Small Unilamellar Phospholipid Vesicles Research Articles

Skeletal Muscle Myosin Is Procoagulant By Binding Factor XI Via Its A3 Domain and Enhancing Factor XI Activation By Thrombin

Blood coagulation mechanisms play key roles in health and disease. Pilot studies using selected human plasmas showed the potential associations of plasma skeletal muscle myosin (SkM) isoforms and phenotypes with pulmonary embolism and thrombin generation, suggesting SkM may contribute to blood coagulation reactions in plasma. Here we report that ex vivo studies of the coagulability of fresh flowing human blood over SkM-coated surfaces showed that an anti-factor XI (FXI) mAb, but not an anti-tissue factor mAb, inhibited clot formation, indicating that FXI is an essential contributor for the normal observed procoagulant response of blood during its exposure to immobilized SkM. This raised the question of whether procoagulant SkM's requirement for FXI involves direct or indirect effects on FXI.To assess direct interactions between SkM and FXI, Bio-Layer Interferometry (BLI) (Octet Red system) was used to record kinetics for binding of soluble FXI to immobilized SkM. BLI data showed that FXI bound to SkM with a Kd of 0.2 nM (k on= 2.92x10 6 M -1s -1 and k off=9.25x10 -3 s -1) (Fig. 1A). In contrast, prekallikrein (PK) did not bind to the SkM (Fig.1A), indicating the specificity of SkM for binding FXI. The anti-FXI mAb1A6, which recognizes the Apple (A)3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying SkM binds the FXI A3 domain.Studies using purified clotting factors were made to identify which FXI-related activities might be affected by SkM. When FXI activation by thrombin was evaluated under conditions where polyphosphate (PolyP) 100-mer and 700-mer enhance FXI activation, SkM concentration-dependently enhanced FXI activation by thrombin (Fig. 1B). Whereas alkaline phosphatase destroyed PolyP's ability to stimulate FXI activation by thrombin, it did not cause a reduction of SkM's ability to enhance FXI activation, indicating SkM's activity is independent of PolyP-like sequences in SkM. Small unilamellar phospholipid vesicles (20% phosphatidylserine (PS) / 80% phosphatidylcholine) did not affect FXI activation by thrombin; furthermore, reagents that neutralize procoagulant PS, i.e., lactadherin, annexin V, and phospholipase A2, did not affect SkM's enhancement of FXI activation by thrombin, indicating that this activity is not due to anionic phospholipids linked to SkM.The effects of SkM on FXI autoactivation and FXI activation by FXIIa were evaluated. As is well known, PolyP and some other anionic reagents, e.g., nucleic acid polymers, enhance not only FXI activation by thrombin but also FXI autoactivation and FXI activation by FXIIa. However, SkM did not significantly affect FXI autoactivation or FXI activation by factor XIIa, further emphasizing that SkM's enhancement of FXI activation by thrombin is not due to any PolyP-like compounds and that it is a unique property of procoagulant SkM. This also suggests that SkM has a unique mechanism for its procoagulant activity on FXI activation which is limited to the thrombin positive feedback loop.To evaluate further the basis for interactions between FXI and SkM, we employed FXI- PK chimeras because BLI binding studies showed that, in contrast to FXI, PK did not bind to SkM. Recombinant FXI proteins in which each of the four A domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding A domain from PK and were used to identify the site of factor XI to interact with SkM for FXI activation by thrombin. The FXI chimera with the substitution of the PKA1 domain was not activated by thrombin, which is consistent with the fact that the FXI A1 domain is an interactive site for thrombin. Thrombin activation of the two FXI chimeras (FXI/PKA3 and FXI/PKA4) with substitutions of either the A3 or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain (FXI/PKA2) did not reduce the enhancement of activation by thrombin compared to wild type FXI. Furthermore, mAb1A6, which recognizes the A3 domain and which inhibited the prothrombotic activity of fresh blood flowing over a SkM-coated surface, potently inhibited FXI binding to SkM in BLI studies. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains of FXI.In summary, we found that SkM's ex vivo procoagulant activity requires FXI, that SkM enhances FXI activation by thrombin and this requires FXI's A3 and A4 domains, and that SkM's high affinity binding of FXI requires the FXI A3 domain (Fig. 1C). [Display omitted] DisclosuresGruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment. Gailani: Anthos Therapeutics: Consultancy; Aronora: Membership on an entity's Board of Directors or advisory committees; Bayer Pharma: Consultancy; Bristol Myer Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Charge Recombination Kinetics of Bacterial Photosynthetic Reaction Centres Reconstituted in Liposomes: Deterministic Versus Stochastic Approach

