Liposomal heme as oxygen carrier under semi-physiological conditions orientation study of heme embedded in a phospholipid bilayer by an electrooptical method

The meso-tetra(α,α,α,α(o-pivalamidophenyl))porphinato iron-mono(1-lauryl-2-methylimidazole) complex embedded in the bilayer of dimyristoylphosphatidylcholine (liposomal heme) binds molecular oxygen reversibly at pH 7 and 37°C. Orientation of the iron porphyrin complex in the phospholipid bilayer was studied by electric birefringence and dichroism. It was observed that both the phospholipid bibilayer of liposome and the porphyrin plane are oriented nearly in parallel to the electric field. Therefore the angle between the porphyrin plane and the bilayer is considered to be practically small.     

Liposomal heme as oxygen carrier under semiphysiological condition

To prepare a potent synthetic oxygen carrier in aqueous media, the iron(II) picket fence porphyrin complex with one hydrophobic imidazole was incorporated into a lipid bilayer of phosphatidylcholine. The incorporation was confirmed by gel permeation chromatography and ultracentrifugation, which indicates the complex being trapped in the multilayer liposome. The liposomal iron(II) porphyrin complex could bind molecular oxygen reversibly in neutral aqueous media and in the serum of a rat blood at 25°C.     

Correct interpretation of heme protein spectra allows distinguishing between the heme and the protein dynamics

It is shown by using the vibronic approach that the iron displacement out of the porphyrin plane in deoxyheme proteins intermixes the porphyrin pi and axial iron-histidine sigma electronic subsystems. This intermixing explains the substantial coupling of the iron-histidine vibration to the heme Soret excitation, the appearance of the iron-histidine band in the corresponding resonance Raman spectra, and a number of other experimental data, including the dependence of the iron-histidine vibrational frequency on the extent of the iron displacement out of the porphyrin plane. This dependence implies that there is an anharmonic coupling between the corresponding vibrations, which is shown to be the cause of the specific temperature dependence of the iron-histidine band. The anharmonic coupling and the dependence of the dipole transition moment of the charge transfer optical absorption band III on the iron-porphyrin distance cause the anomalous temperature and pressure dependencies of this band. It is shown that the change in both the magnitude and the distribution of the iron-porphyrin distance is expected to affect the band III intensity. Consequently, the stationarity of the band III intensity can be considered as a signature of the stationarity of the iron-porphyrin distance and its distribution in deoxyheme proteins, whereas the band III position and width could be also affected by the change in the protein electric field, caused by the protein globule dynamics.     

Harmonic and anharmonic dynamics of Fe-CO and Fe-O(2) in heme models

We present density-functional molecular dynamics simulations of FeP(Im)(AB) heme models (AB = CO, O(2), Im = imidazole) as a way of sketching the dynamic motion of the axial ligands at room temperature. The FeP(Im)(CO) model is characterized by an essentially upright FeCO unit, undergoing small deviations with respect to its linear equilibrium structure (bending and tilting up to 10 degrees and 7 degrees, often occur). The motion of the carbon monoxide ligand is found to be quite complex and fast, its projection on the porphyrin plane sampling all the porphyrin quadrants in a short time ( approximately 0.5 ps). Simultaneously, the imidazole ligand rotates slowly around the Fe-N(epsilon) bond. In contrast to carbon monoxide, the oxygen ligand in FeP(Im)(O(2)) prefers a conformation where the projection of the O-O axis on the porphyrin plane bisects one of the porphyrin quadrants. A transition to other quadrants takes place through an O-O/Fe-N(p) overlapping conformation, within 4-6 ps. Further details of these mechanisms and their implications are discussed.     

Electric field-induced redistribution and postfield relaxation of epidermal growth factor receptors on A431 cells

The lateral mobility of the epidermal growth factor (EGF) receptor in the plane of the plasma membrane of cultured A431 cells was investigated using direct and indirect fluorescent probes to measure the generation and relaxation of electric field-induced receptor asymmetry. A steady electric field of 15 V/cm for 30 min at 23 degrees C induced a redistribution of the unoccupied EGF receptor such that there was approximately a three-fold higher concentration of receptors at the cathode-facing pole. After termination of the field, the unoccupied receptors back diffused at 37 degrees C with a rate corresponding to a diffusion coefficient of 2.6-3.5 X 10(-10) cm2/s. No diffusion was detected at 4 degrees C. Formation of the hormone-receptor complex is known to induce receptor clustering and internalization. By inhibiting internalization with metabolic poisons, we were able to study the cell surface mobility of clusters of the hormone-receptor complex. The same degree of asymmetry was induced when the occupied receptor was exposed to an electric field and the rate of back diffusion of clusters of the hormone-receptor complex corresponded to a diffusion coefficient of 0.68-0.95 X 10(-10) cm2/s. Although the unoccupied receptor is somewhat more mobile than the hormone-receptor complex, it was still far less mobile than one would predict for an unconstrained protein imbedded in a phospholipid bilayer.     

