Solubilization of Phospholipids by Chaotropic Ion Solutions

Solubilization of Phospholipids by Chaotropic Ion Solutions

(Parte 1 de 3)

Printed ln U,XA Vol. 258 No. 14 Issue of July ?5, p. 8733-8738,1983

Solubilization of Phospholipids by Chaotropic Ion Solutions* (Received for publication, February 7, 1983)

Phosphatidylcholine (PC) dissolves in solutions of concentrated neutral trichloroacetate and tribromo- acetate, anions known to be extremely chaotropic. So- lubilization of egg yolk PC occurs in Na-trichloroace- tate between 2 and 3 M and in Na-tribromoacetate between 1.5 and 2 M. The resulting optically clear solutions were found by gel exclusion chromatography to consist of micelles containing of the order of 10’ lipid molecules which, according to 31P NMR, are undergoing rapid, isotropic motion. Dipalmitoyl-PC is not solubilized below its normal phase transition tem- perature, although it remains in solution if first dis- solved above that temperature; transition temperature and enthalpy change are both drastically depressed in 3 M Na-trichloroacetate. Na-trichloroacetate (3 M) also dissolves sphingomyelin but not phosphatidyletha- nolamine, acidic phospholipids, or egg PC-cholesterol mixtures of more than 3% cholesterol. Destabilization of the lamellar phase of egg PC rel-

ative to the micellar phase is promoted by trichloroac- etate binding to and intercalating between the lipid molecules. Trichloroacetate was found to bind to egg PC liposomes with an affinity constant of about 1 M-I, so that nearly 1:1 association between egg PC and trichloroacetate is predicted at the concentration at which ~lubili~tion occurs. A surface area dilation by an amount (50+%) consistent with formation of mi- celles was confirmed by surface tension isotherms of egg PC on 3 M Na-trichloroacetate. The chaotropic effect may synergize binding by reducing the energy cost of the exposure of hydrophobic portions of lipid that is necessitated by the small radius of curvature of micelles. Several chaotropic agents less potent than trichloroacetate and tribromoacetate did not solubilize egg PC.

Chaotropic ions decrease hydrophobic interactions between apolar molecules by destabilizin~ the quasicrystalline struc- ture of water (1). These ions have been used for the dissocia- tion of enzyme complexes (2) as well as solubilization and isolation of membrane-bound proteins (3-5). Although phos- pholipid bilayers are stabilized by hydrophobic interactions, chaotropic reagents have not been used for the complete solubilization of membranes. Instead, detergents or organic solvents are usually used to solubilize lipids or natural mem- branes.

Here we report that powerful chaotropic reagents, such as trichloroacetat,e or tribromoacetate, at high concentrations, indeed solubilize some phospholipids. Such reagents may be

* This investigation was supported by National Institutes of Health Research Grant GM-28404. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with

18 U.S.C. Section 1734 solely to indicate this fact.

useful for solubilization and isolation of membrane compo- nents, as well as membrane reconstitution. This phenomenon is also of interest from the point of view of lipid chemistry because the solubilization of lipids by chaotropes, relative to that by detergents, is selective.

C~emica~-Chemicals were obtained from the following compa- nies: Sigma, egg yolk phosphatidylcholine, dipalmitoyl-DL-phospha- tidylcholine, bovine brain sphingomyelin, phosphatidylglycerol from egg yolk phosphatidylchoiine, cholesterol, dicetyl phosphate, stea- rylamine, and fluorescein isothiocyanate-dextran; Avanti Biochemi- cals Inc., Birmingham, AL, bovine brain p~osphatidy~serine; Calbi- ochem-Behring, Escherichia coli phosphatidylethanolamine; East- man, N,N‘-dioctadecyloxacarbocyanine-p-toJuenesulfonate; Mal- Iinckrodt, trichloroacetic acid; Aldrich, tribromoacetic acid; Hach Chemical Go., Ames, IA, calcein; New Engfand Nuclear, [l,2-3H] cholesterol. Preparations of PC,’ PS, and SM gave single spots on thin layer chromatography. PE and PG were also almost pure by silica gel thin layer chromatography criteria, although these two preparations showed faint, tailing. Concentrations of phospholipids were determined by a modi~cation of the Bartlett procedure (6). Sodium trichloroacetate and sodium tribromoacetate were prepared by neutralizing trichloroacetic acid and tribromoacetic acid with sodium hydroxide slowly enough that the temperature of the solution did not rise appreciably.

Nuclear Magnetic Resonance Me~ure~ent-~lPt’H) NMR spectra were obtained at 109.16 MHz on a JEOL FX-270 under full proton decoupling. Egg PC at 50 mM was dispersed in 10 mM Mops buffer (pH 7.0) and 50% D20 with or without 3 M Na-trichloroacetate. ~e~uremerat af ~urb~ity Change of Liposomes-The turbidity of lipid dispersed in solutions of differing concentrations of chaotropic and other ions was used as a measure of the solubilization of lipids.

