Vibrational Spectroscopy 75 (2014) 86–92

Cardiolipin containing liposomes are fully ionized at physiological pH.An FT-IR study of phosphate group ionization

Dmitry Malyshka, Leah A. Pandiscia, Reinhard Schweitzer-Stenner *Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA

A R T I C L E I N F O

Article history:Received 9 June 2014Received in revised form 11 October 2014Accepted 11 October 2014Available online 18 October 2014

Keywords:CardiolipinIR-specroscopyPhosphocholineProtonation statesNormal mode analysis

A B S T R A C T

Cardiolipin containing liposomes serve as model systems for probing cytochrome c binding to the innermembrane of mitochondria. In order to interpret binding isotherms properly it is important to identifythe protonation state of the two phosphate groups of this phospholipid. Thus far, the literature proposedconflicting views based on experimentally and computationally support for both the fully protonated andsemi-protonated state as the physiologically dominant species. In order to clarify this issue we measuredthe FT-IR spectra of 100% 1,102,20-tetraoleyl cardiolipin (TOCL), 20% TOCL/80% 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) and 100% DOPC as a function of pH between 2 and 11. While the integratedintensities of all DOPC bands were found to be independent of pH within purely statistical variations, weidentified specific spectral regions in the TOCL spectrum for which the intensities either decrease and orincrease with decreasing pH below pH 4. It was obvious that these regions contain contributions fromPO4

� and P¼O stretching modes, respectively. We confirmed this with DFT-based normal modecalculations for a truncated TOCL head group, which revealed that the spectra regions between 1050 and1100 cm�1 as well as between 1200 and 1300 cm�1 can be ascribed as a superpositions of bands mostlyassignable to collective CH deformation modes and PO4 stretching modes. Our results strongly suggestthat the protonation of the phosphate groups occur in the acidic region and that they exist in their fullyionized state at physiological pH.

ã 2014 Elsevier B.V. All rights reserved.

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1. Introduction

The anionic phospholipid cardiolipin (CL, Fig. 1) has attracted alot of interest from biophysically oriented researchers over a ratherlong period of time. The main reason is its biological relevance; thislipid is found in the inner mitochondrial membrane and in mostbacteria [1]. In the former it is known to bind to the electron carriercytochrome c and to regulate the formation of supercomplexes [2].Besides serving as a quasi receptor for cytochrome c, it can servealso as a substrate for this protein once it acquires peroxidaseactivity. This change of functionality facilitates the release of theprotein from the space between inner and outer membrane, thusallowing it to initiate a biochemical cascade which eventually leadsto the release of the protein and its involvement in mitochondrialapoptosis [3]. In order to study the reaction of cytochrome c withthe inner membrane in quantitative terms, binding studies

Abbreviations: CL, cardiolipin; TOCL, tetraoleyl cardiolipin; DOPC, dioleyl-sn-glycero-phosphocholine.* Corresponding author. Tel.: +1 215 895 2268; fax: +1 215 895 2216.E-mail address: rschweitzer-stenner@drexel.edu (R. Schweitzer-Stenner).

http://dx.doi.org/10.1016/j.vibspec.2014.10.0030924-2031/ã 2014 Elsevier B.V. All rights reserved.

frequently utilize CL-containing liposomes as a model systemfor this membrane [4]. Studies conducted thus far reveal a rathercomplex picture with multiple binding sites of the proteinexhibiting rather different modes of binding (electrostatic,hydrophobic, hydrogen bonding, protein insertion into themembrane) [5].

In order to arrive at a comprehensive and valid theory of allthese experimental data, however, a specific issue needs to beclarified, namely the protonation state of the two phosphates. Onewould generally expect them to be both deprotonated at neutralpH, at which most experiments with CL-containing liposomes havebeen carried out. However, whether or not this is indeed the case isa matter of debate. Early studies indicate that the two phosphoricacid groups act as strong acids, which implies their deprotonationat neutral pH [6]. A phase transition of CL in the acidic regionreported by Seddon points into the same direction [7]. Inves-tigations by Kates et al., however, point into the opposite direction.These authors claim two rather distinguishable protonationprocesses, one with pK = 2.8 and a second one varying between7.5 and 9.5 [8]. This result is corroborated indirectly bycomputational studies of Lemmin et al. who claimed based onthe results of molecular dynamics (MD) simulations that CL with a

Fig. 1. Structure of tetraoleoyl cardiolipin.