Is flexoelectricity the coupling factor between chemical energy and osmotic work in the pump? A model of pump

The following pump model is proposed. A gate is responsible for pump specificity. The actual driving force of the transport of ions against the electrochemical potential gradient is the electric field originating from an altered curvature of the phospholipid bilayer around the pump. The physical origin of this curvature-induced electric field arises from a basic liquid crystal property of lipid bilayers called flexoelectricity. Alterations occurring in phospholipid bilayer arrangement are due to changed conformation of protein; the main energy source of this change is ATP. Consequently, the energy of ATP is transformed, in our pump model, into osmotic work in following steps: ATP + protein (conformation I)----protein (conformation II)----alterations in phospholipid bilayer arrangement----electric field----active transport of ions. This model is the most simple one. In Na, K-pump there is a bidirectional ion transport. In our model of Na, K-pump three conformational states of pump proteins and two different electric fields formed sequentially in opposite directions are supposed.     

The effects of A23187 on the phospholipid phase transition of large unilamellar vesicles (LUVs) as detected by ultrasound spectroscopy

The effect of the hydrophobic Ca2+ ionophore, A23187, on the phospholipid dynamics of large unilamellar vesicle (LUVs: 4: 1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG] membranes, as a function of A23187 content, was investigated using techniques sensitive to the phospholipid phase transition. The ultrasonic absorption per wavelength, alpha lambda, was determined with a double crystal acoustic interferometer, as a function of temperature and frequency for LUVs in the vicinity of their phospholipid phase transition. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) were also employed to probe the thermodynamics and molecular environment of the hydrocarbon side chains. With increasing A23187 content, the phase transition temperature (Tm) of the LUV suspensions remained near 42.0 degrees C, while the amplitude of alpha lambda at the phase transition increased dramatically. At Tm the relaxation frequency, where alpha lambda max occurs, decreased with A23187 content, suggesting that the relaxation rate of the event responsible for the absorption of ultrasound decreased. The ESR studies showed no change in the fluidity of the bilayer with the inclusion of 2 and 5 mol% A23187 in the C-12 region of the bilayer. Therefore, A23187 in LUV membranes slows the structural relaxation of the hydrocarbon side chains of the phospholipid bilayer at the phase transition.     

Development of magnetically aligned phospholipid bilayers in mixtures of palmitoylstearoylphosphatidylcholine and dihexanoylphosphatidylcholine by solid-state NMR spectroscopy

This study reports the solid-state NMR spectroscopic characterization of a long chain phospholipid bilayer system which spontaneously aligns in a static magnetic field. Magnetically aligned phospholipid bilayers or bicelles are model systems which mimic biological membranes for magnetic resonance studies. The oriented membrane system is composed of a mixture of the bilayer forming phospholipid palmitoylstearoylphosphatidylcholine (PSPC) and the short chain phospholipid dihexanoylphosphatidylcholine (DHPC) that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approx. 75% aqueous). Traditionally, the shorter 14 carbon chain phospholipid dimyristoylphosphatidylcholine (DMPC) has been utilized as the bilayer forming phospholipid in bicelle studies. Alignment (perpendicular) was observed with a PSPC/DHPC q ratio between 1.6 and 2.0 slightly above T(m) at 50 degrees C with (2)H and (31)P NMR spectroscopy. Paramagnetic lanthanide ions (Yb(3+)) were added to flip the bilayer discs such that the bilayer normal was parallel with the static magnetic field. The approx. 1.8 (PSPC/DHPC) molar ratio yields a thicker membrane due to the differences in the chain lengths of the DMPC and PSPC phospholipids. The phosphate-to-phosphate thickness of magnetically aligned PSPC/DHPC phospholipid bilayers in the L(alpha) phase may enhance the activity and/or incorporation of different types of integral membrane proteins for solid-state NMR spectroscopic studies.     

