Concentration dependence of nitroxyl spin probes in liposomal solution: electron spin resonance and overhauser-enhanced magnetic resonance studies

In this work, the detailed studies of electron spin resonance (ESR) and overhauser-enhanced magnetic resonance imaging (OMRI) were carried out for permeable nitroxyl spin probe, MC-PROXYL as a function of agent concentration in liposomal solution. In order to compare the impermeable nature of nitroxyl radical, the study was also carried out only at 2?mM concentration of carboxy-PROXYL. The ESR parameters were estimated using L-band and 300?MHz ESR spectrometers. The line width broadening was measured as a function of agent concentration in liposomal solution. The estimated rotational correlation time is proportional to the agent concentration, which indicates that less mobile nature of nitroxyl spin probe in liposomal solution. The partition parameter and permeability values indicate that the diffusion of nitroxyl spin probe distribution into the lipid phase is maximum at 2?mM concentration of MC-PROXYL. The dynamic nuclear polarization (DNP) parameters such as DNP factor, longitudinal relaxivity, saturation parameter, leakage factor and coupling factor were estimated for 2?mM MC-PROXYL in 400?mM liposomal dispersion. The spin lattice relaxation time was shortened in liposomal solution, which leads to the high relaxivity. Reduction in coupling factor is due to less interaction between the electron and nuclear spins, which causes the reduction in enhancement. The leakage factor increases with increasing agent concentration. The increase in DNP enhancement was significant up to 2?mM in liposomal solution. These results paves the way for choosing optimum agent concentration and OMRI scan parameters used in intra and extra membrane water by loading the liposome vesicles with a lipid permeable nitroxyl spin probes in OMRI experiments.     

Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine. Spin label and titration studies

Electrometric titrations and spin label data demonstrate changes in the experimentally determined apparent pK of an ionizable drug in the presence of membranes. This effect is attributed to the difference in partition coefficients for the charged and uncharged forms of the drug. Investigation of the binding of a local anesthetic, tetracaine, to egg phosphatidylcholine membranes indicates that the drug apparent pK decreases in the presence of membranes, the decrease being a function of membrane concentration. The agreement between titration and spin label studies is very good and could be simulated by calculating membrane-bound and free populations of charged and uncharged tetracaine from the independently-measured partition coefficients for the two forms.     

Partitional and motional properties of a spin-labeled daunomycin in lipid bilayers. An ESR study.

The partition coefficient of a spin-labeled daunomycin (DAU-SL) in dimyristoylphosphatidylcholine membrane has been determined using the electron spin resonance (ESR) method. The experiment was carried out as a function of temperature between 5 degrees C and 35 degrees C, giving partition coefficients between 2 and 6 without abrupt change at the phase transition. The thermodynamic parameters on transferring the DAU-SL from the aqueous phase to the lipid bilayer were also calculated. The calculated values are: delta H = 6.11 kcal/mol and delta S = 23 cal/K mol. The partitioning of the DAU-SL and its motion in the membrane were investigated in a wide range of pH (4-10.3). The data show that pH has no effect on partitioning of the DAU-SL which suggest that the drug exists in the uncharged form in the bilayer.     

Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine spin label and titration studies

Electrometric titrations and spin label data demonstrate changes in the experimentally determined apparent pK of an ionizable drug in the presence of membranes. This effect is attributed to the difference in partition coefficients for the charged and uncharged forms of the drug. Investigation of the binding of a local anesthetic, tetracaine, to egg phosphatidylcholine membranes indicates that the drug apparent pK decreases in the presence of membranes, the decrease being a function of membrane concentration. The agreement between titration and spin label studies is very good and could be simulated by calculating membrane-bound and free populations of charged and uncharged tetracaine from the independently-measured partition coefficients for the two forms.     

A novel steroidal spin label for membrane structure studies: synthesis and applications

2,2,6,6-Tetramethyl piperidine-N-oxyl nitroxyls are known to partition between aqueous and lipid phases, thus serving as probes to study membrane dynamics. The synthesis of a novel steroidal spin label, 3alpha-hydroxycholan-24-yl-(2",2",6",6"-tetramethyl-N-oxyl)p iperidyl butan-1',4'-dioate, containing 2,2,6,6-tetramethylpiperidine-N-oxyl moiety covalently bonded to the side chain in 3,24-caprostan-diol has been described. The localization of this spin label in model biomembranes has been studied by using electron spin resonance, differential scanning calorimetry, and 1H and 31P NMR spectroscopic techniques. Its applicability in studying the phase transition properties of model membrane L-alpha-dipalmitoyl phosphatidyl choline in the presence and absence of drugs has been described by using electron spin resonance. The label has also been used to study the permeability of epinephrine into membrane. The results have shown the applicability of the spin label as a potential spin probe in the study of biomembranes.     