D. Malyshka et al. / Vibrational Spectroscopy 75 (2014) 86–92 87

mixed protonation state can explain multiple biophysical proper-ties of CL-rich membrane portions [9]. Results from ab initiocalculations for the CL head groups suggest a stabilization by abicyclic resonance structure formed by the two phosphate groupsand adjacent hydroxyl groups. This result is to some extentplausible, since one can well imagine that the double ionized stateof the phospholipid would be subject to rather strong repulsiveinteractions between the two phosphate groups. However, arecently conducted classical titration study by Olofsson and Sparron two synthetic cardiolipins, i.e., 1,102,20-tetradecanoyl cardiolipin

Fig. 2. Decomposed spectra of 100% cardiolipin liposomes (top) and 100% phosphatidylchones used for the fitting model for the respective liposome spectra across the pH rang

and 1,102,20-tertraoctadecenoyl reestablished the view of a strongdibasic acid [10].

Even though a majority of studies is in favor of the dominance ofthe double ionized state at physiological pH, further clarification iswanted, since the knowledge of the protonation state is pivotal forthe interpretation of binding data. The coexistence of single anddouble protonated states could explain the existence of two modesof cytochrome c binding with different binding affinities [5]. Here,we report the result of a comparatively simple FT-IR study. Wemeasured and analyzed the FT-IR spectra of liposomes formed with100% 1,102,20-tetraoleyl cardiolipin (TOCL) and 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC), respectively, and with a20:80 mixture of TOCL and DOPC in the wavenumber regionbetween 1000 and 1300 cm�1 as a function of pH between 2.0 and12.0. At physiological pH, bands appearing in this spectral windoware generally assigned to symmetric and antisymmetric combi-nations of PO stretching modes. However, it is unclear to whatextent bands assignable to these vibrations are overlapping withbands from coupled CH bending modes [11]. The experimentallyobserved spectra were decomposed into Gaussian bands. We foundthat only a subset of the thus obtained bands exhibit a pHdependence in the acidic region, indicating that a least part of theirintensity originates from phosphates’ stretching vibrations. The

oline liposomes (bottom) at a pH of 7. The decomposition bands shown here are thee.

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experimental work is accompanied by normal mode calculationsfor different protonation states of the phosphatase-containinghead of CL. Based on experimental and computational findings weidentified the wavenumber positions of symmetric and antisym-metric PO stretching modes of the ionized phosphate groups aswell as of the P¼O stretching mode and of a mixed C—P stretch andCPO bending mode of the fully protonated state. Our data confirmthe notion that the two phosphate groups are both ionized atneutral pH. Hence, different protonation states cannot be invokedfor the interpretation of binding data for cytochrome c – (CL-containing) liposome interactions.

2. Material and methods

2.1. Liposome preparation

Both 1,102,20-tetraoleyl cardiolipin (TOCL) and 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) were purchased from AvantiPolar Lipids. 100% TOCL and 20% TOCL/80% DOPC liposomes wereprepared by adopting the protocol of Hanske et al. [12]. After beingdissolved in a 2:1 methanol/chloroform mixture in a round bottomflask, the solutions were rotovaped for half an hour until a dry lipidfilm formed before being placed in a dessicant overnight. The filmwas rehydrated with a 25 mM HEPES buffer to achieve a final lipidconcentration of 10 mM. The resulting solution was sonified in anice bath for an hour at 100 W. The sonified sample was centrifugedat 12,000 rpm for 40 min to remove traces of titanium from thesonifier tip. The supernatant was extracted and left to stabilizeovernight. All liposome solutions were stored under nitrogen toprevent oxidation. The pH of all solutions was adjusted by addingsmall aliquots of 1.25 M hydrochloric acid (HCl) or 1.25 M sodiumhydroxide (NaOH), and then measured with an accumet1 pro pHprobe. The target pH range was 5–12 for DOPC and 2–12 for TOCL.The total lipid concentration for all experiments was 10 mM. Wechecked our CL-solution with light scattering and found that ourpreparation produced a rather homogeneous liposome populationwith an average diameter of ca. 60 nm. This value puts theliposome population into the small unilamellar liposome category(SUV). It lies at the lower end of the 50–150 nm range, which isconsidered to produce the most stable liposomes [13]. The size

