Fluorescence lifetimes of diphenylhexatriene-containing probes reflect local probe concentrations: Application to the measurement of membrane fusion

An important process in the life of a cell is fusion between cellular membranes. This is the process by which two cellular compartments surrounded by different membranes join to become a single compartment surrounded by a single membrane, without significant loss of compartment contents. To demonstrate fusion, the cell biophysicist must demonstrate all three critical aspects of the process: (1) mixing of membrane components, (2) mixing of compartment contents; and (3) retention of compartment contents. Most commonly, accomplishing this involves the use of fluorescence probes. The general theme to the methods described involves some form of concentration-dependent quenching. An unique method developed in our laboratory utilizes the concentration dependence of the fluorescence lifetime of a phosphatidylcholine containing carboxyethyl diphenylhexatriene at position 2 and palmitic acid at position 1 of glycerol (DPHpPC). The fluorescence lifetime of this molecule and that of its parent fluorophore diphenylhexatriene (DPH) shorten dramatically as their two-dimensional concentrations in a membrane increase. This lifetime quenching can be described by dimer formation that reduces the symmetry of the DPH excited state. This phenomenon allows one to use the fluorescence lifetime to gain insight into the local concentration of probe in microscopic regions of a membrane. One application of this is in distinguishing lipid transfer between the outer leaflets of two contacting membrane bilayers from fusion between these membranes that leads to mixing of lipids in both the inner and outer leaflets of the membrane bilayers. This allows a single measurement to demonstrate fusion between membrane pairs.Abbreviations PEG poly(ethylene glycol) - Na₂EDTA ethyiene-diamine-tetraacedic acid, disodium salt - LUV large, unilamellar vesicles made by rapid extrusion technique - DPH 1,6-diphenyl-trans-1,3,5-hexatriene - DPHpPC 1-palmitoyl-2-[[[2-[4- (phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]oxy]carbonyl]-3-sn-phosphatidylcholine - DPPC 1,2-dipalmitoyl-3-sn-phosphatidylcholine - PA palmitic acid - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE - Rh-PE N-(lissamine Rhodamine B sulfoyl)-PE - R₁₈ octadecyl Rhodamine B chloride - ANTS 1-aminonaphthalene-3,6,8-trisulfonic acid - DPX N,N-p-xylylene-bis(pyradinium bromide)     

A method for quantitative interpretation of fluorescence detection of poly(ethylene glycol)-mediated 1-palmitoyl-2-[[[2-[4-(phenyl-trans-1,3,5-hexatrienyl) phenyl]ethyl]oxyl]carbonyl]3-sn-phosphatidylcholine (DPHpPC) transfer and fusion between phospholipid vesicles in the dehydrated state

A method has been developed for calculating the expected fluorescence lifetime of the DPH _p PC probe distributed between different membrane environments. We show how this method can be used to distinguish between lipid transfer and fusion between large unilamellar vesicles occurring in the presence of poly(ethylene glycol) (PEG). This application of the calculation took into consideration the heterogeneity of microenvironments experienced by the probe in a sample containing vesicle aggregates of different sizes. Assuming that the aggregate size distribution was a delta function of the aggregate size, comparison of the calculated and observed lifetimes yielded an estimate of the vesicle aggregate size. For vesicles of varying compositions in the presence of dehydrating concentrations of PEG, this method suggested that only small aggreggates formed. For vesicles that could be demonstrated by other means not to have fused, the data were consistent with lipid transfer occurring only between the outer leaflets of two to four vesicles, even at high PEG concentrations. For vesicles that could be demonstrated to fuse by contents mixing and size changes, the fluorescence lifetime data were consistent with lipid transfer between both the inner and the outer leaflets of two to four fused vesicles. At very high PEG concentrations, where extensive rupture and large, multilamellar products were previously observed, the lifetime data were consistent with much more extensive lipid transfer within larger aggregates. The agreement of predictions made on the basis of lifetime measurements with other observations attests to the validity of the fluorescence lifetime method. In addition, the model and data presented here provide evidence that fusion occurs between small numbers of PEG-aggregated vesicles before the removal of PEG.     