Although turbidity falls with the size of the scattering particles, it also depends upon the refractive index of the medium. The latter changes with solute concentration, so it is necessary to eliminate the effect of refractive index to obtain a measure of particle size alone. This was done as follows. Turbidity depends upon refractive index as the square of the difference between that of the particle and that of the medium (7). For most solutions, the refractive index is an ap- proximately linear function of solute concentration. Thus, in the absence of changes in the properties of the scattering unit, turbidity should depend upon solute concentration (C,) according to 7, = 7,”

- C,/Co)2, where T, is the measured turbidity, iW is the turbidity of the sample in pure solvent, i.e. water, and Co is the solute concentra- tion at which particle and medium refractive indices are identical. This relationship can be used to eliminate the effect of refractive index on turbidity, as well as small variations in the turbidities of samples in water, by dividing the experimentally determined values (io&) at various C, by the factor ~,(1 - C,/C,)’, to give turbidity normalized to the refractive index of the water, i.e. T,,,,~~= T,~~/T,(I ; C6/C0),? A plot of rnOm versus C, should yield a line of zero slope If there IS no change in particle size. A negative slope would indicate a decrease in particle size. C, was obtained by extrapolation of refractive indices from literature values (81, which are very close to linear

The abbreviations used are: egg PC, egg yolk phosphatidylcholine;

DPPC, dipaimitoylphosphatidylcholine; PS, phosphatidylserine; PE, phosphati~ylethanolam~ne; PG, phosphatidylglycerol; SM, sphingo- myelin; DCP, dicetyl phosphate; SA, stearylamine; DSC, differential scanning calorimetry; GdnHC1, guanidine hydrochloride; Mops, 4- morpholinepropanesulfonic acid.

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8734 Lipid Solubi~~~ut~o~ by Chaotropes functions of concentration, or from our measurements obtained with an Abbe refractometer. Although there are a number of approxima- tions used in this procedure, it can clearly differentiate between multilayered liposomes and micelles, so it is a useful parameter.

Phospholipids at 5 mM were hydrated in water and diluted with various solutes containing 5 mM EDTA to a final concentration of 0.25 mM. Turbidity was measured in a spectrophotometer (Coleman Model 124) at 400 nm.

Differential Scanning Cubrimetry-A 50 mM dispersion of DPPC liposomes was prepared by simple hydration at 50 “C. Twenty pl of this suspension was mixed with 20 p1 of Na-trichloroacetate solution in a test tube and the mixture was briefly warmed to 60 “C. An aliquot of 20 pl was transferred to a DSC sample pan. The reference pan contained an equal volume of water. The heating rate was 2.5 “C per min. T, denotes the temperature of onset of the phase transition.

Relative enthalpy was determined from the area of the peak. Electrophoresis of Liposomes-Liposomes were prepared in water and buffer and electrolytes were added subsequently. Samples con- tained 0.1 mM PC, 1.0 mM Mops buffer (pH 7.0),0.5 mM EDTA, and 0.1 M NaCl or Na-trichloroacetate. A Rank Brothers apparatus (Cam- bridge, U. K.) was used according to procedures described previously

(9). Surface Tension Measurement-Surface tension was measured by the Wilhelmy hanging plate method (10). Egg PC (3.1 mM) dissolved in hexane/ethanol(31) was added in lh-pl portions to the surface of the water or a solution of 3 M Na-t~chloroacetate, into which a platinum wire, hanging from an electrobalance, was dipped. Hexane was purified by passage over silica gel. Ethanol was treated with activated charcoal and distilled. Distilled water was charcoal-filtered and redistilled. The trough was a standard 90-m Petri dish. Column Chromatography-A Sepharose CL-GB column (I1 X 430 m) was equilibrated with 10 mM Mops-buffered (pH 7.0) 3 M Na- trichloroacetate and pretreated with egg PC. One hundred pl of 20 mM egg PC with 8 pCi/ml of f3H]cholesterol in 3 M Na-trichloroace- tate was applied to the column and eluted with 3 M Na-trichloroace- tate containing 10 mPrI Mops. Fractions of about 500 gl were collected, 100 gl of which was assayed for phosphorus content. A second 100-gl aliquot was counted in a liquid scintillation counter. Blue dextran (average M, = 2,0,0) was used to determine the column void

volume, V,. Fluorescein isothiocyanate-dextran (M, = 148,0, 39,0, and 20,0) was used to calibrate the included volume. Calcein

(M, = 622) was used to determine the total volume.

