Polydispersity of aggregates formed by the polyene antibiotic amphotericin B and deoxycholate. A spin label study.

The amphotericin B-deoxycholate (AB-DOC) system (1:2, mole basis) was studied with regard to its organizational properties making use of spin label ESR spectra. The spectra of a fatty acid spin label intercalated in AB-DOC preparations revealed two components, one strongly (S) and one weakly (W) immobilized. Spectral subtractions indicated that S corresponds to label in mixed AB-DOC aggregates while W is due to label in deoxycholate micelles. This situation, coexistence of different aggregates, is similar to that found in systems consisting of bile salts and phospholipids. The DOC/AB mole ratio in the mixed aggregate is highest when pure DOC micelles are present. Dilution leads to disappearance of the latter and to continuous loss of DOC from AB-DOC accompanied by an increase in size and decrease in solubility of the aggregates, as verified by filtration and centrifugation experiments. The results indicate that AB-DOC systems are polydisperse. Since amphotericin B preparations having different organizational properties display different toxic and therapeutic effect, the study of amphotericin B aggregates should help in understanding these phenomena at a molecular level.

Thermal transitions of DMPG bilayers in aqueous solution: SAXS structural studies.

Dimyristoylphosphatidylglycerol (DMPG) has been extensively studied as a model for biological membranes, since phosphatidylglycerol is the most abundant anionic phospholipid in prokaryotic cells. At low ionic strengths, this lipid presents a peculiar thermal behavior, with two sharp changes in the light scattering profile, at temperatures named here T(on)(m) and T(off)(m). Structural changes involved in the DMPG thermal transitions are here investigated by small angle X-ray scattering (SAXS), and compared to the results yielded by differential scanning calorimetry (DSC) and electron spin resonance (ESR). The SAXS results show a broad peak, indicating that DMPG is organized in single bilayers, for the range of temperature studied (10-45 degrees C). SAXS intensity shows an unusual effect, starting to decrease at T(on)(m), and presenting a sharp increase at T(off)(m). The bilayer electron density profiles, obtained from modeling the SAXS curves, show a gradual decrease in electron density contrast (attributed to separation between charged head groups) and in bilayer thickness between T(on)(m) and T(off)(m). Results yielded by SAXS, DSC and ESR indicate that a chain melting process starts at T(on)(m), but a complete fluid phase exists only for temperatures above T(off)(m), with structural changes occurring at the bilayer level in the intermediate region.

Probing DMPG vesicle surface with a cationic aqueous soluble spin label.

A small, highly aqueous soluble, deuterated, cationic spin label, 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (dCAT1), was used to directly monitor the negatively charged DMPG vesicle surface in order to test a recent suggestion (Riske et al., Chem. Phys. Lipids, 89 (1997) 31-44) that alterations in the surface potential accompanied apparent phase transitions observed by light scattering. The temperature dependence of the label partition between the lipid surface and the aqueous medium indicated an increase in the surface potential at the gel to liquid-crystal transition, supporting the previous suggestion. Results at the phase transition occurring at a higher temperature were less definitive. Although some change in the dCAT1 ESR spectra was observed, the interpretation of the phenomena is still rather unclear. DMPG surface potentials were estimated from the dCAT1 partition ratios (surface label moles/total label moles), using a simple two-sites model, where the electrostatic potential is zero everywhere but at the vesicle surface, and the interaction between the spin label and the membrane surface is chiefly electrostatic. The Gouy-Chapman-Stern model predicts surface potentials similar to those observed, although the measured decrease in the surface potential with ionic strength is somewhat steeper than that predicted by the model.

How melatonin interacts with lipid bilayers: a study by fluorescence and ESR spectroscopies.

ESR spectra of spin labels placed at the membrane surface and at different depths of the bilayer core, and melatonin fluorescence in the presence of lipid vesicles, suggest an average shallow position for the hormone in the membrane. However, according to the melatonin ability to cross lipid bilayers, nitroxides placed deep in the bilayer were able to quench the melatonin fluorescence. Melatonin membrane partition coefficients were calculated for bilayers in different packing states, and similar and rather high values were found. The data presented here may be quite important to the understanding of melatonin physiological actions at the membrane level.