Dynamics in Diether Lipid Bilayers and Interdigitated Bilayer Structures Studied by Time-Resolved Emission Spectra, Decay Time and Anisotropy Profiles

We present a comparative fluorescence spectroscopic investigation of diacyl and diether phosphatidylcholine vesicles using different probes with well-defined localization within either the hydrophilic headgroup region or the hydrophobic part of the bilayer. Time-resolved emission spectra have been used to characterize the solvent relaxation behavior in both symmetric and asymmetric diether and diacyl phosphatidylcholines. It is shown that time-resolved emission spectra of Prodan (6-propionyl-2-(dimethylamino)-naphthalene) and its long-alkyl chain derivative Patman (6-palmitoyl-2-[[trimethylammoniumethyl]methylamino]-naphthalene chloride) are a sensitive tool for the detection of differences in the micropolarities and viscosities at the hydrophobic/hydrophilic membrane interface of diether and diacyl lipids, respectively. Moreover, a new approach for the detection of interdigitated bilayers is discussed. It relies on the construction of anisotropy and decay time profiles for the set of n-anthroyloxy fatty acids and is compared with an older fluorescence assay based on intensity measurements only. The shape of plots of the fluorescence steady-state anisotropy versus the position of the chromophore (anthracene-9-carboxylic acid) combined with fluorescence lifetime measurements can be used to differentiate among non-fully, and mixed interdigitated gel phase structures and to predict structures for new lipid species.     

2-Naphthol-phosphatidylethanolamine: A fluorescent phospholipid analogue for excited-state proton transfer studies in membranes

The fluorescence properties of the phospholipid derivative,N-[1-(2-naphthol)]-phosphatidylethanolamine (NAPH-PE), have been studied by steady-state and time-resolved fluorescence techniques. The new probe is a naphthol adduct of phosphatidylethanolamine. The emission spectrum of the fluorescent phospholipid depends on the pH and on the proton acceptor concentration as expected for a typical two-state excited-state proton transfer reaction. In ethanol solutions at an apparent pH of 6.7 and in the presence of acetate anion (0.14M), a biexponential decay is obtained from global analysis of the data. The lifetimes, ₁=3.9 ns and ₂=6.2 ns. are constant across the spectral region 350–460 nm. The decay-associated spectra and the species-associated spectra reproduce well the profiles reported for a two-state excited-state proton transfer reaction. The fluorescent phospholipid has been incorporated into dimyristoyllecithin and dipalmitoyllecithin vesicles. Although lower proton transfer is found, the reaction appears to be dependent on the gel-to-liquid-crystalline phase transition of the lipid membrane. In addition, the steady-state anisotropy of NAPH-PE measured as a function of temperature trace the phase transition of the two vesicle systems. Thus, it is shown that the physical state of the bilayer affects a reaction which takes place at the membrane surface. In the presence of acetate ions (0.3M), global analysis, performed in terms of fluorescence decay parameters, recovers preexponential coefficients that are consistent with an excited-state proton transfer reaction. The short lifetime drops from 3.9 to 0.44 ns without significant changes of the longer-lifetime component.     

Pressure Effects on Submicrosecond Phospholipid Dynamics Using a Long-Lived Fluorescence Probe

The effects of applied external hydrostatic pressure on submicrosecond lipid motions in DPPC⁴ bilayers have been examined using coronene (a long-lived planar fluorescent molecule) and DPH. Steady-state fluorescence emission anisotropy (EA) values () obtained for probe-labeled DPPC SUVs measured at different fixed temperatures above T _c as a function of increasing hydrostatic pressure reveal pressure-induced lipid phase transition profiles. For coronene-labeled samples, the observed lipid melt profiles are broad and shifted to higher midpoint EA pressure values (P _1/2) compared with corresponding DPH-labeled SUVs at the same temperature. The data suggest lipid motions occurring on the submicrosecond time scale, detected only by using a long-lived fluorescence probe, which occur well above the normally reported fluid–gel lipid phase transition. Slopes of the pressure-to-temperature equivalence plots (dP _1/2/dT = 39 bar/K) obtained for DPH-or coronene-labeled DPPC SUVs are identical within experimental error and reflect probe independence. For DPH, the slope of the P _1/2(T) plot provides the expected phase transition phospholipid volume change. However, intercept values (at P _1/2 = 1 bar) or apparent phase transition temperatures obtained from the equivalence plots for the two probes are not equal. Differences appear to arise due to the very disparate fluorescence lifetime values of the two probes, which result in rotational sensitivity of coronene to gel lipid volume fluctuations occurring during the extended time window provided by coronene fluorescence.     

