Determination of partition and lateral diffusion coefficients of ubiquinones by fluorescence quenching of n-(9-anthroyloxy)stearic acids in phospholipid vesicles and mitochondrial membranes

The quenching of fluorescence of n-(9-anthroyloxy)stearic acids and other probes by different ubiquinone homologues and analogues has been exploited to assess the localization and lateral mobility of the quinones in lipid bilayers of model and mitochondrial membranes. The true bimolecular collisional quenching constants in the lipids together with the lipid/water partition coefficients were obtained from Stern-Volmer plots at different membrane concentrations. A monomeric localization of the quinone in the phospholipid bilayer is suggested for the short side-chain ubiquinone homologues and for the longer derivatives when cosonicated with the phospholipids. The diffusion coefficients of the ubiquinones, calculated from the quenching constants either in three dimensions or in two dimensions, are in the range of (1-6) X 10(-6) cm2 s-1, both in phospholipid vesicles and in mitochondrial membranes. A careful analysis of different possible locations of ubiquinones in the phospholipid bilayer, accounting for the calculated diffusion coefficients and the viscosities derived therefrom, strongly suggests that the ubiquinone 10 molecule is located within the lipid bilayer with the quinone ring preferentially adjacent to the polar head groups of the phospholipids and the hydrophobic tail largely accommodated in the bilayer midplane. The steady-state rates of either ubiquinol 1-cytochrome c reductase or NADH:ubiquinone 1 reductase are proportional to the concentration of the quinol or quinone substrate in the membrane. The second-order rate constants appear to be at least 3 orders of magnitude lower than the second-order constants for quenching of the fluorescent probes; this is taken as a clear indication that ubiquinone diffusion is not the rate-determining step in the quinone-enzyme interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains

Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.     

Partition of proteins in aqueous two-phase systems containing charged dextran or hydrophobic PEG

Summary The electrochemical effect of a charged dextran derivative and the hydrophobic effect of hydrophobic chain PEG derivative on partitioning of six types of proteins in PEG/dextran aqueous two-phase systems were investigated- When 1. 6%(w/w)DEAE-dextran was present in the system,the partition coefficient decreased quickly with increasing pH value;when 0. 4% (w/w)PEG pentadecanoic acid ester was present in the system, the partition coefficient of protein with strong hydrophobicity was greatly increased. The experimental results show that the influence of hydrocarbon chain PEG derivative on partition coefficient is closely related to the hydrophobicity of proteins.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: A comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of ζ-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.     

Bilayer interaction and localization of cell penetrating peptides with model membranes: a comparative study of a human calcitonin (hCT)-derived peptide with pVEC and pAntp(43-58)

Cell-penetrating peptides (CPPs) are able to translocate problematic therapeutic cargoes across cellular membranes. The exact mechanisms of translocation are still under investigation. However, evidence for endocytic uptake is increasing. We investigated the interactions of CPPs with phospholipid bilayers as first step of translocation. To this purpose, we employed four independent techniques, comprising (i) liposome buffer equilibrium dialysis, (ii) Trp fluorescence quenching, (iii) fluorescence polarization, and (iv) determination of zeta-potentials. Using unilamellar vesicles (LUVs) of different phospholipid composition, we compared weakly cationic human calcitonin (hCT)-derived peptides with the oligocationic CPPs pVEC and penetratin (pAntp). Apparent partition coefficients of hCT-derived peptides in neutral POPC LUVs were dependent on amino acid composition and secondary structure; partitioning in negatively charged POPC/POPG (80:20) LUVs was increased and mainly governed by electrostatic interactions. For hCT(9-32) and its derivatives, D values raised from about 100-200 in POPC to about 1000 to 1500 when negatively charged lipids were present. Localization profiles of CPPs obtained by Trp fluorescence quenching were dependent on the charge density of LUVs. In POPC/POPG, hCT-derived CPPs were located on the bilayer surface, whereas pVEC and pAntp resided deeper in the membrane. In POPG LUVs, an increase of fluorescence polarization was observed for pVEC and pAntp but not for hCT-derived peptides. Generally, we found strong peptide-phospholipid interactions, especially when negatively charged lipids were present.     

