The technique of fluorescence recovery after photobleaching is used to perform an extensive study of the lateral diffusion of a phospholipid probe in the binary mixture dimyristoylphosphatidylcholine/cholesterol, above the melting temperature of the phospholipid. In the regions of the phase diagram where a single liquid phase exists, diffusion can be quantitatively described by free volume theory, using a modified Macedo-Litovitz hybrid equation. In the liquid-liquid immiscibility region, the temperature dependence of the diffusion coefficient is in excellent agreement with current theories of generalized diffusivities in composite two-phase media. A consistent interpretation of the diffusion data can be provided based essentially on the idea that the primary effect of cholesterol addition to the bilayer is to occupy free volume. On this basis, a general interpretation of the phase behavior of this mixture is also proposed.

The influence of cholesterol on the phase behavior of glycerophospholipids and sphingomyelins was investigated by spin-label electron spin resonance (ESR) spectroscopy. 4-(4,4-Dimethyl-3-oxy-2-tridecyl-2-oxazolidinyl)butanoic acid (5-SASL) and 1-stearoyl-2-[4-(4,4-dimethyl-3-oxy-2-tridecyl-2-oxazolidinyl)butanoy l]-sn- glycero-3-phosphocholine (5-PCSL) spin-labels were employed for this purpose. The outer hyperfine splitting constants, Amax, measured from the spin-label ESR spectra as a function of temperature were taken as empirical indicators of cholesterol-induced changes in the acyl chain motions in the fluid state. The Amax values of 5-PCSL exhibit a triphasic dependence on the concentration of cholesterol for phosphatidylcholines and bovine brain sphingomyelin. We interpret this dependence as reflecting the existence of liquid-disordered, ld, liquid-ordered, lo, and coexistence regions, ld + lo. The phase boundary between the ld and the two-phase region and the boundary between the lo and the two-phase region in the phosphatidylcholine-cholesterol systems coalesce at temperatures 25-33 degrees C above the main-chain melting transition temperature of the cholesterol-free phosphatidylcholine bilayers. In the case of bovine brain sphingomyelin, the ld-lo phase coalescence occurs about 47 degrees C above the melting temperature of the pure sphingomyelin. The selectivity of interaction of cholesterol with glycerophospholipids of varying headgroup charge was studied by comparing the cholesterol-induced changes in the Amax values of derivatives of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine spin-labeled at the fifth position of the sn-2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)

The fluorescence deplorarization associated with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene is used to monitor changes in fluidity accompanying the gel-liquid crystalline phase transition in phosphatidylcholine dispersions. In this way, the parameters of the phase transition are determined for both large, multilamellar liposomes and small, single-lamellar vesicles composed of three mixtures of phosphatidycholines: dimyristoyl-dipalmitoyl, dimyristoyl-distearoyl, and dioleyl-dipalmitoyl. Phase diagrams for these mixed-lipid vesicles are constructed by plotting the delimiting temperatures of the phase transition vs. the lipid compostion of the vesicle. The phase diagrams are interpreted to suggest that the miscibilities of the lipids studied are markedly different in small, single-lamellar vesicles and large multilamellar liposomes. These results are discussed in terms of the effects of high curvature on the structure of biological membranes.

The fluid-phase behavior of binary mixtures of cholesterol with phosphatidyichoilnes is investigated using magnetic resonance methods. Phospholipid biradicals provide the electron spin resonance spectroscopic resolution of two Immiscible fluid phases in the dlpalmitoylphosphatidylcholinecholesterol system. Isotropic chemical shifts of the phospholipid carbonyl carbons in binary mixtr with cholesterol measured using solid-state high-resolution nuclear magnetic resonance methods furnish evidence for a putative hydrogen bond between the 3P-hydroxyl of cholesterol and the sn-2 carbonyl of the phospholipid. The location in the bilayer of cholesterol in the two fluid phases is determined by measuring spin label-enhanced spin-lattice relaxation rates of the 13C nuclei of both the phospholipid and cholesterol molecules. These results suggest, in a time-averaged sense, that in the cholesterol-poor fluid phase the cholesterol molecule essentially spans the bilayer, whereas in the cholesterol-rich fluid phase the molecule is present in both monolayers of the bilayer. Fig. 1B. The phase diagrams for mixtures of cholesterol with other phospholipids, differing in both the acyl chain and the headgroup composition, have the same overall shape and consist of three one-phase regions and two two-phase regions as shown in Fig. 1B (8). The phases are denoted solid-ordered (so), liquid-ordered (4o), and liquiddisordered (4d). In the two-phase regions there is coexistence of the so and 4o phases or of the two fluid phases, 4 and eo.The occurrence of fluid-phase immiscibility has been suggested by a triphasic dependence on cholesterol concentration of the hyperfine interactions of phospholipid spin labels incorporated into cholesterol-containing phospholipid bilay- ers (6, 8).In this communication, we present magnetic resonance evidence further substantiating cholesterol-induced fluidphase immiscibility and suggesting the existence of hydrogen bonding between cholesterol and phospholipid molecules.We conclude by proposing a bilayer model specifying the time-averaged location of cholesterol in the Ed and 4o phases.MATERIALS AND METHODS Materials. Unlabeled phospholipids were purchased from Avanti Polar Lipids (Birmingham, AL). Phospholipid monoradicals and 'IC-enriched phospholipids were synthesized as described (8). The biradical was synthesized by acylation of sn-glycero-3-phosphocholine with the corresponding fatty acid spin labels (11).ESR Spectroscopy. The ESR spectra were recorded on an X-band Varian E-line spectrometer. The sample preparation protocols and the details of instrumentation are as described earlier (8). The monoradical spin label concentrations used were 1 mol % unless otherwise specified. The biradical concentrations were 0.5 mol %. NMR Spectroscopy. 13C NMR spectra were recorded at 90 MHz on a Nicolet NT-360B spectrometer equipped with a variable-temperature magic-angle spinning probe from Doty Scientific (Columbia, SC). The samples contained in zirconium rotors were spun at speeds between 1 and 3 kHz, whi...

