Studies on the aggregation behaviour of pegylated human red blood cells with the Zeta sedimentation technique.

Covalent binding of poly(ethylene glycol), abbreviated as PEG, to red blood cells (RBC) surface leads to masking of the RBC blood group determinants and the PEG layer on the cell surface sterically hinders RBC-RBC and RBC-plasma protein interactions. We cross-linked linear mPEG-SPA of various molecular mass (2000, 5000, 20000) to washed human RBC under varying incubation ratios polymer to RBC. The electrophoretic mobility (EM) of the modified RBC decreases with increasing of chain length and concentration of PEG up to 50%. It may reflect the alteration in the surface layer thickness and friction. The aggregation behaviour of the pegylated RBC was studied with the Zeta sedimentation technique modifying the cell-cell interactions pressing them toward each other under centrifugal forces of various magnitudes. As a rule at low centrifugation forces the increase in chain length and concentration of PEG linked to RBC surface reduces the dextran-induced aggregation probably via elevation of the steric repulsion, which counteracts the depletion force generated by the free polymer. This effect was reversed to some extent by elevation of free dextran concentration and centrifugation forces. If cell-cell polymer bridging starts playing a role under these conditions requires further experimental and theoretical investigations.

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research.

The interest in the analysis of lipids and phospholipids is continuously increasing due to the importance of these molecules in biochemistry (e.g. in the context of biomembranes and lipid second messengers) as well as in industry. Unfortunately, commonly used methods of lipid analysis are often time-consuming and tedious because they include previous separation and/or derivatization steps. With the development of "soft-ionization techniques" like electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF), mass spectrometry became also applicable to lipid analysis. The aim of this review is to summarize so far available experiences in MALDI-TOF mass spectrometric analysis of lipids. It will be shown that MALDI-TOF MS can be applied to all known lipid classes and the characteristics of individual lipids will be discussed. Additionally, some selected applications in medicine and biology, e.g. mixture analysis, cell and tissue analysis and the determination of enzyme activities will be described. Advantages and disadvantages of MALDI-TOF MS in comparison to other established lipid analysis methods will be also discussed.

Fusion, leakage and surface hydrophobicity of vesicles containing phosphoinositides: influence of steric and electrostatic effects.

Calcium and lanthanum ion-induced fusion of lipid vesicles containing phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP2) or phosphatidylinositol-3,4,5-trisphosphate (PIP3) and its associated membrane properties, e.g., surface dielectric constant and vesicle leakage, were studied by fluorescence methods. The presence of poly-phosphorylated phosphoinositides (PPI) in lipid vesicles enhanced fusion, depending on the PPI phosphorylation level and the PPI concentration, as determined by the lipid mixing assay. This correlation held even at physiologically relevant small concentrations of PPI in vesicle membranes. However, the presence of nonphosphorylated PI inhibited fusion due to the steric effect of the inositol ring. The cation threshold concentration for the lipid mixing of vesicles made of mixtures of phosphatidylserine (PS) with PI increased with increasing PI contents. For all vesicle systems studied, a decrease in vesicle surface dielectric constant and an increase in vesicle leakage accompanied fusion. The presence of the nonphosphorylated inositol ring in PI did not interfere with the changes in the surface dielectric constant caused by fusogenic cations. Therefore, we deduce that the reduction of the surface dielectric constant is a necessary condition for membrane fusion to occur but it does not correlate with membrane fusion when interacting membranes are blocked for close approach as by the nonphosphorylated inositol ring.

CsCl as an auxiliary reagent for the analysis of phosphatidylcholine mixtures by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) is mainly used for protein and peptide analysis. However, there is growing evidence that also phospholipids like phosphatidylcholines (PC) can be easily analyzed by MALDI-TOF MS. In MALDI-TOF methodology, the sample is cationized by the addition of inorganic ions. This process is strongly dependent on the corresponding ion concentration. In biological samples various cations are present (mainly H+, Na+ and K+) and, therefore, a mixture of different adducts is formed. Since phospholipids exhibit a wide distribution of different fatty acid residues a considerable peak overlap may occur. This is a major problem since the peak assignment in a mixture will be often unclear. In this paper we demonstrate that this problem can be easily overcome by mixing the analyte with caesium chloride (CsCl). This yields naturally non-occurring Cs+ adducts that are apparent due to the large shift of the molecular mass. The proposed method facilitates the clear assignment of most peaks. Besides that, we will show that CsCl can also be used for the determination of the relative fatty acid composition of a given PC sample. For this purpose naturally occurring mixtures of PCs as well as organic extracts of human lipoproteins-that are mainly composed of PC and sphingomyeline-are used.