Biosynthetic incorporation of fluorescent carbazolylundecanoic acid into membrane phospholipids of LM cells and determination of quenching constants and partition coefficients of hydrophobic quenchers

A fluorescence quenching method was developed for determining partition coefficients and diffusional rates of small molecules in cell membranes. This method involves quenching the fluorescence of carbazole-labeled membranes by hydrophobic molecules that partition into membranes. Cell membrane phospholipids of mouse LM cells in tissue culture were biosynthetically labeled with the carbazole moiety by supplementing the growth media with 11-(9-carbazolyl)undecanoic acid. Plasma membranes, microsomes, and mitochondria were isolated free of nonmembranous neutral lipids, and the incorporation of the fluorescent probe was characterized. Quenching studies of the carbazole moiety by a series of N-substituted picolinium perchlorate salts showed that the carbazole moiety was located in the hydrophobic interior of the membrane bilayer. The carbazole fluorescence also was quenched by the hydrophobic quenchers lindane, methoxychlor, and 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene, indicating that these compounds partitioned into the membrane. Stern-Volmer quenching constants determined by fluorescence lifetime and intensity measurements were identical, as expected for dynamic quenching. The effects of different lipid compositions on quenching constants and partition coefficients were determined by comparing different membrane fractions. These parameters also were measured in membranes from cells in which the phospholipid composition was altered by substituting ethanolamine for choline in the growth medium. Changes in the lipid composition produced changes in the bimolecular quenching constants. For example, bimolecular quenching constants for 1,1-dichloro-2,2-bis(rho-chlorophenyl)ethylene were higher in mitochondrial membranes than in plasma membranes and microsomes. They were also higher in dispersions made from membrane phospholipids as compared with intact membranes or total lipid dispersion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of the lateral diffusion coefficients of ubiquinones in lipid vesicles by fluorescence quenching of 12-(9-anthroyl)stearate

The lateral diffusion coefficients of some ubiquinone homologues have been measured in phospholipid vesicles exploiting the fluorescence quenching of the probe 12-(9-anthroyl)stearate by the quinones. Diffusion coefficients higher than 10(-6) cm2 X s-1 have been found at 25 degrees C, compatible with the localization of the ubiquinones in the low-viscosity midplane region of the bilayer.     

Dynamic quenchers in fluorescently labeled membranes. Theory for quenching in a three-phase system.

The theory for quenching of fluorescently labeled membranes by dynamic quenchers is described for a three-phase system: a fluorescently labeled membrane, a nonlabeled membrane, and an aqueous phase. Two different experimental protocols are possible to determine quenching parameters. Using the first protocol, partition coefficients and bimolecular quenching constants were determined for a hydrophobic quencher in carbazole-labeled membranes in the presence of an unlabeled reference membrane. These parameters determined for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE) using this three-phase analysis were in good agreement with values determined by a two-phase analysis without the reference lipid. Hence, the theory was verified. In the second protocol, the quencher partition coefficient was determined for unlabeled membranes in the presence of a carbazole-labeled reference membrane. Partition coefficients for DDE determined by this method were the same as partition coefficients determined for carbazole-labeled membranes using the two-phase analysis. The greater ease in determining partition coefficients and bimolecular quenching constants by the three-phase analysis and, in particular, the ability to determine the partition coefficient in unlabeled membranes make the three-phase analysis especially useful. This method was used to study the effect varying the membrane lipid composition has on the partition coefficient. The data indicate that partition coefficients of DDE in fluid membranes are not dramatically dependent upon polar head group composition, fatty acid composition, or cholesterol content. However, partitioning into gel-phase lipids is at least 100-fold less than fluid-phase lipids.     

Adenine nucleotide translocase greatly increases the partition of trinitrophenyl-ATP into reduced Triton X-100 micelles.

