Influence of Eugenol on the Organization and Dynamics of Lipid

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Influence of Eugenol on the Organization and Dynamics of Lipid Membranes: A Phase-Dependent Study Geetanjali Meher, and Hirak Chakraborty Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03595 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 11, 2018

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Influence of Eugenol on the Organization and Dynamics of Lipid Membranes: A Phase-Dependent Study

Geetanjali Meher and Hirak Chakraborty* School of Chemistry, Sambalpur University, Jyoti Vihar, Burla, Odisha – 768 019, India

*Address

correspondence to Hirak Chakraborty, [email protected], Phone: +91-8008716419

E-mail:

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ACS Paragon Plus Environment

[email protected]

or

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ABSTRACT

Eugenol is known for its antimicrobial effects against microorganisms responsible for infectious diseases in human, food borne pathogens and oral pathogens. In spite of several reports on the antimicrobial function of eugenol by modulating the structural properties of cell membrane there was no direct information on the influence of eugenol in the lipid membrane. In this work, we explored the effect of eugenol on organization and dynamics of large unilamellar vesicles (LUVs) of DMPC using the intrinsic fluorescence of eugenol and an extrinsic hydrophobic probe, DPH, in varying phases. The organization and dynamics of the bilayers of DMPC vesicles were monitored utilizing varieties of steady state and timeresolved fluorescence measurements. Our results show that eugenol stabilizes the gel phase and elevates the phase transition temperature of DMPC in a concentration dependent fashion. Fluorescence lifetime measurements demonstrate that higher eugenol-induced water penetration was observed in fluid phase membrane. Time-resolved anisotropy measurements demonstrate that eugenol reduces the semi-angle of DPH wobbling-in-cone in gel phase membranes, whereas the semi-angle remains unaffected in fluid phase membrane. This implies that the eugenol further orders the gel phase membrane and this could be the plausible reason for eugenol-dependent elevation of phase transition temperature of DMPC. We envisage that these results will contribute important information in understanding the interaction of eugenol with biological membranes.

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INTRODUCTION

Eugenol (4-allyl-2-methoxyphenol, chemical structure shown in scheme 1) is an aromatic hydroxyphenyl propene present abundantly in Syzygium aromaticum, which is commonly known as clove.1 Eugenol is the major constituent of clove oil that is present between 9.4 and 14.7 gm per 100 gm of fresh plant material.2-3 Clove oil is extensively used to treat many diseases including acne, asthma, rheumatoid arthritis and various allergies.4-5 In addition, clove oil is known for its antimicrobial effects against microorganisms responsible for infectious diseases in human, food borne pathogens and oral pathogens.1 The hydroxyl group of eugenol is sometimes attributed to its antimicrobial activities.6 Eugenol could also act by the disruption of cytoplasmic membrane, which increases nonspecific permeability and modulating the transport of ions and ATP.7-8 This has been further shown that eugenol alters the fatty acid profile of the cell membrane of different bacteria.9

Scheme 1. Chemical Structure of Eugenol Though there are several reports on the antimicrobial function of eugenol by modulating the structural properties of cell membrane, but there is limited information on how eugenol modulates the lipid membrane.7, 10-13 An interesting work to study the effect of eugenol with lipid monolayers at the air-water interface suggests that interaction is being modulated by the lipid composition of the monolayer.14 Therefore it is of utmost importance to understand the effect of eugenol in synthetic lipid membranes that provides the opportunity to explore the mechanism of membrane modulating properties of any additive.15-16 Generally, membrane lipids in physiologically relevant condition exist in liquid-crystalline (fluid) phase, but sometimes lipids may present in gel phase.17 Therefore, the gel to fluid