Sonochemistry of nitrone spin traps in aqueous solutions. Evidence for pyrolysis radicals from spin traps

When argon-saturated aqueous solutions of alpha-phenyl-N-tert-butylnitrone (PBN) were sonicated, the spin adducts PBN-Phenyl (Ph), PBN-X, and PBN-H were observed. It can be inferred that PBN-Ph and -X arise from spin adducts of thermal decomposition products of PBN induced by the high temperature due to ultrasonic cavitation. The ESR signal of PBN-H was observed at a lower PBN concentration than those of PBN-Ph and PBN-X. The ratios of ESR intensity of PBN-H to those of PBN-Ph and PBN-X increased with the final temperatures of the cavitation bubbles created by different rare gases. The spin adducts of methyl and tert-butyl radicals from the pyrolysis of PBN, induced by the high temperatures due to cavitation, were found from spin trapping experiments in which 3,5-dibromo-2,6-dideuterio-4-nitrosobenzene sulfonate was used as a spin trap. Similar spin adducts induced by pyrolysis were also observed in sonicated aqueous solutions of other nitrone spin traps, such as alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, and alpha-(4-nitrophenol) N-tert-butylnitrone. The greater the hydrophobicity of the spin traps, as measured by the 2-octanol/water partition coefficients, the lower the concentration of spin trap at which methyl radicals generated by thermal decomposition of the spin trap can be observed. The present results indicate that the nonvolatile, highly hydrophobic spin traps accumulate preferentially in the interfacial region of cavitation bubbles where they undergo thermal decomposition during cavitation to produce the radicals.     

Reappraisal of the electron spin resonance spectra of maleimide and iodoacetamide spin labels in erythrocyte ghosts.

The effects of sulfhydryl inhibitors (iodoacetamide and N-ethylmaleimide) on the electron spin resonance spectra of two maleimide and two iodoacetamide spin labels in erythrocyte ghosts were found to correlate with their relative “lipid”/water partition coefficients. But the spectral characteristics of the maleimide spin labels, and their ghost concentrations after iodoacetamide inhibition, are not consistent with the hypothesis that interprets their spectra solely on the basis of a heterogenous membrane distribution. An alternative hypothesis is suggested which is compatible with relative “lipid solubilities” and the iodoacetamide inhibition spectra.     

Localization of spin labels in oat leaf protoplasts

An assay based on light-mediated oxidation was used to determine whether specific spin labels were partitioned throughout the protoplast or retained in the plasmalemma of Avena sativa L. cv. Garry and Park. Many classes of spin label were tested, including phospholipids, fatty acid, fatty acid methyl ester, maleimide, iodoacetamide, short chain hydrocarbon, androstane, 2,2,6,6-tetramethyl-4-aminopiperidinooxyl (TEMPAMINE) and 2,2,6,6-tetramethylpiperidinooxyl (TEMPO). All except the phosphotidylcholine spin label were found to partition throughout the cell. The phosphotidylcholine spin label may have been selectively retained in the plasmalemma.     

N-2-(2-ethoxycarbonyl-propyl) alpha-phenylnitrone: an efficacious lipophilic spin trap for superoxide detection.

The spin trap N-2-(2-ethoxycarbonyl-propyl) alpha-phenylnitrone, EPPN 1, synthesized by methods previously described, has been purified by recrystallization. A measure of its octanol-phosphate buffer partition coefficient (P(oct) = 29.8) indicated that EPPN was quite lipophilic, yet it could be easily solubilized in water up to 60 mmol L(-1). Although this nitrone was unsuitable for detecting hydroxyl radical, it efficiently trapped several carbon-centered radicals as well as superoxide in aqueous media, without yielding any artifactual signals. Kinetic studies of the superoxide adduct decay gave rate constants k(D) of 2 x 10(-3) and 2.1 x 10(-3) s(-1) at pH 5.6 and pH 7, respectively. EPPN can be considered as an easily prepared and highly pure spin trap, allowing efficient detection of superoxide in aqueous environments.     

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that, for a dipalmitoylphosphatidylcholine (DPPC) membrane whose gel-to-fluid lipid phase transition occurs at 314 K, while the TEMPO spin label is stabilized above the bilayer interface in the gel phase, there is a preferential location of TEMPO below the membrane interface in the fluid phase. For bilayers made of unsaturated lipids, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of the TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.     

Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.     

Spin-label studies of the lipid regions of spinach thylakoids and a detergent-derived oxygen-evolving Photosystem II preparation

We have compared the fluidity of thylakoid membranes with the membrane present in a Triton X-100-derived, oxygen-evolving Photosystem II (PS II) preparation using two different spin labels. Data obtained with 2,2,6,6-tetramethylpipiridine-N-oxyl (TEMPO) shows that the PS II preparation contains less fluid membrane than the thylakoid. The TEMPO partition parameter (f) is about 2.5-times greater for the thylakoids at 6 mg chlorophyll/ml than for the PS II preparation at the same chlorophyll concentration. Similarly, the rotational correlation time, τ, of TEMPO residing in the membrane of the PS II preparation is about 2-times longer than the τ for TEMPO in the thylakoid membrane. A spin label which partitions more completely into the bilayer, 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl (7N14), indicates a much greater fluidity in the thylakoid membrane than the membrane of the PS II preparation. The PS II preparation appears to have a hydrocarbon phase which approaches the rigid limit of EPR detectable motion. These results are discussed in terms of possible lipid depletion in the PS II preparation and in terms of lateral heterogeneity of hydrocarbon fluidity in the thylakoid membrane caused by the lateral heterogeneity in protein components.     

XSophe-Sophe-XeprView. A computer simulation software suite (v. 1.1.3) for the analysis of continuous wave EPR spectra

The XSophe-Sophe-XeprView computer simulation software suite enables scientists to easily determine spin Hamiltonian parameters from isotropic, randomly oriented and single crystal continuous wave electron paramagnetic resonance (CW EPR) spectra from radicals and isolated paramagnetic metal ion centers or clusters found in metalloproteins, chemical systems and materials science. XSophe provides an X-windows graphical user interface to the Sophe programme and allows: creation of multiple input files, local and remote execution of Sophe, the display of sophelog (output from Sophe) and input parameters/files. Sophe is a sophisticated computer simulation software programme employing a number of innovative technologies including; the Sydney OPera HousE (SOPHE) partition and interpolation schemes, a field segmentation algorithm, the mosaic misorientation linewidth model, parallelization and spectral optimisation. In conjunction with the SOPHE partition scheme and the field segmentation algorithm, the SOPHE interpolation scheme and the mosaic misorientation linewidth model greatly increase the speed of simulations for most spin systems. Employing brute force matrix diagonalization in the simulation of an EPR spectrum from a high spin Cr(III) complex with the spin Hamiltonian parameters g(e) = 2.00, D=0.10 cm(-1), E/D = 0.25, A(x) = 120.0, A(y) = 120.0, A(z) = 240.0 x 10 (-4) cm(-1) requires a SOPHE grid size of N = 400 (to produce a good signal to noise ratio) and takes 229.47 s. In contrast the use of either the SOPHE interpolation scheme or the mosaic misorientation linewidth model requires a SOPHE grid size of only N = 18 and takes 44.08 and 0.79 s, respectively. Results from Sophe are transferred via the Common Object Request Broker Architecture (CORBA) to XSophe and subsequently to XeprView where the simulated CW EPR spectra (1D and 2D) can be compared to the experimental spectra. Energy level diagrams, transition roadmaps and transition surfaces aid the interpretation of complicated randomly oriented CW EPR spectra and can be viewed with a web browser and an OpenInventor scene graph viewer.     