In this theoretical work, we analyse the kinetics of charge recombination reaction after a light excitation of the Reaction Centres extracted from the photosynthetic bacterium Rhodobacter sphaeroides and reconstituted in small unilamellar phospholipid vesicles. Due to the compartmentalized nature of liposomes, vesicles may exhibit a random distribution of both ubiquinone molecules and the Reaction Centre protein complexes that can produce significant differences on the local concentrations from the average expected values. Moreover, since the amount of reacting species is very low in compartmentalized lipid systems the stochastic approach is more suitable to unveil deviations of the average time behaviour of vesicles from the deterministic time evolution.

Preparation of ready-to-use small unilamellar phospholipid vesicles by ultrasonication with a beaker resonator

Lipid vesicles are widely used as models to investigate the interactions of proteins, peptides, and small molecules with lipid bilayers. We present a sonication procedure for the preparation of well-defined and ready-to-use small unilamellar vesicles composed of phospholipids with the aid of a beaker resonator. This indirect but efficient sonication method does not require subsequent centrifugation or other purification steps, which distinguishes it from established sonication procedures. Vesicles produced by this method reveal a unimodal size distribution and are unilamellar, as demonstrated by dynamic light scattering and 31P nuclear magnetic resonance spectroscopy, respectively.

Counting and dynamic studies of the small unilamellar phospholipid vesicle translocation with single conical glass nanopores.

Phospholipid vesicles are ubiquitous cellular organelles that perform vital functions including materials transport and information transmission and have found promising biomedical applications. Although the transmembrane translocation (via nanopores) of phospholipid vesicles, especially small unilamellar phospholipid vesicles (SUVs), is recognized to be very important for these processes and applications, the details and dynamics remain not very clear. Herein, we use single conical glass nanopores as a model platform to systematically investigate the translocation dynamics of SUVs (∼50-60 nm in diameter) through small nanopores with orifice diameters ranging from ∼14 to 72 nm. Dynamic translocation of individual SUVs one by one through the nanopores was clearly observed and was analyzed by the occurrence of periodic oscillation in ionic current blockage signal under a negatively applied voltage. Translocation behaviors of the SUVs, in terms of magnitude and duration of ionic current blockage signal, varied and can be modulated by changing nanopore size, solution pH, vesicle concentration, applied voltage, and inner surface charge properties of the nanopores. The translocation rate of the SUVs through an ∼72 nm nanopore is typically on a time scale of a few seconds (per SUV translocation event) and found nonlinearly proportional to the concentration of the SUVs. Moreover, the electrophoretic force has been verified as a main force to drive the SUVs through the nanopore since there is a nearly linear relationship between the current blockage frequency of SUVs translocation and the applied bias potentials ranging from -0.6 to -1 V. The findings provide fundamental insights into the translocation and interactions of SUVs with nanopores, and the reported nanopore platform may find potential useful bioapplications in single-cell and single-vesicle studies.

Synthesis of amphiphilic polyphosphoester ionomers for surface modification of small unilamellar phospholipid vesicles

Synthesis of amphiphilic polyphosphoester ionomers for surface modification of small unilamellar phospholipid vesicles

PEGylated lipids impede the lateral diffusion of adsorbed proteins at the surface of (magneto)liposomes

Protein binding to nanoparticles is a crucial issue in biomedicine, as it triggers their clearance from the bloodstream after intravenous injection. Many techniques are available for measuring strong protein binding interactions, but weak dynamic interactions are more difficult to assess. To tackle the latter problem, in the present work, cytochrome c was chosen as a representative model of a water-soluble protein and the adsorbing particulates were either small unilamellar phospholipid vesicles or 14 nm diameter solid superparamagnetic iron oxide cores onto which a phospholipid bilayer was strongly chemisorbed (so-called magnetoliposomes). Incorporation of cytochrome c oxidase into the phospholipid bilayer allowed the association of cytochrome c with the surface of the particles to be measured with high sensitivity by VIS-spectrophotometry. The impact of enzyme density as well as some of the physical features of the PEG corona (degree of PEGylation and PEG chain length) adjacent to the surface of the lipid structures on the overall kinetics was also investigated.