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phase transition or chain melting has been the most extensively studied lipid phase transition.18 The phase transition temperature mainly depends on the hydrophobic chain length of the lipid; number and position of double bond(s) in the hydrophobic chain. Metal ions are known to elevate the phase transition temperature19 whereas most of the organic additives reduce the phase transition temperature by destabilizing the hydrophobic chain packing of the gel phase lipid membranes.20-21 The biological role of membrane lipids has become immensely important with the progress in biophysics, chemistry and genetics and the biological role of lipids hinges on the lipid phase. In this paper, we have explored the effect of eugenol on the phase transition temperature of DMPC and studied the effect of eugenol on organization and dynamics of the membrane in varying phases utilizing varieties of steady state and time-resolved fluorescence spectroscopic methods. To explore the organization and dynamics of the membrane, we have utilized the fluorescence property of eugenol and membrane probe like DPH. DPH is a rod like molecule and known to be located in the hydrophobic region of the bilayer, with an average distance from the bilayer center of ~ 7.8 Å.22 The fluorescence properties of DPH have been extensively used to gauge the organization and dynamics of hydrophobic region of lipid membranes.23-24 The fluorescence lifetime measurements of eugenol and DPH provide the information of region specific environmental polarity of the membrane.25-26 In this work, we have utilized fluorescence lifetime measurements of eugenol and DPH to determine the effect of eugenol on the environmental polarity of DMPC membrane in varying phases. The time-resolved fluorescence anisotropy measurements of DPH and semi-angle of DPH wobbling-in-cone calculation provided the effect of eugenol on the packing of the hydrophobic region in varying phases.

Altogether, our present work demonstrates that

eugenol raises the phase transition temperature of DMPC membranes and further offers a clear picture of the effect of eugenol on the membrane organization and dynamics of DMPC bilayer in gel and fluid phases.

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MATERIALS AND METHODS

Materials Eugenol and DPH were obtained from Sigma Chemical Co. (St. Louis, MO) and Molecular Probes/Invitrogen (Eugene, OR), respectively. Sodium dihydrogen phosphate (NaH2PO4, 2H2O) and disodium hydrogen phosphate (Na2HPO4) were obtained from Merck, India.

1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was obtained from Avanti

polar Lipids (Alabester, AL). Spectroscopic grade DMSO was purchased from Spectrochem (India). All other chemicals used in the work were of the highest available purity. Water was purified through Millipore (Bedford, MA) Milli-Q water purification system.

Sample Preparation We have used large unilamellar vesicles (LUVs, diameter ~100 nm) of DMPC as the membrane systems for all measurements. The concentration of DMPC was kept constant at 400 µM in all experiments. The eugenol concentration has been varied from 0-60 µM so that the highest eugenol to lipid ratio is ~7 (for 50 µM eugenol), but actual concentration of eugenol in the membrane depends on its partition coefficient. We have used DPH to probe the hydrophobic region of the membrane and DPH concentration was kept constant at 2 µM (0.5 mol% with respect to lipid concentration) to minimize the probe induced alteration of membrane structure. DMPC was dissolved in chloroform and air dried to make a thin film. The film was kept in vacuum desiccator for complete removal of chloroform. The DMPC film was hydrated (swelled) by adding 10 mM phosphate buffer of pH 7.4. The sample was vortexed for 1hr for uniform dispersion of lipids. The entire hydration process was carried out at 30 °C to maintain the temperature above the phase transition temperature of DMPC. LUVs with a diameter of 100 nm were prepared by the extrusion technique using Avanti Mini-Extruder (Alabester, AL) as described previously.27-28

Background samples were

prepared the same way except that the probes were omitted. Stock solutions of eugenol and DPH were prepared in DMSO, and small aliquots of stock solutions were added to working solutions. The amount of DMSO was always less than 5


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1% (v/v), and this amount of DMSO had no detectable effect on further experiment. Required amount of eugenol stock solution in DMSO was added to the prepared LUVs to study the effect of eugenol on membrane organization and dynamics. We avoided co-drying as eugenol is liquid.

Steady State Fluorescence Measurements Steady state fluorescence measurements were performed with Hitachi F-7000 (Japan), spectrofluorometer using 1 cm x 1 cm quartz cuvette. Eugenol was excited at 280 nm and fluorescence emission was monitored from 295 to 450 nm. Excitation and emission slits with a nominal band pass of 5 nm were used for all measurements. Fluorescence anisotropy measurements of DPH were performed using the same instrument keeping excitation wavelength at 360 nm and emission was monitored at 428 nm and anisotropy values were calculated using the following equation:29

r

IVV  G  IVH IVV  2G  IVH

(1)

where G=IHV/IHH, (grating correction or G-factor), IVV and IVH are the measured fluorescence intensities with the excitation polarizer vertically oriented and the emission polarizer vertically and horizontally oriented, respectively.