Fig. 3. Integrated intensity of the bands of the 100% phosphatidylcholine liposomes plott1060 cm�1 (white circle), 1095 cm�1 (black triangle), 1076 cm�1 (white triangle), 1218 c

distribution obtained from the light scattering measurement isshown in Fig. S1 of the Supporting information. The intactness ofthe prepared liposomes could be demonstrated by their capabilityto bind oxidized cytochrome c (Pandiscia and Schweitzer-Stenner,unpublished).

2.2. FT-IR-spectroscopy

All spectra were obtained with a PerkinElmer Spectrum One FT-IR spectrometer. In the current study, we focus on the spectralregion between 1000 and 1300 cm�1, which contains bandsassignable to vibrations of the phosphate groups. A transmissioncell made of two barium fluoride crystals with 50 mm Teflon spacerwas used to hold the sample while 32 background corrected scanswere taken over a span of about 2 min.

3. Results and discussion

3.1. IR spectroscopy and spectral analysis

To elucidate the degree of phosphate head group protonation ofCL and PC, we focused on the part of the spectrum between1000 and 1300 cm�1. Generally, a rather broad band in the regionbetween 1200 and 1300 cm�1 is assigned to asymmetric POOstretch combinations, whereas the broad and asymmetric bandbetween 1000 and 1100 cm�1 is attributed to the symmetric POOstretch [11]. Fig. 2 exhibits these spectral regions for 100% TOCLand 100% DOPC, respectively. The spectra were recorded at pH 5.0.TOCL has a phosphate concentration twice that of DOPC, and it isreflected in its spectrum being roughly twice as intense. DOPC andTOCL spectra measured at different pH were self-consistentlyfitted with a spectral model comprising of a minimal amount ofGaussian bands for which we kept the wavenumber positions andhalfwidths constant using our program Multifit [14]. These bandsare shown in Fig. 2. Wavenumber positions, half widths andintegrated intensities are listed in Table S1. The thus performedspectral decomposition should be regarded as a minimal model,which cannot fully account for the spectral density in theinvestigated wavenumber region. However, as we see below, thedecomposition allowed us to narrow the spectral region which is

ed against the pH. The six bands shown here are centered at 1042 cm�1 (black circle),m�1 (black square), and 1242 cm�1 (white square).

Fig. 4. Integrated intensity of bands constituting the IR spectrum of 100% cardiolipin liposomes plotted as a function of pH. The band intensities were obtained from a spectraldecomposition of spectra measured between 1000 and 1300 cm�1. The bands are labeled as follows: 1017 cm�1 (black circle), 1047 cm�1 (white circle), 1074 cm�1 (blacktriangle, down), 1094 cm�1 (white triangle, up), 1145 cm�1 (black square), 1164 cm�1(white square), 1176 cm�1 (black diamond), 1193 cm�1(white diamond) and 1214 cm�1

(black triangle, up).

D. Malyshka et al. / Vibrational Spectroscopy 75 (2014) 86–92 89

dominated by contribution assignable to P—O and P¼O stretchingmodes.

