Central Asian cobra venom cytotoxins-induced aggregation, permeability and fusion of liposomes

The processes of membrane aggregation, permeability and fusion induced by cytotoxins from Central Asian cobra venom were investigated by studying optical density of liposome samples, permeability of liposome membranes for ferricyanide anions and exchange of lipid material between the membranes of adjacent liposomes. Cytotoxins Vc5 and Vc1 were found to induce aggregation of PC + CL and PC + PS liposomes. Cytotoxin Vc5 increased also the permeability of the liposomes for K3[Fe(CN)6] and enhanced their fusion. Cytotoxin Vc1 increased membrane permeability and enhanced fusion of PC + CL samples only. The changes in membrane permeability and fusion were found to occur within a single value of cytotoxin concentrations. The fusogenic properties of the cytotoxins studied are supposed to be due to the ability to dehydrate membrane surface and to destabilize the lipid bilayer structure. Fusion probability is largely defined by the phospholipid composition of the membranes. A model of interaction of cytotoxins with cardiolipin-containing membranes is offered.     

Perturbation of binary phospholipid mixtures by melittin: a fluorescence and raman spectroscopy study

The effect of melittin on different binary mixtures of phospholipids has been studied by polarization of DPH fluorescence in order to determine if melittin can induce phase separation. Since the interaction between lipids and melittin is sensitive to both electrostatic and hydrophobic forces, we have studied the effect of the acyl chain length and of the polar head group of the lipids. In spite of the difference of the chain length between dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), no phase separation occurs in an equimolar mixture of these lipids in presence of melittin. However, when the charged lipid dipalmitoylphosphatidylglycerol (DPPG) is mixed with either DPPC or DSPC, the addition of melittin leads to phase separation. The DSPC/DPPG/melittin system, which shows a very complex thermotropism, has also been studied by Raman spectroscopy using DPPG with deuteriated chains in order to monitor each lipid independently. The results suggest that the higher affinity of melittin for DPPG leads to a partial phase separation. We propose the formation of DPPG-rich domains perturbed by melittin and peptide-free regions enriched in DSPC triggered by the head group charge and chain-length differences.     

Protein rotational diffusion measurements on the interaction of bee venom melittin with bacteriorhodopsin in lipid vesicles

The rotational diffusion of bacteriorhodopsin reconstituted into dimyristoylphosphatidylcholine vesicles was measured by the technique of flash-induced transient dichroism. In the presence of melittin, a cell lysing peptide from honey bee (Apis mellifera) venom, dose-dependent loss of rotational mobility was observed. Chemically modified melittin derivatives, in which free amine groups were either acetylated or succinylated, were impaired in their ability to induce immobilisation of bacteriorhodopsin. Bacteriorhodopsin reconstitutions of differing lipid/protein ratio were tested and it was found that the bacteriorhodopsin immobilisation phenomena depended on the melittin/protein ratio, not the melittin/lipid ratio. This suggests that melittin produces its effect via direct interaction with bacteriorhodopsin. A mechanism is proposed in which the aggregation of bacteriorhodopsin is induced by electrostatic attraction between its anionic surface moieties and the highly cationic C-terminal segment of melittin.     

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, Tm. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T greater than Tm, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. At low melittin content, phospholipid-to-peptide molar ratios, Ri greater than 30, heterogeneous systems are observed, the new structures coexisting with the original ones. For lipids in the fluid phase and Ri greater than 12, the complexes formed are large unilamellar vesicles of about 1300 +/- 300 A diameter and showing on freeze-fracture images rough fracture surfaces. For lipids in the gel phase, T less than Tm after passage above Tm, and for 5 less than Ri less than 50, disc-like complexes are observed and isolated. They have a diameter of 235 +/- 23 A and are about one bilayer thick; their composition corresponds to one melittin for about 20 +/- 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. For high amounts of melittin, Ri less than 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide co-micelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: vesicularization of multibilayers; fusion of small lipid vesicles; fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.     

