Is trehalose special for preserving dry biomaterials?

Simple sugars, especially disaccharides, stabilize biomaterials of various composition during air-drying or freeze-drying. We and others have provided evidence that direct interaction, an interaction that we believe is essential for the stabilization, between the sugar and polar groups in, for example, proteins and phospholipids occurs in the dry state. Some researchers, however, have suggested that the ability of the sugar to form a glass is the only requirement for stabilization. More recently, we have shown that both glass formation and direct interaction of the sugar and headgroup are often required for stabilization. In the present study, we present a state diagram for trehalose glass and suggest that the efficacy of this sugar for stabilization may be related to its higher glass transition temperatures at all water contents. We also show that trehalose and trehalose:liposome preparations form trehalose dihydrate as well as trehalose glass when rehydrated with water vapor. Formation of the dihydrate sequesters water, which might otherwise participate in lowering the glass transition temperature to below ambient. Because samples remain in the glassy state at ambient temperatures, viscosity is high and fusion between liposomes is prevented.     

Effect of molecular structure and water content on the dielectric relaxation behaviour of amorphous low molecular weight carbohydrates above and below their glass transition

The dielectric relaxation behaviour of several amorphous low molecular weight carbohydrates and their 10% w/w water mixtures has been studied in the supercooled liquid and glassy regions in the frequency range 100 Hz to 100 kHz. The dry carbohydrates show a primary alpha-relaxation (activation energy 250-405 kJ mol(-1)) at temperatures above the calorimetric glass transition temperature, Tg, and, in most cases, a secondary beta-relaxation (activation energy 42-55 kJ mol(-1)) at sub-Tg temperatures. Whilst D-mannose showed a beta-relaxation similar in strength to D-glucose, its deoxy sugar, L-rhamnose showed a relatively weak beta-relaxation. This indicates that the hydroxymethyl group influences relaxation in carbohydrate glasses. Addition of water shifted the alpha-relaxations to lower temperatures and increased the strength of the beta-relaxations. In glucitol this resulted in a merging of the alpha- and beta-relaxations. The beta-relaxation increased in strength and decreased in temperature for the series of water mixtures: D-glucose, maltose, and maltotriose.     

Characterization of molecular mobility in seed tissues: an electron paramagnetic resonance spin probe study.

The relationship between molecular mobility (tauR) of the polar spin probe 3-carboxy-proxyl and water content and temperature was established in pea axes by electron paramagnetic resonance (EPR) and saturation transfer EPR. At room temperature, tauR increased during drying from 10(-11) s at 2.0 g water/g dry weight to 10(-4) s in the dry state. At water contents below 0.07 g water/g dry weight, tauR remained constant upon further drying. At the glass transition temperature, tauR was constant at approximately 10(-4) s for all water contents studied. Above Tg, isomobility lines were found that were approximately parallel to the Tg curve. The temperature dependence of tauR at all water contents studied followed Arrhenius behavior, with a break at Tg. Above Tg the activation energy for rotational motion was approximately 25 kJ/mol compared to 10 kJ/mol below Tg. The temperature dependence of tauR could also be described by the WLF equation, using constants deviating considerably from the universal constants. The temperature effect on tauR above Tg was much smaller in pea axes, as found previously for sugar and polymer glasses. Thus, although glasses are present in seeds, the melting of the glass by raising the temperature will cause only a moderate increase in molecular mobility in the cytoplasm as compared to a huge increase in amorphous sugars.     