Anomalous excitonic CD of meso-tetrakis(3-N-methylpyridiniumyl)porphyrin bound to poly[d(A-T)(2)

When meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (m-TMPyP) formed a complex with poly[d(A-T)(2)], an intense bisignate excitonic CD in the Soret absorption region was observed. The excitonic CD of the m-TMPyP-poly[d(A-T)(2)] complex is unique in that no other combination of the related porphyrin, namely, meso-tetrakis(n-N-methylpyridiniumyl)porphyrin (where n = 2, 4), and polynucleotide including calf thymus DNA, poly[d(G-C)(2)], poly[d(I-C)(2)], and poly(dA).poly(dT), exhibits a comparable CD spectrum. From the [drug]/[DNA] ratio-dependence of the intensity and the shape of the CD spectrum, this porphyrin species is assigned to an extensively aggregated form. The extensively aggregated porphyrin disperses in 1 h after mixing to form moderately stacked porphyrin at a low mixing ratio. The magnitude of linear dichroism of the extensively aggregated porphyrin was small and the sign was negative in the Soret band, which indicated that the molecular plane of porphyrin in the complex is strongly tilted. On the other hand, the molecular plane of porphyrin is almost parallel to the DNA base plane (perpendicular to the DNA helix axis) in the moderately stacked form.     

Electro-osmosis at the surface of phospholipid bilayer membranes

The electro-osmotic velocity is the velocity of a fluid near an interface produced by an electric field parallel to a surface. The velocity adjacent to fixed phospholipid bilayer membranes was measured by observing the velocity of small vesicles suspended in the fluid. The charge densities of the bilayers ranged from 0 to 1 electronic charge per lipid and experiments were performed at temperatures above and below the transition temperature of the phospholipid bilayer in 1, 10 and 100 mM NaCl solutions. The Helmholtz-Smoluchowski equation correctly predicted the electro-osmotic velocity from the known value of zeta potential of the phospholipid bilayer.     

The use of proteinases to determine the topological location of cytochrome P-450 in vesicles derived from smooth endoplasmic reticulum of rat liver.

1. The phospholipid bilayer of intact vesicles from smooth endoplasmic reticulum is impermeable to macromolecules. Specific and non-specific proteinases were used to investigate the site of membrane proteins in the transverse plane of the bilayer. 2. When two proteinases were used in conjunction, denaturing effects additional to proteolysis were observed on cytochrome P-450 content and glucose 6-phosphatase activity which did not depend on the integrity of the phospholipid bilayer. 3. When lipid peroxidation was inhibited, these effects were not observed.     

Structure and dynamics of dioxygen bound to cobalt and iron heme

In this study we use ab initio molecular dynamics simulations to analyze the structure and dynamics of the oxygen ligand in models of the oxymyoglobin active site and its cobalt-substituted analog. Our calculations are performed for iron-porphyrin and cobalt-porphyrin complexes with imidazole and oxygen as axial ligands, and we investigate the effect of the distal histidine in the structure and dynamics of the metal-oxygen unit (MeO(2), Me = Fe, Co). We find that the interaction between the distal histidine and the oxygen ligand is stronger for the cobalt complex than for the iron one, consistent with the superoxide ion character of the bound O(2). The dynamics of the O(2) ligand can be described as oscillations of the O-O axis projection on the porphyrin plane within a porphyrin quadrant combined with frequent jumps from one quadrant to another. However, the ligand motion is significantly faster for CoO(2) compared to FeO(2). As a result, the iron complex shows localized ligand sites, whereas for cobalt several configurations are possible. This gives support to the highly dynamic motion of the oxygen ligand found in several experiments on cobalt oxymyoglobin and model complexes and underlines the higher mobility of the CoO(2) fragment compared to FeO(2).     

Study of the specific heme orientation in reconstituted hemoglobins

NMR studies of the recombination reaction of apohemoglobin derivatives with natural and unnatural hemes and of the heme-exchange reaction for reconstituted hemoglobin have revealed that the heme is incorporated into the apoprotein with stereospecific heme orientations dependent upon the heme peripheral 2,4-substituents and the axial iron ligand(s). Heme orientations also depend on whether recombination occurs at the alpha or beta subunit and on whether or not the complementary subunit is occupied by the heme. In the recombination reaction with the azido complex of deuterohemin, the alpha subunit of the apohemoglobin preferentially combines with the hemin in the "disordered" heme orientation, whereas protohemin is inserted in either of two heme orientations. Mesohemin inserts predominantly in the "native" heme orientation. For the beta subunit, specific heme orientation was also encountered, but the specificity was somewhat different from that of the alpha subunit. It was also shown that the specific heme orientation in both subunits is substantially affected by the axial heme ligands. These findings imply that apohemoglobin senses the steric bulkiness of both the porphyrin 2,4-substituents and the axial iron ligands in the heme-apoprotein recombination reaction. To gain an insight into the effect of the protein structure, the heme reconstitution reaction of semihemoglobin, demonstrating that the heme orientation in the reconstituted semihemoglobin with the azido-deuterohemin complex was in the native form, was also examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of functional electron transfer between membrane biological elements in a two-dimensional lipidic structure at the electrode interface