A study of protein dynamics from anisotropy decays obtained by variable frequency phase-modulation fluorometry: internal motions of N-methylanthraniloyl melittin

Internal motions of melittin and its lipid complexes were studied by anisotropy decays determined by frequency-domain fluorometry. A covalent anthraniloyl probe was attached, probably to lysine-21. The emission spectra indicate that the anthraniloyl moiety is exposed to solvent in both monomeric and tetrameric forms and is present at the lipid-water interfacial region in the lipid complexes. The fluorescence intensity decay of melittin in solution and its lipid complexes was characterized by three lifetimes. The lifetimes were near 1-2 ns, 6-7 ns and 10 ns. At increased temperatures there was an increase in the amplitude of the intermediate lifetime and a decrease in that of the longer lifetime. For all the melittin systems, at least three correlation times were required to fit the anisotropy data. Of the three correlation times, the shortest correlation time represents the local motions of the probe, while the longest represents global motions of the whole molecule. The intermediate correlation time probably represents the dynamics of domains/helices within the molecule. The melittin monomer is highly flexible, with greater than 90% of its anisotropy being lost by the local motions. Even though it is well organized (greater than 75% helical), the tetramer is still a highly flexible molecule, with 70% of its anisotropy being lost by the local motions. The internal motions of melittin decrease upon binding to lipids and are sensitive to the phase state of the lipid complexes.     

Structural changes of liposome phospholipid packing induced by cytotoxin of the Central Asia cobra venom

Liposomes and proteoliposomes obtained from rat brain were used; structural changes induced by Vc5 cytotoxin (CT) from Central Asia cobra venom have been studied by the EPR method using spin probes (5-, 10-, or 12-doxylstearic acid). The addition of CT to liposome samples, containing spin probes resulted in the appearance of a new EPR signal in the initial spectrum (samples without CT), typical of probes with strongly retarded mobility. The presence of hydrophobic interaction between the CT molecules and spin labelled fat acids permits the assumption that CT molecules in liposomes trap both lipid probes and phospholipids localized in the reach of action of hydrophobic forces. CT may be supposed to induce formation in membranes of liposomes with domain structures. As a result of hydrophobic interaction with CT molecules both the phospholipid and lipid probe mobility in the domain is substantially less than that in liposome regions free of CT molecules. Due to this, a new signal appears in the initial EPR spectrum of the spin probes. An analysis of the dependence of the probe order parameter value on CT concentration in samples has suggested that CT act uniformly along the membrane lipid profile with a certain CT concentration range. At high concentrations CT molecules cannot penetrate the lipid region deep enough, due to mutual electrostatic repulsion and steric factors at membrane surface. As a result, structural changes involve regions adjacent to the membrane surface only.     

A deuterium and phosphorus-31 nuclear magnetic resonance study of the interaction of melittin with dimyristoylphosphatidylcholine bilayers and the effects of contaminating phospholipase A2

The interaction of bee venom melittin with dimyristolphosphatidylcholine (DMPC) selectively deuteriated in the choline head group has been studied by deuterium and phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy. The action of residual phospholipase A2 in melittin samples resulted in mixtures of DMPC and its hydrolytic products that underwent reversible transitions at temperatures between 30 and 35 degrees C from extended bilayers to micellar particles which gave narrow single-line deuterium and phosphorus-31 NMR spectra. Similar transitions were observed in DMPC-myristoyllysophosphatidylcholine (lysoPC)-myristic acid mixtures containing melittin but not in melittin-free mixtures, indicating that melittin is able to stabilize extended bilayers containing DMPC and its hydrolytic products in the liquid-crystalline phase. Melittin, free of phospholipase A2 activity, and at 3-5 mol% relative to DMPC, induced reversible transitions between extended bilayers and micellar particles on passing through the liquid-crystalline to gel phase transition temperature of the lipid, effects similar to those observed in melittin-acyl chain deuterated dipalmitoylphosphatidylcholine (DPPC) mixtures [Dufourc, E. J., Smith, I. C. P., & Dufourcq, J. (1986) Biochemistry 25, 6448-6455]. LysoPC at concentrations of 20 mol% or greater relative to DMPC induced transitions between extended bilayers and micellar particles with characteristics similar to those induced by melittin. It is proposed that these melittin- and lysoPC-induced transitions share similar mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification from bee venom of melittin devoid of phospholipase A2 contamination

Conditions for the inactivation of phospholipase A₂ which contaminates melittin preparations were studied. A method for the purification of that peptide from bee venom is proposed. It gives, with a high recovery, a product devoid of phospholipase A₂ activity. In the first step, the venom is fractionated by gel filtration. Then the phospholipase A₂ still present in the melittin fraction is destroyed by sequential sulfitolysis and cyanogen bromide cleavage. This leaves the melittin intact. The final cation-exchange chromatography yields an homogeneous melittin preparation as analyzed by gel filtration, reverse-phase HPLC, and amino acid analysis.     