Lipid analysis of human HDL and LDL by MALDI-TOF mass spectrometry and (31)P-NMR.

The analysis of HDL and LDL is important for the further understanding of atherosclerosis because changes of the protein and lipid moieties occur under pathological conditions. Because destruction of lipids leads to the formation of well-defined products such as lysophospholipids or chlorohydrins, methods that allow their fast and reliable determination would be useful. In this study, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for the analysis of the lipid composition of human lipoproteins. These data were compared with high resolution (31)P-NMR spectroscopy. Differences between LDL and HDL in sphingomyelin and phosphatidylcholine content could be monitored by NMR and mass spectrometry, and differences with respect to the extraction efficiency were found by MALDI-TOF MS. Additionally, treatment of LDL with hypochlorite and phospholipase A(2) resulted in marked changes (formation of chlorohydrines and lysolipids). Lysophosphatidylcholines were detectable by both methods, whereas MALDI-TOF MS failed to detect chlorohydrines of phospholipids. We conclude that MALDI-TOF MS provides rapidly a reliable lipid profile of lipoproteins. However, a previous lipid separation must be performed to detect lipid oxidation products. NMR can be directly applied, but suffers from lower sensitivity, and provides only limited information on fatty acid composition.

Interaction of Zn2+ with phospholipid membranes.

To characterize the specificity of zinc binding to phospholipid membranes in terms of headgroup structure, hydration and phase behavior we studied the zwitterionic lipid 1-palmitoyl-2-oleoyl-phosphatidylcholine as a function of hydration at 30 degreesC in the presence and absence of ZnCl2. Zinc forms a 2:1-1:1 complex with the lipid, and in particular with the negatively charged phosphate groups. Zn2(+)-bridges between neighboring lipid molecules stabilize the gel phase of the lipid relative to the liquid-crystalline state. Upon Zn2+ binding the C-O-P-O-C- backbone of the lipid headgroup changes from a gauche/gauche into the trans/trans conformation and it loses roughly 50% of the hydration shell. The ability of the Zn2(+)-bound phosphate groups to take up water is distinctly reduced, meaning that the headgroups have become less hydrophilic. The energetic cost (on the scale of Gibbs free energy) for completely dehydrating the lipid headgroups is decreased by approximately 10 kJ/mole in the presence of Zn2+. The interaction of phospholipid headgroups with Zn2+ is conveniently described by a hydrated zinc-phosphate complex the key energy contribution of which is more covalent than electrostatic in nature. Dehydration of phospholipid headgroups due to complexation with zinc cations is suggested to increase fusogenic potency of lipid membranes. Zinc appears to be one of the most potent divalent cation in inducing membrane fusion.

The effect of Zn(2+) on the secondary structure of a histidine-rich fusogenic peptide and its interaction with lipid membranes.

Membrane fusion between uncharged lipid vesicles can be triggered by the peptide sequence 'B18' from the fertilization protein 'bindin', but it only proceeds efficiently in the presence of Zn(2+) ions. We studied (i) the interaction of Zn(2+) with the fusogenic peptide B18, (ii) the binding of B18 to 1-palmitoyl-2-oleoylglycero-3-phosphocholine (POPC), and (iii) the ternary system POPC/B18/Zn(2+). The complex formation of Zn(2+) with the central histidine-rich motif of B18 appears to shift the secondary structure away from a beta-sheet towards an alpha-helical conformation. Here we observe for the first time an essentially alpha-helical structure of the peptide when immersed in POPC bilayers which appears to represent its functional fusogenic state. Infrared linear dichroism suggests a peripheral, oblique insertion mode of B18, mediated by the hydrophobic patches along one side of the amphipathic peptide. Furthermore, the hydration level of the peptide is reduced, suggesting that the hydrophobic region of the bilayer is involved in the lipid/peptide interactions. The hydration capacity of the POPC/B18/Zn(2+) system is distinctly smaller than that of POPC/Zn(2+) without peptide. The accompanying decrease in the number of tightly bound water molecules per lipid can be interpreted as a reduction in the repulsive 'hydration' forces, which usually prevent the spontaneous fusion of lipid vesicles. Binding of the B18 peptide in the presence of Zn(2+) effectively renders the membrane surface more hydrophobic, thus allowing fusion to proceed.