Solubility of Phospholipids in Various Chaotropes-Lipo- somes prepared from egg yolk phosphatidylcholine were mixed with several concentrations of various chaotropic re- agents. Turbidities were determined and converted to relative turbidities as described under “Materials and Methods” using the following values of C,: Na-tribromoacetate, 5.5 M; KI, 7.4 M; Na-trichloroacetate, 7.8 M; GdnHCl, 9.2 M; KSCN, 9.3 M; urea, 17.5 M; KN03, 19.0 M. Fig. 1 shows that chaotropic reagents such as KSCN, KI, KN03, urea, and GdnHCl did not reduce the normalized turbidity of liposome suspensions. In anticipation of subsequent results and for simplicity, we describe a dramatic reduction of turbidity as “dissolution.” Thus, these solutions do not “dissolve” egg PC (nor did solutions of NaCl, KC1, and sodium acetate; data not shown). In fact, the general trend was to increase T~~~~, suggesting that some aggregation occurs. The effect was smaller with the weak chaotropes I- and SCN-; these might be expected to bind weakly to liposomes and thereby reduce aggregation. On the other hand, the more potent chaotropic reagents, Na- trichloroacetate and ~a-tribromoacetate, did dissolve egg PC. Dissolution is rapid; it is apparently complete after a few seconds of vortexing. Although normalized turbidity of lipo- somes in these solutions increased slightly at low concentra- tion, it decreased drastically at molar concentrations. Nothing in these solutions was visible under the phase microscope. Furthermore, completely uniform fluorescence was observed under the fluorescence microscope when the liposomes con- tained 2.5% of the fluorescent lipid analog, N,N’-dioctadec- yloxacarbocyanine-p-toluenesulfonate. In accord with the previously observed difference in chaotropic potency (1,12), Na-tribromoacetate more effectively solubilized egg PC than did Na-trichloroacetate.

The possibility that egg PC was degraded in Na-trichlo- roacetate was tested. After egg PC liposomes were incubated in 3 M Na-trichlor~acetate for 1 day at room temperature or for 30 min at 70 “C, phospholipids were extracted from solu- tions and examined by thin layer chromatography. We ob- served a single spot, the RF of which was the same as the original egg PC, and no sign of decomposition products.

Differential Sol~bi~i~y of Various Phosphol~pids in Chao- tropic Solutions-Next we examined the solubilizing effect of

Na-trichloroacetate on phospholipids other than egg PC. Ta- ble I shows that sphingomyelin, as well as egg PC, was solubilized by Na-trichloroacetate. The third column of Table I shows the relative turbidity calculated on the assumption that refractive indices of all lipids are the same as that of egg PC, namely 1.48. In the case of DPPC, if the sample had passed through the phase transition, that is, if it once expe- rienced the liquid crystalline state in the presence of Na- trichloroacetate, it dissolved. In contrast to these neutral phospholipids, acidic phospholipids could not be solubilized by 3 M Na-trichloroacetate under our experimental condi- tions. High turbidities of 3 M Na-trichloroacetate solutions of these lipids might be due to aggregation of liposomes. Fig. 2 shows that there is a sharp increase in the turbidity of egg PC-cholesterol mixtures in Na-trichloroacetate as the cholesterol content exceeds 3 mol %. Because there is no corresponding change in the turbidity of the same lipid mix- tures in water and because a large change occurs with a relatively small increase in cholesterol content, this indicates a reduction in liposome solubility.

Fig. 3 shows the phosphorus NMR spectrum of egg PC in 3 M Na-trichloroacetate. It consists of a single sharp absorp- tion, corresponding to rapid, isotropic motion of lipid mole- cules. Such motion suggests that the structural unit of lipid organization is quite small, such as a monomer, micelle, or very small liposome. This result is thus consistent with the r-“

0-51 \ i I

Concentration of 80krt0S ( M )

FIG. 1. The effect on the turbidity of egg PC liposomes of various solutions of chaotropes. Egg PC liposomes, 5 mM in water, were diluted with various concentrations of chaotropic reagents to yield a final egg PC concentration of 0.25 mM. All samples included 5 mM EDTA to prevent aggregation of liposomes by possible multi- valent cation impurities. After incubation at room temperature for 30 min, turbidities were determined at 400 nm. Normalized turbidities were cafculated as described under “Materials and Methods.” Sym- bols are as follows: 0, Na-tribromoacetate, 0, Na-trichloroacetate; A, KSCN, A, GdnHC1; KI; V, KNOa; and 0, urea.

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Lipid Solubilization by Chaotropes 8735

Solubilization of various phospholipids by Nu-trichloroacetate

Lipid dispersions (5 mM phospholipid) were prepared by hydration of a lipid film with water using a vortex mixer. The dispersions were then diluted with 5 mM EDTA (pH 7.0) or 3 M Na-trichloroacetate containing 5 mM EDTA. The final phospholipid concentration was 0.25 mM except in the case of egg PC/DCP (row 2) where the PC concentration was 1.0 mM. After a 30-min incubation at room tem- perature, the turbidity of the samples was measured at 400 nm. One set of DPPC samples (row 6) was incubated at 60 “C for a few minutes before incubation at room temperature. Normalized turbidity was calculated as described under “Materials and Methods,” the refractive index of all lipids being taken as 1.48.

(Parte 1 de 3)

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