Influence of class B scavenger receptors on cholesterol flux across the brush border membrane and intestinal absorption

To learn more about how the step of cholesterol uptake into the brush border membrane (BBM) of enterocytes influences overall cholesterol absorption, we measured cholesterol absorption 4 and 24 h after administration of an intragastric bolus of radioactive cholesterol in mice with scavenger receptor class B, type 1 (SR-BI) and/or cluster determinant 36 (CD36) deleted. The cholesterol absorption efficiency is unaltered by deletion of either one or both of the receptors. In vitro determinations of the cholesterol uptake specific activity of the BBM from the mice reveal that the scavenger receptors facilitate cholesterol uptake into the proximal BBM. It follows that cholesterol uptake into the BBM is not normally rate-limiting for the cholesterol absorption process in vivo; a subsequent step, such as NPC1L1-mediated transfer from the BBM into the interior of the enterocyte, is rate-limiting. The absorption of dietary cholesterol after 4 h in mice lacking SR-BI and/or CD36 and fed a high-fat/high-cholesterol diet is delayed to more distal regions of the small intestine. This effect probably arises because ATP binding cassette half transporters G5 and G8-mediated back flux of cholesterol from the BBM to the lumen of the small intestine limits absorption and causes the local cholesterol uptake facilitated by SR-BI and CD36 to become rate-limiting under this dietary condition.

Lateral Proton Transfer between the Membrane and a Membrane Protein

Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (approximately 7 x 1010 s(-1) M(-1), compared to the expected value of (1-2) x 10(10) s(-1) M(-1)), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to approximately 3 x 10(13) s(-1) M(-1), which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.

Membrane-bound alpha-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles.

We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (alphaS) bound to small unilamellar phospholipid vesicles, rodlike SDS micelles, or lipid bicelles. By measuring distances as long as approximately 7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that alphaS does not retain a "hairpin" structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the interhelix linker. While the possibility of local helix discontinuities in the structure of membrane-bound alphaS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.

ABCG1 and ABCG4 are coexpressed in neurons and astrocytes of the CNS and regulate cholesterol homeostasis through SREBP-2

Here, we describe the initial characterization of Abcg4(-/-) mice and identify overlapping functions of ABCG4 and ABCG1 in the brain. Histological examination of tissues from Abcg4(+/-)/nlsLacZ and Abcg1(+/-)/nlsLacZ mice demonstrates that coexpression of Abcg4 and Abcg1 is restricted to neurons and astrocytes of the central nervous system (CNS). Interestingly, Abcg4 mRNA is undetectable outside the CNS, in contrast with the broad tissue and cellular expression of Abcg1. We also used primary astrocytes, microglia, neurons, and macrophages to demonstrate that the expression of Abcg1, but not Abcg4, is induced after the activation of liver X receptor. Cellular localization studies demonstrated that both proteins reside in RhoB-positive endocytic vesicle membranes. Furthermore, overexpression of either ABCG1 or ABCG4 increased the processing of sterol-regulatory element binding protein 2 (SREBP-2) to the transcriptionally active protein, thus accounting for the observed increase in the expression of SREBP-2 target genes and cholesterol synthesis. Consistent with these latter results, we show that the expression levels of the same SREBP-2 target genes are repressed in the brains of Abcg1(-/-) and, to a lesser extent, Abcg4(-/-) mice. Based on the results of the current study, we propose that ABCG1 and ABCG4 mediate the intracellular vesicular transport of cholesterol/sterols within both neurons and astrocytes to regulate cholesterol transport in the brain.