Time-resolved Fluorescence Measurements Fluorescence lifetimes were calculated from time-resolved fluorescence intensity decays using IBH 5000F Nano LED equipment (Horiba, Edison, NJ) with Data Station software in the time-correlated single photon counting (TCSPC) mode. A pulsed lightemitting diode (LED) was used as the excitation source. This LED generates optical pulse at 281 nm (for exciting eugenol) and 340 nm (for exciting DPH) with pulse duration 1.2 ns and are run at 1 MHz repetition rate. The Instrument Response Function (IRF) was measured at the respective excitation wavelength using Ludox (colloidal silica) as scatterer. To optimize the signal-to-noise ratio, 10,000 photon counts were collected in the peak channel. All 6


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experiments were performed using emission slits with bandpass of 6 nm. Data were stored and analyzed using DAS 6.2 software (Horiba, Edison, NJ). Fluorescence intensity decay curves were deconvoluted with the instrument response function and analyzed as a sum of exponential terms: 𝐹 𝑡 =

𝑛 𝑖

𝛼𝑖 𝑒𝑥𝑝(−𝑡/𝜏𝑖 )

A considerable plot was obtained with random deviation about zero with a minimum χ2 value of 1.2 or less. Intensity averaged mean lifetimes τavg for tri-exponential decays of fluorescence were calculated from the decay times and pre-exponential factors using the following equation.29 (𝛼 1 𝜏 12 +𝛼 2 𝜏 22 +𝛼 3 𝜏 32 ) 𝜏𝑎𝑣𝑔 = (𝛼 1 𝜏 1 +𝛼 2 𝜏 2 +𝛼 3 𝜏 3 )

(2)

where, αi is the fraction that shows i lifetime.

Anisotropy Decay Measurements In time-resolved anisotropy measurements, emission was collected at directions parallel (I║) and perpendicular (I⊥) to the polarization of the vertically polarized excitation beam. Anisotropy was calculated as:25

(3)

where G(λ) is the geometry factor at the wavelength λ of emission. The G factor of the emission collection optics was determined from the emission collected at directions parallel (I║) and perpendicular (I⊥) to the polarization of the horizontally polarized excitation beam. The time-resolved anisotropy decay was analyzed based on the following model: I║(t) = I(t)[1+2r(t)]/3 (4)

I(t) = I(t)[1-r(t)]/3

where I║(t) and I⊥(t) are the decays of the parallel (║) and perpendicular (⊥) components of emission. The equation for time-resolved fluorescence anisotropy can be expressed as a sum to exponential decay: 7


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𝑟 𝑡 = 𝑟0

𝛽𝑖 exp⁡ (−𝑡 ɸ ) 𝑖

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(5)

where ϕi and βi represent the ith rotational correlation time and the corresponding preexponential factor in the experimental decay such that Σβi = 1. ‘r0’ represents the anisotropy at zero time (initial anisotropy). The initial anisotropy at zero time for DPH was used as 0.36.30 The goodness of the fit of a given set of observed data and the chosen function was evaluated by the reduced χ2, which is around 1.0 to 1.15.

Calculation of Semi-angle of Wobbling-in-cone The extent of probe wobbling within the membrane depends on the fluidity (order) of the membrane. The wobbling motion can be modeled as wobbling-in-cone, whose semi angle (θ) can be calculated using the Kinosita model.31-32 The semi angle of wobbling is given by: 1 1 𝑟 𝜃 = 𝑐𝑜𝑠 −1 [1/2{(1 + 8( ∞ 𝑟0 )2 )2 − 1}] (6) where θ = 90° indicates free rotation. r0 represents the anisotropy at zero time (initial anisotropy) in the anisotropy decay experiment and r∞ = r0 × βs, where βs is the pre-exponential factor corresponding to the higher correlation time (slowly decaying component). Higher θ corresponds to the unrestricted rotation of the probe, while lower θ corresponds to the crowded or hindered rotation of the probe. The r0 value was kept fixed at 0.36 for DPH30 while analyzing the fluorescence anisotropy decay kinetics using eq (6).

RESULTS In spite of several results indicating the antimicrobial activities of eugenol by modulating the properties of membrane, there is no study till date to show the effect of eugenol on the physical properties of the membrane. In this work, we have studied the effect of eugenol in membrane organization and dynamics using several steady state and time-resolved fluorescence methods in both gel and fluid phases.

