EPR investigations of phase transitions in amphiphilic systems using hydrophilic spin probes

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EPR investigations of phase transitions in amphiphilic systems using hydrophilic spin probes

D.D. Lasič, M. Schara

To cite this version:

D.D. Lasič, M. Schara. EPR investigations of phase transitions in amphiphilic sys- tems using hydrophilic spin probes. Journal de Physique, 1982, 43 (11), pp.1653-1657.

�10.1051/jphys:0198200430110165300�. �jpa-00209546�

EPR investigations ^{of phase} transitions in amphiphilic systems

using hydrophilic spin probes

D. D. Lasi010D and M. Schara

J. Stefan Institute, University ^E. Kardelj ^of Ljubljana, Ljubljana, Yugoslavia (Reçu ^le 13 janvier 1981, révisé le 13 avril 1982, accepte ^le 8 juillet 1982)

Résumé. ^- La technique ^{de sonde EPR a été} utilisée pour étudier les transitions coagel-gel ^et gel-cristal liquide

dans trois systèmes amphiphiles. ^{On a} déterminé le partage ^des molécules sondes hydrophiles ^{entre l’eau et l’envi-}

ronnement lipidique. ^{Dans la} phase coagel les ^{molécules sondes sont} exclues du lipide cristallisé tandis que dans la phase liquide-cristalline ^{elles sont} dissoutes dans les deux milieux. Une nouvelle transition de phase ^{est observée}

à ~ 120 °C.

Abstract. ^- The EPR spin probe technique ^{was used to} study coagel-gel ^and gel-liquid crystal phase transitions in three amphiphilic systems. ^The partition ^{of the} small, hydrophilic spin probe molecules between the lipid ^and

water environment was measured. In the coagel phase ^the spin probe ^{molecules were} excluded from the crystallized lipid, while in the liquid crystalline phase they ^{were dissolved} in both environments. A new phase transition was

observed at ~ 120 °C.

Physics ^Abstracts

61.30E - 64.70E

1. Introduction. ^- The lamellar amphiphilic sys- tems undergo ^several phase transitions. At high temperatures ^{there is a} liquid crystalline phase ⁱⁿ

which the lipid molecules with melted hydrocarbon

chains are oriented perpendicular ^{to the} bilayer planes. ^The phase transition to the gel phase ^occurs

at Tc. ^This phase is characterized either by ^{bi- or} monolayered [1] ] equidistant lipid lamellae with the

lipid molecules still perpendicular ^to the lamellae.

However, the chains are rigid. ^Below T’ c ^the coagel phase begins ^to appear. This phase ^{is a} heterogeneous

mixture of hydrated lipid crystals, ^with rigid ^mole-

cules tilted in bilayers, ^{and a diluted water solution}

of lipids. ^In analogy ^{with the} recrystallization ^process, by ^which impurities ^are excluded from the crystals,

one might expect ^a change in the distribution coef- ficient K ⁼ CL/Cw (CL ^and Cw ^are the concentrations of the spin probe ^{in the} lipid ^{and water) of} spin probes

which are soluble in both phases ^at higher tempe-

ratures. In this study ^several spin probes with diffe- rent solubility characteristics were used.

2. Experimental. ^- The labelled amphiphilic pha-

ses : KPW (potassium palmitate/water ^{- 70} weight %

of lipid), ^NaPW (sodium palmitate/water ^{- 65 w} J/§

of lipid), ^{LW I} (egg yolk lecithin/water ^{- 60 w} ;’% ^of lipid), ^{LW II} (egg yolk lecithin/water ^{- 75} w % ^of lipid) and the solution of liposomes (egg yolk ^leci-

thin/water ^{- 1 w} % ^of lipid) ^were prepared ^{as des-}

cribed elsewhere [2, 3]. ^The phase transition tempera-

tures from our measurements are given later in the text. In the KPW system spin probes ^TEMPO (1-oxyl-2,2,6,6,-tetramethylpiperidine), ^TEMPOL (TEMPO-4-ol), ^TEMPON (TEMPO-4-on), ^CES (TEMPO-4-cyanoetoxy) ^{and DTBN} (di-t-butylni- troxide) ^{were used. In the} NaPW, ^{LW I and LW II} system only ^the spin probe ^{TEMPO was used. In}

LW I, ^{LW II} and in the solution of liposomes ^the

influence of the paramagnetic broadening agent K3Fe(CN)6 ^{was also measured.}

EPR spectra ^{of the} samples ⁱⁿ the sealed glass capillaries (1 ^{mm inner} diameter) ^were recorded in a

Varian E9 EPR spectrometer ^{with an added} tempera-

ture controller.