The presence of adenine nucleotide translocase (ANT) was found to greatly enhance the partitioning of the ATP analog 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) into reduced Triton X-100 micelles. The protein's effect was studied through the quenching of fluorescence of purified ANT, irreversibly inhibited by carboxyatractyloside (CAT), solubilized in reduced Triton X-100 micelles. The dependence of quenching of the protein's time-resolved tryptophan fluorescence on TNP-ATP concentration was measured and found to follow a Stern-Volmer mechanism. However, the calculated quenching constant was too large to be accounted for by the aqueous TNP-ATP concentration. Experiments were therefore conducted to determine the partitioning of the quencher between the three phases present: aqueous, protein-free micelle, and protein micelle; a system also described by the equation of Omann, G. M., and M. Glaser (1985. Biophys. J. 47:623-627.). By measuring the dependence of the apparent quenching rate constant on the protein concentration and protein/micelle ratios, this equation was used to calculate both the quencher partition coefficient into protein-free micelles (Pm) and into protein-micelles (Ppm), as well as the bimolecular quenching rate constant (kpm) in protein micelles. From the quenching experiments, kpm = 5.0 x 10(8)M-1s-1,Pm = 290 and pyrene quenching experiment to be 325, and by a rapid filtration experiment to be 450. Clearly, the presence of the integral membrane protein ANT-CAT in reduced Triton X-100 micelles greatly increases the partition of TNP-ATP into the micelle. ANT alters the properties and thus, the structure of the detergent micelle, which has direct implications for the use of detergent micelles as a model system for membrane proteins and may indicate that analogous effects occur in the mitochondrial membrane.     

Partitioning of triphenylalkylphosphonium homologues in gel bead-immobilized liposomes: chromatographic measurement of their membrane partition coefficients

Unilamellar liposomes of small or large size, SUVs and LUVs, respectively, were stably immobilized in the highly hydrophilic Sepharose 4B or Sephacryl S-1000 gel beads as a membrane stationary phase for immobilized liposome chromatography (ILC). Lipophilic cations of triphenylmethylphosphonium and tetraphenylphosphonium (TPP+) have been used as probes of the membrane potential of cells. Interaction of TPP+ and triphenylalkylphosphonium homologues with the immobilized liposomal membranes was shown by their elution profiles on both zonal and frontal ILC. Retardation of the lipophilic cations on the liposome gel bed was increased as the hydrophobicity of the cations increased, indicating the partitioning of lipophilic cations into the hydrocarbon region of the membranes. The cations did not retard on the Sepharose or Sephacryl gel bed without liposomes, confirming that the cations only interact with the immobilized liposomes. Effects of the solute concentration, flow rate, and gel-matrix substance on the ILC were studied. The stationary phase volume of the liposomal membranes was calculated from the volume of a phospholipid molecule and the amount of the immobilized phospholipid, which allowed us to determine the membrane partition coefficient (KLM) for the lipophilic cations distributed between the aqueous mobile and membrane stationary phases. The values of KLM were generally increased with the hydrophobicity of the solutes increased, and were higher for the SUVs than for the LUVs. The ILC method described here can be applied to measure membrane partition coefficients for other lipophilic solutes (e.g., drugs).     

Use of holographic laser interferometry to study the diffusion of polymers in gels

The aim of this study was to demonstrate the potential for holographic interferometry to be used for diffusion studies of large molecules in gels. The diffusion and partitioning of BSA (67,000 g/mol) and pullulans (5,900-112,000 g/mol) in agarose gel were investigated. The gel diffusion coefficients obtained for BSA were higher when distilled water was used as a solvent compared to those obtained with 0.1 M NaCl as the solvent. Furthermore, the gel diffusion coefficient increased with increasing BSA concentration. The same trend was found for liquid BSA diffusion coefficients obtained by DLS. BSA partition coefficients obtained at different agarose gel concentrations (2-6%, w/w) decreased slightly with increasing gel concentration. However, all BSA gel diffusion coefficients measured were significantly lower than those in pure solvent and they decreased with increasing agarose concentration. The gel diffusion coefficients obtained for pullulans decreased with increasing pullulan molecular weight. The same effect from increased molecular weight was seen in the liquid diffusion coefficients measured by DLS. The pullulan partition coefficients obtained decreased with increasing molecular weight. However, pullulans with a larger Stokes' radius than BSA had partition coefficients that were higher or approximately the same as BSA. This implied that the pullulan molecules were more flexible than the BSA molecules. The results obtained for BSA in this study agreed well with other experimental studies. In addition, the magnitude of the relative standard deviation was acceptable and in the same range as for many other methods. The results thereby obtained showed that holographic interferometry is a suitable method for studying diffusion of macromolecules in gels.     