Membrane phase is an important determinant of

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membrane physical properties.17 The gel phase is more ordered as the lipid acyl chains are extended in all trans conformation, whereas the fluid phase is disordered due to gauche conformation of the lipid acyl chains.

Partitioning of Eugenol in lipid membranes The intrinsic fluorescence property of eugenol has been utilized to study the partitioning of eugenol in DMPC membranes. Eugenol shows a single fluorescence peak at 318 nm, while exciting at 280 nm. The fluorescence peak of eugenol can be attributed to its phenolic ring. The fluorescence peak of eugenol shifts to the blue edge (at 312 nm) in presence of DMPC membranes. Figure 1 shows the normalized fluorescence spectra of eugenol in buffer and in presence of DMPC membranes at 10 °C. The 6-nm blue shift indicates the partitioning of eugenol into a relatively hydrophobic environment of lipid bilayer.

Figure 1. Plot of normalized fluorescence spectrum of eugenol in absence (red) and in presence (blue) of DMPC membranes. Blue shift of 6 nm in emission maxima indicates the partitioning of eugenol in hydrophobic region of the DMPC membrane. All experiments were carried out in 10 mM phosphate buffer of pH 7.4 at 10 °C temperature. The eugenol and lipid concentrations were kept constant at 5 μM and 400 μM, respectively and excitation wavelength was fixed at 280 nm. See Materials and Methods section for more details.

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Effect of Eugenol in phase transition temperature of DMPC Generally, the organic additives reduce the phase transition temperature of lipid. The temperature dependent fluorescence anisotropy of DPH can be successfully used to evaluate the phase transition temperature of DMPC.33 Figure 2 shows the plots of DPH anisotropy in DMPC membranes as a function of temperature in absence and in presence of 20, 40 and 60 µM of eugenol. The phase transition temperature of DMPC obtained from our measurement is 23.7 °C, which is similar to the literature value of phase transition temperature of DMPC (23.9 °C).34 The phase transition temperature of DMPC increases by ~1 °C in presence of 60 µM eugenol. The enhancement of phase transition temperature induced by eugenol could be due to the parallel orientation of eugenol molecules with the hydrophobic tails of DMPC, resulting in more compact packing of DMPC bilayer. This result is quite significant to understand the interaction of eugenol with the lipid membranes.

Figure 2. Plot of change in fluorescence anisotropy of DPH in DMPC membranes as a function of temperature. The inflection point of the plot gives the phase transition temperature of DMPC. The fluorescence anisotropy of DPH in DMPC membranes was measured in absence (black) and in presence of 20 µM (green), 40 µM (blue) and 60 µM (red) eugenol. Inset shows the phase transition temperature of DMPC in absence and in presence of various concentrations of eugenol. DPH was excited at 360 nm and fluorescence anisotropy was measured at 428 nm. All measurements were carried out in 10 mM phosphate buffer of pH 7.4. Concentration of DMPC was kept constant at 400 µM for all measurements. Data points shown are means ± S.E of at least three independent measurements. See materials and methods section for more details. 10


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Fluorescence lifetime of Eugenol in varying phases of lipid Fluorescence lifetime of any fluorophore provides information about the polarity of its neighboring environment.35-36 We have measured the fluorescence lifetime of eugenol at its varying concentration in gel (10 °C) and fluid phase (37 °C) of DMPC membranes. Figures 3A and B show the lifetime decay profile of 10 and 60 µM eugenol in gel and fluid phases of DMPC membranes, respectively. The fluorescence lifetime of eugenol enhances in presence of DMPC membranes (data not shown) due to partitioning into a more hydrophobic environment. It is imperative from the structure of eugenol that it might be partitioning near the interfacial region of the membrane. The fluorescence lifetime of eugenol in presence of fixed concentration of DMPC (400 µM) decreases with increasing concentration of eugenol in both phases (see Table1). The fluorescence lifetime of eugenol in both phases as a function of eugenol concentration has been shown in Figure 3C. Our results show that higher concentration of eugenol makes the probe environment relatively more polar in both gel and fluid phases, though the extent is much higher in fluid phase. The increase in polarity of the environment might be attributed to the water penetration at the depth of the membrane, where eugenol resides.

