CoenzymeQ10 localizations in model membranes. A Langmuir monolayer study

Biophysical Chemistry 207 (2015) 74–81

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CoenzymeQ10 localizations in model membranes. A Langmuir monolayer study Willy Nerdal b, Torill Regine Sandvik Nilsen b, Signe Steinkopf a,⁎ a b

Department of Biomedical Laboratory Science, Bergen University College, Inndalsveien 28, N-5020 Bergen, Norway Department of Chemistry, University of Bergen, Allégaten 41, N-5007 Bergen, Norway

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• At biological level, the CoQ10 causes looser lipid packing in the DPPC monolayer. • At biological level, the CoQ10 induces domain formation in the POPS monolayer. • At biological level, the PB lipids camouflage the effects of CoQ10. • At biological level, the CoQ10 causes closer lipid packing in the DEPC monolayer.

a r t i c l e

i n f o

Article history: Received 9 July 2015 Received in revised form 15 September 2015 Accepted 15 September 2015 Available online xxxx Keywords: Monolayer DPPC POPS PB DEPC

a b s t r a c t The interaction of coenzyme Q10 (CoQ10) in a monolayer of 1,2-dipalmitoyl-sn-glysero-3-phospho-L-choline (DPPC), in a monolayer of 1,2-dierucoyl-sn-glysero-3-phospho-L-choline (DEPC), in a monolayer of 1palmitoyl-2-oleoyl-sn-glysero-3-phospho-L-serine (POPS) and in a monolayer of total lipid extract from pig brain (PB) has been investigated by using the Langmuir monolayer technique. Surface pressure (π)-mean molecular area (mma) isotherms have been measured for pure lipid monolayers and lipid monolayers with 0.5, 1.0, 2.0, 5.0 and 10.0 mol% CoQ10 concentrations. At the biological concentration (1.0–3.0 mol%) of CoQ 10 , intercalation of CoQ 10 occurs in the lipid acyl chains of DPPC, POPS and PB monolayers. Above the biological concentration of CoQ10, the CoQ10 molecule induces domain formation in the monolayers of DPPC, POPS and PB lipids. The DEPC monolayer behavior deviates from the other lipids in this study. At 2.0 mol% the CoQ10 promotes very dense lipid packing, and the CoQ10 molecule is located parallel to the DEPC acyl chains at all concentrations. © 2015 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. E-mail address: [email protected] (S. Steinkopf).

http://dx.doi.org/10.1016/j.bpc.2015.09.003 0301-4622/© 2015 Elsevier B.V. All rights reserved.

Coenzyme Q10 (CoQ10) or ubiquinone, plays an important role in lipid and mitochondrial membranes [1] by taking part in redox reactions and is a key component of the electron transport chain and ATP production. The reduced form, ubiquinol, is an antioxidant [2]

W. Nerdal et al. / Biophysical Chemistry 207 (2015) 74–81

that protects the lipid membranes from lipid peroxidation [3] and the cell from oxidative stress [4] and regenerates vitamin-E, a potent antioxidant [5]. Lack of CoQ10 is thought to be involved in neurodegenerative diseases such as Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis [6–8] in that an increase in free radical production seems to be important in the development of these diseases [9]. Further, CoQ10 deficiency might lead to cardiac diseases [10–12]. CoQ10 contains a redox-active quinone (head) and a polyisoprene chain of ten isoprene units and displayed together with lipids employed in this study in Scheme 1. Scheme 2 shows the two redox stated of the quinone unit in CoQ10 and Scheme 3 some of the suggested locations of CoQ10 in the lipid bilayer. Knowing the location of CoQ10 in membranes is essential for understanding the function of CoQ10 and this has been a matter of dispute for more than a decade. The poly-isoprene chain will promote molecular interactions with hydrophobic molecules of low polarity [13] so that CoQ10 will predominantly locate within the hydrophobic region of the bilayer and cause the redox active quinone to be a diffusible part of the membrane. As the quinone head group is involved in electron and proton transfers, it is a water-seeking group. Several reports have been published using phospholipid bilayers as membrane models. The interpretations of the findings are contradictory. NMR studies employing suspension [14] vesicles and bicelles [15] demonstrate that in these model systems of a phospholipid bilayer the ~2 mol% CoQ10 is