Preservation of dried liposomes in the presence of sugar and phosphate

It has been well established that sugars can be used to stabilize liposomes during drying by a mechanism that involves the formation of a glassy state by the sugars as well as by a direct interaction between the sugar and the phospholipid head groups. We have investigated the protective effect of phosphate on solute retention and storage stability of egg phosphatidylcholine (egg PC) liposomes that were dried (air-dried and freeze-dried) in the presence of sugars and phosphate. The protective effect of phosphate was tested using both glucose (low T(g)) and sucrose (high T(g)) by measuring leakage of carboxyfluorescein (CF), which was incorporated inside the vesicles. Liposomes that were dried with glucose or phosphate alone showed complete leakage after rehydration. However, approximately 30% CF-retention was obtained using mixtures of phosphate and glucose. Approximately 75% CF-retention was observed with liposomes that were dried with sucrose. The solute retention further increased to 85% using mixtures of phosphate and sucrose. The pH of the phosphate buffer prior to drying was found to have a strong effect on the solute retention. Fourier transform infrared spectroscopy studies showed that phosphate and sugars form a strong hydrogen bonding network, which dramatically increased the T(g). The HPO(4)(2-) form of phosphate was found to interact stronger with sugars than the H(2)PO(4)(-) form. The increased solute retention of liposomes dried in the sugar phosphate mixtures did not coincide with improved storage stability. At temperatures below 60 degrees C the rate of solute-leakage was found to be strikingly higher in the presence of phosphate, indicating that phosphate impairs storage stability of dried liposomes.     

Fusion of phosphatidylethanolamine-containing liposomes and mechanism of the L alpha-HII phase transition

The initial kinetics of fusion and leakage of liposomes composed of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) have been correlated with the phase behavior of this lipid. Gagné et al. [Gagné, J., Stamatatos, L., Diacovo, T., Hui, S. W., Yeagle, P., & Silvius, J. (1985) Biochemistry 24, 4400-4408] have shown that this lipid is lamellar (L alpha) below 20 degrees C, is hexagonal (HII) above 70 degrees C, and shows isotropic 31P NMR resonances at intermediate temperatures. This isotropic state is also characterized by complex morphological structures. We have prepared DOPE-Me liposomes at pH 9.5 and monitored the temperature dependence of the mixing of aqueous contents, leakage, and changes in light scattering upon reduction of the pH to 4.5. At and below 20 degrees C, where the lipid is in the L alpha phase, there is very little aggregation or destabilization of the liposomes. Between 30 and 60 degrees C, i.e., where the lipid is in the isotropic state, the initial rates of liposome fusion (mixing of aqueous contents) and leakage increase. At temperatures approaching that where the hexagonal HII phase transition occurs, the initial rates and extents of fusion decrease, whereas leakage is enhanced. Similar results were found for dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine (2:1) liposomes. These results clearly establish a common mechanism between the appearance of the isotropic state (between the L alpha and HII phases) and the promotion of liposome fusion. We propose a simple model to explain both the observed behavior of phosphatidylethanolamine-containing membranes with respect to liposome fusion and/or lysis and the beginning of the L alpha-HII phase transition.     

The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions

Raffinose family oligosaccharides (RFO) have been implicated as protective agents in the cellular dehydration tolerance, especially of many plant seeds. However, their efficacy in stabilizing membranes during dehydration has never been systematically investigated. We have analyzed the effects of sucrose, raffinose, stachyose, and verbascose on liposome stability during air-drying. With increasing degree of polymerization (DP), the RFO were progressively better able to stabilize liposomes against leakage of aqueous content and against membrane fusion after rehydration. Indeed, there was a very tight linear correlation between fusion and leakage for all RFO. These data indicate that increased protection of liposomes against leakage with increasing DP is due to better protection against fusion. This is in accord with the higher glass transition temperature of the longer chain oligosaccharides. Further evidence for the influence of glass transitions on membrane stability in the dry state was provided by experiments testing the temperature dependence of membrane fusion. During incubation at temperatures up to 95 degrees C for 2 h, fusion increased less with temperature in the presence of higher DP sugars. This indicates that RFO with a higher glass transition temperature are better able to protect dry membranes at elevated temperatures. In addition, Fourier-transform infrared (FTIR) spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry liposomes in the presence of all investigated sugars. However, the RFO became slightly less effective with increasing chain length, again pointing to a decisive role for preventing fusion. A direct interaction of the RFO with the lipids was indicated by a strong effect of the sugars on the phosphate asymmetric stretch region of the infrared spectrum.     