Incorporation of highly purified melittin into phosphatidylcholine bilayer vesicles

Melittin free of phospholipase A2 was prepared. In the absence of salt this highly pure protein starts to aggregate in solution at a protein concentration of Cp greater than 10(-3) M. In high salt solution (2 M) aggregation starts at Cp greater than 10(-6) M. This was determined from the blue shift of the intrinsic fluorescence of the protein. Reinvestigation of the quenching behaviour clearly shows that self-aggregation cannot be deduced from quenching experiments using nitrate or 2,2,6,6-tetramethylpiperidine-1-oxyl as quencher. The incorporation of melittin into phosphatidylcholine bilayer vesicles was studied by fluorescence quenching and by energy-transfer experiments using 2- and 6-anthroyloxypalmitic acid as acceptor and peptide tryptophan as donor. Incorporation of melittin into small unilamellar vesicles was found to be reduced below the lipid phase transition temperature, Tt, whereas it incorporates and distributes more randomly above Tt. Cooling the temperature below Tt after incubation at T greater than Tt leads to a deeper incorporation of the peptide into the lipid bilayer due to electrostatic interaction between the lipid phosphate groups and the positively charged amino acids. This stabilizing effect is lost above Tt and melittin is extruded to the polar phase. Quenching experiments support this finding. EPR measurements clearly demonstrate that even in the presence of high amounts of melittin up to 10 mol% with respect to the lipid broadening of the phase transition curves was only observed with fatty acid spin labels, where the doxyl group is localized near the bilayer surface. The order degree of the inner part of the bilayer remains almost unchanged even in the presence of high melittin content.     

Bilayer-stabilizing properties of myelin basic protein in dioleoylphosphatidylethanolamine systems

31P-NMR and X-ray diffraction techniques are used to study the comparative ability of myelin basic protein (MBP) vs. other basic proteins to convert hexagonal (HII) phases to stable lamellar (L alpha) structures. Pure dioleoylphosphatidylethanolamine (DOPE) at pH 9 and 7, and mixtures of DOPE/phosphatidylserine (PS) (95:5 and 80:20% w/w) at pH 7 were employed for this investigation. The polymorphic behavior of the lipid suspensions was evaluated in the presence and absence of several basic proteins (MBP, calf thymus histone, lysozyme, melittin) and the cationic polypeptide, polylysine (PL). Each of the proteins and PL was capable of binding the pure DOPE HII phase at pH 9 but with varying morphological consequences, i.e., lamellar stabilization (MBP, histone, PL), formation of new protein-DOPE HII phases (lysozyme) or lipid disordering/vesiculation (melittin). Reduction to pH 7 resulted in the dissociation of protein from DOPE - with the exception of melittin - and the reformation of a pure lipid HII phase. Additions of PS to DOPE at pH 7 facilitated protein binding, but among the proteins examined, only MBP was capable of converting the lipid suspension into a stable multilamellar form. Differences in the lipid morphology produced by each protein are discussed in terms of protein physicochemical characteristics. In addition, a possible relationship between MBP-lipid interactions and the stability of myelin sheath lipid multilayers is inferred from the significant bilayer-stabilizing capacity of MBP.     

Melittin binding to mixed phosphatidylglycerol/phosphatidylcholine membranes

The binding of bee venom melittin to negatively charged unilamellar vesicles and planar lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) was studied with circular dichroism and deuterium NMR spectroscopy. The melittin binding isotherm was measured for small unilamellar vesicles containing 10 or 20 mol % POPG. Due to electrostatic attraction, binding of the positively charged melittin was much enhanced as compared to the binding to neutral lipid vesicles. However, after correction for electrostatic effects by means of the Gouy-Chapman theory, all melittin binding isotherms could be described by a partition Kp = (4.5 +/- 0.6) x 10(4) M-1. It was estimated that about 50% of the total melittin surface was embedded in a hydrophobic environment. The melittin partition constant for small unilamellar vesicles was by a factor of 20 larger than that of planar bilayers and attests to the tighter lipid packing in the nonsonicated bilayers. Deuterium NMR studies were performed with coarse lipid dispersions. Binding of melittin to POPC/POPG (80/20 mol/mol) membranes caused systematic changes in the conformation of the phosphocholine and phosphoglycerol head groups which were ascribed to the influence of electrostatic charge on the choline dipole. While the negative charge of phosphatidylglycerol moved the N+ end of the choline -P-N+ dipole toward the bilayer interior, the binding of melittin reversed this effect and rotated the N+ end toward the aqueous phase. No specific melittin-POPG complexes could be detected. The phosphoglycerol head group was less affected by melittin binding than its choline counterpart.     