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located close to the bilayer terminal methyl of the phospholipid acyl chains, i.e. in the plane between the two leaflets of the bilayer and parallel to the bilayer plane. This conclusion is further supported by results from differential scanning calorimetry (DSC) studies [16–18] and neutron diffraction [19]. However, conclusions of results from fluorescence anisotropy [20] are that the benzoquinone ring interacts with phospholipid polar head groups, and the hydrophobic isoprene unit of CoQ10 is anchored in the hydrophobic bilayer mid-plane, parallel to the lipid acyl chains. At a 0.05–0.5 mol% level CoQ10 is suggested located parallel to the lipid acyl chains in a 13C NMR study [21]. This result is supported by a recent 13C MAS study of CoQ10 interaction with 1-palmitoyl-2oleoyl-sn-glysero-3-phospho-L-serine (POPS) bilayer [22] where the 2–10 mol% CoQ10 is found to be located at the phosphate group close to the serine head group, indicating location parallel to the lipid acyl chains. Further, the results from this study show that the presence of CoQ10 induce lipid phases of higher molecular mobility and such a phenomenon could be part of the mechanism that carries the ubiquinol moiety to locations in the membrane where lipid peroxidation is about to take place. CoQ10 is also suggested to be organized in a separate phase [23] or with the isoprene chain anchored/localized in the center of the bilayer with the quinone head group in the polar region [24]. Further, in a DMPC bilayer (short acyl chains) study [25], the CoQ10 was found to be localized both in the bilayer mid-plane and in the polar region of the bilayer. These various locations suggested for CoQ10

Scheme 1. Upper row (left to right): DPPC, POPS, DEPC and CoQ10. Lower row (left to right). Displays major lipids in PB: A phospholipid with ethanolamine head group (PE), cholesterol, a ceramide, a sphingomyelin and a ganglioside with an oligosaccharide head group. The molecular structures are shown without hydrogen atoms.

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Scheme 2. CoQ10 is a redox agent in the transport of electrons between enzymes embedded in the inner mitochondrial membrane. Upper: oxidized form. Lower: reduced form.

incorporation into model membranes might be affected by both concentration of CoQ10 and the type of lipid used as model membrane as well as the oxidation state of CoQ10 [26]. Since the results of studies with models of the lipid bilayer and the composition of lipids forming a lipid bilayer diverge with respect to localization of CoQ10 in membranes, lipid monolayer films has also been used as model membranes to monitor the electrochemistry and localization of CoQ10 [27,28]. One study led to the conclusion that CoQ10 when mixed with PC molecules forms aggregates or “pseudo-ring” conformations parallel to the saturated acyl-chains in a chain-length dependent manner [29]. The Langmuir monolayer film balance technique is a method chosen by several

authors due to the possibility to study the intercalation of CoQ10 in lipids at both hydrophobic (tail) and hydrophilic (head) environment [26,30– 32]. From Langmuir monolayer studies, it is found that CoQ1–10 is expelled from the DPPC [31] monolayer and the extent of this expulsion is dependent of the isoprenoid chain-length [32]. That CoQ10 is located above the DPPC monolayer (5:1 mixture) is also found in a recent study [26], where the authors suggest CoQ10 locations in the DPPC monolayer to be dependent on the redox processes. One is located between the choline head-groups in contact with the subphase and a second location is into the DPPC matrix close to the phosphate group, and a third above the acyl-chains of DPPC. However, many studies where carried out employing monolayer of DPPC, a neutral, saturated lipid. To our knowledge, only one work is considering other lipids than DPPC, namely POPS-lipids with negatively charged head-group with one unsaturated acyl-chain, and that work is a bilayer study [22]. In the present work, we have studied the intercalation of 0.5, 1.0, 2.0, 5.0 and 10.0 mol% CoQ10 into monolayers of each of the four lipids DPPC, POPS, total lipid extract from pig brain (PB) and DEPC. The manufacturer supplies a sample content of total lipid extract from pig brain of PE (16.7%), PS (10.6%), PC (9.6%), PA (2.8%), PI (1.6%) and “other” (58.7%). In addition, with support from the 13C NMR spectra in a study of cisplatin interaction with lipid bilayer of total lipid extract from pig brain, this lipid extract is also found to contain ∼20% cholesterol, ∼10% sphingomyelin as well as ∼20% ganglioside and less than 10% ceramide, thus, triglycerides are not found in this lipid extract [33]. DEPC is a non-biological lipid often employed in the laboratory, e.g. in NMR studies of peptides/proteins in lipid bilayers [34]. The chosen concentrations of CoQ10 include the biological level of 1–3 mol% CoQ10 in lipid membranes. At the biological concentration (1.0–3.0 mol%) of CoQ10, intercalation of CoQ10 occurs in the lipid acyl chains of DPPC, POPS and PB monolayers. Above the biological concentration of CoQ10, the CoQ10 molecule induces domain formation in the monolayers of DPPC, POPS and PB lipids. The DEPC monolayer behavior deviates from the other lipids in this study. At 2.0 mol% the CoQ10 promotes very dense lipid packing, and the CoQ10 molecule is located parallel to the DEPC acyl chains at all concentrations. 2. Experimental 1,2-dipalmitoyl-sn-glysero-3-phospho-L-choline (DPPC), 1,2dierucoyl-sn-glysero-3-phospho-L-choline (DEPC) and 1-palmitoyl-2oleoyl-sn-glysero-3-phospho-L-serine (POPS) and total lipid extract from pig brain (PB) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL). The mean molecular weight of a PB lipid is estimated to 827 g/mol [33]. The lipids were kept in the dark as chloroform solutions