Fluorescence lifetime characterization of bacteria using total lifetime distribution analysis with the maximum entropy method

Total lifetime distribution analysis was employed to obtain fluorescence lifetime profiles of the intrinsic fluorescence ofPseudomonas fluorescens, Escherichia coli, Bacillus subtilis, andStaphylococcus epidermidis. The lifetimes were measured using a multiharmonic Fourier transform phase-modulation fluorometer which can simultaneously measure the phase shift and demodulation at many modulation frequencies. The 364-nm line from an argon-ion laser and the 325- and 442-nm lines from a helium-cadmium laser were used for sample excitation. Broad emission windows were used to capture as much of the bacterial emission as possible for the lifetime measurements. The maximum entropy method was used to recover lifetime profiles from the multifrequency phasemodulation data. At all three excitation wavelengths, the bacteria exhibited three lifetime components, in the ranges of 0.5-1, 2–3, and 4–8 ns. Using 325-nm excitation, a fourth component, in the range of 9–14 ns, was recovered in all of the bacteria; using 364-nm excitation, the fourth component was resolved only in the two Gram-negative bacteria (P. fluorescens andE. coli). Excitation at 364 nm provided the most reproducible lifetime profiles and showed some differences among the four bacteria.     

Effect of some statin group of drugs on the phase profile of liposomal membrane – a fluorescence anisotropy study

Using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, we have investigated the effect of some statin group of drugs, widely used in hyperlipidemia, on the phase transition of model membranes such as dipalmitoyl phosphatidylcholine (DPPC) liposomes. The nature of the changes in the fluorescence anisotropy values suggested that the drugs simvastatin and mevastatin fluidized the membrane both before and after the phase transition temperature (T _m), whereas atorvastatin fluidized the membrane below T _m but rigidified the same above T _m, i.e., introduced an intermediate fluid condition within the lipid matrix. We have calculated the changes in van’t Hoff enthalpy values associated with the phase transitions due to chain melting in all the cases, and observed that the values of enthalpy decreased with increase in drug concentrations. In order to get a better insight, the fraction of motionally restricted lipid molecules was determined.     

A Novel Squarylium Dye for Monitoring Oxidative Processes in Lipid Membranes

A novel squaraine probe SQ-1 has been found to be appropriate for monitoring the peroxidation processes in membrane systems. Formation of free radicals was triggered by methemoglobin (metHb) or cytochrome c (cyt c) binding to the model lipid membranes composed of zwitterionic lipid phosphatidylcholine (PC) and anionic lipid cardiolipin (CL). Protein association with the lipid vesicles was followed by drastic quenching of SQ-1 fluorescence. The observed spectral changes were suppressed in the presence of free radical scavengers, butylated hydroxytoluene (BHT) and thiourea (TM) suggesting that SQ-1 decolorization can be attributed to its reactions with lipid radicals.     

Miscibility phase diagrams of giant vesicles containing sphingomyelin

Saturated sphingomyelin (SM) lipids are implicated in lipid rafts in cell plasma membranes. Here we use fluorescence microscopy to observe coexisting liquid domains in vesicles containing SM, an unsaturated phosphatidylcholine lipid (either DOPC or POPC), and cholesterol. We note similar phase behavior in a model membrane mixture without SM (DOPC/DPPC/Chol), but find no micron-scale liquid domains in membranes of POPC/PSM/Chol. We delineate the onset of solid phases below the miscibility transition temperature, and detail indirect evidence for a three-phase coexistence of one solid and two liquid phases.     

Fluorescence Studies on New Potential Antitumoral Benzothienopyran-1-ones in Solution and in Liposomes

Fluorescence properties of four new potential antitumoral compounds, 3-arylbenzothieno[2,3-c]pyran-1-ones, were studied in solution and in lipid membranes of dipalmitoyl phosphatidylcholine (DPPC), egg yolk phosphatidylcholine (Egg-PC) and dioctadecyldimethylammonium bromide (DODAB). The 3-(4-methoxyphenyl)benzothieno[2,3-c]pyran-1-one (1c) exhibits the higher fluorescence quantum yields in all solvents studied. All compounds present a solvent sensitive emission, with significant red shifts in polar solvents for the methoxylated compounds. The results point to an ICT character of the excited state, more pronounced for compound 1c. Fluorescence (steady-state) anisotropy measurements of the compounds incorporated in liposomes of DPPC, DODAB and Egg-PC indicate that all compounds have two different locations, one due to a deep penetration in the lipid membrane and another corresponding to a more hydrated environment. In general, the methoxylated compounds prefer hydrated environments inside the liposomes. The 3-(4-fluorophenyl)benzothieno[2,3-c]pyran-1-one (1a) clearly prefers a hydrated environment, with some molecules located at the outer part of the liposome interface. On the contrary, the preferential location of 3-(2-fluorophenyl)benzothieno[2,3-c]pyran-1-one (1b) is in the region of lipid hydrophobic tails. Compounds with a planar geometry (1a and 1c) have higher mobility in the lipid membranes when phase transition occurs.     