The change in

fluorescence lifetime (difference between fluorescence lifetime in presence of highest and lowest concentration of eugenol) is almost double for fluid phase membranes compared to that of gel phase (Figure 3C inset). Water partitioning in the gel phase membrane is not plausible as eugenol stabilizes gel phase of DMPC membranes. Hence, the small decrease in fluorescence lifetime of eugenol in gel phase could be due to more interfacial location of eugenol in gel phase membranes, whereas the significant decrease of fluorescence lifetime in fluid phase could be attributed to the water penetration in the membrane in presence of eugenol.

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Figure 3. Plot of time-resolved fluorescence intensity decay of 10 µM (red) and 60 µM (green) eugenol in presence of DMPC at (A) 10 °C, gel phase and (B) 37 °C, fluid phase. Instrument response functions (IRF) for gel and fluid phases are shown in black with the respective fluorescence intensity decay profile. Representative residual plots are shown at the bottom of the fluorescence intensity decay plot. (C) Mean fluorescence lifetime of eugenol in 400 µM DMPC is shown as a function of eugenol concentration at 10 °C (gel phase, red) and 37 °C (fluid phase, black). A 281 nm LED was used to excite eugenol and corresponding emission was monitored at 315 nm. All measurements were carried in 10 mM phosphate buffer of pH 7.4. Concentration of DMPC was kept constant at 400 µM for all measurements. Maximum change in eugenol fluorescence lifetime in gel (red) and fluid (black) phase has been shown in the inset. Data points shown are means ± S.E. See materials and methods section for more details.

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Fluorescence lifetime of DPH in varying phases of DMPC membrane: Effect of Eugenol The fluorescence lifetime of DPH is sensitive to the polarity at the hydrophobic region of the membrane as DPH resides ~7.8 Å from the center of the bilayer.22 The reduction of mean fluorescence lifetime of DPH can be owed to the water penetration at the depth of the membrane, where DPH is located. To evaluate the effect of eugenol on the modulation of the hydrophobic region of the DMPC membranes in gel and fluid phases, we have measured fluorescence lifetime of DPH in absence and in presence of various concentrations of eugenol. Figure 4A and B show the fluorescence decay profile of DPH in absence and in presence of 50 µM eugenol in gel and fluid phases, respectively. Figure 4C shows the plot of average fluorescence lifetime of DPH as a function of eugenol concentration in both phases and the corresponding maximum changes have been shown in Figure 4C inset. Our results show that the change in fluorescence lifetime of DPH is being more affected in fluid phase compared to that of gel phase. The small change in DPH fluorescence lifetime in gel phase could be attributed to the relatively polar unsaturated alkyl chain of eugenol in the vicinity of DPH, which makes the probe environment relatively polar. Interestingly, fluorescence lifetime measurements of DPH further support that the effect of eugenol is more pronounced in fluid phase compared to that in gel phase. As a result, the change in average fluorescence lifetime is almost double for fluid phase membranes.

Time-resolved fluorescence anisotropy measurement of DPH in varying phases of DMPC membrane: Effect of Eugenol The time-resolved fluorescence anisotropy measurements of DPH provide insight into the rotational dynamics of the probe in the membrane environment.37-38 We have measured the time-resolved anisotropy of DPH in both gel and fluid phases in presence of various concentrations of eugenol. Figure 5A and B show the anisotropy decay for DPH in absence and in presence of 20, 40 and 60 µM eugenol concentration in gel and fluid phases, respectively. The anisotropy decay has been analyzed using eqn (5), considering a bi-exponential function and the results are shown in Table-2. It is evident from Table-2 that both slow and fast components of rotational correlation time are faster in gel phase compared to that in the fluid phase, which seems to be anomalous. Interestingly, the pre-exponential factor associated to the slow 13


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Figure 4. Plot of time-resolved fluorescence intensity decay of DPH in absence (red) and in presence of 50 µM (green) eugenol in presence of DMPC at (A) 10 °C, gel phase and (B) 37 °C, fluid phase. Instrument response functions (IRF) for gel and fluid phases are shown in black with the respective fluorescence intensity decay profile. Representative residual plots are shown at the bottom of the fluorescence intensity decay plot. (C) Mean fluorescence lifetime of DPH in 400 µM DMPC is shown as a function of eugenol concentration at 10 °C (gel phase, red) and 37 °C (fluid phase, black). A 340 nm LED was used to excite DPH and corresponding emission was monitored at 428 nm. All measurements were carried in 10 mM phosphate buffer of pH 7.4 using 2 µM DPH. Concentration of DMPC was kept constant at 400 µM for all measurements. Data points shown are means ± S.E. See materials and methods section for more details.