Scheme 3. Some suggested locations for CoQ10 in the lipid bilayer.

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at −20 °C. CoenzymeQ10 (CoQ10) powder was obtained from Sigma Chemical Co. (St. Louis, MO). Stock solutions (2 mg/ml) of lipids and CoQ10 were prepared in chloroform from Fisher Scientific Co. (Strasbourg, France). Working solutions of lipids (1 mg/ml) and lipid with 0.5, 1.0, 2.0, 5.0 and 10.0 mol% of CoQ10 were dissolved in chloroform. The HEPES buffer (5.0 mM) was from Fisher Scientific Co. Aliquots of NaOH or HCl of analytical grade was added to the HEPES buffer to obtain pH 7.40. Water with low ionic concentration (18.2 MΩ/cm) was used. A KSV Minitrough (Helsinki, Finland) of dimensions 75 (w) × 364 (l) × 5 (h) mm was used in the study of CoQ10 and lipid monolayer at the air/water interface. The trough was filled with HEPES buffer (5.0 mM, pH 7.40) and all the experiments were carried out at room temperature (22 °C). A sample volume of 25 μl was carefully spread on the aqueous surface with a Hamilton syringe. The chloroform was allowed to evaporate for 5 min before the compression started. During compression, the barrier speed was running at 5 mm/min. The surface pressure Π was determined using the Wilhelmy plate method. The lift-off areas were defined by the mean molecular area (mma) when the surface pressure [34] has reached 1 mN/m. The isotherms in Figs. 2–6 show the surface pressure as a function of the mma during compression. All experiments were repeated three times, the mean values and SD's were calculated by Excel (Microsoft, USA). The molecular models were generated by the Spartan `14 software (Wavefunction, Irvine, Ca).

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Fig. 1. The isotherms of pure lipid monolayers. The isotherm of DPPC is shown in a blue line, while the isotherms of POPS and DEPC are shown in red and purple lines, respectively. The isotherms of total lipid extract from pig brain (PB) and CoQ10 are shown in green and turquoise lines, respectively (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

3. Results 3.2. DPPC 3.1. Pure lipids The mma's at lift-off, at 20 mM/m, at 30 mN/m, and at the collapse point are summarized in Table 1 and presented in Figs. 1–5. The surface pressures of 20 and 30 mN/m are selected as this range corresponds to the surface pressure of a biological cell. In Fig. 1, the monolayer of pure CoQ10 shows the presence of both a gaseous phase and a LE phase that undergoes multilayer formation at a surface pressure of 11.6 mN/m. The course of the isotherms of DPPC, POPS and PB has been described elsewhere in the literature [35–37]. The isotherm of the monolayer of DEPC shows higher mma's throughout all phases compared to the other investigated lipids, and collapses at approximately the same surface pressure as the PB monolayer.