Diazeniumdiolate reactivity in model membrane systems

The effect of small unilamellar phospholipid vesicles on the acid-catalyzed dissociation of nitric oxide from diazeniumdiolate ions, R 1R 2N[N(O)NO] −, [ 1: R 1 = H 2N(CH 2) 3-, R 2 = H 2N(CH 2) 3NH(CH 2) 4-; 2: R 1 = R 2 = H 2N(CH 2) 3-; 3: R 1 = n-butyl-, R 2 = n-butyl- NH 2 + (CH 2) 6-; 4: R 1 = R 2 = nPr-] has been examined at pH 7.4 and 37 °C. NO release was catalyzed by anionic liposomes (DPPG, DOPG, DMPS, POPS and DOPA) and by mixed phosphatidylglycerol/phosphatidylcholine (DPPG/DPPC and DOPG/DPPC) covesicles, while cationic liposomes derived from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic liposome DMPC did not significantly affect the dissociation rates of the substrates examined. Enhancement of the dissociation rate constant in DPPG liposome media (0.010 M phosphate buffer, pH 7.4, 37 °C) at 10 mM phosphoglycerol levels, ranged from 37 for 1 to 1.2 for the anionic diazeniumdiolate 4, while DOPA effected the greatest rate enhancement, achieving 49-fold rate increases with 1 under similar conditions. The observed catalysis decreases with increase in the bulk concentration of electrolytes in the reaction media. Quantitative analysis of catalytic effects has been obtained through the application of pseudo-phase kinetic models and equilibrium binding constants at different liposome interfaces are compared. The stoichiometry of nitric oxide release from 1 and 2 in DPPG/DPPC liposome media has been obtained through oxyhemoglobin assay. DPPG = 1,2-dipalmitoyl- sn-glycero-3-[phospho- rac-(1-glycerol)], DOPG = 1,2-dioleoyl- sn-glycero-3-[phospho- rac-(1-glycerol)], DMPS = 1,2-dimyristoyl- sn-glycero-3-[phospho- l-serine], POPS = 1-palmitoyl-2-oleoyl- sn-glycero-3-[phospho- l-serine], DOPA = 1,2-dioleoyl- sn-glycero-3-phosphate; DPPC = 1,2-dipalmitoyl- sn-glycero-3-phosphocholine, DMPC = 1,2-dimyristoyl- sn-glycero-3-phosphocholine, DOTAP = 1,2-dioleoyl-3-trimethylammonium-propane.

Characterization of Supported Membranes on Topographically Patterned Polymeric Elastomers and Their Applications to Microcontact Printing

This article describes the fluorescence microscopy and imaging ellipsometry-based characterization of supported phospholipid bilayer formation on elastomeric substrates and its application in microcontact printing of spatially patterned phospholipid bilayers. Elastomeric stamps, displaying a uniformly spaced array of square wells (20, 50, and 100 mum linear dimensions), are prepared using poly(dimethyl)siloxane from photolithographically derived silicon masters. Exposing elastomeric stamps, following UV/ozone-induced oxidation, to a solution of small unilamellar phospholipid vesicles results in the formation of a 2D contiguous, fluid phospholipid bilayers. The bilayer covers both the elevated and depressed regions of the stamp and exhibits a lateral connectivity allowing molecular transport across the topographic boundaries. Applications of these bilayer-coated elastomeric stamps in microcontact printing of lipid bilayers reveal a fluid-tearing process wherein the bilayer in contact regions selectively transfers with 75-90% efficiency, leaving behind unperturbed patches in the depressed regions of the stamp. Next, using cholera-toxin binding fluid POPC bilayers that have been asymmetrically doped with ganglioside Gm1 ligand in the outer leaflets, we examine whether the microcontact transfer of bilayers results in the inversion of the lipid leaflets. Our results suggest a complex transfer process involving at least partial bilayer reorganization and molecular re-equilibration during (or upon) substrate transfer. Taken together, the study sheds light on the structuring of lipid inks on PDMS elastomers and provides clues regarding the mechanism of bilayer transfer. It further highlights some important differences in stamping fluid bilayers from the more routine applications of stamping in the creation of patterned self-assembled monolayers.

