Partition coefficients of fluorescent probes with phospholipid membranes

A method for determination of membrane partition coefficients of five fluorescent membrane probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), p-((6-phenyl)-1,3,5-hexatrienyl) benzoic acid (DPH carboxylic acid), 3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid (DPH propionic acid), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and N-4-(4-didecylaminostyryl)-N-methylpyridinium iodide (4-di-10-ASP), was developed utilizing the fluorescence enhancement of a constant probe concentration by titration with excess phospholipid liposomes. The partition coefficients of DPH, DPH carboxylic acid, DPH propionic acid, TMA-DPH and 4-di-10-ASP into dipalmitoylphosphatidylcholine membranes were determined to be 1.3.10(6), 1.0.10(6), 6.5.10(5), 2.4.10(5) and 2.8.10(6) respectively. Knowledge of the partition coefficients may help select a lipid concentration for membrane studies that necessitate a probe's dominant incorporation into membranes.     

Novel fluorescent phospholipids for assays of lipid mixing between membranes

A series of fluorescent phospholipids has been synthesized, by a general and versatile procedure, with various fluorescent groups attached to the methyl-terminal half of one acyl chain in an otherwise normal phospholipid structure. Phospholipids labeled with (dialkylamino)coumarin moieties, and to a slightly lesser extent those labeled with a bimane group, exhibit a strong and stable blue fluorescence in phospholipid dispersions that is relatively insensitive to the physical state of the lipid phase. The fluorescence of these labeled phospholipids is efficiently quenched by resonance energy transfer to lipids labeled with a [[(dimethylamino)phenyl]azo]phenyl or a methyl(nitrobenzoxadiazolyl)amino group when these acceptors are incorporated into the same bilayer as the donor species. Acyl chain labeled phospholipid probes, both of whose chains are at least sixteen carbons in length, exchange extremely slowly between lipid vesicles (less than 1% exchange/h). These properties allow various donor-acceptor combinations of probes to be employed in sensitive and reliable assays of lipid mixing accompanying membrane fusion. We demonstrate that, in two particularly demanding applications (assays of the calcium-mediated coalescence of phosphatidylserine vesicles and of the proton-triggered coalescence of phosphatidylethanolamine vesicles), some combinations of acyl chain labeled probes offer substantial advantages over the commonly used N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine/N-(l issamine rhodamine B sulfonyl)phosphatidylethanolamine pair to monitor accurately the progress of lipid mixing between vesicles.     

Partition of CoQ-homologs in lipid membranes

After studies on incorporation of Coenzyme-Q-homologs into mitochondrial membranes, we have undertaken the same investigation on phospholipid vesicles in order to know the behavior of different Ubiquinones in the biphasic system lipid membrane/H2O. Using concentrations of egg lecithin corresponding to the phospholipid content of 1 mg mitochondrial protein, it was found that some homologs are partitioned in lipid vesicles nearly the same as they are incorporated by mitochondria, while others behave differently. On the basis of the results it is possible to calculate approximately partition coefficients for each Q-homolog. This work shows that also membrane structure, besides the partition membrane/water, affects the incorporation of Ubiquinones.     

Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide gamma-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10-40 degrees C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol dramatically decreases partition (2100 falls to 100, at 10 degrees C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.     

Photoconversion of Lysotracker Red to a green fluorescent molecule

Dear Editor： Lysotracker Red DND-99 （Invitrogen-Molecular Probes） is a fluorophore in the form of a conjugated multi-pyrrole ring structure containing a weakly basic amine that selectively accumulates in acidic compartments and exhibits red fluorescence （excitation： 577 nm, emission： 590 nm） （Figure 1A）. It is structurally related to Lysotracker Green （Figure 1B） but has an additional pyrrole ring in conjugation with the primary structure, which produces a longer wavelength emission. Lysotracker Red is commonly used in multicolor imaging studies as a lysosomal marker to determine intracellular localization of a protein of interest by fluorescence and confocal microscopy [1-5] and is recommended by the manufacturer for this application. While using Lysotracker Red to study the localization of a protein fused to green fluorescent protein （GFP）, we observed an additional strong green fluorescent signal that colocalized with Lysotracker Red. After careful examination, however, we noted that the added green signal appeared only after illumination of the cells by a standard 100W mercury epi-fluorescence light source equipped with a 560/40 excitation filter （Leica TX2）. Prior to exposing the field to broadband excitation light, it was possible to visualize by confocal scanning （488nm excitation line） cells that exclusively expressed GFP. Remarkably, after exposure to broadband excitation light, green fluorescence appeared in all cells irrespective of GFP expression, displayed signal intensity similar to that of GFP, and colocalized with Lysotracker Red （Figure S1）.[第一段]     

Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide γ-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10–40°C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol dramatically decreases partition (2100 falls to 100, at 10°C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.     

