Robert Bittman and Lea Blau QueensCollege of CUNY Flushing, New yo,k 11367
Kinetics of Solute Permeability in Phospholipid Vesicles
A series of experiments for undergraduate laboratory courses concerning the isolation, separation, detection, and determination of fatty acid composition of egg-yolk phosoholioids has been described recentlv in this Journal (I. >). students in the biochemistry laboratory course here have oerformed an exneriment involving - the kinetics of passive diffusion of solutes across artificial membranes formed by dispersing phospholipids in aqueous medium. This experiment may be combined with the series described earlier. thus extending the laboratorv scheme on the chemistry of egg-yolk lipid;. The experiment may also be performed as a special project (independent of a series on lipid chemistry), introducing students to the topic of solute permeation through biological membranes. Introduction to Trans-Membrane Solute Transport
The mechanisms of solute permeation through cell membranes and the relation of membrane com~onentsto membrane-related functions such as transport are subjects of intense current interest. Cell membranes contain specific proteins that facilitate the uptake and transport of certain oolar solutes across the membrane hv binding solute molecules and shielding their polar groups from the hydrocarbon-like membrane interior.' Trans-membrane diffusion allows solutes to enter and leave the cell and thus makes possible the regulation of the internal environment, the maintenance of metabolism, and, in excitable membranes (as in nerve conduction), the generation of ion gradients. Thus, the general ability of cells to function is dependent on solute permeation through biological membranes. Small solutes can also enter or leave the cell by the mechanism of simple diffusion. The rate of simple diffusion of nonelectrolytes across cell membranes is believed to be governed by partitioning into the membrane (which depends on equilibrium parameters of partition between solute and the membrane) and diffusion into the membrane interior (3, 4).
Most of the lipid component of biological membranes is believed to be present in the form of a bilayer. The lipid bilayer is thought to provide a matrix, within which a t least some interspersed proteins and lipids are capable of rapid diffusion in the plane of the membrane (5-7).Studies of permeation in lipid bilayers show that bilayer membranes are permeable to water and small, neutral nonpolar molecules and that the diffusion of solutes across hilayers depends on the structure and dynamics of the lipid molecules comprising the hilayer, e.g., on the degree of molecular packing and thermal mobility of the hydrocarbon chains
'Recent reviews of uptake of salutes via transport systems are available. See, for example, Lehninger, A. L., "Biochemistry," 2nd Ed., Worth Publishers, Ine., New York, 1975, chap. 28; Stryer, L., "Biochemistry," W. H. Freeman & Ca., San Francisco, 1975, chap. 32; Christensen, H. N., "Biological Transport," Benjamin, Menla Park, Calif., 1974; Bronner, F., and Kleinzeller, A. (Editors),"Current Topics in Membranes and Transport,'' Vol. 5, Academic Press, New York, 1974; Epstein, W., in "Biochemistry of Cell Walls and Membranes-Biochemistry Series One, Vol. 2, MTP International Review of Science," (Editor: Fox, C. F.), University Park Press, Baltimore, 1975, pp. 249-278.
and on the charge of the polar head group of the phospholipid (8, 9). Membrane permeability to solutes is characterized by a permeability coefficient, P
*
Ac
where 1IA dnldt is the molar flux of solute across unit surface area of the membrane, and Ac is the difference in osmolarity of the solute in the solutions across the membrane. P depends on the solute diffusion coefficient, the memhrane thickness, and on the solute concentration in the memhrane relative to the aqUQOUSmedium ( 1 0 ) . Many cells behave as osmometers. When exposed to a hypotonic medium (a solution that has a lower osmotic pressure than the cytoplasm of the cell), they swell, i.e., water moves into the cell to restore osmotic equilibrium between the cell and the external medium. A decrease in volume (shrinking) occurs when cells are exposed to a bypertonic medium. Osmoticallv induced volume changes are conveniently measured by light-scattering or turbidity changes. Turbidity changes can be measured on a conventional spectrophotometer, using wavelengths where absorption arises from light scattering only (e.g., A > 450 nm). Osmotically Induced Volume Changes of Phospholipid Vesicles
Multiwalled phospholipid vesicles, or liposomes, form spontaneously when phospholipids are dispersed in aqueous solution. Electron microscopy shows that liposomes consist of multiconcentric shells of lipid bilayer membranes (8, 11). A volume of aqueous solution is contained between each single bilayer membrane. Liposomes are a model system for the studv of cell oermeabilitv because thev act as osmometers and their large surface areas make them well suited for permeability studies. The permeability of solutes across liposomal bilayers can be monitored spectrophotometricallv. I t is found that the chanee in lioosome volume. AV, is proportional to the change in the reciprocal of the absorbance, A(llA), where absorbance arises from light scattering (12). Osmotically induced shrinking of liposomes is accom~aniedbv an increase in absorbance. and osmotically indiced swekng by a decrease in absorbance (12). In the ex~erimentdescribed here. the initial rate of liposome swelling, dVldt, arising from glycerol or thiourea entry is determined from the initial rate of the reciprocal of the absorbance change, d(1lA)ldt. I t should be noted that study of osmotic volume changes in liposomes is complicated by the presence of multiple bilayers and marked heterogeneity with respect to size and surface area. Since size distribution varies from one preparation of liposomes to another, and since d(l/A)/dt decreases with increasing lipid concentration (131, comparisons of the initial rates of solute permeation into cholesterol-free and cholesterol-containing liposomes may be made by measuring d(l/A)/dt a t a given total lipid concentration and similar initial absorbance.