Restricted Active Site Docking by Enzyme-bound Substrate Enforces the Ordered Cleavage of Prothrombin by Prothrombinase

The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg(320) site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg(271) site binds to the active site poorly in an interaction that is approximately 600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg(271) at the active site of prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the V(max) for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by prothrombinase.

Surface functionalization of magnetoliposomes in view of improving iron oxide-based magnetic resonance imaging contrast agents: Anchoring of gadolinium ions to a lipophilic chelate

Small unilamellar phospholipid vesicles containing the phosphatidylethanolamine–diethylene triamine pentaacetic acid (PE–DTPA) conjugate as one of the building stones were constructed. The ability of these colloids to complex gadolinium(III) ions at the surface of both the inner and outer bilayer shells was verified using a colorimetric method with Arsenazo III as a dye indicator. On incubation of these functionalized vesicles with magnetoliposomes (MLs, nanometer-sized magnetite cores encapsulated in a phospholipid bilayer), PE–DTPA percolates into the ML coat. The PE–DTPA content could be fine-tuned by varying the conjugate concentration in the donor vesicles. In the experimental conditions applied, up to 500 Gd 3+ ions were immobilized per ML colloid. The resulting ML–Gd 3+ complexes might have great potential, for example, as a novel magnetic resonance imaging contrast agent.

Membrane Dynamics of the Amphiphilic Siderophore, Acinetoferrin

Acinetobacter haemolyticus is an antibiotic resistant, pathogenic bacterium responsible for an increasing number of hospital infections. Acinetoferrin (Af), the amphiphilic siderophore isolated from this organism, contains two unusual trans-2-octenoyl hydrocarbon chains reminiscent of a phospholipid structural motif. Here, we have investigated the membrane affinity of Af and its iron complex, Fe-Af, using small and large unilamellar phospholipid vesicles (SUV and LUV) as model membranes. Af shows a high membrane affinity with a partition coefficient, K(x)= 6.8 x 10(5). Membrane partitioning and trans-membrane flip-flop of Fe-Af have also been studied via fluorescence quenching of specifically labeled vesicle leaflets and (1)H NMR line-broadening techniques. Fe-Af is found to rapidly redistribute between lipid and aqueous phases with dissociation/partitioning rates of k(off) = 29 s(-1) and k(on) = 2.4 x 10(4) M(-1) s(-1), respectively. Upon binding iron, the membrane affinity of Af is reduced 30-fold to K'(x) = 2.2 x 10(4) for Fe-Af. In addition, trans-membrane flip-flop of Fe-Af occurs with a rate constant, k(p) = 1.2 x 10(-3) s(-1), with egg-PC LUV and a half-life time around 10 min with DMPC SUV. These properties are due to the phospholipid-like conformation of Af and the more extended conformation of Fe-Af that is enforced by iron binding. Remarkable similarities and differences between Af and another amphiphilic siderophore, marinobactin E, are discussed. The potential biological implications of Af and Fe-Af are also addressed. Our approaches using inner- and outer-leaflet-labeled fluorescent vesicles and (1)H NMR line-broadening techniques to discern Af-mediated membrane partitioning and trans-membrane diffusion are amenable to similar studies for other paramagnetic amphiphiles.

Binding of Substrate in Two Conformations to Human Prothrombinase Drives Consecutive Cleavage at Two Sites in Prothrombin

Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombinase by using a series of recombinant derivatives: wild type prothrombin (II(WT)) contained both cleavage sites; II(Q271) contained a single cleavable site at Arg(320); II(Q320) and II(A320) contained a single cleavable site at Arg(271); and II(QQ) was resistant to cleavage. Cleavage at Arg(320) in II(Q271) could account for the initial cleavage reaction leading to the consumption of either plasma prothrombin or II(WT), whereas cleavage at Arg(271) in either II(Q320) or II(A320) was found to be approximately 30-fold slower. Equivalent kinetic constants were obtained for three of the four possible half-reactions. Slow cleavage at Arg(271) in intact prothrombin resulted from an approximately 30-fold reduction in V(max). Thus, the observed pathway of bond cleavage by prothrombinase can be explained by the kinetic constants for the four possible individual cleavage reactions. II(Q320) was a competitive inhibitor of II(Q271) cleavage, and II(QQ) was a competitive inhibitor for each reaction with K(i) approximately K(m). The data are inconsistent with previous proposals and suggest a model in which substrates for each of the four possible half-reactions bind in a mutually exclusive manner and with equal affinity to prothrombinase in a cleavage site-independent way. Despite equivalent exosite binding interactions between all four possible substrates and the enzyme, we propose that ordered bond cleavage results from the constraints associated with the binding of substrates in one of two conformations to a single form of prothrombinase.