Association of lysozyme with phospholipid vesicles is accompanied by membrane surface dehydration.

Lysozyme is a globular protein which is known to bind to negatively charged phospholipid vesicles. In order to study the relationship between binding of the protein and the subsequent destabilization of the phospholipid vesicles a set of experiments was performed using phospholipid monolayers and vesicles. Using microelectrophoresis the binding of lysozyme to phospholipid vesicles made of PS was determined. At low ionic strength and mild acidic pH of the solution lysozyme reduced the magnitude of the negative zeta potential of PS vesicles at lower concentrations compared to neutral pH and high ionic strength. In contrast, the bound fraction of lysozyme to PS vesicles was nearly constant at acidic and neutral pH. At low pH, the binding of lysozyme was accompanied by a strong aggregation of the vesicles. Lysozyme binding to PS vesicles is accompanied by its penetration into the PL monolayer. This was measured by surface tension and film balance measurements at low pH and low ionic strength. The interaction of lysozyme with negatively charged vesicles lead to a decrease of the vesicle surface hydration as measured by the shift of the emission peak of the fluorescent probe DPE. The binding of bis-ANS increased at low pH after addition of lysozyme to the vesicles. This indicates that more hydrophobic patches of the lysozyme-PS complex are exposed at low pH. At low pH the binding process of lysozyme to PS vesicles was followed by an extensive intermixing of phospholipids between the aggregated vesicles, accompanied by a massive leakage of the aqueous content of vesicles.

Binding of phosphatidic acid to the protein-tyrosine phosphatase SHP-1 as a basis for activity modulation.

Activation of the SH2 domain-possessing protein-tyrosine phosphatase SHP-1 by acidic phospholipids as phosphatidic acid (PA) has been described earlier and suggested to participate in regulation of SHP-1 activity toward cellular substrates. The mechanism of this activation is poorly understood. Direct binding of phosphatidic acid to recombinant SHP-1 could be demonstrated by measuring the extent of [(14)C]PA binding in a chromatographic assay, by measuring the extent of binding of SHP-1 to PA-coated ELISA plates or silica beads (TRANSIL), and by spectroscopic assays employing fluorescently labeled PA liposomes. In addition to PA, phosphatidylinositol 3,4, 5-trisphosphate (PIP3), dipalmitoylphosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, and phosphatidylserine (PS) were found to bind to SHP-1, albeit to a lesser extent. A high-affinity binding site for PA and PIP3 was mapped to the 41 C-terminal amino acids of SHP-1. This site was absent from the related protein-tyrosine phosphatase SHP-2 and conferred activation of SHP-1 by PA toward two different substrates at low lipid concentrations. A SHP-1 mutant missing this binding site could, however, still be activated toward phosphorylated myelin basic protein as a substrate at high PA concentrations. This activation is likely to be mediated by a second, low-affinity binding site for PA in the N-terminal part of SHP-1 within the SH2 domains. High-affinity phospholipid binding to the C-terminus of SHP-1 may present a specific mechanism of regulating activity and/or cellular localization.

Ultrastructural characterization of peptide-induced membrane fusion and peptide self-assembly in the lipid bilayer.

The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn(2+), which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into beta-sheet amyloid fibrils, whereas in the presence of Zn(2+) it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn(2+) does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an alpha-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn(2+) is to promote this local helical structure in B18 and to prevent its inactivation as beta-sheet fibrils.

Investigation of phospholipid area compression induced by calcium-mediated dextran sulfate interaction.