The results of a calorimetric and fluorescent probe study of the thermotropic behavior of various types of dispersions of dipalmitolphosphatidylcholine bilayer vesicles are reported. Bangham-type, multilamellar vesicles exhibit tow distinct phase transitions at 34.6 and 41.2 degrees C. On the other hand, single-lamellar spherical vesicles appear to exhibit a single transition at 37 degrees C. The single-lamellar vesicles are thermodynamically unstable below 27 degrees C and slowly transform into a multilamellar structure with a single phase transition of 41.2 degrees C. These transformed structures resemble, but are not identical with, Bangham-type vesicles. An experimentally testable thermodynamic and kinetic model based upon these results is developed.

The fluorescence depolarization associated with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene is used to monitor the changes in fluidity accompanying the gel-liquid crystalline phase transition in synthetic phosphatidycholine dispersions. The parameters of the phase transition are determined for both large, multilamellar liposomes and small, single-lamellar vesicles. These parameters are compared with those obtained using other techniques. In addition, the data are interpreted in terms of two limiting molecular models, which in turn offer insight into the structural differences between multilammelar liposomes and small vesicles.

Examination of the thermotropic behavior of aqueous dispersions of dipalmitoylphosphatidylcholine-cholesterol mixtures by high-sensitivity scanning calorimetry has revealed that the phospholipid gel to liquid-crystalline phase transition consists of two components. One, a relatively sharp transition centered at 39.6-40.7 degrees C, exhibits a transition enthalpy change which decreases linearly with increasing cholesterol content, approaching zero at a cholesterol content of about 25 mol %. The other, a broad, lower intensity transition centered at approximately 41.5 degrees C for cholesterol concentrations of 20 mol %, displays an enthalpy change which is maximal at about 20-25 mol % cholesterol and which decreases as the cholesterol content decreases to zero or increases above 25 mol %. The origin of these two transitions is discussed in terms of a separation of these lipid mixtures into cholesterol-rich and cholesterol-poor domains.

The effect of cholesterol on the acyl chain order of three glycerophosphocholines with 14, 16, and 18 carbons per acyl chain, namely, di(14:0)PC, di(16:0)PC, and di(18:0)PC, above the gel to liquid-crystalline phase transition temperature was investigated by using 2H nuclear magnetic resonance spectroscopy. Average acyl chain lengths were calculated from the segmental order parameters (Smol) for the sn-1 and the sn-2 chains in the absence of cholesterol and at 3:1, 2:1, and 1:1 mole ratios of phospholipid-cholesterol. The three binary mixtures of cholesterol with phosphatidylcholines are in the liquid-ordered (lo) phase. For all the three phosphatidylcholine-cholesterol systems, the distance from the carbonyl groups to the terminal methyl groups is shorter than the length of the cholesterol molecule. A molecular model for the lo phase consistent with these observations has in a statistical sense a part of each cholesterol molecule in one monolayer extending into the other monolayer. This results in a packing arrangement akin to that in interdigitated systems. On the basis of the effect of cholesterol on phospholipid acyl chain orientational order, it is suggested that the liquid-disordered (ld) phase at low cholesterol concentrations corresponds to a packing mode in which the cholesterol molecule spans the entire transbilayer hydrophobic region. A molecular mechanism is proposed in which increasing the concentration of cholesterol has the effect of stretching the acyl chains of phospholipids by increasing the population of trans conformers up to a stage where the hydrophobic length is considerably longer than the cholesterol molecule. Beyond this concentration, the partially interdigitated phase forms.(ABSTRACT TRUNCATED AT 250 WORDS)