polarized and depolarized dynamic light scattering

polarized and depolarized dynamic light scattering

7 Pages · 2005 · 1.21 MB · English

*Centro de Quimica Fisica Molecular, 1096 Lisboa Codex, Portugal; and tInstitute of Physical Chemistry, University of Uppsala,. 751 21 Uppsala 150 s-1, which is close to the calculated value for the rod with this dimension. Abbreviations used: ILT, inverse Laplace transformation; DLS, dynamic lig

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Rodlike cholesterol micelles in aqueous solution studied using polarized and depolarized dynamic light scattering Miguel A R B Castanho,* Wyn Brown, and Manuel J E Prieto* *Centro de Quimica Fisica Molecular, 1096 Lisboa Codex, Portugal; and tInstitute of Physical Chemistry, University of Uppsala, 751 21 Uppsala, Sweden ABSTRACT Micelles of cholesterol in aqueous solution have been investigated using polarized and depolarized dynamic light scattering They are shown to be highly extended and characterized by a narrow size distribution It is shown that a rodlike model is applicable with length, L = 580 nm Determination of the rotational diffusion coefficient by analysis of the autocorrelation function gave a value of e = 150 s1, which is close to the calculated value for the rod with this dimension Depolarized dynamic light scattering measurements as a function of angle gave a value of 1 10 s' INTRODUCTION Cholesterol is a sterol widely known for its biochemical role in biological membranes (for a review see, eg, Yea gle, 1985), its interaction with serum lipoproteins (see, eg, Yeagle et al, 1982) and with bile salts (eg, Mazer et al, 1980) Moreover, studies of cholesterol aggregation in aqueous medium are essential for an understanding of the occurrence of some pathological situations such as the formation of cholesterol gallstones in bile (Small, 1967; Lonsdale, 1968) and atherosclerotic lesions (Small, 1970; Small and Shipley, 1974) Cholesterol gallstones result from the supersaturation of bile (Ad mirand and Small, 1968) (which is a solution composed mainly of three kinds of lipids: bile salts, lecithins, and cholesterol) with cholesterol The excess cholesterol will precipitate (Mazer et al, 1977) forming microcrystals that may start, or enlarge, a process of Epitaxy (Lons dale, 1968) (growth of one crystal on a substrate of an other) If this saturation takes place in the blood, choles terol accumulates in the atherosclerotic lesions forming thermodynamically stable systems (Small and Shipley, 1974) with other lipids (mainly cholesterol esters and phospholipids) The physical state of these lipids in rela tion to the biochemical environment can be understood on the basis of their interaction with an aqueous system (Small and Shipley, 1974) Knowledge of the structure and physicochemical properties of cholesterol aggre gates in aqueous medium is thus essential for an under standing of these pathologies Aqueous solutions of cholesterol have been little in vestigated in spite of the fact that knowledge of choles terolcholesterol and cholesterolwater interactions is es sential to a deeper understanding of complex systems as those involving lipids and/or proteins and/or sterols and water Haberland and Reynolds (1973) examined choles terol aggregates in water employing a dialysis technique Correspondence should be sent to Wyn Brown, Institute of Physical Chemistry, University of Uppsala, Box 532, 751 21 Uppsala, Sweden Abbreviations used: ILT, inverse Laplace transformation; DLS, dynamic light scattering and demonstrated that there is a reversible monomer micelle equilibrium, with a critical micelle concentra tion of 2540 nM (25°C) and a solubility limit of 47,gM using their method of preparation A rodlike shape for the micelle was advanced to explain the relationship be tween aggregate dimensions ( < 1,000 A in its largest di mension as deduced from dialysis experiments) and its molecular weight (2 I0, from ultracentrifugation ex periments) A sidebyside stacking of the ring systems was proposed to account for the high interaction energy between the aggregated monomers Later, Gilbert et al (1975) continued this type of investigation and con cluded that the unique structure of the hydrophobic por tion of cholesterol allows a particularly favorable pack ing of water molecules around the sterol in an aqueous phase and that some repulsion between polar groups is required to account for the existence of micelles of finite size An xray study on crystals of cholesterol monohydrate grown from acetonewater solutions (Craven, 1976) supported the ability of cholesterol to stack sidebyside in bilayers of 339 A thickness In this work, the structure and some physicochemical properties of cholesterol micelles prepared from acetone water solutions, followed by ultrasonification, are eluci dated using dynamic light scattering measurements The investigation forms part of a wider program directed to studies of the interaction between cholesterol and po lyene antibiotics, which are only effective against cells having sterolcontaining membranes (Bolard, 1986) One of the published models for the interaction of fili pin, a polyene antibiotic, and cholesterol (Elias et al, 1979) proposes an association of filipin with the sterol in a 1:1 stoichiometry at the membrane surface, in an aqueous environment The only method developed so far to study the interaction between antibiotics and ster ols (Norman et al 1972) was recently shown to be incor rect (Castanho et al, 1992) and so this remains an open field The results presented here also have significance for an understanding of the dynamics of cholesterol ex Biophys J Biophysical Society Volume 63 December 199214551461 00063495/92/12/1455/07 $200 00063495/92/12/1455/07 $200 1 455 change between vesicles (Bruckdorfer and Sherry, 1984), a process commonly used in the preparation of artificial membranes In addition, the ability of choles terol to form "pools" in artificial bilayers of phospho lipids (Rogers et al, 1979) instead of a random distribu tion, can only be elucidated if the fundamental choles terolcholesterol interactions are clarified The results from dynamic light scattering described below, giving the translational and rotational diffusion coefficients, strongly suggest that cholesterol forms long rodshaped micelles EXPERIMENTAL Materials and preparation Cholesterol was obtained from Sigma Chemical Co (St Louis, MO) and used as received after its purity had been checked by thin layer chromatography on silica plates, using a mixture chloroform:acetone (98:2) as eluent TrisHCI was purchased from B D H (London) and used as received to prepare the buffer (TrisHCI 50 mM, pH 74, NaCl 10 mM) NaCl (p a) and acetone (p a) were from Merck (Darm stadt, Germany) Micelle preparation To a given volume of buffer at 850C, with vigorous but nonturbulent stirring, small volumes (no more than 1/20 of the total volume of buffer) of cholesterol solutions in acetone (0685 mM) were injected with a syringe Use of small volumes prevents the effervescence of ace tone, so avoiding losses of cholesterol that would otherwise dry and remain on the walls of the container One minute was the minimum time interval between two additions to prevent acetone accumulating in the solution and boiling locally This was controlled measuring the temperature of the mixture; as acetone has a boiling point of 5620C, accumulation of acetone would decrease the temperature After com plete addition of the cholesterol solution in acetone to the buffer, the aqueous suspensions of cholesterol were left during 15 min at 850C with vigorous stirring, so that no significant amount of acetone re mained in solution The final concentration of acetone in the aqueous solutions of cholesterol was controlled by UV absorption Acetone in water has an absorption maximum at 265 nm (e = 185 M' cm') (Hayes and Timmons, 1971) Measurements were carried out in a PerkinElmer A15 spectrophotometer, using 10cm path cuvettes, in dicating that the molar percentage of acetone in the final solutions was <0001%

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