3. Results. ^- X band EPR spectra ^{of the} spin probe TEMPO in the KPW amphiphilic system ^are shown in figure ^{1. In the} liquid crystalline phase (T > 48°C) anisotropic spectra ^{are observed. In} the gel phase (48 > T > 30 OC) ^the splitting ^{of the} high field line (m ^{= -} 1) disappears ^{and a broad} high ^field peak ^{is observed. In the} coagel phase (T ³⁰ OC) ^the hyperfine ^{lines narrow} and the value of the isotropic hyperfine splitting (a) ^increases.

In figure ² the detailed change ^{of the} anisotropic spectra around 120°C is shown. The temperature

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphys:0198200430110165300

1654

Fig. ^{1. -} X band EPR spectra of TEMPO in the KPW

amphiphilic system.

Fig. ^{2. - X band EPR} spectra of TEMPO in the liquid crystalline phase ^{of KPW} system.

Fig. ^{3. - The} temperature dependence ^{of the order para-}

meter in the liquid crystalline phase of the KPW system.

Fig. ^{4. - X band EPR} spectra of TEMPO in the NaPW

amphiphilic system.

dependence of the order parameter (S) ^is plotted ⁱⁿ figure ^3. Figure ⁴ represents ^the spectra ^{of TEMPO} in NaPW. Here Tc ^{= 80 °C and} T’ c ^{= 60 °C. In the} liquid crystalline phase ^the anisotropy ^{of the} spectra

was again ^observed only ^{in the m = - 1} hyperfine

line. This line _merges into a single line in the gel phase. ^{In the} coagel phase ^{the two} component spec- trum is observed. The spectrum ^is composed ^{from the} hydrophobic ^and hydrophilic component. ^{The inten-}

sity ^{of the} hydrophobic component ^decreases by lowering ^the temperature. ^{The X band} spectra ^of TEMPOL in KPW are shown in figure ^{5. In the} liquid crystal ^a narrow, nearly isotropic spectrum ^is

Fig. ^{5. - X band EPR} spectra of TEMPOL in the KPW system.

Fig. ^{6. -} Q ^{band EPR} spectra of TEMPOL in the KPW system.

observed. In the gel phase ^this spectrum ^broadens and in the coagel phase ^the spectrum separates ^into

two components characteristic for _{the pure} lipid ^and

water at that temperature. Again, by lowering ^the temperature ^the intensity ^{of the} hydrophobic compo- nent decreases. Q ^band spectra ^{are shown in} figure ^6.

In the liquid crystalline phase ^an isotropic spectrum is observed while a two component spectrum appears in the gel phase. ^{In the} coagel the exclusion of the spin probes ^{from the} lipid ^{is observed.} Figures ^{5 and 6}

show typical hysteresis. ^The intensity ^{ratio of the} hydrophobic ^to hydrophilic spectral component decreases by lowering ^the temperature. ^{The varia-} tion is not reversible as the samples ^were kept ^for

Fig. ^{7. -} Temperature dependence ^{of the} isotropic hyper-

fine splitting ^of spin probe TEMPOL in the KPW system in the X and Q ^{band. The} samples ^{were heated to 100 °C}

and the measurements were taken on cooling.

JOURNAL DE PHYSIQUE. ^- T. 43, N° 1 I, ^{NOVEMBRE 1982}

Fig. ^{8. - EPR} spectra of TEMPO dissolved in LW I

amphiphilic system : a) ^{T =} 18.2 °C, b) ^{T = -} 13.8 °C, and c) ^{T = - 13.5} °C, presence of 1 ^m mole/I K3Fe(CN)6.

15 min. at each temperature. ^{In these} experiments

water freezes constantly ^{at -} 18 °C, ^{as confirmed}

with the separate experiments using pure ^water solu- tion of this spin probe ⁱⁿ equal capillaries. ^In figure ⁷

the temperature dependence ^{of the} isotropic hyper-

fine splittings of TEMPOL in KPW system ^{in X and} Q ^{band are} shown. The samples ^{were heated to}

100 °C and the measurements taken on cooling. ^Due

to different time scales of these experiments ^{the two} component spectrum appears ^at T° ^{in the} Q ^{band and}

at T’ c in the X band. In figure ^{8 the EPR} spectra ^of TEMPO dissolved in LW I amphiphilic system ^are shown. In the liquid crystalline phase ^a single ^compo-

nent spectrum ^{is observed} while in the coagel phase

a two component spectrum appears. In this system the hydrophobic component ^{does not} disappear by lowering ^the temperature. Using ^water soluble para-

magnetic ^{ions the water} component ^{of the} spectrum is bleached and a single component spectrum ^is observed (Fig.. 8c).