First synthesis of a fully active spin-labeled peptide hormone.

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. The ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac0-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac. Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for alpha-MSH.

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides.

Similar to melanocyte stimulating hormone (alpha-MSH), its potent and long-acting analogue, [Nle(4), D-Phe(7)]alpha-MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides. Correlation times, calculated from the electron paramagnetic resonance signal of Toac bound to the peptides, and Toac-Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium.

Contributions to the Gaussian line broadening of the proxyl spin probe EPR spectrum due to magnetic-field modulation and unresolved proton hyperfine structure.

A simple expression is derived to compute the total Gaussian linewidth of a Voigt line that is broadened by sinusoidal magnetic-field modulation as follows: delta HPPG(Hm)2 = delta HPPG(0)2 + kappa 2Hm2, where delta HPPG(Hm) is the Gaussian linewidth observed with an modulation amplitude Hm/2 and delta HPPG(0) is the Gaussian linewidth in the limit of zero modulation. The field modulation contributes an additional Gaussian broadening of kappa Hm, where kappa is a constant, which adds in quadrature to delta HPPG(0) to give the total Gaussian linewidth. Denoting the overall linewidth of the Voigt line in the absence of modulation broadening by delta HPP0(0), it is shown, both by analytical means and by spectral simulation, that the constant kappa is equal to 1/2 in the limit of Hm << = delta HPP0(0); however, using values of Hm as large as delta HPP0(0) leads to only minor departures from kappa = 1/2. The formulation is valid both for Lorentzian and Voigt lines and is tested for 2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-carboxylic acid (3-carboxy proxyl) in CCl4 and in aqueous buffer. This spin probe was studied because the proxyl group is the only major spin-probe moiety whose Gaussian linewidth had not been characterized in the literature. For 3-carboxy proxyl, it is found that delta HPPG(0) = 1.04 +/- 0.01 G independent of solvent polarity. Precision values of the 14N hyperfine coupling constant for 3-carboxy proxyl at 9.5 degrees C are as follows: 14.128 +/- 0.001 G in CCl4 and 16.230 +/- 0.002 G in aqueous buffer. The temperature dependence of delta HPPG(0) and the 14N hyperfine coupling constant are reported as empirical equations. Results of the present work taken together with previously published data permits accurate correction for the effects of inhomogeneous broadening due to unresolved hyperfine structure and modulation broadening for the majority of spin probes in common use.

Correlation between the effects of a cationic peptide on the hydration and fluidity of anionic lipid bilayers: a comparative study with sodium ions and cholesterol.

The cationic tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) is known to interact with anionic vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), partially penetrating the lipid membrane. In the lipid liquid crystal phase, phospholipid derivatives spin labeled at the different C-atoms along the acyl chain, show that the peptide increases the bilayer packing at all depths. Parallel to that, there is an increase in the probe's isotropic hyperfine splittings, indicating that the peptide significantly decreases the membrane hydrophobic barrier. Accordingly, it is suggested that the increase in membrane packing yielded by alpha-MSH is partly due to a greater level of interchain hydration. This result is compared to the increase in packing and decrease in polarity yielded by cholesterol, and the absence of structural or polar alterations with Na+. The latter result shows that the peptide effect is not related to an increase of positive charges at the anionic vesicle surface. Alterations on the lipid bilayer polar profile measured by the nitroxide hyperfine splitting z component in frozen samples are shown to be different from those obtained at room temperature. However, it is shown here that a certain correlation can be drawn between the increase in polarity measured in frozen samples and the packing effect caused by the different molecules in the lipid gel phase.

Melanotropic peptides-lipid bilayer interaction. Comparison of the hormone alpha-MSH to a biologically more potent analog.