A high-sensitivity differential scanning calorimetric study of the interaction of melittin with dipalmitoylphosphatidylcholine fused unilamellar vesicles

High-sensitivity differential scanning calorimetry has been used to examine the interaction of bee venom melittin with dipalmitoylphosphatidylcholine fused unilamellar vesicles. Experiments were performed under conditions for which melittin in solution is either monomeric (in low salt) or tetrameric (in high salt). It was found that under both sets of conditions melittin abolishes the pretransition at a relatively high lipid-to-protein molar incubation ratio, Ri (about 200) and that at intermediate values of Ri it broadens the main transition profile and reduces the transition enthalpy. This provides evidence which suggests that melittin is at least partially inserted into the apolar region of the bilayer. Evident at low values of Ri are two peaks in the lipid thermal transition profiles, which may arise from a heterogeneous population of lipid vesicles formed through fusion induced by melittin, or by lipid phase separation. For those profiles which exhibited only one peak, transition enthalpies, normalized to those of the lipid in the absence of the protein, are plotted vs. the bound protein-to-lipid molar ratios for the experiments performed under the conditions which give monomeric and tetrameric melittin in solution. These plots yield straight lines, the slopes of which give the number of lipid molecules each protein molecule excludes from participating in the phase transition. These were found to be 9.9 +/- 0.7 and 4.1 +/- 0.5 for monomeric and tetrameric melittin, respectively. The results are discussed in terms of possible models for the binding of melittin to phospholipid vesicles. For simple hexagonal packing of lipid molecules, incorporation as an aggregate is favored when melittin is tetrameric in solution, whereas incorporation as a monomer is favored when melittin is monomeric in solution. For low-salt solutions, evidence is obtained for the contribution of free melittin to lipid fusion, perhaps by the formation of protein bridges between apposed vesicles.     

Facilitation of phospholipase A2 activity by mastoparans, a new class of mast cell degranulating peptides from wasp venom

The effect of mastoparan, Ile-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-Lys-Ile-LeuNH2, and related peptides on the release of arachidonic acid from egg yolk lecithin liposomes, rat peritoneal mast cells, and cultured human fibroblasts was studied. In unsonicated liposomes, labeled with 1-stearoyl-2[1-14C]arachidonyl-sn-glycero-3-phosphocholine, 5 X 10(-5) M mastoparan caused a 12-, 15-, and 50-fold increase in the production of arachidonic acid catalyzed by phospholipase A2 from bee venom, eastern diamondback rattlesnake and porcine pancreas, respectively. The stimulant effect of mastoparan and related peptides was dose-dependent and further enhanced by sonication of liposomes. In contrast, melittin, while stimulating the production of arachidonic acid by phospholipase from bee venom, was inactive with the rattlesnake and pancreatic enzymes. Melittin was also only weakly active with liposomes containing stearic acid in place of arachidonic acid. Like melittin, mastoparans stimulated phospholipase activity in tissue homogenates and caused a dose-dependent release of arachidonic acid from rat peritoneal mast cells and cultured human fibroblasts prelabeled with [14C]arachidonic acid. The heptapeptide fragments mastoparan 1-7 and mastoparan 8-14, and succinylated mastoparan were ineffective. The results suggest that mastoparan and related peptides in insect venoms act, at least in part, by stimulating phospholipase activity.     