The association of anionic polyelectrolytes such as dextran sulfate (DS) to zwitterionic phospholipid surfaces via Ca(2+) bridges results in a perturbation of lipid packing at physiologically relevant Ca(2+) concentrations. Lipid area compression was investigated in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar bilayer dispersions by (2)H-NMR and in monolayer studies. Binding of DS to DMPC surfaces via Ca(2+) results in denser lipid packing, as indicated by higher lipid chain order. DMPC order parameters are homogeneously increased throughout the lipid bilayer. Higher order translates into more extended hydrocarbon chains and decreased average lipid area per molecule. Area compression is reported as a function of DS concentration and molecular weight. Altering the NaCl and Ca(2+) concentrations modified electrostatic interactions between DS and phospholipid. A maximal area reduction of DeltaA = 2.7 A(2) per DMPC molecule is observed. The lipid main-phase transition temperature increases upon formation of DMPC/Ca(2+)/DS-complexes. Lipid area compression after addition of DS and Ca(2+) to the subphase was also observed in monolayer experiments. A decrease in surface tension of up to 3.5 mN/m at constant molecular area was observed. DS binds to the lipid headgroups by formation of Ca(2+) bridges without penetrating the hydrophobic region. We suggest that area compression is the result of an attractive electrostatic interaction between neighboring lipid molecules induced by high local Ca(2+) concentration due to the presence of DS. X-ray diffraction experiments demonstrate that DS binding to apposing bilayers reduces bilayer separation. We speculate that DS binding alters the phase state of low-density lipoproteins that associate with polyelectrolytes of the arterial connective tissue in the early stages of arteriosclerosis.

Annexin V interaction with phosphatidylserine-containing vesicles at low and neutral pH.

Annexin V belongs to a class of Ca2+-binding proteins for which different functions in the cell are discussed, e.g., involvement in exocytosis, inhibition of protein kinase C, and calcium channel activity in cartilage matrix vesicles. All these functions are related to the ability of the annexins to bind to acidic phospholipids. In this study, the interaction of annexin V with large unilamellar vesicles (LUV) prepared from phosphatidylserine (PS) at low pH was compared to that at neutral pH. Annexin V strongly binds to PS LUV at low pH, whereas at neutral pH 20-100 microM Ca2+ are required to induce binding. This is caused by the different electric charge of the protein. The binding ability of the PS LUV for annexin V is higher at low pH. Binding of annexin V induces dehydration of the vesicle surface and a decrease of the lateral diffusion within the bilayer. While this dehydration is due to vesicle contact at pH 4, at pH 7.4 it is due to surface covering by annexin V. Annexin V promotes the phospholipid intermixing between LUVs at pH 5 and below but inhibits it at pH 7.4. A substitution of up to 80% of the PS by the uncharged phosphatidylcholine does not impair the extent of phospholipid intermixing at pH 4. The high binding capacity of PS LUV, the disappearance of the inhibiting action, and a calculated increase of the annexin V hydrophobicity make it likely that annexin V is able to penetrate into the membrane at low pH. At neutral pH, annexin V molecules act as steric barriers, preventing close apposition of two vesicles. At pH 5, annexin V lowers the threshold concentration of the Ca2+-induced phospholipid intermixing. Such a promotion is well-known for annexin VII (synexin). The effect may be related to the isoelectric points of the two annexins which have been reported as 4.8 (annexin V) and 7.0 (annexin VII), respectively.

Beta-blockers inhibit the modification of low-density lipoproteins by sodium hypochlorite in vitro.

The effect of beta-blockers (alprenolol, oxprenolol, atenolol, acebutolol) and the non-steroidal anti-inflammatory drug, diclofenac, on modification of low-density lipoproteins (LDL) by sodium hypochlorite (NaOCl) was investigated in vitro. Beta-blockers and diclofenac inhibit the formation of thiobarbituric acid reactive substances in LDL modified by NaOCl. Beta-blockers, but not diclofenac, inhibit the hypochlorite-induced aggregation of LDL which was determined by photon correlation spectroscopy. The intracellular accumulation of cholesterol esters in J774 macrophages is inhibited by addition of beta-blockers, but not diclofenac, to LDL prior to the addition of NaOCl. The modification inhibiting effect of beta-blockers is inversely correlated to the binding capabilities of these substances to LDL which were assessed by laser electrophoresis. Inhibition of LDL modification in vivo by beta-blockers may reduce the risk of atherosclerosis and, therefore, compensate for the cholesterol-raising effect of these drugs in human plasma.

