Interaction of the local anaesthetic heptacaine with dipalmitoylphosphatidylcholine liposomes: a densimetric study.

Interaction of the local anaesthetic heptacaine, monohydrochloride of [2-(heptyloxy)-phenyl]-2-(1-piperidinyl)-ethyl ester of carbamic acid, with multilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes in aqueous solution with high excess of water has been studied by means of density measurements in the scanning regime in the main phase transition region. The anaesthetic decreased the temperature of main phase transition. The molar partition coefficients of heptacaine between aqueous phase, liquid crystal and gel phases of DPPC have been determined from a combination of phase transition data obtained by densimetry with a DPPC/heptacaine phase diagram published in the literature. The saturation of heptacaine concentration in liposomes has been observed at higher total amount of anaesthetic. The partial specific volume of heptacaine located in DPPC bilayers is slightly lower than in the aqueous phase.     

Interaction of fluorinated ether anesthetics with artificial membranes.

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T1) measurements are consistent with substantial immobilization of the lipid-bound anesethetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

A solid-solution theory of anesthetic interaction with lipid membranes: temperature span of the main phase transition

Anesthetics (or any other small additives) depress the temperature of the main phase transition of phospholipid bilayers. Certain anesthetics widen the temperature span of the transition, whereas others do not. The widening in a first-order phase transition is intriguing. In this report, the effects of additive molecules on the temperature and its span were explained by the solid-solution theory. By assuming coexistence of the liquid-crystal and solid-gel phases of lipid membranes at phase transition, the phase boundary is determined from the distribution of anesthetic molecules between the liquid-crystal membrane versus water and between the solid-gel membrane versus water. The theory shows that when the lipid concentration is large or when the lipid solubility of the drug is large, the width of the transition temperature increases, and vice versa. Highly lipid-soluble molecules, such as long-chain alkanols and volatile anesthetics, increase the width of the transition temperature when the lipid:water ratio is large, whereas highly water-soluble molecules, such as methanol and ethanol, do not. The aqueous phase serves as the reservoir for anesthetics. Depletion of the additive molecules from the aqueous phase is the cause of the widening. When the reservoir capacity is large, the temperature width does not increase. The theory also predicts asymmetry of the specific heat profile at the transition.     

Interaction of fluorinated ether anesthetics with artificial membranes

Fluorine-19 nuclear magnetic resonance spectroscopy is applied to the study of the environment of dipalmitoyl phosphatidylcholine-bound fluorinated ether anesthetics (enflurane, fluoroxene and methoxyflurane) both below and above the lipid gel to liquid crystal phase transition temperature. Line widths and spin-lattice relaxation time (T₁) measurements are consistent with substantial immobilization of the lipid-bound anesthetic molecules. Heating anesthetic/lipid mixtures above the lipid transition temperature leads to narrowing of the lipid-bound anesthetic fluorine resonances accompanied by little or no change in anesthetic fluorine-19 chemical shifts, suggesting that although the mobility of the bound anesthetic increases at the higher temperature, the nature of the anesthetic-lipid interaction changes little as a result of this phase change. Differential scanning calorimetric studies of the effects of these anesthetics on the phase transition behavior of the phospholipid indicate that the regions of the bilayer in which volatile anesthetics partition at lower concentrations are different from the regions in which they partition at higher concentrations.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

Pressure effects on the structure and phase behavior of DMPC-gramicidin lipid bilayers: a synchrotron SAXS and 2H-NMR spectroscopy study

The influence of Gramicidin D (GD) incorporation on the structure and phase behavior of aqueous dispersions of DMPC lipid bilayers has been studied using small-angle x-ray scattering (SAXS) and (2)H-NMR spectroscopy. The experiments covered a temperature range from -10 degrees C to 60 degrees C and a pressure range of 0.001-4 kbar. Pressure was used to be able to tune the lipid bilayer conformational order and phase state and because high pressure is an important feature of certain natural biotopes. The data show that, depending on the GD concentration, the structure of the temperature- and pressure-dependent lipid phases is significantly altered by the insertion of the polypeptide, and a p,T-phase diagram could be obtained for intermediate GD concentrations. Upon gramicidin insertion, a rather narrow fluid-gel coexistence regions is formed. Two gel phases are induced which are different from those of the pure lipid bilayer system and which separate at low temperatures/high pressures. For both the temperature- and pressure-induced fluid-to-gel transition, a similar pseudocritical transitional behavior is observed, which is even more pronounced upon incorporation of the peptide.     

Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases.

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, Re. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the 'equilibrium partition model', the dependence of the 'solubilizing detergent concentration' on the lipid concentration intersects with the lipid axis at -1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical ('solubilizing') effective ratio depends upon the nature of detergent less than any of these two factors.     

Correlations for the partition behavior of proteins in aqueous two-phase systems: effect of overall protein concentration

The effect of protein concentration in partitioning in PEG/salt aqueous two-phase systems has been investigated. PEG 4000/phosphate systems in the presence of 0% w/w and 8.8% w/w NaCl have been evaluated using amyloglucosidase, subtilisin, and trypsin inhibitor. Also, a PEG 4000/phosphate system with 3% w/w NaCl was used for alpha-amylase. The concentration of the protein in each of the phases affected its partition behavior. The pattern for the individual proteins was dependent on their physicochemical properties. In the top phase, maximum protein concentration was determined mainly by a steric exclusion effect of PEG, and hydrophobic interaction between PEG and proteins. In the bottom phase, maximum concentration was determined mainly by a salting-out effect of the salts present. As the ionic strength was increased in the systems the concentration in the top phase increased for all proteins. In the bottom phase an increase in ionic strength increased the salting-out effect. Amyloglucosidase had a very low maximum concentration in the PEG-rich top phase which was probably due to its large size (steric exclusion) and low hydrophobicity, and a high concentration in the salt-rich bottom phase due to its high hydrophilicity. In the case of subtilisin and trypsin inhibitor, their high concentrations in the top phase were due to their hydrophobic nature (hydrophobic interaction with PEG) and small size (negligible steric exclusion). The maximum concentration in the bottom phase for trypsin inhibitor was lower than that of subtilisin which was probably due to its higher hydrophobicity and, hence, a stronger salting-out effect. The protein concentration in each of the two phases was correlated with a "saturation"-type equation. The partition coefficient could be satisfactorily predicted, as a function of the overall protein concentration, by the ratio between the "saturation" equations of the two individual phases. Better correlations were obtained when an empirical sigmoidal Boltzmann equation was fitted to the data, since in virtually all cases the partition coefficient is constant at low protein concentration (true partitioning) and changes to a different constant value at a high overall protein concentration. (c) 1996 John Wiley & Sons, Inc.     

Fundamental studies of biomolecule partitioning in aqueous two-phase systems

Phase diagram data at 4 degrees C was determined for the aqueous two-phase systems composed of polyethylene glycol, dextran, and water. The Flory-Huggins theory of polymer thermodynamics was used to correlate partitioning of biomolecules in these aqueous two-phase systems resulting in a simple linear relationship between the natural logarithm of the partition coefficient and the concentration of polymers in the two phases. This relationship was verified by partitioning a series of dipeptides which differ from one another by the addition of a CH(2) group on the c-terminal amino acid residue and by utilizing a set of low-molecular-weight proteins. The slope of the line could be expressed in terms of the interactions of the biomolecule with the phase forming polymers and water. The main result for the dipeptides was that knowledge of the partition coefficient in any of the PEG/dextran/water systems, regardless of polymer molecular weight, enabled prediction of the coefficient in all of the systems. The dipeptides were also used for determination of the Gibbs free energy of transfer of a CH(2) group between the phases. This quantity was correlated with polymer concentration, thus establishing a hydrophobicity profile for the PEG/ dextran/water systems. The methodology for predicting dipeptide partition coefficients was extended to proteins, where it was found that low-molecular-weight proteins gave a linear relationship with the tie line compositions of a phase diagram.     