The isotherms of DPPC are shown in Fig. 2. At lift-off, in the gaseous phase, the isotherms with 0.5 and 5.0 mol% CoQ10 show lower mma than the isotherm of pure DPPC, while the isotherms with 1.0, 2.0 and 10.0 mol% CoQ10 show increased mma. All isotherms of DPPC, except the isotherm with 10.0 mol% CoQ10, show the presence of a LE–LC phase coexistence. The isotherm with 2.0 mol% CoQ10 shows the broadest region of LE–LC phase coexistence (Δmma = 26.3 Å2). Further, a kink in the isotherm with 10.0 mol% CoQ10 is observed at a surface pressure of 18.5 mN/m and this isotherm shows highest mma through all phases. POPS. The isotherms of POPS are shown in Fig. 3, where the isotherms with CoQ10 show higher mma's in the gaseous and liquid states

Table 1 Mean molecular area (Å2) at lift-off, at Π = 20, at Π = 30 and at collapse as well as % change with the presence of CoQ10 in monolayers of DPPC, POPS, PB and DEPC. Mean values of three parallels of the lipid monolayers are shown. The mean values of the collapse point pressure are shown and the SDs are included. Lipid

DPPC DPPC w/0.5% Q10 DPPC w/1% Q10 DPPC w/2% Q10 DPPC w/5% Q10 DPPC w/10% Q10 POPS POPS w/0.5% Q10 POPS w/1% Q10 POPS w/2% Q10 POPS w/5% Q10 POPS w/10% Q10 PB PB w/0.5% Q10 PB w/1% Q10 PB w/2% Q10 PB w/5% Q10 PB w/10% Q10 DEPC DEPC w/0.5% Q10 DEPC w/1% Q10 DEPC w/2% Q10 DEPC w/5% Q10 DEPC w/10% Q10

Mean molecular area (Å2)

Π(mN/m)

Relative Change in % Lift-off

Π = 20

Π = 30

−9.9 1.1 5.9 −7.2 13.1

−2.4 2.4 14.6 5.73 33.0

3.1 −0.4 17.6 4.8 24.7

3.6 1.8 21.8 6.1 19.8

1.6 −8.2 −2.3 −2.7 −3.9

−6.7 −9.2 −9.3 −10.8 −8.9

8.0 13.5 10.4 12.0 10.9

−3.1 7.5 5.4 10.2 −0.5

−10.3 −4.6 −11.1 −5.7 −11.3

10.2 18.2 21.8 11.0 14.9

1.1 −1.9 1.8 8.5 12.0

7.2 −1.2 −0.7 12.9 8.1

9.9 0.0 −1.3 12.2 6.3

13.2 1.1 −0.9 12.0 7.7

−3.1 2.6 2.8 3.5 5.2

2.1 3.2 −8.5 14.7 5.6

3.2 5.8 −8.9 11.5 4.5

6.9 8.4 −5.4 9.0 4.8

6.0 9.8 −4,7 7.0 3.2

11.4 3.4 3.9 −2.4 −2.2

Lift-off

Π = 20

Π = 30

Collapse

Collapse

112.8 ± 1.2 101.6 ± 1.8 114.0 ± 0.5 119.4 ± 2.7 104.7 ± 3.5 127.5 ± 3.0 126.5 ± 1.8 117.8 ± 1.9 114.8 ± 1.8 114.7 ± 3.5 112.9 ± 2.0 115.3 ± 5.3 92.6 ± 1.4 93.6 ± 4.3 90.8 ± 2.3 94.3 ± 3.5 100.5 ± 1.3 103.7 ± 2.0 117.0 ± 3.2 119.44 ± 1.9 120.8 ± 2.6 107.1 ± 2.2 134.2 ± 6.3 123.5 ± 9.2

50.6 ± 0.7 49.4 ± 0.4 51.8 ± 0.7 58.0 ± 2.6 53.5 ± 0.1 67.3 ± 4.0 62.4 ± 0.7 67.4 ± 1.2 70.8 ± 1.3 68.9 ± 2.8 69.7 ± 2.2 69.2 ± 1.7 58.2 ± 1.1 62.4 ± 0.9 57.5 ± 1.3 57.8 ± 1.3 65.7 ± 0.9 62.9 ± 2.0 71.0 ± 1.3 73.3 ± 1.0 75.1 ± 1.3 64.7 ± 0.6 79.2 ± 2.4 74.2 ± 1.7

45.4 ± 0.9 46.8 ± 1.1 45.2 ± 0.5 53.4 ± 1.6 47.6 ± 1.3 56.6 ± 0.7 55.7 ± 0.8 54.0 ± 0.5 59.9 ± 1.6 57.7 ± 2.5 61.4 ± 8.8 55.4 ± 1.2 52.3 ± 1.2 57.5 ± 0.7 52.3 ± 1.2 51.6 ± 0.7 58.7 ± 1.3 55.6 ± 1.7 61.0 ± 0.7 65.2 ± 0.8 66.1 ± 0.9 57.7 ± 0.7 66.5 ± 1.8 63.9 ± 1.1