Figs. 3 and 4 displays the integrated intensities of the obtainedbands of DOPC and TOCL as a function of pH. Within the limit ofexperimental accuracy, the DOPC band intensities do not show anypH-dependence. This is expected since the protonation anddeprotonation of the acidic and alkaline part of the head groupof this zwitterionic lipid occur respectively at extreme pH notcovered by our experimental data [15]. The small variations of thedata points can be considered as a measure of their statisticaluncertainty. For TOCL, however, the situation is different. Weidentified bands at 1094 cm�1, 1073 cm�1 and 1213 cm�1, theintensity of which decreases as function of pH below pH 5, where

Fig. 5. Difference spectra across the acidic pH as compared to neutral pH for 100% cardioby subtracting the spectrum of 100% CL liposomes measured at pH 2 (solid), pH 3 (dottedsame liposomes at neutral pH. The inset shows the original spectra.

two bands at 1016 cm�1 and 1192 cm�1 become concomitantlymore intense. There is no significant pH-dependence of the TOCLspectrum above pH 7.0. Thus our data do not provide any evidencefor a half-protonated state of the CL head group being populated atneutral pH [8]. All the other bands are either pH independent orexhibit only a weak pH-dependence, which indicates minorcontributions of PO vibrations to their normal modes.

The pH-dependence of the TOCL spectrum in the acidic region isfurther illustrated in Fig. 5, which displays the difference betweenthe spectrum measured at pH 7 and that measured at the indicatedpH. The positive signals in these difference spectra represent thebands whose intensity is reduced toward acidic pH, whereas thenegative peaks indicate contribution from the protonated state,

lipin liposomes between 1000 and 1300 cm�1. The spectra shown here are obtained), pH 4 (dashed), and pH 5 (dashed and dotted) from the spectrum obtained for the

Fig. 6. FT-IR absorbance spectra of 20% TOCL/80% DOPC liposomes measured at different pH values between 2 and 7. The individual spectra are plotted as follows: pH 2 (solid),pH 3 (dotted), pH 4 (short dash), pH 5 (dashed and dotted), and pH 7 (long dash).

Fig. 7. Structure of the bi-phosphate head group of ionized (6a) and protonatedcardiolipin (6b) obtained from DFT based geometry optimization. These structureswere used to calculate the normal modes discussed in the text.

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which increase with decreasing pH. It makes sense to assign theformer to symmetric and antisymmetric combinations of POO�

stretching modes and the latter to modes of the protonated lipidwhich involves P¼O stretching. A more detailed assignment isgiven based on the normal mode analysis described below.

The inner mitochondrial membrane has a mixture of phos-pholipids, with the cardiolipin concentration standing roughly at20% [16]. Therefore the question arises whether liposomes with a100% CL are truly representative of what one can expect on thesurface of the mitochondrial inner membrane. In principle, onemay argue that if mixed protonation states are not obtained on asurface with the high charge density of the 100% TOCL liposomes,one would not expect them to occur if only 20% of the liposomesurface is occupied with TOCL. However, since size and curvature ofmembranes are affected by the charge distribution [17], wedecided to check 20% TOCL/80% DOPC liposomes even though weexpected that the above influence of phosphate protonation wouldbe difficult to discern owing to the low TOCL content. We thereforemeasured the IR absorption spectra of 20% TOCL/80% DOPCliposomes as a function of pH in the acidic region. The thusobtained spectra are shown in Fig. 6. Due to the low concentrationof TOCL, the respective difference spectra (Fig. S2) are very noisy.We did not observe any noticeable spectral changes in pH 4–7 range. At pH 3 and 2, however, the intensity of the high energypart of the low wavenumber band (below 1100 cm�1) decreases, asobtained for 100% TOCL. The underlying bands are assignable tocombination of POO� – stretching vibrations to be specified below.Expected spectral changes in the regions below 1050 cm�1 and inthe region between 1250 and 1300 cm�1 seem to be much weaker.However, as indicated in Fig. 5, spectra changes in the regionbetween 1050 and 1100 cm�1 are clearly dominant, so that thedifficulty to detect the other changes in the spectra of the 20%TOCL/80% DOPC mixture is not too surprising.