The phase behavior of mixed aqueous dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phases and transition sequences for aqueous dispersions of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) have been studied by differential scanning calorimetry, dynamic x-ray diffraction, freeze-fracture electron microscopy, 31P-nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The results have been used to construct a dynamic phase diagram of the binary mixture as a function of temperature over the range 20 degrees-90 degrees C. It is concluded that DPPC and 1,2-DPG form two complexes in the gel phase, the first one with a DPPC/1,2-DPG molar ratio of 55:45 and the second one at a molar ratio of approximately 1:2, defining three different regions in the phase diagram. Two eutectic points are postulated to occur: one at a very low 1,2-DPG concentration and the other at a 1,2-DPG concentration slightly higher than 66 mol%. At temperatures higher than the transition temperature, lamellar phases were predominant at low 1,2-DPG concentrations, but nonlamellar phases were found to be predominant at high proportions of 1,2-DPG. A very important aspect of these DPPC/1,2-DPG mixtures was that, in the gel phase, they showed a ripple structure, as seen by freeze-fracture electron microscopy and consistent with the high lamellar repeat spacings seen by x-ray diffraction. Ripple phase characteristics were also found in the fluid lamellar phases occurring at concentrations up to 35.6 mol% of 1,2-DPG. Evidence was obtained by Fourier transform infrared spectroscopy of the dehydration of the lipid-water interface induced by the presence of 1,2-DPG. The biological significance of the presence of diacylglycerol in membrane lipid domains is discussed.     

Hydroxypropyl cellulose/poly(ethylene glycol)-co-poly(propylene glycol) aqueous two-phase systems: system characterization and partition of cells and proteins.

Novel aqueous polymeric two-phase systems are described. These systems are formed by mixing hydroxypropyl cellulose (molecular mass 100,000, trade name Klucel L) with poly(ethylene glycol)-co-poly(propylene glycol) copolymer [molecular mass 6,500, poly(propylene glycol) content 50% w/w, trade name Pluronic P105], in a saline buffer. The phase diagram was measured and the interfacial tensions, phase separation times, and lower phase viscosities of three phase systems having constant Pluronic P105 concentration but varying in Klucel L concentration were determined. The partition behavior of a representative cell, bacterium, and protein and the affinity ligand-mediated alteration in the partition behavior of a protein from a yeast extract protein mixture were also characterized. The results suggest that Klucel L/Pluronic P105 phase systems may be cost-effective substitutes for, or complements to, existing aqueous polymeric phase systems. The physical characterization and representative partition data reported here should facilitate application of these new systems.     

Partition of chlorpromazine into lipid bilayer membranes: the effect of membrane structure and composition

Partition coefficients, kp, of chlorpromazine between the aqueous phase and lipid bilayer vesicles were determined as function of drug concentration, lipid chain length, cholesterol content and temperature encompassing the range of the lipid phase transition. Radioactivity and absorption measurements were performed to determine the kp values. Up to a concentration of 3 . 10(-5) M, the partition coefficient is independent of chlorpromazine concentration, whereas it decreases drastically at higher chlorpromazine concentrations, at which membrane lysis is observed. Membrane structure is not disturbed at less than 3 . 10(-5) M chlorpromazine, as was concluded from electron paramagnetic resonance studies measuring TEMPO partitioning and order degree. However, the lipid phase-transition temperature decreases and is broadened at higher chlorpromazine concentrations. From fluorescence measurements, we conclude the formation of chlorpromazine micelles at concentrations higher than 5 . 10(-5) M in chlorpromazine in the absence of lipids and the formation of mixed micelles in the presence of lipids. The effect of lipid chain length on kp values was investigated. The partition coefficient decreases from 8100 in dilauroyl- to 3400 in dipalmitoylphosphatidylcholine vesicles, both at 50 degrees C, that is, above their corresponding phase-transition temperature tt. At t less than tt the kp values are strongly reduced, by at least a factor of 10, depending on lipid chain length and membrane composition. It is possible to establish a lipid phase-transition curve from the temperature-dependent measurements of the kp values. Cholesterol within the lipid membrane strongly decreases kp. At 20 mol% cholesterol in dipalmitoylphosphatidylcholine membranes, the partition coefficient is reduced from 3400 to 2300. This value is well comparable to the kp value obtained in erythrocyte ghosts. In contradiction to earlier experiments by Conrad and Singer (Biochemistry 20 (1981) 808-818), this value in a biological membrane could be obtained by the hygroscopic desorption as well as the centrifugation method. From our experiments we are justified in further considering artificial bilayer membranes as models for biological membranes.     