39.4 ± 1.2 40.8 ± 1.0 40.1 ± 0.8 48.0 ± 1.8 41.8 ± 1.4 47.2 ± 4.7 52.4 ± 0.3 47.0 ± 0.7 50.0 ± 1.2 46.6 ± 1 49.4 ± 1.3 46.5 ± 0.5 46.8 ± 1.6 53.0 ± 0.5 47.3 ± 0.5 46.4 ± 0.5 52.4 ± 1.4 50.4 ± 1.8 53.1 ± 1.9 56.3 ± 0.9 58.3 ± 1.7 50.6 ± 3.1 56.8 ± 1.3 54.8 ± 2.6

51.1 ± 3.4 51.9 ± 3.0 46.9 ± 4.0 49.9 ± 4.3 49.7 ± 1.7 49.1 ± 2.2 36.2 ± 0.5 39.9 ± 0.7 42.8 ± 1.8 44.1 ± 0.6 40.2 ± 1.0 41.6 ± 1.7 42.5 ± 1.3 41.2 ± 3.5 43.6 ± 2.6 43.7 ± 1.2 44.0 ± 1.7 44.7 ± 2.8 41.2 ± 2.1 45.9 ± 1.5 42.6 ± 2.8 42.8 ± 5.7 40.2 ± 0.6 40.3 ± 3.8

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(Π)Collapse

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Fig. 2. The isotherms of DPPC. The isotherm of pure DPPC is shown in a blue line. The isotherms of DPPC with 0.5 and 1.0 mol% CoQ10 are shown in red and green lines, respectively. The isotherm with 2.0 mol% CoQ10 is shown in a purple line, while the isotherms with 5.0 and 10.0 mol% CoQ10 are shown in turquoise and orange lines, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

than the isotherm of pure POPS. The isotherms with CoQ10 are all concurrent until a surface pressure of 17.4 mN/m where the isotherms with 0.5 and 10.0 mol% CoQ10 show an inflection point. Upon further compression of the barriers, the isotherm with 0.5 mol% CoQ10 becomes concurrent with the pure POPS isotherm. At a surface pressure of 30 mN/m, the isotherms with 0.5 and 10.0 mol% CoQ10 both show lower mma's than the isotherm of pure POPS, while the other CoQ10 containing isotherms show higher mma's. The isotherm of pure POPS collapses at the lowest surface pressure, while all the CoQ10 containing isotherms show collapse points at higher pressures, most pronounced in the isotherm with 2.0 mol% CoQ10. 3.3. Total lipid extract from pig brain

Fig. 4. The isotherms of total lipid extract from pig brain, (PB). The isotherm of pure PB is shown in a blue line. The isotherms of PB with 0.5 and 1.0 mol% CoQ10 are shown in red and green lines, respectively. The isotherm with 2.0 mol% CoQ10 is shown in a purple line, while the isotherms with 5.0 and 10.0 mol% CoQ10 are shown in turquoise and orange lines, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.0 mol% CoQ10. Further, the surface pressure at the collapse point of the isotherm with 0.5 mol% CoQ10 is reduced, while the other isotherms with CoQ10 collapse at higher pressures compared to the isotherm with pure PB. The most striking result is the presence of a plateau at a surface pressure of 14.8 mN/m in the isotherm with 10.0 mol% CoQ10. Upon further compression of the barriers, the plateau disappears at a surface pressure of 19.5 mN/m. 3.4. DEPC

The isotherms of PB are shown in Fig. 4 and the isotherms with 0.5, 5.0 and 10.0 mol% CoQ10 show increased mma's through all phases compared to the isotherm of pure PB and the isotherms with 1.0 and

The isotherms of DEPC are shown in Fig. 5. The isotherm with 2.0 mol% CoQ10 shows lower mma through all phases compared to the pure monolayer of DEPC, while the other CoQ10 containing isotherms show increased mma. The most pronounced increase in mma is observed in the gaseous and liquid phases of the isotherm with 5.0 mol%