The preservation of liposomes by raffinose family oligosaccharides during drying is mediated by effects on fusion and lipid phase transitions

Raffinose family oligosaccharides (RFO) have been implicated as protective agents in the cellular dehydration tolerance, especially of many plant seeds. However, their efficacy in stabilizing membranes during dehydration has never been systematically investigated. We have analyzed the effects of sucrose, raffinose, stachyose, and verbascose on liposome stability during air-drying. With increasing degree of polymerization (DP), the RFO were progressively better able to stabilize liposomes against leakage of aqueous content and against membrane fusion after rehydration. Indeed, there was a very tight linear correlation between fusion and leakage for all RFO. These data indicate that increased protection of liposomes against leakage with increasing DP is due to better protection against fusion. This is in accord with the higher glass transition temperature of the longer chain oligosaccharides. Further evidence for the influence of glass transitions on membrane stability in the dry state was provided by experiments testing the temperature dependence of membrane fusion. During incubation at temperatures up to 95 °C for 2 h, fusion increased less with temperature in the presence of higher DP sugars. This indicates that RFO with a higher glass transition temperature are better able to protect dry membranes at elevated temperatures. In addition, Fourier-transform infrared (FTIR) spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry liposomes in the presence of all investigated sugars. However, the RFO became slightly less effective with increasing chain length, again pointing to a decisive role for preventing fusion. A direct interaction of the RFO with the lipids was indicated by a strong effect of the sugars on the phosphate asymmetric stretch region of the infrared spectrum.     

Low amounts of sucrose are sufficient to depress the phase transition temperature of dry phosphatidylcholine, but not for lyoprotection of liposomes

Disaccharides such as sucrose and trehalose play an important role in stabilizing cellular structures during dehydration. In fact, most organisms that are able to survive desiccation accumulate high concentrations of sugars in their cells. The mechanisms involved in the stabilization of cellular membranes in the dry state have been investigated using model membranes, such as phosphatidylcholine liposomes. It has been proposed that the lyoprotection of liposomes depends on the depression of the gel to liquid-crystalline phase transition temperature (T(m)) of the dry membranes below ambient and on the prevention of membrane fusion by sugar glass formation, because both lead to leakage of soluble content from the liposomes. Since fusion is prevented at lower sugar/lipid mass ratios than leakage, it has been assumed that more sugar is needed to depress T(m) than to prevent fusion. Here, we show that this is not the case. In air-dried egg phosphatidylcholine liposomes, T(m) is depressed by >60 degrees C at sucrose/lipid mass ratios 10-fold lower than those needed to depress fusion to below 20%. In fact, T(m) is significantly reduced at mass ratios where no bulk sugar glass phase is detectable by Fourier transform infrared spectroscopy or differential scanning calorimetry. A detailed analysis of the interactions of sucrose with the P=O, C=O, and choline groups of the lipid and a comparison to published data on water binding to phospholipids suggests that T(m) is reduced by sucrose through a "water replacement" mechanism. However, the sucrose/lipid mass ratios necessary to prevent leakage exceed those necessary to prevent both phase transitions and membrane fusion. We hypothesize that kinetic phenomena during dehydration and rehydration may be responsible for this discrepancy.     

Preservation of dried liposomes in the presence of sugar and phosphate

It has been well established that sugars can be used to stabilize liposomes during drying by a mechanism that involves the formation of a glassy state by the sugars as well as by a direct interaction between the sugar and the phospholipid head groups. We have investigated the protective effect of phosphate on solute retention and storage stability of egg phosphatidylcholine (egg PC) liposomes that were dried (air-dried and freeze-dried) in the presence of sugars and phosphate. The protective effect of phosphate was tested using both glucose (low T_g) and sucrose (high T_g) by measuring leakage of carboxyfluorescein (CF), which was incorporated inside the vesicles. Liposomes that were dried with glucose or phosphate alone showed complete leakage after rehydration. However, approximately 30% CF-retention was obtained using mixtures of phosphate and glucose. Approximately 75% CF-retention was observed with liposomes that were dried with sucrose. The solute retention further increased to 85% using mixtures of phosphate and sucrose. The pH of the phosphate buffer prior to drying was found to have a strong effect on the solute retention. Fourier transform infrared spectroscopy studies showed that phosphate and sugars form a strong hydrogen bonding network, which dramatically increased the T_g. The HPO₄²⁻ form of phosphate was found to interact stronger with sugars than the H₂PO₄⁻ form. The increased solute retention of liposomes dried in the sugar phosphate mixtures did not coincide with improved storage stability. At temperatures below 60 °C the rate of solute-leakage was found to be strikingly higher in the presence of phosphate, indicating that phosphate impairs storage stability of dried liposomes.     