3.2. Normal mode analysis

We performed DFT calculations on the head group of the lipid(Fig. 7) in vacuo on a B3LYP 6-31G** level of theory. After ageometry optimization a normal mode calculation was carried out.The wavenumbers of all modes obtained were real, indicating thatthe optimized structure belongs to a minimum of the energy

surface. The calculations were carried out with the program TITANfrom Schrödinger, Inc. This procedure yielded 28 vibrations in awindow between 1000 and 1600 cm�1

. Fig. 8 depicts the spectraldensity of the vibrational spectrum, i.e., the number of modes in awavenumber interval of 20 cm�1. Regions containing phosphategroup vibrations are marked gray. Most of the obtained modes aremore or less delocalized combinations of CH bending, CH2 and CH3

deformation modes with some admixtures from C—O and C—C

Fig. 8. Spectral density representation for the wavenumber interval between 1000 and 1600 cm�1 as obtained from the normal mode calculation of the compounds in Fig. 5.The ordinates display the number of modes obtained in 20 cm�1 intervals the lower ends being indicated on the abscissas (1500 cm�1 means all modes between 1500 and1599 cm�1 were counted). The upper panel exhibits the mode distribution for the ionized and the lowerpanel the mode distribution for the fully protonated compound. Barsin black represent combinations of CH deformation and C—C stretching modes. Gray bars represent modes with a substantial contributions from P—O, P¼O and C—Pstretching modes.

D. Malyshka et al. / Vibrational Spectroscopy 75 (2014) 86–92 91

stretching and OH bending modes. Stretching vibrations of the twophosphate groups are relatively localized. The wavenumbers oftwo nearly unmixed antisymmetric POO stretching modes of thetwo phosphate groups were obtained as 1307 and 1303 cm�1.There is relatively limited overlap with CHn deformation modes atthe high energy side, while some overlap with non-phosphategroup modes is predicted for the low energy side of these twobands. The situation is less clear with regard to the symmetricPOO-stretching vibrations. We found five normal modes between1090 and 1130 cm�1 with admixtures from OPO symmetricstretching vibrations. This region is somewhat separated from abroad spectrum of partially highly delocalized CHn-deformationmodes.

A comparison of this normal mode analysis with the decom-posed IR spectra suggests that the band at 1213 cm�1 results froman overlap of the two barely interacting antisymmetric POOstretching modes. Bands at lower and higher wavenumbers areassignable to combinations of CH deformation modes. The bands at

1073 and 1094 cm�1 should both be related to POO symmetricstretching modes. The occurrence of two pH dependent bands is ingood agreement with the results of our normal mode calculationswhich suggest that POO symmetric stretch-containing modes arespread over a much larger spectral interval than POO antisymmet-ric modes.

We also performed a DFT and subsequent normal modecalculation for the fully protonated species, which is also shownin Fig. 7. The spectral density distribution is depicted in the lowerpanel of Fig. 8. We found three modes with contributions from P¼Ostretch at wavenumbers of 1314, 1316 and 1330 cm�1. The P¼Omodes are more delocalized than their counterparts of the fullyionized phosphate groups. The mode exhibiting a wavenumber of1314 cm�1 is an in-phase and the corresponding mode at1330 cm�1 an out-of-phase combination of the two P¼O stretchingmodes. Based on the pH dependence displayed in Fig. 4 we have toassign the band at 1192 cm�1 to an overlap of these two modes,from which the out-of-phase combination can be expected to be

92 D. Malyshka et al. / Vibrational Spectroscopy 75 (2014) 86–92

more intense in an IR spectrum. Thus, we obtained the band(s)assignable to P¼O modes at slightly lower wavenumbers than thebands resulting from antisymmetric POO modes, while the DFTcalculations suggest the opposite behavior. To rationalize thisdiscrepancy one has to consider the fact that the wavenumbers ofP¼O stretching modes stretch over a very broad region between1130 and 1260 cm�1, depending on the chemical environment. Ourexperimental value of 1192 cm�1 lies well in this region [18]. On thecontrary, the wavenumbers of the asymmetric and symmetricPOO� stretching modes exhibit a much less pronounced variation[19–21]. The band at 1016 cm�1, the intensity of which increasesalso with decreasing pH, cannot be assigned to a mode with P¼Ostretching content. Our normal mode calculations rather suggestthat it should be assigned to a mixture of PH bending and P—O, O—C and C—C stretching vibrations with a normal mode frequency of1029 cm�1. This band is of course absent in the spectrum of theionized state of the peptide.