Long-lived fluorescence probes for studying lipid dynamics: A review

A great many studies have focused on the heterogeneous packing of lipids in the bilayer matrix. However, less attention has been directed toward the temporal aspects of these lipid-lipid interactions. Studies of lipid packing fluctuations, or gel-fluid exchange, using fluorescence probe methodologies have been limited. This limitation arises from thesubmicrosecond time scale over which the fluctuations are expected to occur. Traditionally, dynamic studies of lipid bilayers have been restricted to the nanosecond time regime, and the submicrosecond time window has not been explored in any great depth by fluorescence methods, although persistent lipid dynamics has been evident. Probes with long fluorescence lifetimes (several hundred nanoseconds) have the potential to expand this important time window, providing information on gel-fluid exchange rates and insights into how important biological effectors such as proteins, cholesterol, and anesthetics affect or modulate these fluctuations. Using the long-lived fluorescence probe coronene, combined with time-resolved fluorescence methods geared toward microheterogeneity, we present a view of bilayer dynamics in an alternate time domain. Fluorescence probes are expected to inhabit an equilibrium between fluid and gel environments. Some probes remain in their respective environments throughout their excited-state lifetime, while others reside in surroundings that will change (i.e., melt). Long-lived fluorescence membrane probes can provide direct estimates of submicrosecond lipid fluctuation or melt rates. Simple Landau modeling leads to adistribution of melt rates and provides an attractive alternative to a simplercompartmental model where a unique lipid fluctuation of gel-fluid exchange rate is measured. Thedistribution model is probe independent (defined by thermodynamic quantities) and can be applied generally to the rotational motions of fluorescence probes embedded in the lipid bilayer.Abbreviations DMPC l--dimyristoylphosphatidylcholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DPPC l--dipalmitoylphospha-tidylcholine - DSC differential scanning calorimetry - EA fluorescence emission anisotropy - LUV large unilamellar vesicles - SUV small unilamellar vesicles - T_c lipid phase transition temperature     

Time-resolved emission spectra and anisotropy profiles for symmetric diacyl- and dietherphosphatidylcholines

The solvent relaxation behavior of Patman (6-palmitoyl-2-[[2-(trimethylammonium) ethyl]methylamino]naphthalene chloride) was investigated in small unilamellar vesicles composed of symmetric diacyl( 1,2-dipalmitoylphosphatidylcholine; DPPC) and diether lipids (l,2-dihexadecylphosphatidylcholine; DHPC), calculating time-resolved emission spectra (TRES) and correlation functions. Both the steady-state spectra as a function of temperature and excitation wavelength and the TRES of Patman in DPPC are blue-shifted compared to those in DHPC. The solvent relaxation at three temperatures above and below the phase transition is considerably faster in DHPC than in DPPC. As the steady-state anisotropies of Patman and TMA-DPH [l-(4-trimethylammoniumphenyl)-6-phenyl-l,3,5-hexatriene] are similar in both lipids as a function of both temperature and emission wavelength, we conclude that the introduction of ether linkages allows more efficient water penetration in the glycerol region, leading to a more polar environment and therefore faster solvent relaxation of the incorporated dyes. Using a series ofn-(9-anthroyloxy) fatty acids (n = 2, 3, 6, 9, 12; 16-AP), we show that anisotropy profiles can be used to distinguish between noninterdigitated (DPPC) and fully interdigitated (DHPC) gel-phase structures. 16-(9Antroyloxy) palmitic acid (16-AP) is an especially useful probe exhibiting pronounced differences in the steady-state anisotropies in non- and fully interdigitated gel phases.     