Fig. 3. The isotherms of POPS. The isotherm of pure POPS is shown in a blue line. The isotherms of POPS with 0.5 and 1.0 mol% CoQ10 are shown in red and green lines, respectively. The isotherm with 2.0 mol% CoQ10 is shown in a purple line, while the isotherms with 5.0 and 10.0 mol% CoQ10 are shown in turquoise and orange lines, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. The isotherms of DEPC. The isotherm of pure DEPC is shown in a blue line. The isotherms of DEPC with 0.5 and 1.0 mol% CoQ10 are shown in red and green lines, respectively. The isotherm with 2.0 mol% CoQ10 is shown in a purple line, while the isotherms with 5.0 and 10.0 mol% CoQ10 are shown turquoise and orange lines, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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CoQ10. However, in the solid state, the isotherms with 0.5 and 1.0 mol% CoQ10 show the highest mma's and collapse at higher pressures than the other isotherms. The interaction between pure lipid monolayer and lipid monolayer with CoQ10, are analyzed by calculation of mma's for a mixed two component system using the additivity rule [38,39].

area occupied by the choline head group is identical in both DPPC and DEPC, the observed increased mma of DEPC can be explained by the differences in the acyl chains. The DEPC lipid contains two identical unsaturated acyl chains with 22 carbon atoms with the double bond positioned at C-13 to C-14 that occupies larger mma's than the other investigated lipids.

A12 ¼ N1 A1 þ N2 A2

4.2. DPPC

ð1Þ

where N1A1 and N2A2 are the mole fractions multiplied with the mma per molecule of the pure components at the identical surface pressure. A12 is the mma per molecule of the mixed monolayer of the two components system. Fig. 6 shows the plot of average area per molecule versus mol% of CoQ10 present in the monolayer at a surface pressure of 10.0 mN/m, below the pressure where CoQ10 forms multilayers. The straight line in each plot represents the calculated curve for an ideal non-interacting two components system and the experimental values are indicated by dots. None of the mma's of the investigated lipid monolayers change linearly with the CoQ10 mol% and deviations from ideal miscibility of two components in a mixture, has been calculated from the Gibb's free energy of mixing by use of Simpsons rule according to the Goodrich approach [40]. The largest difference in Gibb's free energy of mixing in the lipid monolayers is found at 2.0 mol% CoQ10 . The results are 19.5 ± 1.8 J/mole , 9.4 ± 2.6 J/mole , 3.7 ± 1.4 J/mole and -9.8 ± 1.0 J/mole for the monolayers of DPPC, POPS, PB and DEPC, respectively.

4. Discussion 4.1. Pure lipids The isotherms of monolayers of DPPC, POPS and PB have been explained elsewhere [35–37,41], and will not be discussed in detail here. However, the lipids behave differently upon increased surface pressures (Fig. 1). The monolayer of CoQ10 form multilayers, while the monolayer of DPPC shows the presence of a LE–LC coexisting phases. The isotherms of PB, POPS and DEPC follow the same course, where the isotherm of DEPC shows looser monolayer packing through all phases. Since the

By comparing the DPPC with CoQ10 isotherms with the isotherm of pure DPPC (Fig. 2), the CoQ10 appears to interfere with the DPPC lipids. The presence of 0.5 mol% CoQ10 (below the biological concentration) in DPPC monolayer induces closer packing of the lipids, as demonstrated by lowered mma's through all phases. Further, the negative Gibb's free energy of mixing,ΔGM, (data not shown) indicate attractive forces between the DPPC lipids and the CoQ10 molecules. Furthermore, the DPPC LE–LC coexisting phases present indicates that the CoQ10 interacts with the choline head group. The LE–LC coexisting phases are also present in the isotherms with 1.0, 2.0 and 5.0 mol% CoQ10. This is in accord with the finding that DPPC lipid phase transition is unaffected by low CoQ10/DPPC ratio [11,19]. At 1.0 and 2.0 mol% CoQ10 (at biological concentration) the mma's increase in the gaseous and liquid phases compared to pure DPPC. Further,Δ GM, is positive, indicating repulsive forces between the CoQ10 molecules and the DPPC lipids and that the CoQ10 acyl chain is oriented parallel to the DPPC acyl chains. The mma of the isotherm with 5.0 mol% CoQ10 (above the biological concentration) is reduced through all phases compared to the isotherm with 2.0 mol% CoQ10. It is conceivable that the CoQ10 molecules will dimerize/aggregate at this concentration parallel to the acyl chains induced by repulsive forces between the CoQ10 molecules and the DPPC lipids (Δ GM is positive). Increasing the CoQ10 concentration to 10.0 mol%, the mma increases and the LE–LC coexisting phases disappears. This indicates that the CoQ10 molecules also intercalate into the acyl chain region of the DPPC monolayer. Further, the observed kink at a pressure of 18.5 mN/m suggests segregation of DPPC lipids and CoQ10 molecules. The CoQ10 molecules could induce domain formation (at 10.0 mol%) by promoting regions of the monolayer to be enriched in CoQ10 molecules and DPPC lipids, respectively. For instance, it has been found

