquidcrystalline phases (14), the gel-to-liquid-crystal phase transition temperature, T , is 43.4 * C and the pretransition is at 33.3 * C. Below the pretransition and under high hydration conditions, the DHPC gel phase is characterized by interdigitated alkyl chains. Calorimetric experiments (7) have shown that the presence of long-chain ketones in DHPC bilayers results in a slight depression of the gel-to-liquid crystal transition temperature and more marked changes in the thermodynamic properties of the pretransition. It was concluded that long chain ketones are incorporated in the DHPC bilayers.


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The temperature dependence of the C = Ο stretching bands in the infrared spectra of 9HP incorporated in DHPC bilayers, containing different amounts of cholesterol, is shown in Figure 1. For the sample containing no cholesterol (Figure 1 A), the C = Ο groups of 9HP give a band at 1719 cm" at temperatures below T ; upon heating above T , a band appears at -1700 cm" , evident as a broad shoulder. The same pattern is observed in the sample containing 8 mol % cholesterol (Figure IB). However, in this case the temperature at which the band at -1700 c m ' appears is shifted to lower values (between 35 and 39 * C) compared with the cholesterol-free sample (Figure 1 A). 1

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At 29 mol % cholesterol below T there are three C = Ο bands at 1698,1706 and 1719 c m ' (Figure 1C). In this case, heating above T results in broadening of the 1719 c m ' band and decrease in intensity of the other two bands; these remain present but only evident as an asymmetry to lower frequency on the strong 1719 c m ' band. In the spectra of the sample containing 45 mol % cholesterol (Figure ID) three bands also appear at the same frequencies as in Figure 1C. The difference is that the bands at 1698 and 1706 cm" (corresponding to hydrogen bonded C = 0 groups) are much more intense at temperatures below T . As in the case of the intermediate cholesterol concentration, heating above T induces significant decreases in their intensity such that they are only observed as a weak shoulder (Figure ID). c

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4. MUGA AND CASAL

1

Figure 1: Infrared spectra (1800 to 1600 cm" ) of 9-heptadecanone (9HP) incorporated in hydrated bilayers of DHPC containing 0 mol % (A), 8 mol % (Β), 29 mol % ( Q and 45 mol % cholesterol (D) at the indicated temperatures.


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FTIR SPECTROSCOPY IN COLLOID AND INTERFACE SCIENCE

Taken together, the spectra shown in Figure 1 indicate that hydrogen bonding to the C = Ο group of 9HP is dependent on cholesterol concentration and temperature. Incorporation of cholesterol into DHPC bilayers at temperatures below T induces hydrogen bonding to the C = Ο group of 9HP demonstrated by increasing intensity of the bands at 1698 and 1706 cm" . In the liquid-crystalline phase, i.e., at temperatures above T , the extent of hydrogen bonding is also dependent on cholesterol concentration; an increase in hydrogen bonding is observed for the samples with 0 and 8 mol % cholesterol (Figures 1A and IB) while the opposite is found for the samples with 29 and 45 mol % cholesterol (Figures 1C and ID). c

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The results presented so far indicate that the incorporation of cholesterol modifies the extent of hydrogen bonding between the C = Ο group of an aliphatic ketone (9HP) and a proton donor. The lipid molecule, DHPC, does not contain proton donors and therefore there is no possibility of lipid-induced hydrogen bonding to the guest ketone. Therefore, hydrogen bonding to this C= Ο group is either to the solvent (D 0) or to the OH group at position 3 of cholesterol. To shed light on the question of which molecule serves as the proton donor we performed a series of control experiments. The sample containing 45 mol % cholesterol, chosen as the one inducing the largest changes, was spread as a film on a CaF crystal and subjected to dehydration by circulating dry nitrogen at 28 * C. The spectra recorded during dehydration are shown in Figure 2A. Clearly, as dehydration proceeds the intensity of the bands due to hydrogen bonded C = Ο groups (at 1698 and 1706 cm" ) decreases. In the absence of D 0 there are no bands due to hydrogen bonded C = Ο groups. Furthermore, as shown in Figure 2B these changes are completely reversible; upon exposure of the film to an atmosphere containing D 0 , the bands at 1698 and 1706 c m ' reappear. Also, in the infrared spectrum of 9HP dissolved in η-octane there is only one C = Ο stretching band at 1720 cm' , and addition of cholesterol such that its concentration is twelve times that of 9HP does not induce changes in the C = Ο stretching band of 9HP. From these control experiments, we conclude that cholesterol does not form hydrogen bonds directly with theC = 0 group of 9HP. We therefore interpret the effects observed on the C = Ο stretching bands of 9HP (Figure 1) as arising from 9HP - solvent (D 0) contact. 2