Fluorescence Studies on Potential Antitumoral Heteroaryl and Heteroannulated Indoles in Solution and in Lipid Membranes

Fluorescence properties of three potential antitumoral compounds, a 3-(dibenzothien-4-yl)indole 1, a phenylbenzothienoindole 2 and a 3-(dibenzofur-4-yl)indole 3, were studied in solution and in lipid aggregates of dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE) and egg yolk phosphatidylcholine (Egg-PC). The 3-(dibenzofur-4-yl)indole 3 exhibits the higher fluorescence quantum yields in all solvents studied (0.32 ≤ Φ_F ≤ 0.51). All the compounds present a solvent sensitive emission, with significant red shifts in alcohols. The results point to an ICT character of the excited state, more pronounced for compound 1. Fluorescence (steady-state) anisotropy measurements of the compounds incorporated in lipid aggregates of DPPC, DOPE and Egg-PC indicate that the three compounds are deeply located in the lipid bilayer, feeling the difference between the rigid gel phase and fluid phases.     

Interaction of dipalmitoyl phosphatidylcholine (DPPC) liposomes and insulin

Insulin, a peptide that has been used for decades in the treatment of diabetes, has well-defined properties and delivery requirements. Liposomes, which are lipid bilayer vesicles, have gained increasing attention as drug carriers which reduce the toxicity and increase the pharmacological activity of various drugs. The molecular interaction between (uncharged lipid) dipalmitoyl phosphatidylcholine (DPPC) liposomes and insulin has been characterized by using Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction. The characteristic protein absorption band peaks, Amide I (at about 1660?cm^?1) and Amide II band (at about 1546?cm^?1) are potentially reduced in the liposome insulin complex. Wide-angle x-ray scattering measurements showed that the association of insulin with DPPC lipid of liposomes still maintains the characteristic DPPC diffraction peaks with almost no change in relative intensities or change in peak positions. The absence of any shift in protein peak positions after insulin being associated with DPPC liposomes indicates that insulin is successfully forming complex with DPPC liposomes with possibly no pronounced alterations in the structure of insulin molecule.     

Polarized fluorescence and absorption spectroscopy of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester. A new and lipid bilayer-spanning probe

We report on the properties of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester (Rh101C₃₂Rh101) in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and in liquid solvents. The results are compared with those of rhodamine 101 octadecanyl ester (Rh101C₁₈). Both molecules are solubilized in the lipid bilayer and the Rh101 moieties are anchored in the lipid-water interface, so that the electronic transition dipole moments (S ₀ S ₁) are oriented preferentially in the plane of the bilayer. At low concentrations of the dyes in lipid bilayers of DOPC, the fluorescence relaxation is single exponential with a lifetime of =4.9±0.2 ns. The relative fluorescence quantum yield of _C32/_C18 0.95 in DOPC vesicles. These results strongly suggest that only a small fraction of the Rh101C₃₂Rh101 molecules are quenched, by, for example, intra- or intermolecular dimers in the ground state at mole fractions of less than 0.1% in the lipid bilayers. For Rh101C₃₂Rh101 in lipid vesicles, the steady-state and time-resolved fluorescence anisotropies are compatible with efficient intramolecular electronic energy transfer. It is concluded that nearly every Rh101C₃₂Rh101 molecule is spanning across the lipid bilayer of DOPC.     

DMANS——一种新型荧光闪烁体染料的寿命与能量转移的研究

本工作对一种新型荧光闪烁体染料——4-二甲胺基—4''硝基芪(DMANS)在不同介质中的光谱行为和能量转移进行了研究,发现介质的极性大小以及介质和染料间的能量转移对该闪烁体染料的荧光量子产率和闪烁发光延迟具有重大影响。研究对选择基体材料和合理组成闪烁材料配方有一定的参考价值。     

Diphenylhexatriene-phosphatidylcholine fluorescence in POPC vesicles: Comparison of the exponential-series and the maximum-entropy methods

We have investigated the time-resolved fluorescence of diphenylhexatriene (DPH) covalently linked to phosphatidylcholine (PC) in palmitoyloleoylglycerophosphocholine (POPC) vesicles with special consideration of the comparison of two methods for distributional lifetime analysis: the exponential-series method (ESM) and the maximum-entropy method (MEM). Generally, both methods were found to reveal equivalent results on high-quality data. Different are the shapes of the recovered distributions (symmetry and width) as well as the time effort for the numerical analysis.     