Fig. 6. The plot of mma of the individual lipid monolayer versus the mol% of CoQ10 at a surface pressure of π = 10.0 mN/m. The solid line represents the ideal curve for non-interacting two components system. The dotted lines represent the observed mma's.

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from differential scanning calorimetry [16] that CoQ10 at a level of 20 mol% in DPPC bilayer causes the coenzyme to be dispersed as a separate phase sandwiched between the leaflets of the bilayer. Further, above 5.0 mol% [17], the CoQ10 is found to induce phase separation in mixtures of derivatives of phosphatidylcholine. 4.3. POPS In all monolayers of POPS (Fig. 3), the CoQ10 molecules induces looser lipid packing in the gaseous and liquid phases until a pressure of 17.4 mN/m. This indicates that the CoQ10 molecules are present in the monolayer. However, at this pressure, a plateau is appearing in the isotherm with 0.5 mol% CoQ10. At a surface pressure of 25.6 mN/m, this isotherm is concurrent with the pure POPS isotherm, indicating that the CoQ10 molecules are forced out of the POPS monolayer probably in the methyl terminal acyl-chain region of the monolayer. Increasing the pressure above 25.6 mN/m, the isotherm with 0.5 mol% CoQ10 shows lower mma than the pure POPS isotherm, indicating closer packing of the lipids. This could be due to hydrophobic interactions between the CoQ10 molecules and the methyl groups of the acyl chains. Support for this interpretation is from a solid-state NMR [22] study where the presence of CoQ10 induced enhanced mobility of the acyl methyl groups of POPS. In the isotherms with 1.0 and 2.0 mol% CoQ10, the mma's increase through all phases indicating that CoQ10 is intercalated into the monolayers, and that the intercalation has a stabilizing effect on the lipid packing. This is supported by the observed increase of the collapse points compared to the isotherm of pure POPS. The isotherms with 5.0 and 10.0 mol% CoQ10 (above the biological concentrations) both show a kink at a surface pressure of 17.4 mN/m and could result from formation of CoQ10 rich and POPS rich regions in the monolayers. Further, these isotherms show lower mma's than the isotherms with 1.0 and 2.0 mol% CoQ10 in the liquid and solid phases, and the mma of the isotherm with 10.0 mol% CoQ10 is lower than for the pure POPS isotherm in the solid phase. These observations also support formation of CoQ10 aggregates. The ΔGM, is positive for all the CoQ10 concentrations in the POPS monolayer, indicating repulsive forces between the CoQ10 and the POPS molecules. The presence of domains formed by CoQ10 interaction with POPS have been found in solid-state 31P-NMR spectra of POPS bilayer with CoQ10, where three phases with different phosphate mobility [22] appeared. 4.4. Total lipid extract from pig brain All isotherms with CoQ10 and PB are shown in Fig. 4. At 0.5 mol% CoQ10, the isotherm shows increased mma's through all phases compared to the isotherm of pure PB. This indicates that CoQ10 intercalates between the PB acyl chains. TheΔGM, is positive due to repulsive forces between the CoQ10 molecules and the lipids. On the other hand, the isotherms with 1.0 and 2.0 mol% CoQ10 (at biological concentrations) are similar to the isotherm of pure PB. This indicates that the CoQ10 molecules at these concentrations induce closer packing of the PB lipids. TheΔGM, is positive for the isotherm with 1.0 mol% CoQ10 and negative for the 2.0 mol% CoQ10, indicating repulsive and attractive forces between the CoQ10 molecules and the lipids, respectively. Intercalation of CoQ10 into the monolayer results in mma's similar to monolayer of pure PB and could be explained by a camouflaging effect of lipids with large head groups present in the PB monolayer, like gangliosides or sphingolipids [41], and this is most effective at 2.0 mol% CoQ10. The isotherm with 5.0 mol% CoQ10 shows increased mma through all phases compared to the isotherms with lower concentrations of CoQ10. This indicates that the CoQ10 intercalates into the monolayer and that the CoQ10 concentration is above the camouflaging capability of the PB monolayer. TheΔGM, is positive at this concentration and at 10.0 mol% CoQ10, indicating that the forces between the CoQ10 molecules and the PB lipids are repulsive. The isotherm with 10.0 mol% CoQ10 shows increased mma in the gaseous and in the liquid phases compared to all