Sendai virus induced leakage of liposomes containing gangliosides

Sendai virus induced liposome leakage has been studied by using liposomes containing a self-quenching fluorescent dye, calcein. The liposomes used in this study were prepared by a freeze and thaw method and were composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine (1:2.60:1.48 molar ratio) as well as various amounts of gangliosides and cholesterol. The leakage rate was calculated from the fluorescence increment as the entrapped calcein leaked out of the liposomal compartment and was diluted into the media. It was shown that the target liposome leakage was virus dose dependent. Trypsin-treated Sendai virus in which the F protein had been quantitatively removed did not induce liposome leakage, indicating that the leakage was a direct result of F-protein interaction with the target bilayer membrane. The activation energy of this process was approximately 12 kcal/mol below 17 degrees C and approximately 25 kcal/mol above 17 degrees C. Gangliosides GM1, GD1a, and GT1b could serve as viral receptor under appropriate conditions. Liposome leakage showed a bell-shaped curve dependence on the concentration of ganglioside in the liposomes. No leakage was observed if the ganglioside content was too low or too high. Inclusion of cholesterol in the liposome bilayer suppressed the leakage rate of liposomes containing GD1a. It is speculated that the liposome leakage is a consequence of fusion between Sendai virus and liposomes.     

Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature

We have examined whether there is a relationship between the lamellar-hexagonal phase transition temperature, TH, and the initial kinetics of H+- and Ca2+-induced destabilization of phosphatidylethanolamine (PE) liposomes. The liposomes were composed of dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine (EPE), or phosphatidylethanolamine prepared from egg phosphatidylcholine by transesterification (TPE). These lipids have well-spaced lamellar-hexagonal phase transition temperatures (approximately 12, approximately 45, and approximately 57 degrees C) in a temperature range that allows us to measure the initial kinetics of bilayer destabilization, both below and above TH. The liposomes were prepared at pH 9.5. The TH of EPE and TPE was measured by using differential scanning calorimetry, and it was found that the TH was essentially the same at low pH or at high pH in the presence of 20 mM Ca2+. At temperatures well below TH, either at pH 4.5 or at pH 9.5 in the presence of Ca2+, the liposomes aggregate, leak, and undergo lipid mixing and mixing of contents. We show that liposome/liposome contact is involved in the destabilization of the PE liposomes. The temperature dependence of leakage, lipid mixing, and mixing of contents shows that there is a massive enhancement in the rate of leakage when the temperature approaches the TH of the particular PE and that lipid mixing appears to be enhanced. However, the fusion (mixing of aqueous contents) is diminished or even abolished at temperatures above TH. At and above the TH, a new mechanism of liposome destabilization arises, evidently dependent upon the ability of the PE molecules to adapt new morphological structures at these temperatures. We propose that this destabilization demarks the first step in the pathway to the eventual formation of the HII phase. Thus, the polymorphism accessible to PE is a powerful agent for membrane destabilization, but additional factors are required for fusion.     