Partition of DDT in synthetic and native membranes

Partition of DDT (2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane) was determined in artificial and native membranes. Partition in egg phosphatidylcholine of about 260 000 is independent of temperature over the range from 10 to 40 degrees C, in which the lipid is in the liquid-crystalline state. Incorporation of 50 mol% cholesterol decreases DDT partition to about 120 000. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in DDT partitioning. Partition decreases symmetrically in the temperature ranges to both sides of the phase transition. The insecticide is preferentially accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 336 000, 180 000 and 88 000 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. Partition values in native membranes decrease sequentially as follows: sarcoplasmic reticulum, mitochondria, myelin, brain microsomes and erythrocytes. This sequence is similar to that observed in related liposomes of total extracted lipids, although the absolute partitions showed decreased values. Partition of DDT in native membranes exhibits a negative temperature coefficient not apparent in related lipid dispersions. The effect of intrinsic membrane cholesterol on partition of DDT was also investigated.     

Partition of malathion in synthetic and native membranes

Partition coefficients of [14C]malathion in model and native membranes are affected by temperature, cholesterol content, and lipid chain length. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10-40 degrees C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol severely decreases partition and practically abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in malathion partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 225, 135 and 48 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. Partition values in native membranes decrease sequentially as follows: sarcoplasmic reticulum, mitochondria, brain microsomes, myelin and erythrocytes. This dependence parallels the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values.     

Rotational decoupling between the hydrophilic and hydrophobic regions in lipid membranes

Cells use homeostatic mechanisms to ensure an optimal composition of distinct types of lipids in cellular membranes. The hydrophilic region of biological lipid membranes is mainly composed of several types of phospholipid headgroups that interact with incoming molecules, nanoparticles, and viruses, whereas the hydrophobic region consists of a distribution of acyl chains and sterols affecting membrane fluidity/rigidity related properties and forming an environment for membrane-bound molecules such as transmembrane proteins. A fundamental open question is to what extent the motions of these regions are coupled and, consequently, how strongly the interactions of phospholipid headgroups with other molecules depend on the properties and composition of the membrane hydrophobic core. We combine advanced solid-state nuclear magnetic resonance spectroscopy with high-fidelity molecular dynamics simulations to demonstrate how the rotational dynamics of choline headgroups remain nearly unchanged (slightly faster) with incorporation of cholesterol into a phospholipid membrane, contrasting the well-known extreme slowdown of the other phospholipid segments. Notably, our results suggest a new paradigm in which phospholipid dipole headgroups interact as quasi-freely rotating flexible dipoles at the interface, independent of the properties in the hydrophobic region.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes

1. A reversible light-induced enhancement of the fluorescence of a “hydrophobic fluorophore”, 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1′-ethylene-2,2′-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response.

2. This effect is decreased under phosphorylating conditions (presence of ADP, P_i, Mg²⁺), and completely inhibited by the uncoupler of phosphorylation NH₄Cl (5–10 mM), as well as the ionophores nigericin and gramicidin-D (both at 5 · 10⁻⁸ M). The MgCl₂ concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a “high energy intermediate”. The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the “high energy state” of the thylakoid membrane rather than a terminal phosphorylation step.

3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fluorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H⁺ uptake per se because (a) the pH dependence of H⁺ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H⁺ uptake, reduces anthroyl stearate fluorescence.

Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate or Diquat catalyzed electron flow and is the first “non-amine” fluorescence probe utilized for this purpose in chloroplasts.     

Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes.

1. A reversible light-induced enhancement of the fluorescence of a "hydrophobic fluorophore", 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1'-ethylene-2,2'-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response. 2. This effect is decreased under phosphorylating conditions (presence of ADP, Pi, Mg2+), and completely inhibited by the uncoupler of phosphorylation NH4Cl(5-10mM), as well as the ionophores nigericin and gramicidin-D (both at 5 - 10(-8)M). The MgCl2 concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a "high energy intermediate". The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the "high energy state" of the thylakoid membrane rather than a terminal phosphorylation step. 3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fllorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H+ uptake per se because (a) the pH dependence of H+ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H+ uptake, reduces anthroyl stearate fluorescence. Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate of Diquat catalyzed electron flow and is the first "non-amine" fluorescence probe utilized for this purpose in chloroplasts.     