A comparison of calculated and measured wavenumbers revealrather modest scaling factors: 0.9 for P¼Os, 0.92 for theantisymmetric POO modes, 0.92 for their symmetric counterpartsand 0.98 for the P—O stretch – OH bending combination.

Thus far, not much effort has been invested into a vibrationalanalysis of the hydrophilic head group of cardiolipin. Hydratedphosphodiesters usually exhibit bands between 1220 and1230 cm�1 (asymmetric stretch) and 1085–1115 cm�1 (symmetricstretch). Hübner et al. measured the IR spectra of 100% cardiolipinand assigned the peak wavenumbers of the broad bands in the1000–1150 cm�1 and 1200–1250 cm�1 region to the symmetric(1090 cm�1) and antisymmetric (1215 cm�1) modes [11,21]. Ourresults suggest that the peak in the 1200 cm�1 region is indeedclose to the band assignable to the antisymmetric POO vibration,while a somewhat more nuanced picture emerges for thesymmetric stretch combinations, which contributes to severalnormal modes and therefore covers a broader spectral region.Substantial fractions of the two bands do result from ratherdelocalized CH-deformation modes. The interference with all typesof coupled CH bending and deformation modes as well as C—Cstretching modes can be expected to be much pronounced in CLthan in our truncated model system owing to additionalcontribution of the lipid carbon hydrate chains. We performedsome calculations for a larger complex which contained the CLhead and the interface between head groups and the carbonhydrate chains on a HF 6-31G** level of theory and discovered nowa strong overlap between modes of highly coupled CH deformationmodes and e.g., modes with a strong contribution from POO�

antisymmetric stretching modes. The result explains why thebands that we assign to stretching modes of the protonated andionized phosphate groups do not go to zero at neutral (P¼O) andacidic pH (POO). They either overlap with combinations of CHdeformation modes or the phosphate stretching modes constitutejust a fraction of the eigenvectors of the modes, which contributeto this spectral region.

4. Summary

We have measured and analyzed the FT-IR spectra of 100%TOCL, 100% DOPC and 20%TOCL/80% DOPC as a function of pHbetween 1000 and 1300 cm�1. We identified specific bands in thespectrum of TOCL and the TOCL/DOPC mixture which change theirintensity at acidic pH. They are assignable to antisymmetric andsymmetric POO stretching modes (at neutral and alkaline pH) andto the symmetric P¼O stretching modes as well a combination ofPH bending and P—O, O—C and C—C stretching vibrations (in the

acidic region). Results from a DFT based normal mode analysis of atruncated TOCL head group suggest that the bands from thesephosphate group modes are heavily overlapped by bandsoriginating in highly delocalized CH bending vibrations. Whilethe two antisymmetric stretching modes of the TOCL phosphategroups are practically localized, stronger coupling was obtained forthe two symmetric modes. With regard to the protonation stateour data strongly suggest that the two phosphate groups of TOCLare complete ionized at physiological pH, thus corroborating anearlier titration study of Olofsson and Sparr [10]. The distancebetween the two phosphate groups is too large to allow for anystable acid-anion pair which could produce a rather stableprotonation state over a broad range of pH values [22]. Our resultmight be considered surprising because one would normallyexpect that electrostatic interactions between the two ionizedphosphate groups would make a double-ionized state ratherunstable. However, the distance between the two phosphategroups is slightly larger 8 Å. This exceeds the distance between theterminal groups of an extended dipeptide like diglycine for whichwe have obtained substantial screening of Coulomb interactions bywater [23].

Acknowledgement

Dmitry Malyshka acknowledges the receipt of a Maryanoffsummer research stipend after his freshman year. Leah Pandisciawas supported in part by a research fellowship from the College ofArts and Sciences.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.vib-spec.2014.10.003.

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