Preservation of freeze-dried liposomes by trehalose

One of the practical difficulties with the frequently proposed use of liposomes for delivery of water-soluble substances to cells in whole organisms is that liposomes are relatively unstable during storage. We have studied the ability of trehalose, a carbohydrate commonly found at high concentrations in organisms capable of surviving dehydration, to stabilize dry liposomes. With trehalose both inside and outside the bilayer, almost 100% of trapped solute was retained in rehydrated vesicles previously freeze-dried with 1.8 g trehalose/g dry phospholipid. Trehalose is very effective at inhibiting fusion between liposomes during drying, as assessed by freeze-fracture and resonance energy transfer between fluorescent probes incorporated into the bilayer. However, inhibition of fusion alone does not account for the preservation of the dry liposomes, since the concentration of trehalose required to prevent leakage is more than 10-fold that required to prevent fusion. We provide evidence that stabilization of the dry liposomes requires depression of transition temperature and consequent maintenance of the constituent lipids in the dry liposomes in a liquid crystalline phase.     

Effects of the acyl chain composition of phosphatidylcholines on the stability of freeze-dried small liposomes in the presence of maltose.

The effects of the acyl chain composition of phosphatidylcholines (PCs) on the stability of small unilamellar vesicles during freeze-drying and rehydration in the presence of maltose were studied by monitoring the retention of a trapped marker, calcein, in the internal liposome compartment. In dipalmitoyl PC, beta-oleoyl-gamma-palmitoyl-PC and egg yolk PC liposomes, good or fair retentions (>50%) were observed in the presence of maltose, but maltose was ineffective in preserving retention in the dioleoyl PC (DOPC) liposomes (<10%). The extremely low retention in the DOPC liposome was ascribed to neither a formation of the inverted hexagonal phase of the liposomal membrane nor the fusion/aggregation of the liposomes in the drying-rehydration process. Differential scanning calorimetry measurements suggested that interactions of maltose with PC headgroups were essential to stabilizing the dry liposomes. These interactions were significant in the saturated or mixed chain liposomes but were markedly reduced in the DOPC liposomes.     

Avidin–biotin-immobilized liposome column for chromatographic fluorescence on-line analysis of solute–membrane interactions

Unilamellar liposomes with entrapped fluorescent dye calcein were stably immobilized in gel beads by avidin–biotin-binding. The immobilized liposomes remained extremely stable upon storage and chromatographic runs. The immobilized calcein-entrapped liposomes were utilized for fluorescent analysis of solute–membrane interactions, which in some cases are too weak to be detected by chromatographic retardation. A liposome column was used as a sensitive probe to detect the interactions of membranes with pharmaceutical drugs, peptides and proteins. Retardation of the solutes was monitored using a UV detector. Perturbation of the membranes, reflected as leakage of the entrapped calcein by some of the solutes, can thus be detected on-line using a flow-fluorescent detector. For the amphiphilic drugs or synthetic peptides, perturbation of membranes became more pronounced when the retardation (hydrophobicity) of the molecules increased. On the other hand, in the case of positively-charged peptides, polylysine, or partially denatured bovine carbonic anhydrase, significant dye leakage from the liposomes was observed although the retardation was hardly to be measured. Weak protein–membrane interactions can thus be assumed from the large leakage of calcein from the liposomes. This provides additional useful information for solute–membrane interactions, as perturbation of the membranes was also indicated by avidin–biotin-immobilized liposome chromatography (ILC).     

Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix

The aim of this study was to determine whether the combined effect of water activity and temperature on inactivation rates of freeze-dried microorganisms in a lactose matrix could be explained in terms of the glass transition theory. The stabilized glass transition temperature, Tg, of the freeze-dried products was determined by differential scanning calorimetry at two different temperatures, T (20 and 37 degrees C), and different water activities (0.07-0.48). This information served as a basis for defining conditions of T and water activity, which led to storage of the bacteria in the glassy (T < Tg) and nonglassy (T > Tg) states. The rates of inactivation of the dry microorganisms subjected to different storage conditions were determined by plate counts and could be described by first-order kinetics. Rates were analyzed as a function of water activity, storage temperature, and the difference between Tg and T. Inactivation below Tg was low; however, Tg could not be regarded as an absolute threshold of bacteria stability during storage. When the cells were stored in the nonglassy state (T > Tg), inactivation proceeded faster, however, not as rapid as suggested by the temperature dependence of the viscosity above the glass transition temperature. Furthermore, the first-order rate constant, k, was dependent on the storage temperature per se rather than on the temperature difference between the glass transition temperature and the storage temperature (T - Tg).     