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An increase in hydrogen bonding to the 9HP molecule, evidenced from the bands at 1699 and 1706 cm" , may occur due to either water penetration into the lipid bilayer or to translocation of the guest ketone to a region where contact with the bulk water phase is favored. In order to distinguish between these two possibilities we studied systems containing C = 0 groups where translocation is either not possible or at least greatly diminished. This was achieved by incorporating two derivatives of potassium stéarate, namely, 5-oxo potassium stéarate (5-oxo KSA) and 12-oxo potassium stéarate (12-oxo KSA). These molecules contain a carboxylate "head group" and are incorporated in lipid bilayers such that their COO- group is located near the lipid polar group and their chains embedded in the lipid hydrocarbon core (1 7, 18). Thus, the C = Ο groups at position 5 or 12 are 'fixed' allowing a relationship between membrane depth and solvent accessibility to be established. 1



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Figure 6: Infrared difference spectra in the methylene scissoring region of DHPC : 9HP (12:1 molar ratio) bilayers containing (A) 0 mol % cholesterol (B) 8 mol %, (C) 29 mol %, and (D) 45 mol % cholesterol. The corresponding temperatures are the higher values of the -4.0 · C intervals in which subtractions have been performed.


MUGA AND CASAL


temperatures above 28 * C. This coincides with the temperature at which the parameter I^/If decreases for this sample (Figure 5A). Thus, there is a direct correlation between interchain vibrational coupling and I^/If. The same relationship is observed in the sample containing 29 mol %, showing that the ketone may translocate only when the alkyl chains are motionally disordered such that there is no vibrational coupling.


Conclusion From the data presented here several conclusions may be reached regarding the effect of cholesterol on lipid bilayers. It is shown that, even if the presence of cholesterol in bilayers serves to moderate temperature-induced changes, its ability to affect the location of solubilized molecules is highly temperature dependent. We have also shown, in accord with previous work (ll) that the presence of cholesterol in the gel phase results in a larger separation between the lipid polar groups and this in turn allows water to penetrate into the lipid hydrophobic core. This penetration is apparent only at short distances from the lipid-water interface. t

Recent experiments have shown that the non-specific, physical chemical interactions between small hydrophobic, water-insoluble molecules and the hydrocarbon chains of lipid membranes are important determinants of the rate at which these molecules enter cells and are metabolized (3,34). Cholesterol has the capability of modifying these interactions and also increases the affinity of vesicle surfaces for amphiphillic molecules (4) separating the lipid polar groups (55). Cholesterol can modify both the hydrophobic attraction between lipid hydrocarbon chains and electrostatic interactions between lipid polar groups. The influence it has on the location of 9HP reflects this dual effect At low temperature, the "spacer" effect of cholesterol allows the ketone to gain access directly to the lipid-water interface. At high temperatures, a more disordered hydrocarbon core favors the solubilization of the guest molecule. This effect of cholesterol on the location of guest molecules has been cited for chlorophyll a (36) and tetracaine (5) both molecules with specific biological function. In the present work we have been able to observe this effect over the physiological ranges of temperature and cholesterol concentration. Future experiments with other molecules would clarify if this property of cholesterol is generally applicable to other systems and, furthermore, if it extends to interactions between two molecules solubilized in a membrane. Legend of Symbols DHPC: l,2-dihexadecyl-sn-glycero-3-phosphocholine; KSA: potassium stéarate; 5-oxo KSA: 5oxo potassium stéarate; 12-oxo KSA: 12-oxo potassium stéarate; 9HP:9-heptadecanone; T : temperature of the gel-to-liquid crystal phase transition of DHPC bilayers; 43.4ÎC c


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Effect of Cholesterol on Location of Organic Molecules in Lipid Bilayers

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