Phase properties of liquid-crystalline Phosphatidylcholine/Phosphatidylethanolamine bilayers revealed by fluorescent probes.

The mixing properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were examined in liquid-crystalline phase using fluorescent probes incorporated into lipid bilayers. The excimer to monomer (E/M) fluorescence ratio of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (PPC) versus PPC concentration was higher for binary mixtures containing phosphatidylcholine (PC)/phosphatidylethanolamine (PE) (1:1) compared to PC matrix. When POPC was gradually replaced with POPE, the E/M ratio also increased suggesting the enhanced lateral mobility or the lateral enrichment of PPC into domains or both. Evidences for the PE-induced domain formation were further provided by resonance energy transfer between 2-(4, 4-difluoro-5-methyl-4-boro-3a, 4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero- 3-phospho choline and PPC, which was enhanced as a function of PE concentration, and by the polarization of 1,6-diphenyl-1,3, 5-hexatriene. In addition, PE reduced free volume and polarity of lipid bilayers as measured by the emission fluorescence of 1,2-bis PPC and 6-lauroyl-2-dimethylaminonaphthalene. When POPE analogs with a methylated head group instead of normal POPE were used, the diminished effect on the domain formation was shown in the order N-methyl PE > N,N-dimethyl PE. The results suggest that the mixing properties of POPE and POPC are not random but that lipid domains of phospholipids are formed.     

Rotational diffusion of a steroid molecule in phosphatidylcholine-cholesterol membranes: fluid-phase microimmiscibility in unsaturated phosphatidylcholine-cholesterol membranes

Rotational diffusion of androstane spin-label (ASL), a sterol analogue, in various phosphatidylcholine (PC)-cholesterol membranes was systematically studied by computer simulation of steady-state ESR spectra as a function of the chain length and unsaturation of the alkyl chains, cholesterol mole fraction, and temperature for a better understanding of phospholipid-cholesterol and cholesterol-cholesterol interactions. Special attention was paid to the differences in the cholesterol effects on ASL motion between saturated and unsaturated PC membranes. ASL motion in the membrane was treated as Brownian rotational diffusion of a rigid rod within the confines of a cone imposed by the membrane environment. The wobbling rotational diffusion constant of the long axis, its activation energy, and the cone angle of the confines were obtained for various PC-cholesterol membranes in the liquid-crystalline phase. Cholesterol decreases both the cone angle and the wobbling rotational diffusion constant for ASL in all PC membranes studied in this work. The cholesterol effects are the largest in DMPC membranes. An increase of cholesterol mole fraction from 0 to 30% decreases the rotational diffusion constant by a factor of 9-15 (depending on temperature) and the cone angle by a factor of about 2. In dioleoyl-PC membranes, addition of 30 mol % cholesterol reduces both the rotational diffusion constant and the cone angle of ASL by factors of approximately 2.5 and approximately 1.3, respectively, while it was previously found to cause only modest effects on the motional freedom of phospholipid analogue spin probes [Kusumi, A., Subczynski, W. K., Pasenkiewicz-Gierula, M., Hyde, J. S., & Merkle, H. (1986) Biochim. Biophys. Acta 854, 307-317]. It is proposed that fluid-phase microimmiscibility takes place in dioleoyl-PC-cholesterol membranes at physiological temperatures, which induces cholesterol-rich domains in the membrane, partially due to the steric nonconformability between the rigid fused-ring structure of cholesterol and the 30 degrees bend at the C9-C10 cis double bond of the alkyl chains of dioleoyl-PC. The mechanism by which cholesterol influences the lipid dynamics in the membrane is different between saturated and unsaturated PC membranes.     

Interaction of antiaggregant molecule ajoene with membranes

The structure of ajoene, a molecule extracted from garlic, has been studied by ¹H-NMR and its interaction with model membranes by ¹H-, ²H-, ³¹-P-NMR and ESR experiments. This study clearly shows that the ajoene molecule is located deep in the layer and is close to the interlayer medium. Moreover while NMR experiments show that the membrane structure is only slightly affected by the presence of ajoene, ESR experiments reveal significant modifications in phospholipid dynamics. This interaction, observed before with the phenothiazine derivative, promazine, results in an increase of the membrane fluidity in its hydrophobic part and could be related to clinical properties of ajoene.