Multiple binding sites for local anesthetics in membranes: characterization of the sites and their equilibria by deuterium NMR of specifically deuterated procaine and tetracaine

Anesthetics bound to model membranes were observed directly by means of deuterium nuclear magnetic resonance (NMR). The specifically deuterated local anesthetics procaine and tetracaine were synthesized, and their partition coefficients (water:phosphatidylcholine) and pKa values determined. The interaction of these anesthetics with lamellar dispersions of egg phosphatidylcholine was studied by 2H nuclear magnetic resonance and by electron spin resonance (ESR) of a spin-labelled phospholipid at low (5.5) and high (9.5) pH. The ESR experiments suggest that tetracaine intercalates in the membrane and that it equilibrates between water and the phospholipid bilayers of the multilamellar system. The NMR results are consistent with a model where the anesthetic is (1) free in water, (2) weakly bound, and (3) strongly bound to the membrane. A fast exchange exists between the two first sites, but exchange is slow with the third site. Binding of type 3 is observed only at high pH for procaine, whereas it is found both at low and high pH for tetracaine. Calculations of the partition coefficients for the charged and uncharged forms of tetracaine indicate that both sites, 2 and 3, are occupied by the charged form at low pH and by the uncharged form at high pH. The partition coefficient for the weakly bound species was estimated from an analysis of the dependence of line width on the lipid to water ratio. The NMR data suggest that the binding sites for the strongly bound charged and uncharged species are different, the former probably being closer to the membrane-water interface. Estimates of molecular order parameters for the strongly bound species indicate that it is located with its long molecular axis approximately parallel to the director for ordering of the fatty acyl chains. A small increase in lipid ordering by tetracaine is observed at low pH, as evidenced by 2H NMR of the deuterated N-methyl groups of phosphatidylcholine; the reverse occurs at high pH.     

Partitioning of glycomacropeptide in aqueous two-phase systems

The partition behavior of glycomacropeptide (GMP) was determined in polyethylene glycol (PEG) and sodium citrate aqueous two-phase systems (ATPS). It was found that the partitioning of GMP depends on PEG molar mass, tie line length, pH, NaCl concentration and temperature. The obtained data indicates that GMP is preferentially partitioned into the PEG phase without addition of NaCl at pH 8.0. Larger tie line lengths and higher temperatures favor GMP partition to the PEG phase. Furthermore, it was verified that PEG molar mass and concentration have a slight effect on GMP partition. The increase in the molar mass of PEG induces a reduction of the protein solubility in the top PEG rich phase, being shown that the use of PEG1500 is beneficial for the extraction of GMP. A protein recovery higher than 85% was obtained in the top phase of these systems, clearly demonstrating its suitability as a starting point for the separation of GMP.     

Determination of lipid loss during aqueous and phase partition fixation using formalin and glutaraldehyde

Phase partition fixation permits fixation of tissue in a nonaqueous environment, thus eliminating osmotic effects. It was shown in an earlier investigation that retention of protein in liver blocks can be improved by phase partition fixation. By using radioisotopic labeling techniques, the effects of phase partition fixation on lipid retention during fixation, dehydration, and clearing have been determined and compared with those of standard aqueous fixation techniques. In this article we show that retention of total lipid in liver blocks following phase partition fixation using formalin was comparable to or better than that with aqueous formalin fixation and processing. Fixation with glutaraldehyde using phase partition fixation resulted in somewhat greater loss of total lipid than that observed for aqueous buffered glutaraldehyde-fixed blocks.     

A solvent partition method for microscale ganglioside purification

A simple and rapid method for the purification of gangliosides from the total lipid extract of plasma, cells, or tissue is described. The novel component of the method is the partition of the dried total lipid extract in the three-component solvent system consisting of diisopropyl ether, 1-butanol, and 50 mM aqueous NaCl (6/4/5, v/v/v). Gangliosides partition nearly quantitatively into the lower aqueous phase, and other lipids into the upper organic phase, resulting from the mixture of these three solvents. The ganglioside-containing aqueous phase is then freed of salts and other low-molecular-weight impurities by gel filtration. The thin-layer chromatographic patterns of total gangliosides thus obtained are clear and distinct, even when small samples with very low ganglioside concentrations (e.g., 1-ml samples of plasma) are processed by this method. Thus, this new ganglioside purification method is especially applicable to comparative qualitative studies of gangliosides requiring the analysis of multiple small samples.     