Effects of Quenching Mechanism and Type of Quencher Association on Stern-Volmer Plots in Compartmentalized Systems

Fluorescence quenching techniques have been used extensively in recent years to examine reaction rates and the compartmentalization of components in lipid micelles and membranes. Steady-state fluorescence methods are frequently employed in such studies but the interpretation of the resulting Stern-Volmer plots is often hampered by uncertainties regarding the mode of association of the quencher with the lipid structure and the nature of the quenching mechanism. This paper presents a method for simulating steady-state Stern-Volmer plots in two phase systems, and shows how the forms of such plots are influenced by the type of association of the quencher with the membrane or micelle (partition and/or binding) and by the type of quenching mechanism (dynamic and/or static). Comparisons of simulated plots with experimental data must take into account the possible combinations of quencher association(s) and quenching mechanism(s). The methods presented are applicable to synthetic and natural membranes and provide a basis for comparing the quenching of fluorescent molecules in biological membranes of differing composition.     

Apparent monomer activity of saturated fatty acids im micellar bile salt solutions measured by a polyethylene partitioning system

Partitioning of saturated fatty acids between discs of polyethylene film and aqueous buffer has been characterized and subsequently used to measure monomer activities of fatty acids in micellar solutions of bile salt. Partitioning of fatty acids between polyethylene and buffer achieved equilibrium in about 24-48 hr. Partition coefficients for fatty acids 10:0 and 16:0 were essentially independent of concentration, as expected for true partitioning. Experiments with various pH buffers showed that only the protonated form of fatty acids 12:0 and 16:0 participated in partitioning, and the midpoints of the partition coefficients vs. pH curves were 4.5-5.0 and 6.5-7.0, respectively. Experimentally determined partition coefficients at pH 7.4 ranged from 2.03 +/- 0.09 for 9:0 to 56,100 +/- 13,850 for 17:0. The addition of each methylene group increased the partition coefficient by a factor of about 3.75, corresponding to an incremental free energy change for each methylene group of -3433 J.mole(-1) (-820 cal.mole(-1)). Monomer activities of solutions of 14:0 and 16:0 dissolved in 20 mM taurodeoxycholate were linearly dependent on the total fatty acid concentration. 1 mM 14:0 and 16:0 in 20 mM taurodeoxycholate had monomer activities of 1.3 x 10(-5) M and 5.6 x 10(-7) M, respectively. Solutions prepared with a constant concentration ratio of fatty acid to taurodeoxycholate had essentially constant monomer activities between 8 and 20 mM taurodeoxycholate. These studies support the hypothesis that fatty acid interaction with bile acid micelles is similar to a phase distribution system, so that some measurable monomer activity of fatty acid exists in equilibrium with the mass of fatty acid contained in the micelle.     

Interactions of chlorinated hydrocarbon insecticides with membranes

Chlorinated hydrocarbon insecticides quench the fluorescence of N-alkyl derivatives of carbazole. We used phospholipids with covalently attached carbazole as probes for the interactions of chlorinated hydrocarbon insecticides with lipid bilayers, the object being to understand better the toxicities of chlorinated hydrocarbons. Fluorescence quenching measurements revealed the lipid-water partition coefficients of the chlorinated hydrocarbons, their diffusion coefficients in the membranes, and the binding capacities of the membranes for the chlorinated hydrocarbons. Active insecticides were compared with inactive analogues to test whether activities correlated with chlorinated hydrocarbon-membrane interactions. Thus DDT and methoxychlor were compared with inactive DDE, and insecticidal γ-lindane was compared with three less active stereoisomers. The partition coefficients, diffusion coefficients and membrane saturation capacities did not correlate with insecticidal potency. The partition coefficients of these chlorinated hydrocarbons were larger in bilayers containing unsaturated fatty acyl chains as compared to bilayers containing saturated fatty acyl chains. Interestingly, neural membranes are known to contain a large percentage of unsaturated lipids. Our results indicate that the activities of chlorinated hydrocarbons are not a result of specific interactions of these compounds with the lipids of membranes. However, the neurotoxicity of chlorinated hydrocarbons may be amplified by selective partitioning in the unsaturated neural membranes.     