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Role of Cholesterol insolute Permeation
Cholesterol comprises about 40 mole % of the erythrocyte and myelin membrane lipids and is a principal lipid Volume 53, Number 4, April 1976 / 259
component of most plasma memhranes of mammalian cells. Cholesterol is readily incorporated into phospholipid hilayers. Studies with phospholipid-cholesterol bilayer membranes show that cholesterol controls the state of fluidity of the fatty-acyl chains of phospholipids. When phospholipids are present in the liquid-crystalline (or mesomorphic) state, cholesterol restricts the number of possible conformations in the fatty-acyl chains, preventing the bilayer from being too mobile. Decreased motional freedom of many of the methylene groups of the hydrocarbon chains is accompanied by reduction in the rates of solute transport and inhibition of the activity of memhrane-hound enzymes (14. . . 15). . When the ohosoholioid . . . is oresent in the pel ohase (below the liquid-crystalline transition temperature of the o h o s ~ h o l i ~ i dcholesterol ), ~roducesadditional fluiditv in the bilayei (14, 16). ~ h u s , - a thigh cholesterol ~ o n c e ~ t r a tions. most of the fattv-acvl methvlene erouos acauire a stateof fluidity intermkdiaie between that ofthe &id gel ohase (where the chains are fully extended) and that of the more mobile liquid-crystalline phase (where the chains are not fully extended and gauche conformations exist). Bilayer membranes prepared from mixtures of saturated and unsaturated phospholipids in the absence of cholesterol have regions of both solid- and liquid-like domains over a relatively broad temperature ranee (17). Such heteroneneous lipid patches ard not observed when high concenGations of cholesterol are present (18). A consequence of the ability of cholesterol to cause formation of a hbmogeneous, continuous bilayer even when many classes of phospholipids are present (as in natural membranes) is that the permeability barrier of a cholesterol-rich memhrane will be relatively uniform, and the influence of variation of polar head group and fatty-acyl chains of the phospholipids, or of temperature would he only minor. (In fact, there is evidence that cells may alter their memhrane lipid composition so that a constant ohvsical state of the memhrane is maintained (e.g., (IS)). +his change in biological lipid comoosition is achieved bv chanees - in hiosvnthetic h .i d .oathways.) In cholesterol-free memhranes, the coexistence of solid- and liquid-like domains may permit greater compressibility and offer the possibility for rapid insertion of newly synthesized lipid and memhrane protein molecules (20). Structure of Egg-Yolk Phosphatidylcholine The structural formula of ~hos~hatidvlcholine (PC or
" .
i l
RCOCH
I
II
CH,OPOCH,CH~CH,),,
I
0Egg PC, as well as other lipids isolated from natural sources, is a mixture of a number of molecules containing different fatty-acyl chains. The fatty acids esterified at the 1 position are predominantly palmitic (16:O) and stearic (18:O); a t the 2 position, the unsaturated fatty acids oleic (18:l)and linoleic (18:2) predominate, and small amounts of acyl chains containing 20 and 22 carbon atoms with various degrees of unsaturation also occur (21, 22). The presence of unsaturated acyl chains causes hilayer memhranes prepared from egg PC to be in the liquid-crystalline state at room temperature (the gel to liquid-crystalline phase transition temperature of egg PC is approximately -5"). Egg PC has been used frequently to form lipid hilayer membranes because its bilayers resemble the predominant 260 / Journal of Chemical Education
structural feature of most biological memhranes at room temperature, namely a fluid-like lipid bilayer. In the experiment we describe here, a synthetic phospholipid containing myristoyl chains (140) a t the 1 and 2 positions is used in addition to egg PC. The gel to liquid-crystalline phase transition of dimyristoyl PC occurs near room temperature; therefore, the effects of cholesterol on both phospholipid phases can be investigated. Furthermore, because of its saturated hydrocarbon chains, dimyristoyl PC is not readily susceptible to oxidation. Experimental Preparation of Liposomes2 PC, cholesterol, and dicetyl phosphoric acid were obtained from commercial sources (e.g., Sigma Chemical Co., Calhiochemical Corp.). Cholesterol was recrystallized from methanol. The purities of PC and cholesterol preparations were determined by thin-layer chromatography ie.g., (2 1, (141, i23)L3Stock solutions of PC (20 mM), cholesterol (15 mM), and dicetyl phosphoric acid (1 mM) were prepared in chloroform. (A molecular weight of 800 was assumed for PC.) Liposomes were prepared from PC without cholesterol, and with PC:eholesterol molar ratios of 2 : l and 12. Liposomes were prepared by pipetting into 20-ml vials the desired amounts from the stock solutions, chloroform was removed under nitrogen, the lipids were evaporated t o dryness under vacuum, and the thin lipid film was suspended in a solution of 0.03 M KC1 by agitation on a Vortex mixer for 60 s. One glass bead (3 mm diameter) was added per 2 ml of suspending KC1 solution prior to mixing. Egg PC was dispersed a t roam temperature and dimyristoyl PC was dispersed at approximately BODC.The concentration of lipids in liposomes was 8 mM. All liposomes contained 4 mole % of dicetyl phosphoric acid, which was added to confer a net charge on the bilayers. Each student requires -2 ml of each liposome preparation.
Apparatus
.
Soeetronie t .w. e 20 soectrometers are adeouate.. althoueh more rxpensive s p e ~ t n ~ p h o t o m e t rwteuld ri enhnnce the .;en.iriwty and acrumry. For display of the uutput