Phospholipid bilayer formation at a bare Si surface: a time-resolved neutron reflectivitystudy

Neutron reflectivity was applied to monitor in situ the adsorption of small unilamellarphospholipid vesicles on a solid bare hydrophilic Si interface. The obtained reflectivitycurves are consistent with the rupture and fusion model for the adsorption ofphosphatidylcholine vesicles to solid interfaces. The results show details of the adsorbedbilayer system at ångström resolution and indicate the presence of a thin thick water leaflet that separates the bilayer from the Si surface. The resolved structuraldetails provide the basis for further investigation of processes such as adsorption andpenetration of peptides and proteins towards the supported bilayer at high resolution.

ABA-C15: A New Dye for Probing Solvent Relaxation in Phospholipid Bilayers

We synthesized and studied N-palmitoyl-3-aminobenzanthrone (ABA-C 1 5 ), which we proved to be an advantageous new fluorescent phospholipid membrane label. While the absorption of ABA-C 1 5 in protic solvents shows negative solvatochromism, its fluorescence emission is substantially red-shifted when the polarity of the solvent is increased. ABA-C 1 5 is excitable by lasers emitting in the range between 390 and 490 nm; it exhibits reasonable quantum yields in protic solvents and binds with high affinity to small unilamellar phospholipid vesicles. Absorption, steady state fluorescence, and solvent relaxation data indicate that the aminobenzanthrone chromophore is located in the headgroup region of phospholipid bilayers in the liquid crystalline state of small unilamellar vesicles. The solvent relaxation kinetics probed by ABA-C 1 5 in the liquid crystalline state is characterized by three solvent relaxation times in the order of 0.05, 0.2, and 1.5 ns, respectively. We observed that the relative contribution of the 0.05 ns component and the overall Stokes shift became larger with increasing difference between the experimental temperature and the main phase transition temperature; this suggests that the chromophore becomes more accessible by water molecules. In the gel phase, a component faster than 30 ps significantly contributes to the solvent relaxation kinetics. However, the solvent relaxation on the nanosecond time scale appears to be slower than in the liquid crystalline phase. The shape and time evolution of the time-resolved emission spectra suggest that two distinct microenvironments of the dye might be responsible for the atypical solvent relaxation characteristics in the gel phase.

Preparation and analysis of small unilamellar phospholipid vesicles of a uniform size

The interaction of carbonmonoxyhemoglobin and heme with small unilamellar phospholipid vesicles was studied using dynamic light scattering. Addition of carbonmonoxyhemoglobin to dimyristoylphosphatidylcholine:dimyristoylphosphatidylserine small unilamellar vesicles resulted in an increase of average vesicle size from 17.4 to 32.0 nm. Addition of heme to vesicles produced a smaller size increase, from 17.4 to 21.0 nm. Also reported is a method for preparing small unilamellar lipid vesicles of a uniform size, suitable for use in NMR spectroscopy.

Investigation of the spontaneous transferability of a phospholipid-poly(ethylene glycol)-biotin derivative from small unilamellar phospholipid vesicles to magnetoliposomes

High-gradient magnetophoresis was used to monitor the spontaneous transfer between phospholipid vesicles and magnetoliposomes of dipentadecanoylphosphatidylethanolamine (DC 15PE) and of its conjugate with a biotinylated PEG molecule (DC 15-PEG-B). For the latter molecule, a half-time of 70 min was found, whereas DC 15PE did not move. The observed transferability of PEGylated phospholipids offers a unique method for generating Stealth ®-magnetoliposomes.