The KPW amphiphilic system ^{was also} investigat-

ed using TEMPON, ^{CES and DTBN. The first two} spin probes ^{behave similar to} TEMPOL while the last one was similar to TEMPO.

4. Discussion. ^- In the liquid crystalline phase ^of

KPW and NaPW system ^the spin probe ^molecules

are partially ^ordered [2] ^and anisotropic spectra ^are observed. The measured orientational order para- meter S depends, besides the ordering ^{of the} liquid crystal, strongly ^on the chemical nature of probes, temperature ^{as well as} indirectly ^{on the} partition

coefficient K(T). ^In the lamellar phase ^{of KPW} sys- tem TEMPO shows S - 0.25 due to the rapid ^exchan-

ge of spin probes between the lipid environment, ^where they ^have anisotropic spectra ^{with S -} 0.30, ^{and the} isotropic ^water environment [2].

The observed changes ^{in the EPR} high field line

anisotropy ^{in the} temperature range around 120 °C

can be explained by ^the change in S reflected in the splitting AB(m ^{= -} 1) between the outermost extrema of this line

1656

Here ^v is the microwave frequency, h Planck’s constant and fl ^Bohr magneton. ^The motionally ^ave- raged g ^and hyperfine splitting ^tensor values for TEMPO for the case where the magnetic ^{field is} parallel ^and perpendicular ^{to the} bilayer ^director

can be expressed [4] :

and a and g ^{are the} isotropic ^{values of} correspond- ing ^{tensors while} gii and Aii ^{are the tensor} components in the molecular system ^{of the} spin probe. ^The drop

in the S(T) ^curve probably ^{indicates a new} phase

transition at T - 120 °C in this liquid crystalline system for which polymorphism ^{has been} already reported figure ³ [5, 6].

All the spin probes ^behave similarly ^{in the} gel ^and coagel phases. Lineshapes indicate that there is still

an exchanged anisotropic spectrum ^{in the} gel phase.

But the ordering of TEMPO is smaller. For T T’ c

the demixtion begins ^{and the} solubility ^{of the} spin probes ^{in the} lipid ^decreases by ^the decreasing ^tem- perature (K -> 0). This is reflected through ^the super-

imposed spectrum ^{where the} hydrophilic peak ^increa-

ses, i.e. the fraction of spin probe molecules in water increases. Only the smallest molecule ^- DTBN - is an exception ^{since K ’# 0 for} temperatures ^consi- dered in our experiment. The exclusion is not com-

plete ^{and the} spin probe molecules dissolved in lipid

rotate nearly isotropically ^{at - 25 °C with the corre-}

lation time 7 x 10- 10 s. This time increases to 3 ^x 10-9sat - 42 °C.

The same sequence is observed when the NaPW system is cooled (Fig. 4). ^{In the} liquid crystalline phase ^the anisotropic exchanged spectrum ^{is observ-} ed. The gel phase (from ^{80 to 60} °C) ^{has an} exchanged spectrum ^with low S, while in the coagel phase ^there

is a superimposed spectrum ^where larger ^exclusion

of spin probes ^from lipid begins ^{at 27 °C.}

Figure ^{5 shows the} spectra ^of TEMPOL in KPW.

At 50 °C a nearly isotropic spectrum with a =1.579 mT is recorded. This is a consequence of the rapid exchange

of TEMPOL between the compartments ^with

a’o c - ^{1.630 mT and} aiooc ⁼ 1.390 mT. On cooling separation begins ^{at 20 °C and the} increasing hydro- philic ^and decreasing hydrophobic ^EPR peaks ^are distinguished clearly. ^The distribution coefficient

K20oc, calculated from the two peak intensities and

extrapolated ^to 20 °C, with value 2.5 [7] ^{is in} good agreement ^{with the one} calculated and extrapolated

from the high temperature phases using

where fL ^and fW ^{are the fractions of the} spin probe

molecules dissolved in lipid ^{and water} respectively;

aL and aware the hyperfine splittings ⁱⁿ pure solvents.

Below the freezing point ^of water, ^{there is no} signal

with a characteristic lipid environment splitting. ^The

same experiment ^{was also} performed ^{in the} Q ^band (Fig. 4). ^Because of the different time scales the demix- tion was observed at a higher temperature. To merge the high ^field (m ^{= -} 1) ^{line into a} single hyperfine

component ⁱⁿ Q ^{band be} exchange time should be smaller than 7.4 x 10-8 _s, and 1.4 ^x 10 -’ in X band.

