Effect of cholesterol content on the structural and dynamic membrane properties of DMPC/DSPC large unilamellar bilayers.

In this study, we report the effect of cholesterol content on the dynamic and structural properties of a dimyristoyl-phosphatidylcholine and distearoyl-phosphatidylcholine mixture in large unilamellar vesicles. The range of cholesterol concentrations studied varied around approximately 33.3mol%, where it has been postulated that an abrupt change in bilayer organization occurs. Steady-state fluorescence measurements demonstrated a typical behavior; at low temperatures in the main phase transition, the cholesterol concentration did not affect the gel phase, but at 37.5°C (phase coexistence) and in the liquid crystalline phase, the presence of cholesterol produced an increase in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan. The greater effect was observed in the liquid crystalline phase, in which the bilayer became a mixture of fluid-like and liquid-ordered phases. The results obtained at approximately 33.3mol% of Cholesterol demonstrated that the Generalized Polarization of Laurdan, the DPH lifetime, the limiting anisotropy and the rotational correlation time, as well as the fluorescence quenching of DPH by TEMPO, are at maxima, while the fluorescence intensity of dehydroergosterol and the lipid solubility in TritonX-100 are at minima. These results correlate well with the hypothesis of domain segregation in the DMPC/DSPC/Cholesterol LUV system. In this context, we postulate that at 33.3mol% of Cho, the proportion of ordered domains reaches a maximum.

Mechanism of pyrogallol red oxidation induced by free radicals and reactive oxidant species. A kinetic and spectroelectrochemistry study.

Pyrogallol red (PGR) presents high reactivity toward reactive (radical and nonradical) species (RS). This property of PGR, together with its characteristic spectroscopic absorption in the visible region, has allowed developing methodologies aimed at evaluating the antioxidant capacity of foods, beverages, and human fluids. These methods are based on the evaluation of the consumption of PGR induced by RS and its inhibition by antioxidants. However, at present, there are no reports regarding the degradation mechanism of PGR, limiting the extrapolation to how antioxidants behave in different systems comprising different RS. In the present study, we evaluate the kinetics of PGR consumption promoted by different RS (peroxyl radicals, peroxynitrite, nitrogen dioxide, and hypochlorite) using spectroscopic techniques and detection of product by HPLC mass spectrometry. The same pattern of oxidation and spectroscopic properties of the products is observed, independently of the RS employed. Mass analysis indicates the formation of only one product identified as a quinone derivative, excluding the formation of peroxides or hydroperoxides and/or chlorinated compounds, in agreement with FOX's assays and oxygen consumption experiments. Cyclic voltammetry, carried out at different pH's, shows an irreversible oxidation of PGR, indicating the initial formation of a phenoxy radical and a second charge transfer reaction generating an ortho-quinone derivative. Spectroelectrochemical oxidation of PGR shows oxidation products with identical UV-visible absorption properties to those observed in RS-induced oxidation.

Covalently bound ionene polyelectrolyte-silica gel stationary phases for HPLC.

Micelle-mimetic ionene-based stationary phases for high-performance liquid chromatography (HPLC) are prepared by attaching [3,16]- and [3,22]-ionenes to aminopropyl silica through a carbon-nitrogen bond. These [x,y]-ionenes are polyelectrolytic molecules consisting of dimethylammonium charge centers interconnected by alternating alkyl chain segments containing x and y methylene groups, some of which can form aggregate species whose properties mimic those of conventional surfactant micelles. These ionene-bonded stationary phases were characterized using different recommended HPLC test mixtures. Test solute chromatographic behavior on the ionene phases was found to be similar to that of intermediate oligomeric or polymeric C-18 and/or phenyl phases, depending upon the specific test mixture employed. In addition, the phases exhibit significant solute shape recognition ability. The ionene stationary phases were successfully employed for the separation of the components of the recommended ASTM reversed-phase test mixture, as well as for ortho-, meta- and para-disubstituted benzenes and other positional or geometric isomeric compounds. The ionene materials allow for chromatographic separations under either reversed-phase or ion-exchange conditions. The retention mechanism on these multimodal phases can occur by hydrophobic partitioning or electrostatic interactions, depending upon the characteristics of the components of the analyte mixture (neutral or anionic). The effects of alteration of the percent organic modifier, flow rate and temperature of the mobile phase on chromatographic retention and efficiency on these phases were briefly examined.