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component is remarkably larger in gel phase (~60 to 75% compared to ~15% in fluid phase) compared to that in fluid phase. In addition, the value of pre-exponential factor associated to slower rotational coefficient increases with addition of eugenol, whereas no such change has been observed in fluid phase. For better understanding of DPH rotational dynamics in gel and fluid phase membranes and the effect of eugenol on DPH dynamics, we have calculated semiangle of probe wobbling in a cone using Kinosita model.32

Phase dependent semi-angle of DPH wobbling in a cone: Effect of Eugenol The semi-angle of DPH wobbling in a cone has been calculated using eqn. (6) and the cone angle has been plotted as a function of eugenol concentration for gel and fluid phases of DMPC membranes in Figure 5(C). The cone angle of probe wobbling is remarkably small in gel phase (~32°) compared to that in fluid phase (~60°) in absence of eugenol. This smaller semiangle of DPH rotation in gel phase membranes could be attributed to the smaller rotational correlation time in gel phase. In addition, the semi-angle of DPH rotation reduces with the addition of eugenol in gel phase membranes but there is no significant change in fluid phase membrane. This indicates that eugenol orders the membrane in gel phase, resulting in reduction of semi-angle of probe rotation, whereas eugenol does not affect the hydrophobic chain packing in fluid phase. Taken together, the time-resolved fluorescence anisotropy measurements and semi-angle calculation elucidate that eugenol orders the hydrophobic region in the gel phase membranes but does not affect the hydrophobic packing in the fluid phase membranes.

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Figure 5. Plot of time-resolved fluorescence anisotropy decay of DPH in absence (black) and in presence of 20 µM (green), 40 µM (blue) and 60 µM (red) eugenol in presence of 400 µM DMPC at (A) 10 °C, gel phase and (B) 37 °C, fluid phase. The decay profile was best fitted in a two-exponential anisotropy decay model (fitted lines are shown in red through the corresponding data). (C) Wobbling-in-cone angle of DPH in DMPC is shown as a function of eugenol concentration at 10 °C (gel phase, red) and 37 °C (fluid phase, black). A 340 nm LED was used to excite DPH and corresponding emission was monitored at 428 nm. All measurements were carried in 10 mM phosphate buffer of pH 7.4 using 2 µM DPH concentration. Concentration of DMPC was kept constant at 400 µM for all measurements. Data points shown are means ± S.E. See materials and methods section for more details.

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DISCUSSION

The overall goal of this work is to explore the effect of eugenol in the organization and dynamics of gel and fluid phase membranes. The membrane is different from other macromolecular assemblies in biological systems as there is no intermolecular connectivity among the lipid molecules. Hence, the two-dimensional organization of different lipids provides the inherent dynamics along the bilayer normal.31, 39 As a result of this organization, properties such as polarity, order and heterogeneity vary along the bilayer normal. Interestingly, the amino acid distribution of transmembrane proteins is highly asymmetric and hence the membrane organization and dynamics is extremely important to accommodate membrane proteins.40 About 50% of genetically encoded proteins are membrane binding proteins and it is expected that ~50% of cellular reactions take place in the membrane. 41 Because of these facts, it is necessary to have a fine-tuned organization and dynamics of the membrane.