A molecular dynamics study of the bee venom melittin in aqueous solution, in methanol, and inserted in a phospholipid bilayer

The structural properties of melittin, a small amphipathic peptide found in the bee venom, are investigated in three different environments by molecular dynamics simulation. Long simulations have been performed for monomeric melittin solvated in water, in methanol, and shorter ones for melittin inserted in a dimyristoylphosphatidylcholine bilayer. The resulting trajectories were analysed in terms of structural properties of the peptide and compared to the available NMR data. While in water and methanol solution melittin is observed to partly unfold, the peptide retains its structure when embedded in a lipid bilayer. The latter simulation shows good agreement with the experimentally derived ³J-coupling constants. Generally, it appears that higher the stability of the helical conformation of melittin, lower is the dielectric permittivity of the environment. In addition, peptide-lipid interactions were investigated showing that the C-terminus of the peptide provides an anchor to the lipid bilayer by forming hydrogen bonds with the lipid head groups.     

Calcium-regulated interactions of human alpha-lactalbumin with bee venom melittin

Affinity chromatography, fluorescence and circular dichroism spectroscopy methods have been used to study the interaction of melittin, a 26-residue peptide from bee venom, with Ca2(+)-binding alpha-lactalbumin from human milk. It has been revealed that melittin binds to the apo- and acidic states of alpha-lactalbumin while the presence of Ca2+ makes the interaction essentially weaker. The association constant for the complex of melittin with apo-alpha-lactalbumin determined from spectropolarimetric melittin-titration data is 2 X 10(7) M-1. The complexation of alpha-lactalbumin with melittin decreases its affinity to Ca2+ by three orders of magnitude. The interaction of apo-alpha-lactalbumin with melittin causes some changes in the environment of its aromatic amino acid residues and drastically alters the conformation of melittin, increasing its alpha-helical content but leaving its single tryptophan residue accessible to water. In the case of the acidic state of alpha-lactalbumin the interaction does not induce an increase in alpha-helical content of melittin.     

Leakage of internal markers from erythrocytes and lipid vesicles induced by melittin, gramicidin S and alamethicin: a comparative study.

The membrane-disruptive capacities of melittin, derivatised melittins, alamethicin and gramicidin S have been compared for the human erythrocyte membrane and lipid vesicles of three different compositions (phosphatidylcholine, 85% phosphatidylcholine/15% phosphatidylserine, and a lipid analogue of the outer leaflet of the human erythrocyte membrane). The sensitivity to ionic strength, divalent metal ions and polylysine of release of fluorescent markers from liposomes and of haemoglobin from intact erythrocytes has been assayed. Acetyl melittin was found to he more effective than melittin in lysing phosphatidylcholine and phosphatidylcholine/phosphatidylserine vesicles, somewhat less effective in the lipid analogue and markedly less effective in lysing erythrocytes. Succinyl melittin was non-haemolytic, but was able to lyse lipid vesicles at a high concentration. Ca2+ inhibited melittin haemolysis at high ionic strength (150 mM NaCl), but produced a more complex response of stimulation followed by inhibition at low ionic strength. In lipid vesicles, Ca2+ either stimulated melittin lysis or was ineffective. Zn2+ exerted effects similar to Ca2+ with lipid vesicles at approx. 10-fold lower concentration except that a weak inhibition was observed for the erythrocyte membrane lipid analogue at high ionic strength. Polylysine strongly inhibited haemolysis by melittin at low ionic strength, but was ineffective or stimulatory in lipid vesicle lysis. High phosphate concentration also inhibited melittin haemolysis, but again no corresponding effect could he found in any of the lipid vesicle systems. These disparities between effects of melittin on erythrocytes and lipid vesicles support the proposal that melittin-protein interactions are of consequence to its haemolytic action. Similar experiments were performed with gramicidin S and alamethicin in order to compare their lytic properties with those of melittin. It was found that each lysin exhibited its own individual pattern of sensitivity to lipid composition, ionic strength and inhibition by cations. It thus appears likely that the detailed molecular interactions responsible for lysis are significantly different for each of these three agents.     

Investigation of toroidal pore and oligomerization by melittin using transmission electron microscopy

We studied the effects of melittin on various cell wall components and vesicles of various lipid compositions. To interact with the cytoplasmic membrane, melittin must traverse the cell wall, which is composed of oligosaccharides. Here, we found that melittin had a strong affinity for chitin, peptidoglycan, and lipopolysaccharide. We further examined the influence of lipid compositions on the lysis of the membranes by melittin. The result showed that melittin bound better to negatively charged than to zwitterionic lipid vesicles but was more potent at inducing leakage from zwitterionic lipid vesicles. Our studies further indicated that the oligomeric state of melittin varied between tetramers and octamers during the formation of toroidal pores. Dextran leakage experiments confirmed the formation and dimension of these toroidal pores. Finally, transmission electron microscopy revealed that melittin formed pores via peptide oligomerization by the toroidal pore-forming mechanism. The toroidal pores composed of 7-8 nm diameter rings that encircled 3.5-4.5 nm diameter cavities on zwitterionic lipid vesicles.     