Does the photochemical conversion of colchicine into lumicolchicines involve triplet transients? A solvent dependence study.

beta- and gamma-lumicolchicines are photoproducts formed by the cycloisomerization of the tropolone ring of colchicine (COL) alkaloids. The mechanism of the photoconversion, suggested to involve the triplet state, is examined here by studying the effect of the solvent polarity on the lumicolchicine photoisomer ratio. Triplet COL, detected by laser flash photolysis, is quenched by oxygen, but not by transtilbene or 1-methylnaphtalene. Neither the quantum yield of conversion of COL nor the photoproduct ratio was altered by the presence of oxygen. Likewise, energy transfer to COL from triplet acetone produced by either isobutanal/horseradish peroxidase system or tetramethyldioxetane thermolysis failed to provoke photoreaction of COL. Our data argue against the intermediacy of a COL triplet state in the photoisomerization and stress on the role of specific solvent-solute interactions in determining the partitioning of excited singlet state into the beta- and gamma-isomer formation.

Analytical applications and implications of intramolecular micelle-mimetic ionene aggregates.

The synthesis and relevant properties of some cationic polyelectrolytes of the [x,y]-ionene type (i.e., molecules consisting of dimethylammonium charge centers interconnected by alternating alkyl chain segments containing x and y methylene groups) are described. Such hydrophobic ionenes can form intramolecular aggregates and function as micelle-mimetic agents since they mimic the key properties of traditional surfactant micelles (such as hexadecyltrimethylammonium bromide) in many respects. However, the ionenes possess certain important advantages over surfactant micelles. For example, aqueous ionene-containing solutions exhibit much less foaming and analyte molecules can be extracted and recovered from their solutions by use of organic solvents. In this work, some applications of the ionenes in analytical chemistry, such as their use as mobile phase additives for thin-layer chromatographic and capillary electrophoretic separations, as fluorescence enhancement agents, and as a means to catalyze slow spectroscopic derivatization reactions are demonstrated. In addition, other potential advantages concerning the utilization of the micelle-mimetic ionenes in chemical analysis applications are mentioned.

Ruthenium(II) tris(bipyridyl) ion as a luminescent probe for oxygen uptake.

The present work describes an alternative technique for following the rate of oxygen uptake by chemical and enzymatic systems. This method is based on spectrofluorometric monitoring of the well-known quenching effect of molecular oxygen on the emission of the photoexcited [Ru(bpy)3]2+ ion, added to the reaction mixtures. The rate of oxygen consumption determined using the present method agrees with that obtained by conventional polarographic techniques in all of the following systems: ascorbate/Cu2+, glucose/glucose oxidase (EC 1.1.3.4), and propanal/horseradish peroxidase (EC 1.11.1.7); in the last case, agreement was observed both in the presence and absence of serum albumin and of chloroplasts. Spectrofluorometric data for amphotericin autoxidation in dimethyl sulfoxide are in accord with the rate of decay of the ESR signal of a spin trap added to the reaction mixture. The advantages and limitations of the present spectrofluorometric technique relative to conventional polarographic monitoring of dissolved oxygen are discussed.

Interactions of neutral molecules with ionic micelles.

The interactions of neutral molecules with ionic micelles are analyzed. The cell and mass action models are presented in order to provide a semi-quantitative description of the solubilization process. Both approaches are discussed from a thermodynamic and kinetic point of view and the different definitions of solute incorporation constants are also discussed and compared. An extensive compilation of standard free energies of transfer from water to micelles is provided and the basis of the employed methods to obtain them is presented. Several aspects of the solubilization process such as its dynamics, the effect of additives, the probe microenvironment and its dependence with the solute mean occupation number are reviewed and critically discussed. The effect of solute incorporation upon the micelle shape and size is also briefly reviewed.