It is concluded that in the liquid crystalline phase ^the exchange ^rate of TEMPOL molecules between

lipid ^{and water is faster than 1.4 x 10’ S - 1 and}

in the gel phase in the range between ^{1.4 x} 10’

and 7.1 ^x 106 s -1. In the coagel phase it is slower than 7.1 x 106 S-1 and/or impossible ^{to observe} by

our measurements due to the larger distances, ^the

molecules have to diffuse in a given domain in the

heterogeneous phase (Fig. 7).

In the lecithin water system ^{K # 0 was observed}

at all temperatures. ^{This can be} explained by ^{the less}

dense lecithin crystal ^{lattice. In this} system ^{an addi-} tional check was made. Water soluble paramagnetic

ions were dissolved in the system. By ^the exchange

interactions they broaden the signal ^{of the} spin probe dissolved in water. Therefore only ^{the EPR} component ^{of the} spin probe molecules dissolved in the lipid ^are observable (Fig. 8). ^{In the} liquid crystal-

line phase ^{of LW I the} paramagnetic bleaching agent does not alter the spectrum, because its molecules

are too big ^to diffuse into the water layers. However,

in the coagel phase ^{of LW I water} aggregates ^into

microdroplets ⁱⁿ which the paramagnetic ^{ions are}

dissolved and only ^the spectrum ^of spin probes ^dis-

solved in lipid is observed (Fig. 6). This indicates the loss of the homogeneity ^{of the} system.

In the lamellar mesophase ^{LW II} the influence of the bleaching agent ^{was not observed. This mate-} rial and all denser lamellar phases ^{have water} layers ^of

zero thickness [8]. ^{All water molecules are localized} laterally in the free space between the polar ^heads

and do not form a larger ^{amount of} microdroplets

in any quantity, ^at least several hours after the

cooling.

The same experiment ^was also done with the lipo-

some solution. Here the intensity ratio of the water and lipid signals ^{is different because the amount of}

water is much bigger. The observed superimposed spectrum [9] ^{can be} explained ^as follows : all the

spin probes ^{dissolved in} water cannot exchange ^fast enough ^{with the} lipid environment because their

mean free path ^{l’ = 6 Dt, where D} is the translational diffusion constant of the spin probe and t is the EPR

experiment ^time scale, ^{is one order of} magnitude

smaller than the mean distance between the lipo-

somes.

In all the systems ^the temperature ^{of the} coagel- gel phase transition was reversible in the _range of several OC at heating/cooling ^{rates 0.2} OC/min. ^Howe-

ver, the hysteresis of the distribution coefficient is

5. Conclusions. ^- The reversible destruction of the

homogeneity ^{of the} amphiphilic systems ^{at the} coagel- gel phase transition was observed. This phase ^transi-

tion is not sharp; ^{it has a} large coexistence region,

up ^to 30 OC. The exchange ^{rates of the} spin probe

molecules between the lipid ^{and water} environment

were estimated.

which may be related ^{to some} additional structural

changes ^{in this} polymorphic mesophase, ^{was observ-}

ed (Figs. 2, 3).

Acknowledgments. ^- The authors thank Dr. M.

M. Pintar for the critical reading ^of manuscript.

This work supported by the Research Council of Slovenia.

References

[1] VINCENT, ^J. M., SKOULIOS, A., ^Acta Crystallogr. ²⁰ (1966) ^432.

[2] LASI010D, ^D. D., SCHARA, M., ⁱⁿ Magnetic ^{Resonance in}

Colloid and Interface Sciences, ^eds. Fraissard,

J. P., Resing, ^{H. A.} (D. Reidel Publ. Co) 1980, p. 537.

[3] LASIC, ^{D. D.,} SCHARA, M., ^Vest. Slov. Kem. Drus.

26 (1979) ^275.

[4] GRIFFITH, ^O. H., JosT, ^P. C., ⁱⁿ Spin Labelling, ^{ed. Ber-} liner, ^{L. J.} (Academic Press) 1976, p. 453.

[5] ^{DAVIS, J.} H., JEFFREY, K. R., ^Chem. Phys. Lip. ²⁰ (1977) 87.

[6] ^{VAZ, N. A.} P., DOANE, ^J. W., Phys. Rev. Lett. 42

(1979) ^1406.

[7] LASIC, ^D. D., SCHARA, M., Period. Biol. 83 (1981) ^163.

[8] SMALL, ^D. M., BOURGES, M., ^Mol. Cryst. ¹ (1966) ^541.

[9] MCCONNELL, ^H. M., WRIGHT, ^K. L., MCFARLAND, B. G., ^Biochem. Biophys. ^Res. Commun. 47 (1972)

273.