The lipidic phases are one of the most important determinants for lipid

organization and dynamics. In gel phase, the lipid acyl chains are in trans- conformation and form a highly ordered hydrophobic region, whereas in fluid phase the lipid acyl chains are in gauche conformation, which leads to disordered hydrophobic core of the membrane.17 It has been shown that small molecules like general anesthetics modulate the chain order,42 membrane fluidity,43 membrane conductance44 and phase transition temperature.45-47 The gel to fluid phase transition or chain melting has been the most extensively studied lipid phase transition and it is characteristic to length and chemistry of the hydrophobic chain. Organic additives are known to destabilize the packing of the hydrophobic chains leading to decrease in phase transition temperature. Interestingly, eugenol enhances phase transition temperature in a concentration dependent fashion. A previous study has shown that eugenol does not alter the elasticity of liquid condensed phase of 1,2-dipalmitoylphosphatidylcholine (DPPC) monolayer and phase transition occurs at higher surface area compared to control.48 This result supports our observation of eugenol-induced increase in phase transition temperature of DMPC. The phase transition temperature of DMPC changes by ~1 °C in presence of lipid to eugenol ratio ~7, the ratio will be higher as the partition coefficient of eugenol is not as high 17


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as other phenolic compounds.49 Higher phase transition temperature of DMPC in presence of eugenol implies parallel arrangement of eugenol to the hydrophobic tail of the lipid and hence further ordering of the chain packing. We have used eugenol and DPH fluorescence lifetime measurements to understand the change in polarity of the interfacial and hydrophobic region of DMPC membranes induced by eugenol. Figure 3 and Figure 4 clearly demonstrate that the effect of eugenol is more pronounced in fluid phase compared to gel phase. Similar result was observed in DPPC membrane, where eugenol induced marginal change in fluorescence anisotropy of DPH and TMA-DPH in gel phase.50 It needs a special mention that change in eugenol lifetime in gel phase is extremely small, which could be due to more interfacial location of eugenol at higher concentration. The ordering effect of eugenol in gel phase membranes is clearly visible from the time-resolved anisotropy measurements and semi-angle of wobbling-in-a-cone of the probe calculation.

The pre-exponential of the slower

component of the time-resolved anisotropy measurement is much higher in gel phase than that in fluid phase, which signifies that most of the DPH molecules are experiencing restricted rotational movement in the gel phase membranes. Nonetheless, the semi-angle of DPH rotation decreases with increasing concentration of eugenol in gel phase membrane. This suggests that eugenol is aligning parallel to the hydrophobic chain of the lipid and increasing the packing of the gel phase membranes. Taken together, eugenol stabilizes the gel phase and elevates the phase transition temperature of DMPC. Eugenol-induced change in polarity of membrane interface is more significant for fluid phase membranes. In addition, eugenol makes the hydrophobic region of the gel phase membrane more ordered and hence semi-angle of probe rotation becomes extremely restricted, whereas it does not alter the hydrophobic tail packing of the fluid phase membranes. Finally, our present work provides comprehensive information on the effect of eugenol on the organization and dynamics of DMPC membranes in varying phases. The unique ability of eugenol to stabilize the gel phase and elevate phase transition temperature could be explored further to understand its effect in membrane remodeling.

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Table-1. Fluorescence lifetimes of Eugenol with its increasing concentration in varying phases of DMPC membranes

Lipidic Phase

Conc. of Eugenol (μM)