the other isotherms of PB. In the liquid phase, a plateau is appearing at a surface pressure of 14.3 mN/m. The observed plateau could be due to CoQ10 molecules that are forced out of the monolayer. However, the isotherm is not concurrent with the isotherm with pure PB. Upon further compression of the barriers, the isotherm shows increased mma compared to the pure PB monolayer and reduced mma compared with the isotherm with 0.5 mol% CoQ10. This suggests that some CoQ10 molecules remain in the monolayer. Based on the observed results, it seems reasonable to suggest that at 10.0 mol% CoQ10, the CoQ10 molecules have two locations, one in the monolayer, and the other on top of the monolayer. 4.5. DEPC The isotherms with 0.5 and 1.0 mol% CoQ10 in DEPC (Fig. 5), show increased mma through all phases and higher collapse points than the isotherm with pure DEPC, indicating looser lipid packing with enhanced stability due to localization of CoQ10 parallel to the DEPC acyl chains. The Δ GM values are negative for these isotherms. At 2.0 mol% CoQ10, the isotherm shows significantly reduced mma compared to the isotherm of the pure DEPC. Despite a slightly positive ΔGM, the continues shape of the isotherm of this monolayer (Fig. 5) demonstrates that the 2.0 mol% CoQ10 intercalates in the DEPC monolayer. By further increasing the CoQ10 concentration to 5.0 mol%, (above the biological concentration) the mma increases through all phases compared to the isotherms of pure DEPC and with 2.0 mol% CoQ10, and the ΔGM is negative for the isotherm with 5.0 mol% CoQ10, indicating that the CoQ10 molecules intercalates into the monolayer. The isotherm with 10.0 mol% CoQ10 shows reduced mma through all phases compared to the isotherm with 5.0 mol% CoQ10, and ΔGM is negative. An explanation for this observation might be that the CoQ10 molecules undergo dimerization/aggregation in the monolayer. 5. Conclusions The interaction of CoQ10 with lipid monolayers is dependent on the CoQ10 concentration, the lipid head-group charge and size, and the acylchain length and saturation/unsaturation. The 10 isoprene-units chain of CoQ10 can be located parallel or anti-parallel to the phospholipid acyl-chains, and CoQ10 can form aggregates and induce domain formation. All the mentioned localizations are indicated in the studied lipid monolayers. At CoQ10 concentrations lower than the biological level, the CoQ10 intercalates into the monolayers parallel to the acyl chains. The exception is in the POPS monolayer, where CoQ10 (0.5 mol%) induces domain formation. At the biological concentration of CoQ10 (between 1.0 and 3.0 mol%) the CoQ10 is located in between the lipid acyl chains in monolayers of DPPC, POPS and DEPC. The isotherms of PB with CoQ10 at the biological concentrations are nearly identical, due to the camouflaging effect of the lipid mixture of PB. At higher CoQ10 concentrations than the biological (at 5.0 and 10.0 mol% CoQ10), the CoQ10 molecules intercalates between the DPPC acyl chains, while domain formation occurs in the POPS and PB monolayers. At 2.0 mol% CoQ10, the monolayer of DEPC deviates compared to the monolayers of the biological lipids, by promoting a very dense lipid packing. At higher concentrations, such as at 10.0 mol%, the CoQ10 forms aggregates in the DEPC monolayer. Thus, this phospholipid could introduce unwanted effects on membrane peptide/protein structure when studied in a DEPC bilayer. The usefulness of comparing several lipid species as in this study is demonstrated by how the effects on monolayer packing of phospholipid head group, charge, acyl chain saturation/unsaturation can be followed. These results demonstrate that the Langmuir monolayer technique is a powerful tool for investigation of interaction of amphiphilic molecules with lipids, and can also be used as screening of molecular interactions on models of biological membranes, thus, assisting in the selection of lipid bilayer systems to be subjected to an experimental method requiring larger amount of sample like solid-state NMR.

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