Membrane-buffer partition coefficients of tetracaine for liquid-crystal and solid-gel membranes estimated by direct ultraviolet spectrophotometry

The membrane-buffer partition coefficient of tetracaine was measured by direct ultraviolet spectrophotometry in dimyristoylphosphatidylcholine unilamellar liposomes at temperatures above and below the main phase transition. The partition coefficients of uncharged tetracaine to solid-gel (18 degrees C) and liquid-crystal (30 degrees C) membranes were 6.9 x 10(4) and 1.2 x 10(5), respectively. Despite the general assumption that local anesthetic binding to the solid membrane is negligible, this study showed that the solid membrane binding amounts to 57.5% of the liquid membrane binding. Binding of the charged form to the liquid or solid membrane was not detectable under the present experimental condition of 0.03 mM tetracaine bulk concentration. The present method measures metachromasia of local anesthetics when bound to lipid membranes. Its advantage is that the separation of the vesicles from the solution is not required. A linearized equation is presented that estimates the partition coefficient or binding constant graphically from a linear plot of the absorbance data. The method is applicable for estimation of drug partition when a measurable spectral change occurs due to complex formation.     

Characterization of the solubilization of lipid bilayers by surfactants

This communication addresses the state of aggregation of lipid-detergent mixed dispersions. Analysis of recently published data suggest that for any given detergent-lipid mixture the most important factor in determining the type of aggregates (mixed vesicles or mixed micelles) and the size of the aggregate is the detergent to lipid molar ratio in these aggregates, herein denoted the effective ratio, R_e. For mixed bilayers this effective ratio has been previously shown to be a function of the lipid and detergent concentrations and of an equilibrium partition coefficient, K, which describes the distribution of the detergent between the bilayers and the aqueous phase. We show that, similar to mixed bilayers, the size of mixed micelles is also a function of the effective ratio, but for these dispersions the distribution of detergent between the mixed micelles and the aqueous medium obeys a much higher partition coefficient. In practical terms, the detergent concentration in the mixed micelles is equal to the difference between the total detergent concentration and the critical micelle concentration (cmc). Thus, the effective ratio is equal to this difference divided by the lipid concentration. Transformation of mixed bilayers to mixed micelles, commonly denoted solubilization, occurs when the surfactant to lipid effective ratio reaches a critical value. Experimental evaluation of this critical ratio can be based on the linear dependence of detergent concentration, required for solubilization, on the lipid concentration. According to the ‘equilibrium partition model’, the dependence of the ‘solubilizing detergent concentration’ on the lipid concentration intersects with the lipid axis at −1/K, while the slope of this dependence is the critical effective ratio. On the other hand, assuming that when solubilization occurs the detergent concentration in the aqueous phase is approximately equal to the critical micelle concentration, implies that the above dependence intersects with the detergent axis at the critical micelle concentration, while its slope, again, is equal to the critical effective ratio. Analysis of existing data suggests that within experimental error both these distinctively different approaches are valid, indicating that the critical effective ratio at which solubilization occurs is approximately equal to the product of the critical micelle concentration and the distribution coefficient K. Since the nature of detergent affects K and the critical micelle concentration in opposite directions, the critical (‘solubilizing’) effective ratio depends upon the nature of detergent less than any of these two factors.     

X-Ray Diffraction Study in Water of Lipids Extracted from Human Erythrocytes: The Position of Cholesterol in the Lipid Lamellae

Lipids, carefully extracted from fresh human erythrocytes, form liquid-crystalline structures in water. A phase diagram of this system was constructed, characterizing, by X-ray diffraction, the structures which form as a function of concentration of lipid and temperature. One extended range of concentration of the phase diagram, in which a single lamellar phase exists, permitted further analysis of the diffraction data. This phase consists of lipid layers of constant thickness separated by water layers of varying thickness according to the water content of the system. The distribution of the electron density is precisely analyzed and the amplitude of the reflections is, at all concentrations, proportional to the Fourier Transform of an isolated lipid layer. This shows that the lipid layer is filled with the hydro-carbon chains of the phospholipids and is covered on both sides by their hydrophilic groups. Cholesterol, present in high concentration in erythrocyte membranes, is located so that part of its steroid nucleus is between the polar groups of the phospholipid molecules while the rest of the molecule extends into the inner hydrocarbon layer.