The interaction of the native peptide alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more active analog [Nle4, D-Phe7]-alpha-MSH(MSH-I) with lipid vesicles was studied by spin label electron spin resonance (ESR) spectroscopy and circular dichroism (CD). Using spin labels located at the membrane interface and at different depths along the acyl chain, it was shown that the binding of both peptides to the membrane induces tighter lipid packing at all the monitored positions. However, the effect of the analog on the spin label ESR parameters was much more evident, and suggested that it penetrates farthest into the lipid matrix than the native molecule. Lipid partition coefficients were calculated based on the effect the peptides cause on the ESR spectra of spin labels incorporated in the membrane. For the biologically more potent peptide, the partition coefficient was found to be about 4-times greater than that of the native hormone. For the same concentration of peptide bound to the membrane, MSH-I was found to cause a slightly greater effect on the membrane structure than alpha-MSH, in accord with its possible deeper penetration into the bilayer. CD spectra in aqueous solution and in the alpha-helix inducing solvent 2,2,2-trifluoroethanol showed that the two peptides have somewhat different structures in solution, though similar conformational changes occur in both peptides as a result of their interaction with negatively charged vesicles or micelles. The higher peptide-lipid association constant and the deeper penetration of the analog into lipid bilayers could be related to its greater activity and/or prolonged action.

Ortho-aminobenzoic acid as a fluorescent probe for the interaction between peptides and micelles.

Ortho-aminobenzoic acid (o-Abz) has been used as a fluorescent probe in internally quenched fluorescent peptides for continuous protease assays. We investigated the fluorescent properties of the probe in order to verify if it can be used to monitor the interaction of peptides with micelles. Abz-aminoacyl-monomethyl amides (Abz-Xaa-NHCH(3), where Xaa=Arg, Phe, Leu and Glu) were synthesized. Quantum yield, spectral position, anisotropy and lifetime decay were analyzed in the presence and absence of sodium dodecyl sulfate (SDS) micelles. Significant changes in the fluorescence parameters were observed for Abz-Arg-NHCH(3) in comparison to Abz-Glu-NHCH(3), indicating a strong electrostatic component in the compound's interaction with the negative charged micelles. The change in fluorescence parameters, observed when the probe is bound to hydrophobic amino acids Abz-Phe-NHCH(3) and Abz-Leu-NHCH(3), is probably due to insertion of those compounds into micelles. Abz-NHCH(3) fluorescence is less affected by the presence of micelles, indicating that the occurrence of interaction is dependent on the properties of the amino acid to which the fluorophore is attached. The quenching data with acrylamide confirmed these results. Titration curves allowed the estimation of association constants between Abz compounds and SDS, according to a single partition model. Although the results cannot be strictly applied to the titration with charged compounds, it was verified that the association constant for the isolated Abz-NHCH(3) is significantly lower than those for Abz-Phe-NHCH(3) and Abz-Leu-NHCH(3). It is concluded that the Abz group is a sensitive and convenient fluorescent probe to monitor peptide binding to amphiphilic aggregates. That conclusion is supported by measurements with the peptide Abz-Leu-Arg-Phe-NH(2).

Structural and thermal characterization of dioctadecyldimethylammonium bromide dispersions by spin labels.

Dioctadecyldimethylammonium bromide (DODAB) dispersions obtained by simply mixing the amphiphile in water, and by bath-sonication, were investigated by electron spin resonance (ESR) of stearic acids and their methyl ester derivatives, labeled at the 5th and 16th carbons of the acyl chain. The ESR spectra indicate that the non-sonicated dispersions are formed mainly by one population of DODAB vesicles, either in the gel (TT(m)) state. Around T(m) there is a co-existence of the two phases, with a thermal hysteresis of about 3.2 degrees C. In sonicated DODAB dispersions, spin labels indicate two different environments even for temperatures far below T(m): one similar to that obtained with non-sonicated samples, a gel phase, and another one in the liquid-crystalline state. The fluid phase domain present below T(m) could correspond to either the periphery of bilayer fragments, reported to be present in sonicated DODAB dispersions, or to high curvature vesicles.

Effect of aggregation on the kinetics of autoxidation of the polyene antibiotic amphotericin B.