Diffusion and partition of solutes in cartilage under static load

We describe experimental apparatus, methodology and mathematical algorithms to measure diffusion and partition for typical small ionic solutes and inulin (a medium size solute) in statically loaded cartilage. The partition coefficient based on tissue water (K(H(2)O)) of Na(+) increased from 1.8 to 4.5 and for SO(4)(-2) decreased from 0.5 to 0.1, when the applied pressure was raised from zero to 22 atm K(H(2)O) of inulin decreased from 0.3 to 0.05, for an increase in pressure from zero to 11 atm. Our theoretical interpretation of the results is that the partition coefficient can be expressed as a function of fixed charge density (FCD) for both loaded and unloaded cartilage. The partition coefficient shows good agreement with the ideal Gibbs-Donnan equilibrium, particularly when FCD is based on extrafibrillar water (EFW). The diffusion coefficients, D also decreased with an increase in applied pressure; raising the pressure from 0 to 22 atm resulted in the following changes in the values of D: for Na(+) from 2.86 x 10(-6) to 1.51 x 10(-6) cm(2)/s, for SO(4)(-2) from 1.58 x 10(-6) to 7.5 x 10(-7) cm(2)/s, for leucine from 1.69 x 10(-6) to 8.30 x 10(-7) cm(2)/s and for inulin from 1.80 x 10(-7) to 3.30 x 10(-8) cm(2)/s. For the three small solutes (two charged and one neutral) the diffusion coefficient D is highly correlated with the fraction of fluid volume in the tissue. These experimental results show good agreement with the simple model of Mackie and Meares: hence solute charge does not affect the diffusion of small solutes under load. For inulin D & K show some agreement with a modified Ogston model based on two major components, viz., glycosaminoglycans (GAG) and core protein. We conclude that the changes in the partition and diffusion coefficients of small and medium size solutes in statically loaded cartilage can be interpreted as being due to the reduction in hydration and increase in FCD. The change in the latter affects the partition of small ionic solutes and the partition and diffusion of larger molecules. Our results throw light on the ionic environment of chondrocytes in loaded cartilage as well as on the transport of solutes through the matrix.     

Membrane behavior as influenced by partitioning of amphiphiles during drying: a comparative study in anhydrobiotic plant systems

During cellular desiccation, reduction in volume can in principle cause amphiphilic compounds to partition from the cytoplasm into membranes, with structural perturbance as the result. Here, we studied the effect of partitioning of endogenous amphiphiles on membrane surface dynamics in desiccation-tolerant and -intolerant, higher and lower plant systems, using electron paramagnetic resonance (EPR) spin probe techniques. Labeling cells with the amphiphilic spin probe perdeuterated TEMPONE (PDT) enabled partitioning into the various phases to be followed. During drying, PDT molecules preferentially partitioned from the aqueous cytoplasm into the membrane surface and, at advanced stages of water loss, also into oil bodies. There was no specific partition behavior that could be correlated with lower/higher plants or with desiccation-tolerance. In vivo labeling with 5-doxylstearate (5-DS) enabled membrane surface fluidity to be characterized. In hydrated plants, the 5-DS spectra contained an immobile and a fluid component. The characteristics of the immobile component could not be specifically correlated with either lower or higher plants, or with desiccation tolerance. The relative contribution of the fluid component to the 5-DS spectra was higher in lower plants than in higher plants, but considerably decreased with drying in all desiccation-tolerant organisms. In contrast, the proportion of the fluid component in desiccation-sensitive wheat seedling root was higher than that in desiccation-tolerant wheat axis and considerably increased at the onset of water loss. We suggest that partitioning of amphipaths fluidize the membrane surface, but that in desiccation-tolerant systems the membranes are protected from excessive fluidization.     

Solubility of carbon dioxide in lipid bilayer membranes and organic solvents

Partition coefficients of carbon dioxide into lipid bilayers (liposomes) and organic solvents were measured as a function of temperature. The molar partition coefficient of CO2 into liposomes of egg lecithin at 25 degrees C was 0.95 (ml CO2/ml lipid)/(ml CO2/ml saline). The addition of an equimolar amount of cholesterol to the egg lecithin decreased the partition coefficient by about 25%. The partition coefficients for CO2 into liposomes at 25 degrees C were lower than the partition coefficients into octanol (1.3), hexadecane (1.5) and olive oil (1.7). The results are discussed in terms of the solubility-diffusion model of non-electrolyte transport through lipid bilayer membranes.     

Partition of a fluorescent molecule between liquid-crystalline and crystalline regions of membranes

Summary We have determined the partition coefficient of the fluorescent molecule perylene between liquid crystalline and crystalline regions of vesicle membranes formed from binary mixtures of several lipids. We measured the fluorescence intensity of perylene in these vesicles as a function of temperature and used the intensity profiles, together with a theory developed in a previous paper, to determine the partition coefficient defined as the ratio of the concentration of perylene in the liquid-crystalline (fluid) regions of the membrane to the concentration in the crystalline (solid) phase. In vesicles composed of dipalmitoyl phosphatidylcholine/distearoyl phosphatidylcholine (dppc/dspc) mixtures and of dipalmitoyl phosphatidylcholine/dipalmitoyl phosphatidylethanolamine (dppc/dppe) mixtures, the partition coefficient is close to unity. Its value is 1.04±0.18 for dppc/dsp mixtures and 1.10±0.26 for dppc/dppe mixtures. In vesicles composed of dimyristoyl phosphatidylcholine/distearoyl phosphatidylcholine mixtures, the partition coefficient was more difficult to determine and its value ranged from 0.3 to 7.     