Calorimetric studies on dry pectinlyase preparations: impact of glass transition on inactivation kinetics

The glass transition temperature (T(g)) of a dry ultrafiltrated pectinlyase (PL) preparation decreased from 56 to 24 degrees C when water content increased to 20%. The thermal transition temperature (T(p)) for protein denaturation decreased greatly up to 40% moisture; above 40% no further changes in T(p) were observed. In the glassy state, a lag period of approximately 7 days with no PL activity loss was observed; after that, PL activity was lost. Above T(g), the rates of PL inactivation greatly increased. In the glassy state E(a) was 16.6 kJ/mol. When the system was in a higher mobility state (rubbery), E(a) increased to 66.5 kJ/mol.     

Fast laser-induced solute release from liposomes sensitized with photochromic lipid: effects of temperature, lipid host, and sensitizer concentration.

Liposomes of gel-phase phospholipid have been prepared containing a photochromic lipid sensitizer. A fast UV laser pulse isomerizes the sensitizer destabilizing the lipid bilayer structure and causing release of trapped solute. The kinetics of solute release have been investigated as a function of host lipid chain length, sensitizer concentration, and temperature, and the limits of liposome stability have been established. At low concentrations of sensitizer, pulsed laser irradiation induces some solute release when continuous UV illumination is ineffective. Although rates of solute release usually increase with temperature, at low sensitizer concentration in a rigid host, leakage at first increases but then decreases rapidly above a threshold temperature. The results presented are relevant to the design of photostimulated drug delivery systems and to potential applications of photosensitive liposomes as caging agents for biological effectors.     

Membrane fusion and inverted phases

We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates.     

Molecular motions of polysaccharides in the solid state: dextran, pullulan and amylose.

Dextran, pullulan and amylose have been investigated by differential scanning calorimetry, thermogravimetric analysis, dynamic mechanical and dielectric spectroscopy over a wide range of temperatures and frequencies. No melting or glass transition is seen below the range of thermal degradation (about 300 degrees C) for either amylose or pullulan; only dextran shows a Tg at 223 degrees C (delta cp = 0.40 J/g deg). The viscoelastic spectrum of the 'dry' polysaccharides is characterized by a low temperature relaxation that occurs at -94, -73 and -59 degrees C, at 1 kHz, (activation energy 32, 39 and 52 kJ/mol) in dextran, pullulan and amylose respectively and is assigned to small entity local motions of the polysaccharide backbone. Absorbed water strongly modifies the relaxation spectrum, inducing a new relaxation below room temperature and dissipation regions associated with water loss above room temperature. The former appears at temperatures higher than the relaxation characteristic of the dry polymer and moves to lower temperature with increasing water content. In normal 'room humidity' conditions (about 10% absorbed water) the water-induced relaxation, attributed to the motion of complex polymer-water relaxing units, is the only observable feature in the dynamic mechanical and dielectric spectrum below room temperature.     

Effect of phosphatidylglycerol on the incorporation process of cytochrome P-450 in liposomes from dimyristoylphosphatidylcholine

Using the electron paramagnetic resonance (EPR) method with spin-labeled fatty acids and gel-penetrating chromatography, the effect of phosphatidylglycerol on cytochrome P-450 incorporation into liposomes from dimyristoylphosphatidylcholine was investigated. An addition of phosphatidylglycerol caused an increase in the protein content of the proteoliposomes as well as their accelerated formation at temperatures below and above the liposome phase transition temperature (Ts). The dependence of the proteoliposome formation rate on the phosphatidylglycerol content in the liposome mixture is described by complex kinetics with a maximum in the presence of 10 mol.% of the negatively charged phospholipid. The mechanism of proteoliposome formation is discussed in terms of asymmetric distribution and phase state of the phospholipids in the original vesicles.