Effects of non-electrolyte molecules with anesthetic activity on the physical properties of DMPC multilamellar liposomes

The effects of 13 non-electrolytes with moderate anesthetic potency on the order of DMPC liposomes were examined. Changes in order were monitored by steady-state fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPG). At 30 degrees C, all of the compounds tested decreased the DPH steady-state anisotropy (rs), with potencies highly correlated to their oil/water partition coefficients. However, only the most hydrophobic anesthetics decreased TMA-DPH RS. Some of the most hydrophilic compounds, including ethanol and urethane, actually increased TMA-DPH rs, suggestive of an increase in membrane order. The concept of selectivity was borrowed from partitioning theory and used to explain some effects on anesthetic potency of decreasing temperature to 18 degrees C. In the gel as opposed to the liquid crystalline phase, selectivity for decreasing membrane order (as monitored by DPH) markedly increased, suggesting that anesthetic partitioning and/or the site of anesthetic action was occurring in a more hydrophobic domain. The solute-independent difference (or capacity) between two membranes for perturbation was defined as membrane sensitivity. Sensitivity appeared to also decrease with decreasing temperature, despite the decrease in membrane partitioning. This effect is thought to result from the selective delivery of the anesthetic solute to the membrane interior and away from more hydrophilic domains where anesthetics may order membrane structure.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.     

胶原、壳聚糖包覆的小单层脂质体的体外稳定性评价

为了提高小单层脂质体（ＳｍａｌＵｎｉｌａｍｅｌａｒＶｅｓｉｃｌｅｓ,ＳＵＶ）的体外稳定性,以５（６）－羧基荧光素（５（６）－ＣＦ）为荧光探针,研究了脂质体被胶原蛋白和壳聚糖这两种天然生物大分子包覆后的通透性。发现,胶原蛋白、壳聚糖和磷脂酰胆碱（ＰＣ）的重量比分别为２：１和８：１时,已能明显降低脂质体的通透性。并以一种分子内电荷转移化合物３－甲氧基－４＇－Ｎ,Ｎ－二甲氨基黄酮（ＤＭＭＦ）为探针,用荧光偏振法研究了胶原蛋白、壳聚糖和ＰＣ在上述比例下,对脂质体膜流动性的影响,发现脂质体在被包覆前后,其膜的流动性没有明显的改变。可见,胶原蛋白、壳聚糖包覆脂质体能够在基本不干扰脂质体膜流动性的情况下,明显地提高其体外的稳定性     

A method for estimating lateral diffusion coefficients in membranes from steady-state fluorescence quenching studies.

The Stern-Volmer theory, in which the quantum yield ratio (Io/I) depends linearly on the quencher concentration, will typically be inapplicable to fluorescence quenching in membranes. Numerical analysis shows that diffusion-controlled quenching results in a nonlinear concentration dependence for diffusion coefficients less than or of the order of 10(-6) cm2 s-1 and probe fluorescence lifetimes in the region of 10-100 ns. Lateral diffusion coefficients in membranes are typically overestimated an order of magnitude or more by the Stern-Volmer theory. An alternative empirical method is presented, which represents nonlinear concentration curves by a single parameter linear approximation determined by a least-squares analysis. The fitting parameter, P, depends on the interaction distance, the membrane thickness, the maximum extent of quenching and, in the case of biexponential probe fluorescence decay, the fluorescence kinetic parameters. P is presented in tabular form for a useful range of these parameters. The method is used to estimate diffusion coefficients for plastoquinone and plastoquinol from pyrene fluorescence quenching in soya bean phosphatidylcholine liposomes. It is found that the diffusion coefficients are nearly equal and in the region of 1.3-3.5 X 10(-7) cm2 s-1 for interaction radii of 1.5-0.5 nm, respectively.