We have previously studied the autoxidation of the polyene antibiotic amphotericin B (AB). In this paper we describe the dependence of the kinetics of autoxidation on the aggregation state of the antibiotic. Autoxidation, which is involved in drug inactivation and has been suggested to play a role in the mechanism of drug action, was assessed through the reaction of formed radicals with the spin label Tempol (2,2,6,6-tetramethyl-4-hydroxy-N-oxylpiperidine) by following the loss of the electron spin resonance signal, as previously described, and by oxygen consumption. Two types of AB (I and II) were used, the former being obtained by further purification of the latter. The kinetics of autoxidation were compared for aggregates formed by the antibiotic. Differences in aggregation state for both type I and type II AB were observed between monomeric, borax-complexed, and preparations in water containing variable proportions of dimethyl sulfoxide (DMSO) by optical absorption and circular dichroism (CD) spectra. On the other hand, although the suspensions of type I and type II AB in water-10% DMSO did not differ in their optical properties, they could be distinguished by quasielastic light scattering experiments, type II yielding smaller aggregates. It is proposed that the lack of difference in optical and CD spectra are due to the similarity of the microenvironments in both aggregates. In contrast, the borax complex of both type I and type II AB yielded similar optical and CD spectra and quasielastic light scattering behavior, indicating that complexation led to similar aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and time dependence of amphotericin B: deoxycholate aggregation by quasielastic light scattering.

Quasielastic light scattering measurements of amphotericin B (AB):deoxycholate (DOC) preparations provided information about particle size and aggregation as a function of concentration. The data allowed the time dependence of the aggregation to be followed and indicated that the initial rates of the change in average equivalent hydrodynamic diameter increased with decreasing concentration. The results extend the model proposed by Lamy-Freund and co-workers, which describes AB:DOC systems as consisting of AB:DOC mixed aggregates co-existing with pure DOC micelles. Although the AB:DOC aggregates are unstable at all concentrations studied, the rate of aggregation increases by three orders of magnitude as the concentration is reduced from 20 mM (DOC concentration) to the concentration region of DOC micellization. These results are in agreement with the different distribution of AB and DOC in the body of experimental animals, and may be of relevance for the understanding of the serious toxic effects of AB.

Mechanism of inactivation of the polyene antibiotic amphotericin B. Evidence for radical formation in the process of autooxidation.

Radical formation during autooxidation of the polyene antibiotic amphotericin B was monitored by following the kinetics of decay of the ESR signal of a stable nitroxide. The kinetics were seen to depend both on antibiotic and on nitroxide concentration. The radicals formed were studied by spin trapping. Three preparations--clinical Fungizone, amphotericin B suspended in buffer, and amphotericin B in buffer - 10% dimethyl sulfoxide--yielded spin adducts of different nature and/or concentrations. In the absence of dimethyl sulfoxide, amphotericin B yielded at least two carbon-centered radicals whose spectra indicated restricted mobility. This suggests that the radicals arise from the whole amphotericin B molecule located in molecular aggregates present in the preparations, and not from smaller and possibly more water-soluble fragments of the antibiotic. In the presence of dimethyl sulfoxide the spin adducts derived from secondary carbon radicals originated from this solvent. Their spectra were indicative of fast tumbling. Direct evidence for autooxidation was obtained by measuring oxygen consumption. All processes examined occurred at the same time scale observed for drug inactivation, in agreement with the idea that loss of activity is due to antibiotic autooxidation.

Role of soft and hard aggregates in the thermodynamics of lipid dispersions.

We study the thermodynamics of a two-dimensional polydisperse ideal gas model of different species of aggregates. We show that if these aggregates are distinguished not only by their sizes but also by their ability to display shape fluctuations, the system presents dominance of one or other species, depending on the temperature region. This result, which emerges solely from the statistics of the model in total absence of interaggregate interactions, describes well the observed temperature dependence of light scattering in dispersions of dimyristoyl phosphatidylglycerol, a negatively charged lipid.

High melatonin solubility in aqueous medium.