Carcinogenic and non-carcinogenic aromatic hydrocarbons in lipid membranes. A fluorescence study of pyrene and benzo[a]pyrene.

1. The fluorescence behavior of a non-carcinogenic (pyrene) and a carcinogenic (benzo[a]pyrene) aromatic hydrocarbon was examined in the presence of a phospholipid bilayer membrane in the gel phase. 2. The monomer emission spectrum of pyrene in the membrane is very similar to that in micelles indicating a site near the aqueous surface region. Benzo[a[pyrene monomer spectra exhibit a red shift in the membrane relative to aliphatic hydrocarbon solvents. On the basis of spectral shifts in other homogeneous solvents, it is inferred that the carcinogen is located in the upper portion of the membrane acyl region, a more polarizable environment than the hydrocarbon core of the bilayer. 3. Analysis of the excimer to monomer emission intensity ratio as a function of probe molar ratio indicates that pyrene is much less soluble in the membrane than benzo[a]pyrene. 4. These results complement published EPR data which show that carcinogenic aromatic hydrocarbons cause structural changes in the membrane, while non-carcinogenic ones do not. These differences in membrane solubility and ability to alter membrane structure are discussed in the context of the different carcinogenic potencies of the hydrocarbons.

Effects of temperature and lipid composition on the serum albumin-induced aggregation and fusion of small unilamellar vesicles.

Small unilamellar vesicles of egg phosphatidylcholine (PC) or dimyristoylphosphatidylcholine, mixed with small unilamellar vesicles labelled with 2-(10-(1-pyrene)decanoyl)phosphatidylcholine, exhibit a constant average size and excimer to monomer (E/M) ratio for several hours when incubated at pH 3.6 at a temperature higher than the phase transition temperature (Tc) of the lipids. Addition of bovine serum albumin to this system produces a transient turbidity increase, a fast decrease in the E/M ratio, a partial loss of vesicle-entrapped [14C]sucrose and a measurable leak-in of externally added sucrose. Sepharose 4B filtration of the system demonstrates that the E/M ratio decrease is strictly paralleled by the formation of liposomes which exhibit a low E/M ratio and a hydrodynamic radius larger than that of small unilamellar vesicles. These data demonstrate that the E/M ratio decrease can be unequivocally ascribed to a vesicle-vesicle fusion process induced by serum albumin. The rate of serum-albumin induced fusion of small unilamellar vesicles is: (a) maximal at a stoichiometric ratio of approx. 2 albumins per vesicle; (b) sensitive to the nature of the lipid and; (c) not altered when human serum albumin replaces bovine serum albumin. The rate of albumin-induced fusion of dimyristoylphosphatidylcholine small unilamellar vesicles is higher below the Tc of the lipid and increases with temperature above the Tc. The formation of protein-bound aggregates with defined stoichiometries and a high local vesicle concentration, as well as changes in the local degree of hydration, are proposed to be the driving forces for the protein-induced vesicle fusion in this system.

A kinetic and structural study of two-step aggregation and fusion of neutral phospholipid vesicles promoted by serum albumin at low pH.

The addition of bovine serum albumin (BSA) to 25 +/- 5 nm diameter single bilayer phosphatidylcholine (PC) vesicles (SBV) (pH 3.5) gives rise to readily visible transient turbidity. Studies of this system, employing a series of techniques, including time-dependent turbidity changes, membrane filtration, centrifugation, Sepharose chromatography and freeze fracture electron microscopy demonstrated that the process involves aggregation and fusion of the vesicles. At least three distinct time-dependent steps have been characterized: (1) the rapid initial formation (in approx. 5 min) of large aggregates (responsible for the visible turbidity) composed of SBV interconnected by BSA in its F form. The formation of these aggregates may be reversed by raising the pH or adding excess BSA to the system at this stage; (2) spontaneous collapse of these large aggregates, in an irreversible step, to form a heterogeneous population of vesicles; (3) fusion produces as the final product of the process, a relatively homogeneous population of larger (50 +/- 10 nm diamter) vesicles. This system serves as a convenient and simple model system for the detailed study of protein-mediated aggregation and fusion of membranes at the molecular level.