Modification of the lipid-protein interaction in human low-density lipoprotein destabilizes ApoB-100 and decreases oxidizability

The interactions of the lipid and protein moiety of human low-density lipoprotein (LDL) and their influence on the oxidation behavior of LDL were modified using an amphipathic peptide, melittin, as a probe. The interaction of melittin with the LDL phospholipid surface resulted in a destabilization of apolipoprotein B-100 (apoB-100) as monitored by differential scanning calorimetry, while the characteristics of lipid core melting remained nearly unchanged. Binding of melittin caused a restriction of lipid chain mobility near the glycerol backbone, but not in the middle or near the methyl terminus of the fatty acyl chains as observed by electron paramagnetic resonance. Also, upon melittin addition, the level of copper binding to apoB-100 and the oxidizability of LDL by Cu2+ ions were greatly reduced, as indicated by abolished tryptophan fluorescence quenching upon Cu2+ binding and, during oxidation, prolongation of the lag phase of oxidation, attenuated consumption of alpha-tocopherol, and a lowered maximal rate of conjugated diene formation. This reduction of oxidizability could not be reversed by increasing the Cu2+ concentration. It is deduced that interaction of Cu2+ and alpha-tocopherol is required for reductive activation of the metal. It can be abolished by interfering with the interactions between apoB-100 and the lipid moiety of LDL which modifies the conformation of LDL and, as a consequence, hinders copper binding to apoB-100.     

Effective charge of melittin upon interaction with POPC vesicles

The binding of bee venom melittin to small unilamellar vesicles and large nonsonicated multilamellar bilayer membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied by means of circular dichroism, 31P-NMR and electrophoretic mobility. The melittin binding isotherm for small unilamellar vesicles (SUV) could be described by a partition equilibrium with Kp = (6 +/- 1).10(4) M-1. Electrostatic effects were taken into account by means of the Gouy-Chapman theory. Combining the partition equilibrium with the Gouy-Chapman analysis suggested an effective charge for melittin of Zp = 1.9, which is lower than the true electric charge of 5-6. The variation of the 31P-NMR signal of SUV showed the change in potential at the phosphodiester moiety of the lipid upon addition of melittin. This potential change was lower than that for an ion with an electrical charge of 5-6 and corresponded to a charge of 1.5. Electrophoretic mobility measurements with multilamellar vesicles confirmed the charge reduction effect. These experimental results show that the use of the simple Gouy-Chapman theory requires an effective electrical charge of the melittin which is lower than the formal charge.     

Study of the effect of melittin on the thermotropism of dipalmitoylphosphatidylcholine by Raman spectroscopy

The effect of amphiphilic toxin melittin (Mel) on the thermotropic behavior of dipalmitoylphosphatidylcholine (DPPC) has been studied by Raman spectroscopy. The spectra show that for complexes that were incubated above 40 degrees C, melittin does not penetrate DPPC bilayers in the gel state as an intrinsic protein since the conformation of the lipid acyl chains is just slightly perturbed by the toxin. Instead, at the DPPC/Mel molar ratios investigated (Ri = 5 and 15), Raman results suggest the formation of discoidal particles as complexes of apolipoproteins with phosphatidylcholines. These lipid/protein assemblies are characterized by a high conformational order, low intermolecular chain-chain interactions due to the size of the particles, and a low cooperativity of the gel to liquid-crystalline transition. The latter is biphasic for samples studied. It is believed that aggregation of these particles into larger ones occurs when the bilayers become less stable at higher temperature and that melittin is partially embedded into the hydrophobic core of the larger lipid/protein units. The freezing of the dispersion at approximately 0 degrees C also causes a reversible aggregation of the particles that leads to the formation of domains in which the interchain interactions are very similar to that of the pure lipid. The small particles of DPPC/Mel are also metastable, and with time, they form larger aggregates from which melittin is expulsed.