Characterization of dimyristoylphosphatidylcholine liposome aggregates induced by dextran sulfate and La(3+) by fluorescence spectroscopy.

The addition of dextran sulfate (DS) to DMPC vesicles in the presence of di- and trivalent cations leads to a strong aggregation, resulting in a stack-like arrangement of the opposing membrane surfaces as shown by freeze-fracture electron microscopy. The strong aggregation is connected with a lipid mixing process, especially in the presence of La(3+) (measured by the NBD/Rh assay). The extent of lipid mixing depends on the molecular weight of DS and size of the DMPC vesicles. Additionally, a decrease in the surface dielectric constant of DMPC vesicles [measured by the emission shift of the fluorescent probe, dansylphosphatidyl-ethanolamine (DPE)] was observed. A direct dependence on the molecular weight (MW) of DS exists: the higher their MW, the higher the blue emission shift of the DPE probe. The results are discussed in terms of the theory proposed by Ohki and Arnold, which connects the decrease of the surface dielectric constant with the interaction parameters of phospholipid membranes.

Ion-induced fusion of phosphatidic acid vesicles and correlation between surface hydrophobicity and membrane fusion.

Divalent cation (Ca2+ and Mg2+) and hydrogen ion-induced fusions of small unilamellar phosphatidic acid vesicles were studied by the use of fluorescence fusion assay. These fusogenic ions also increased interfacial tension and reduced the surface dielectric constant of phosphatidic acid membranes as the ion concentration increased. A good correlation was found between the threshold of vesicle fusion and the degree of changes in such membrane properties induced by these fusogenic ions. At the fusion threshold point, the increase in interfacial tension and the decreased surface dielectric constant of the membrane were approximately 6 and 14 dyn/cm, respectively, regardless of Ca2+, Mg2+ or H+. Such changes correspond to the increases in surface hydrophobicity of the membrane. As these fusogenic ionic concentrations increased, the electrophoretic mobility of the phosphatidic acid vesicle decreased, because of the binding of ions to negatively charged sites of the membranes. However, the quantities of the reduced negative surface charges were not necessarily in good correlation with the threshold of vesicle fusion. It is suggested that the complexes of the phosphate group with these fusogenic ions are responsible for increases in the surface hydrophobicity of the membrane, which is an important factor for membrane fusion.

Effect of cholesterol, diacylglycerol and phosphatidylethanolamine on PEG 6000 induced lipid mixing and surface dielectric constant of phosphatidylcholine vesicle.

Using the NBD/Rh fluorescence assay the PEG 6000 induced phospholipid mixing of phosphatidylcholine liposomes containing cholesterol, diacylglycerol or phosphatidylethanolamine was measured. All 3 components shifted the PEG 6000 concentration necessary to induce 50% of maximal phospholipid mixing to lower concentrations. After the addition of PEG cholesterol containing PC liposomes exhibit different values of the surface dielectric constant as measured by the stokes shift of the fluorophore dansyl-PE compared to pure PC, whereas in DAG- and PE-containing PC liposomes no differences were observed. It is concluded that the incorporation of cholesterol leads to a different surface dielectric constant after PEG addition. The changed surface dielectric properties are a prerequest for the onset of fusion, as shown by Ohki and Arnold (1990). The incorporation of DAG and PE into PC membranes leads to structural instabilities as proposed by Siegel et al. (1989). This additional structurally unstable region created by molecules like PE or DAG may shift the onset of fusion to lower PEG concentration.

Effect of glycosaminoglycans and PEG on fusion of Sendai virus with phosphatidylserine vesicles.