These studies develop a methodology to form supported phospholipid bilayers at an electrode/solution interface that models biological membrane systems. Two kinds of electrode were used, a planar gold electrode and a microporous aluminium oxide electrode on which octadecanethiol or octadecyltrichlorosilane was self-assembled. The supported lipidic structures were produced by transfer of a phospholipid monolayer by the Langmuir—Blodgett technique or by direct fusion of phospholipid vesicles. Ubiquinone was introduced into the lipidic structures during their formation; electrochemical measurements demonstrated the mobility of ubiquinone along the plane of the bilayer. A membrane enzyme, pyruvate oxidase from E. coli, was successfully incorporated into this artificial bilayer and was found to be able to exchange electrons with ubiquinone present in the bilayer.     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid microsphere containing lipophilic heme: preparation and oxygen transportation under physiological conditions.

Lipophilic heme (1-laurylimidazole-ligated 5,10,15,20-tetrakis(alpha, alpha, alpha, alpha-o- pivalamidophenyl)porphinatoiron(II) complex) is solubilized in lipid (triglyceride) at high concentrations and emulsified with a phospholipid in physiological salt solution, giving a deeply red-colored suspension of lipid microspheres (approx. 250 nm in diameter). The heme forms an oxygen adduct in a similar manner as oxyhemoglobin and the lipid microspheres take up and release oxygen reversibly at 37 degrees C in the aqueous medium. The oxygen-transporting ability is comparable with that of the red blood cell. Intravenous injection of the heme/lipid microsphere solution to rabbits demonstrates that it transports oxygen even in vivo and that it is cleared from the blood stream with a half-life time of approx. 1 h.     

The antimicrobial peptide trichogin and its interaction with phospholipid membranes.

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC), replaced one of the three alpha-aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin-promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.     

Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study.

Purified adrenocortical microsomal cytochromes P-45017 alpha,lyase and P-450C21 were reconstituted with and without NADPH-cytochrome P-450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P-450 ratio of 35 (w/w) by cholate dialysis procedures. Trypsinolysis revealed that a considerable part of each P-450 molecule is deeply embedded in the lipid bilayer, on the basis of the observation of no detectable digestion for P-45017 alpha,lyase and the proteolysis-resistant membrane-bound heavy fragments for P-450C21. Rotational diffusion was measured in proteoliposomes and adrenocortical microsomes by observing the decay of absorption anisotropy, r(t), after photolysis of the heme-CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1-2 ms to a time-independent value r3. Coexistence of a mobile population with an average rotational relaxation time phi of 138-577 microseconds and immobile (phi > or = 20 ms) populations of cytochrome P-450 was observed in both phospholipid vesicles and microsomes. Different tilt angles of the heme plane from the membrane plane were determined in proteoliposomes to be either 47 degrees or 63 degrees for P-45017 alpha,lyase from [r3/r(0)]min = 0.04 and either 38 degrees or 78 degrees for P-450C21 from [r3/r(0)]min = 0.19, when these P-450s were completely mobilized by incubation with 730 mM NaCl. Very different interactions with the reductase have been observed for the two P-450s in proteoliposomes. In the presence of the reductase, the mobile population of cytochrome P-450C21 was increased significantly from 79% to 96% due to dissociation of P-450 oligomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

Red cell membrane lipids in hemoglobinopathies

The complex mixture of lipids and proteins of the red blood cell membrane is well maintained during the life of the cell. Lipid analysis of the red cell reveals hundreds of phospholipid molecular species and cholesterol that differ with respect to their (polar) head group, and (apolar) side chains. These molecules move rapidly in the plane, as well as across the lipid bilayer. This dynamic movement is highly organized. In the plane of the bilayer, areas enriched in certain lipids accommodate protein structure and modulate function. While lipids move across the bilayer, the organization is highly asymmetric. Amino phospholipids are mainly found on the inside and choline containing phospholipids on the outside. Both the composition and organization of the red cell membrane is maintained throughout the life of the red cell by an intricate mechanism that involves enzymes, transporters and cytosolic factors. Key proteins that maintain red blood cell lipid organization have recently been identified. Alterations in these mechanisms, as the result of the globin mutations in sickle cell disease or thalassemia will lead to loss of membrane viability, apoptosis during erythropoiesis, early demise of the cell in the circulation, and when these cells are not removed appropriately their presence has pathologic consequences.