The retinoic-like juvenile hormone controls the looping of left-right asymmetric organs in Drosophila

In vertebrate development, the establishment of left-right asymmetry is essential for sidedness and the directional looping of organs like the heart. Both the nodal pathway and retinoic acid play major and conserved regulatory roles in these processes. We carried out a novel screen in Drosophila to identify mutants that specifically affect the looping of left-right asymmetric organs. We report the isolation of spin, a novel mutant in which the looping of the genitalia and spermiduct are incomplete; under-rotation of the genitalia indicates that spin controls looping morphogenesis but not direction, thus uncoupling left-right asymmetry and looping morphogenesis. spin is a novel, rotation-specific allele of the fasciclin2 (Fas2) gene, which encodes a cell-adhesion protein involved in several aspects of neurogenesis. In spin mutants, the synapses connecting specific neurosecretory cells to the corpora allata are affected. The corpus allatum is part of the ring gland and is involved in the control of juvenile hormone titers during development. Our genetic and pharmacological results indicate that Fas2(spin) rotation defects are linked to an abnormal endocrine function and an elevated level of juvenile hormone. As juvenile hormone is an insect sesquiterpenoid related to retinoic acid, these results establish a new genetic model for studying organ looping and demonstrate an evolutionarily conserved role for terpenoids in this process.     

Environmentally induced post‐spin property changes in spider silks: influences of web type,spidroin composition and ecology

Many spiders use silk to construct webs that must function for days at a time, whereas many other species renew their webs daily. The mechanical properties of spider silk can change after spinning under environmental stress, which could influence web function. We hypothesize that spiders spinning longer‐lasting webs produce silks composed of proteins that are more resistant to environmental stresses. The major ampullate (MA) silks of orb web spiders are principally composed of a combination of two proteins (spidroins) called MaSp1 and MaSp2. We expected spider MA silks dominated by MaSp1 to have the greatest resistance to post‐spin property change because they have high concentrations of stable crystalline β‐sheets. Some orb web spiders that spin three‐dimensional orb webs, such as Cyrtophora, have MA silks that are predominantly composed of MaSp1. Hence, we expected that the construction of three‐dimensional orb webs might also coincide with MA silk resistance to post‐spin property change. Alternatively, the degree of post‐spin mechanical property changes in different spider silks may be explained by factors within the spider's ecosystem, such as exposure to solar radiation. We exposed the MA silks of ten spider species from five genera (Nephila, Cyclosa, Leucauge, Cyrtophora, and Argiope) to ecologically high temperatures and low humidity for 4 weeks, and compared the mechanical properties of these silks with unexposed silks. Using species pairs enabled us to assess the influence of web dimensionality and MaSp composition both with and without phylogenetic influences being accounted for. We found neither the MaSp composition nor the three‐dimensionality of the orb web to be associated with the degree of post‐spin mechanical property changes in MA silk. The MA silks in Leucauge spp. are dominated by MaSp2, which we found to have the least resistance to post‐spin property change. The MA silk in Argiope spp. is also dominated by MaSp2, but has high resistance to post‐spin property change. The ancestry of Argiope is unresolved, but it is largely a tropical genus inhabiting hot, open regions that present similar stressors to silk as those of our experiment. Ecological factors thus appear to influence the vulnerability of orb web spider MA silks to post‐spin property change. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 580–588.     

Spin traps: in vitro toxicity and stability of radical adducts

We have evaluated the effects of DMPO, CMPO, EMPO, BMPO, and DEPMPO on functioning CHO cells and the stability of the radical adducts in the presence of cells. The potential toxic effects of the spin traps were measured by two estimates of cell viability (trypan blue exclusion and colony formation) and one of cell function (rate of oxygen consumption). We also studied the effects of the spin traps on colony formation in a second cell line, 9L tumor cells. Toxicity varied with the type of cell line and the parameter that was measured. In aqueous solutions the order of stability for all spin adducts was SO(3) > OH > CH(3), while in cell suspensions it was SO(3) > OH approximately CH(3). The radical adducts of the new spin traps have significantly increased stability as compared to DMPO. These results indicate that the new spin traps potentially offer increased stability of spin adducts in functioning cells. It also is clear that it is necessary to carry out appropriate studies of the stability and toxicity in the system that is to be studied for any particular use of these spin traps. It then should be feasible to select the spin trap(s) best suited for the proposed study.