The pineal hormone melatonin (5-methoxy-N-acetyl-tryptamine) has been reported to participate in important physiological processes. Although some of its biological actions seem to depend on a protein receptor at the membrane surface, melatonin is known to interact with a large variety of tissues and cells, suggesting that the molecule may not necessarily interact through a specific membrane receptor at a specific cell. Most discussions of melatonin activity have assumed that the molecule is highly hydrophobic. Contrary to belief, the present work shows that melatonin is soluble in a purely aqueous medium up to 5 x 10(-3) M and describes a new method of melatonin preparation which shows the high hydrophilicity of the molecule. The results presented will affect the current biological hypothesis on the need of a melatonin carrier in the blood stream or the mechanisms which allow the hormone to cross the cell membrane and interact at the level of the nucleus.

Spin label and 2H-NMR studies on the interaction of melanotropic peptides with lipid bilayers.

The interaction of the cationic tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more active analog [Nle4, DPhe7]-alpha-MSH with lipid membranes was investigated by means of ESR of spin probes incorporated in the bilayer, and NMR of deuterated lipids. All spin labels used here, stearic acid and phospholipid derivatives labeled at the 5th and 12th position of the hydrocarbon chain, and the cholestane label, incorporated into anionic vesicles of DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) in the liquid-crystalline phase, indicated that both peptides decrease the motional freedom of the acyl chains. No peptide effect was detected with neutral lipid bilayers. Changes in the alpha-deuteron quadrupolar splittings and spin lattice relaxation time of DMPG deuterated at the glycerol headgroup paralleled the results obtained with ESR, showing that the peptides cause a better packing both at the headgroup and at the acyl chain bilayer regions. The stronger effect caused by the more potent analog in the membrane structure, when compared to the native hormone, is discussed in terms of its larger lipid association constant and/or its deeper penetration into the bilayer.

Permeability of pure lipid bilayers to melatonin.

Melatonin, the chief hormone of the pineal gland, has been reported to interact with a variety of different cells. This ubiquitously acting hormone has been found to interact with protein receptors both at the cell membrane and in the nucleus. Moreover, melatonin was recently shown to be a very potent hydroxyl radical scavenger. The present work focuses on the interaction of melatonin with pure lipid bilayers. It is shown that melatonin can cross multilamellar lipid vesicles, which are used here as model systems for the lipid phase of biological membranes. Thus, the data prove that melatonin can easily pass through the cell membrane and bath every part of the cell, as previously suggested in the literature. Melatonin lipid association constant was calculated based on the change of the hormone fluorescence intensity due to its penetration into the hydrophobic lipid phase. Though melatonin was recently shown to be highly soluble in aqueous media, its lipid association constant is rather high, indicating that the biological action of the hormone is likely to be at the membrane level, either via its interaction with membrane receptors, and/or as a lipoperoxidation radical scavenger.

How bradykinin alters the lipid membrane structure: a spin label comparative study with bradykinin fragments and other cations.

Electron spin resonance spectroscopy of several different spin labels was used to comparatively study the interaction of the cationic peptide hormone bradykinin (BK; Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), and some BK fragments (des-Arg(9)-BK, des-Arg(1)-BK, and Arg-Pro-Pro-Gly-Phe or BK(1-5)), with anionic vesicles of dimyristoyl phosphatidylglycerol (DMPG). For temperatures above the lipid gel-liquid crystal thermal transition (T(m) approximately 20 degrees C), membrane-incorporated spin labels indicated that all peptides (total concentration of 10 mol % relative to lipid) interact with the bilayer, turning the membrane less fluid, both at its surface and center, suggesting a partial penetration of the peptides into the membrane core. However, in the lipid gel phase (t < T(m)), BK was found to display a much stronger interaction with the membrane, decreasing the bilayer fluidity. At temperatures around 15 degrees C the BK-DMPG system was found to present a hysteresis, evinced by the different electron spin resonance spectra yielded upon cooling and heating the sample. System reversibility was found at all other temperatures (0-45 degrees C). That effect could not be assigned to the BK higher concentration at the membrane surface, due to its higher net charge (2(+)) compared to the fragments (1(+)), because ten times more des-Arg(9)-BK (100 mol %) yielded opposite result. Further, that was found to be a result rather different from those elicited by the other cations tested: the monovalent Na(+), the divalent Zn(2+), and the peptide pentalysine. The data presented here are discussed in the light of the different BK and BK fragments biological activities.