The fusion of Sendai virus with phosphatidylserine vesicles was monitored by a pyrene-phosphatidylcholine fluorescence assay. A strong influence of pH and ionic strength on the extent of fusion was observed. The negatively-charged polymers (dextran sulfate, heparin and chondroitin sulfate) inhibited the ability of the viruses to fuse with the liposomes. The extent of inhibition, for a given amount (w/v) of the polymers, was the greatest for dextran sulfate followed by heparin and chondroitin sulfate. The extent of inhibition depended on the pH and ionic strength of the solution; the lower the pH of the solution, the more effective the fusion inhibition by the polymers. The molecular weight of dextran sulfate (DS) influenced the inhibition effect, i.e., DS with higher molecular weight exhibited a stronger inhibition effect. The presence of sodium sulfate, even in excess concentration, had no inhibitory effect on fusion. On the other hand, PEG had an opposite effect on fusion compared to the negatively-charged polymers, and it decreased their inhibition effect when both were present in the same media. It is concluded that the inhibition of the fusion activity of Sendai virus results from the adherence of negatively-charged polymers to the virus surface preventing close contacts between the virus and liposome surface.

Influence of polar polymers on the apoprotein region of human serum lipoproteins: an electron paramagnetic resonance (EPR) study.

Electron spin resonance spectroscopy was used for measurements of the surface potential and apoprotein structure of LDL and HDL in the presence of Ca2+ and dextran sulfate, heparin and chondroitin sulfate. A decrease in the absolute values of surface potential of LDL and HDL was observed after addition of Ca2+. In the presence of the negatively charged macromolecules the measured surface potential was less reduced. The spectral properties of a maleimide spin label covalently attached to the apoprotein were changed under conditions of aggregation of LDL induced by dextran sulfate, chondroitin sulfate or heparin in the presence of Ca2+. In the HDL system this effect was only observed for dextran sulfate. The influence of PEG on the spectral parameters of the spin label is dependent on the molecular weight of the polymer. PEG 400 decreased the mobility of the spin-labelled apoprotein region of LDL, whereas PEGs with higher molecular weight only slightly increased the maleimide mobility. On the other hand, the maleimide-labelled apoprotein region of HDL showed a higher sensibility to all PEGs used. Addition of PEG leads to immobilization of apoprotein A.

Dextran sulfate-dependent fusion of liposomes containing cationic stearylamine.

The incorporation of the positively charged stearylamine into phosphatidylcholine liposomes was studied by measuring electrophoretic mobilities. Up to a molar ratio SA/PC = 0.5 an increase of the positive zeta potential can be observed. Addition of the negatively charged macromolecule dextran sulfate leads to a change of the sign of the surface potential of the PC/SA liposomes indicating binding of the macromolecule to the surface. This process is accompanied by an increase in turbidity, which is dependent on the molecular weight of the dextran sulfate and the SA concentration (measured by turbidimetry). Using the NBD/Rh and Pyr-PC fluorescence assays the fusion of SA containing liposomes was investigated. A strong influence of the SA content and molecular weight of dextran sulfate on the fusion extent was observed. The fusion extent is proportional to the SA content in the PC membrane and the molecular weight of dextran sulfate. PC/SA/PE liposomes exhibit a higher fusion extent after addition of dextran sulfate compared to PC/SA liposomes indicating that PE additionally destabilizes the bilayer. Freeze-fracture electron microscopy reveals that the reaction products are large complexes composed of multilamellar stacks of tightly packed, straight membranes and aggregated vesicles. The tight packing of the membranes in the stacks (and the narrow contact of the aggregated vesicles) indicates a strong adherence of opposite membrane surfaces induced by dextran sulfate.

Aggregation of human plasma high density lipoproteins induced by poly(ethylene glycol).

The influence of different surface charge densities (induced by varying pH, addition of positively charged amphiphilic molecules and chemical modification) of high density lipoproteins (isolated by ultracentrifugation) on poly(ethylene glycol) induced aggregation was studied. The effects of different molecular masses of PEG, HDL concentration and the presence of other serum proteins on the PEG mediated aggregation were investigated. The PEG concentration necessary for HDL aggregation is inversely proportional to the used HDL concentration and its molecular weight, and is directly proportional to the presence of other proteins and the magnitude of the negative surface charge density of HDL. The results are in accordance with the predictions of the volume exclusion theory taking into account the influence of repulsive electrostatic forces on the interaction of HDL particles.