α1

τ1

α2

τ2

α3

τ3

τavg

χ2

10

31.01 ±

0.04 ±

61.28 ±

1.06 ±

7.71 ±

3.36 ±

1.14

2.07

0.01

2.05

0.003

0.21

0.03

1.70 ± 0.02

12.70 ±

0.12 ±

77.85 ±

1.08 ±

9.45 ±

2.84 ±

1.09

1.06

0.02

1.16

0.002

0.25

0.03

1.50 ± 0.02

10.15 ±

0.20 ±

81.69 ±

1.15 ±

8.15 ±

2.84 ±

1.07

0.77

0.05

0.86

0.01

0.57

0.03

1.46 ± 0.02

7.40 ±

0.19 ±

83.65 ±

1.16 ±

8.94 ±

2.59 ±

1.04

0.59

0.04

0.90

0.007

0.38

0.02

1.43 ± 0.01

8.33 ±

0.30 ±

81.48 ±

1.20 ±

10.20 ±

2.44 ±

1.00

0.94

0.06

0.65

0.01

0.84

0.06

1.42 ± 0.01

11.04 ±

0.56 ±

82.43 ±

1.30 ±

6.53 ±

2.55 ±

1.06

1.04

0.07

0.74

0.02

0.76

0.03

1.42 ± 0.01

54.89 ±

0.54 ±

25.96 ±

0.90 ±

19.15 ±

3.21 ±

1.89 ±

1.14

5.29

0.02

0.92

0.33

4.37

0.40

0.03

58.22 ±

0.50 ±

21.13 ±

1.19 ±

20.58 ±

2.13 ±

1.70 ±

5.76

0.03

3.15

0.41

3.60

0.19

0.04

67.41 ±

0.55 ±

15.55 ±

1.31 ±

17.04 ±

1.68 ±

1.59 ±

2.95

0.02

2.15

0.37

3.26

0.47

0.05

67.11 ±

0.59 ±

17.54 ±

1.23 ±

15.25 ±

1.78 ±

1.51 ±

3.67

0.02

2.78

0.34

3.21

0.39

0.05

53.01 ±

0.51 ±

30.68 ±

1.04 ±

16.30 ±

1.57±

1.41 ±

13.55

0.08

11.48

0.37

3.28

0.42

0.05

77.79 ±

0.67 ±

10.02 ±

1.21 ±

12.21 ±

1.85 ±

1.33 ±

3.03

0.02

1.96

0.72

4.99

0.36

0.06

20

GEL PHASE

30 40 50 60

10 20

FLUID PHASE

30 40 50 60

19


1.09

1.02

1.06

1.00

1.06

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Table-2. Time-resolved fluorescence anisotropy decay parameters of DPH with increasing concentration of Eugenol in varying phases of DMPC membranes

Lipidic Phase

GEL PHASE

Conc.of Eugenol (μM) 0

β1

Φ1 (ns)

β2

Φ2 (ns)

60.09 ± 0.89

10.26 ± 0.27

39.91 ± 0.89

0.68± 0.01

20

66.52 ± 1.28

6.07 ± 0.65

33.48 ± 1.28

0.61 ± 0.01

40

74.44 ± 0.95

9.66 ± 0.42

25.57 ± 0.95

0.66 ± 0.01

60

74.52 ± 0.71

10.74 ± 0.62

25.49 ± 0.71

0.64 ± 0.02

0

13.27 ± 1.79

15.06 ± 0.57

86.73 ± 1.79

1.76 ± 0.01

20

16.88 ± 0.97

14.43 ± 0.87

83.12 ± 0.97

1.72 ± 0.02

40

13.13 ± 0.24

14.11 ± 0.34

86.87 ± 0.24

1.74 ± 0.001

60

14.76 ± 0.20

13.14 ± 0.44

85.24 ± 0.20

1.7 ± 0.01

r0

0.36

FLUID PHASE

CONCLUDING REMARK

Several steady state and time-resolved fluorescence methods were employed to study the effect of eugenol on large unilamellar vesicles (LUVs) of DMPC at varying phases. Our result indicates that eugenol stabilizes the gel phase of DMPC and increases the phase transition temperature. The change in phase transition temperature is ~1 °C in presence of 100:15 molar ratio of DMPC and eugenol. This change is significant in case of phase transition temperature and considering the lipid to eugenol ratio. The results obtained from the time-resolved fluorescence studies give a comprehensive information of the effect of eugenol in gel and fluid phases of DMPC membranes. Our results show that eugenol further orders the gel phase membrane whereas it destabilizes the fluid phase membrane. Taken

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together, this unique property of eugenol could be exploited to understand the eugenol mediated membrane remodeling.

ACKNOWLEDGMENTS

This work was supported by research grants from the University Grants Commission, New Delhi (File No. F.4-5(138-FRP)/2014(BSR)) and Department of Science and Technology, New Delhi (File No. ECR/2015/000195). H.C. and G.M. thank the University Grants Commission for UGC-Assistant Professor position and UGC-BSR Research Fellowship respectively. We acknowledge Department of Science and Technology, New Delhi and UGC for providing instrument facility to the School of Chemistry, Sambalpur University under the FIST and DRS programs, respectively. We thank Dr. S. N. Sahu and members of Chakraborty laboratory for their comments and discussions.

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For Table of Contents Only

Influence of Eugenol on the Organization and Dynamics of Lipid Membranes: A Phase-Dependent Study Geetanjali Meher and Hirak Chakraborty* School of Chemistry, Sambalpur University, Jyoti Vihar, Burla, Odisha – 768 019, India

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Influence of Eugenol on the Organization and Dynamics of Lipid

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