Subnanosecond fluorescence spectroscopy of human serum albumin as a method to estimate the efficiency of the depression therapy

The efficiency of the therapy of psychiatric diseases is estimated using the fluorescence measurements of the conformational changes of human serum albumin in the course of medical treatment. The fluorescence decay curves of the CAPIDAN probe (N-carboxyphenylimide of the dimethylaminonaphthalic acid) in the blood serum are measured. The probe is specifically bound to the albumin drug binding sites and exhibits fluorescence as a reporter ligand. A variation in the conformation of the albumin molecule substantially affects the CAPIDAN fluorescence decay curve on the subnanosecond time scale. A subnanosecond pulsed laser or a Pico-Quant LED excitation source and a fast photon detector with a time resolution of about 50 ps are used for the kinetic measurements. The blood sera of ten patients suffering from depression and treated at the Institute of Psychiatry were preliminary clinically tested. Blood for analysis was taken from each patient prior to the treatment and on the third week of treatment. For ten patients, the analysis of the fluorescence decay curves of the probe in the blood serum using the three-exponential fitting shows that the difference between the amplitudes of the decay function corresponding to the long-lived (9 ns) fluorescence of the probe prior to and after the therapeutic procedure reliably differs from zero at a significance level of 1% (p < 0.01).     

Fluorescence of organic dyes in lipid membranes: Site of solubilization and effects of viscosity and refractive index on lifetimes

The fluorescence decay of several organic dye molecules intercalated in egg phosphatidylcholine lipid membrane vesicles is consistent with the existence of two or three prominent lifetime components rather than a single continuous distribution of lifetimes. The major lifetime components are identified with different sites of solubilization in the membrane. The variation of the lifetime of the membrane-bound dye was studied as a function of the sucrose concentration, which varied the viscosity and refractive index of the aqueous solution. The combined effect of viscosity and refractive index on the lifetime of the dye was used to identify the site of solubilization of the dye in the membrane. The study was useful to identify dye molecules on the surface which are exposed to the aqueous phase, for which the fluorescence lifetime increased systematically with sucrose (viscosity effect). More importantly, it was possible in a few cases to identify the dye molecules which are oriented in the membrane phase, and the fluorescence lifetime decreased systematically with sucrose (refractive index effect). Anomalous values of order parameters determined from the refractive index effect are explained in terms of an orientational distribution of the linear dye molecule weighted in favor of mutually orthogonal orientations.     

Kinetics and mechanism of electron transfer reaction of single and double chain surfactant cobalt(III) complex by Fe2+ ions in liposome (dipalmitoylphosphotidylcholine) vesicles: effects of phase transition

In this study, we report the kinetics of reduction reactions of single and double chain surfactant cobalt(III) complexes of octahedral geometry, cis-[Co(en)₂(4AMP)(DA)](ClO₄)₃ and cis-[Co(dmp)₂(C₁₂H₂₅NH₂)₂](ClO₄)₃ (en = ethylenediamine, dmp = 1,3-diaminopropane, 4AMP = 4-aminopropane, C₁₂H₂₅NH₂ = dodecylamine) by Fe²⁺ ion in dipalmitoylphosphotidylcholine (DPPC) vesicles at different temperatures under pseudo first-order conditions. The kinetics of these reactions is followed by spectrophotometry method. The reactions are found to be second order and the electron transfer is postulated as outer sphere. The remarkable findings in the present investigation are that, below the phase transition temperature of DPPC, the rate decreases with an increase in the concentration of DPPC, while above the phase transition temperature the rate increases with an increase in the concentration of DPPC. The main driving force for this phenomenon is considered to be the intervesicular hydrophobic interaction between vesicles surface and hydrophobic part of the surfactant complexes. Besides, comparing the values of rate constants of these outer-sphere electron transfer reactions in the absence and in the presence of DPPC, the rate constant values in the presence of DPPC are always found to be greater than in the absence of DPPC. This is ascribed to the double hydrophobic fatty acid chain in the DPPC that gives the molecule an overall tubular shape due to the intervesicular hydrophobic interaction between vesicles surface and hydrophobic part of the surfactant complexes more suitable for vesicle aggregation which facilitates lower activation energy, and consequently higher rate is observed in the presence of DPPC. The activation parameters (ΔS^# and ΔH^#) of the reactions at different temperatures have been calculated which corroborate the kinetics of the reaction.