INFLUENCE OF LYSOZYME ON MONOOLEIN CUBIC PHASE SELF DIFFUSION 1

B.V. Munavirov, I.Y. Desyatnikova, A.V. Filippov

Kazan State University

When placed in water lipids form various types of liquid crystalline phases, among all of them the most
complex ones are probably cubic phases. Cubic phase inner structure is formed by curved lipid bilayers
separating two independent water networks. It makes possible to “fill” lipid cubic phases with substances of
different amphiphilic nature (hydrophobic or hydrophilic) and so they are perspective to be used as in vivo drug
delivery and controlled release systems [1]. Drugs often have protein nature, so it is important to consider the
influence of environment (e.g. temperature, pH) on system condition. It is known, that interaction of protein with
lipids in cubic phases is commonly explained by means of electrostatic (in the case of charged molecules) and
hydrophobic (if protein molecule has hydrophobic parts) interactions [2, 3]. Presence of macromolecules (drugs)
may vary cubic phase symmetry, water channel sizes and permeability, molecule’s diffusion coefficients. Such
two-component system’s stability else may vary in comparison to source pure cubic phase’s. Thus, investigation
of cubic phase properties in the presence of drug is very actual physical problem. Earlier properties of such
systems were studied by means of NMR spectroscopy, electron microscopy, SAXS [3, 4]. In this work we
studied translational mobility of lipid molecules in described systems. Experiments were done by means of
pulsed field gradient 1H NMR, method details are described in literature [5]. Stimulated echo pulse sequence was
used, diffusion time was 7 ms, pulse gradient’s maximum amplitude was 30 Tl/m.
1-MONOOLEOYL-rac-GLYCEROL(C18:1) (monoolein) was used as cubic phase forming lipid. Hen
egg-white lysozyme was chosen as protein, because its properties, conformation, dynamics in solution are well
studied [8]. Materials were purchased from SIGMA CHEMICAL CO., St. Louis, MO, USA and used without
further purification.
Monoolein-water and monoolein-water-lysozyme phase diagrams (fig.1) were taken from literature [1, 6].

a) b)
Fig.1 Studied systems phase diagrams: a) monoolein-water. L2 – inverted
micellar phase, L - lamellar phase, C – cubic phase, HII – inverted hexagonal
phase [1], b) Monoolein-water-lysozyme [6]

Samples were prepared according to methodic described in literature [3, 7]. Lysozyme was dissolved in
0.05M sodium acetate buffer solution, pH=4.5±0.05. Control samples with pure cubic phases were prepared in
D2O. Samples hydration was kept at ≈30% (w/w).
Diffusion decays of cubic phases without protein are described with two diffusion coefficients. Performed
analysis allowed us to conclude that small coefficient relates to lipid while bigger one – to water self-diffusion.
Lipid self-diffusion coefficient is rising with temperature increase.
In the presence of lysozyme, increasing its concentration is seemed to be followed with self-diffusion
coefficient rise in whole studied temperature interval (fig.2).
Lysozyme molecules T2 relaxation time ≤ 1ms, its weight concentration is more than 15 times smaller
than concentration of lipid. Estimations show that in such conditions lysozyme molecule’s protons contribution
is neglible small in comparison with protons of lipid. Observed lipid’s self-diffusion coefficient alteration is
probably related with particular interaction of protein with lipid bilayer. It is known from literature [3], that
lysozyme is globular protein and it weakly interacts with membrane. Else it is known [3] that at certain
concentration level (marked with dotted line on fig.2) it crystallizes in cubic phase’s water channels.

1
Work is supplied by RFFI 05-04-48370 grant
 T=24C
T=30C
T=40C
-11
5,0x10 T=50C
2
T=60C
Ds, м /с
-11
4,0x10

-11
3,0x10

-11
2,0x10

-11
1,0x10

0 2 4 6 8 10
N

Fig.2. Lipid self-diffusion coefficients versus number of lysozyme molecules

per cubic phase unit cell dependencies

We suppose that observed lipid’s self-diffusion coefficient alterations may be explained by one/all of
following effects:
 Lysozyme crystallization is followed with releasing of part of water from its hydrate shell, as a
result bilayer forming lipids hydration level increases.
 Increasing number of lysozyme molecules in cubic phase unit cells changes bilayer curvature,
this is followed by alterations in projection of lipid molecular motion trajectory on direction of
pulse gradient. Changes in bilayer curvature results in increasing of free volume per one lipid
molecule (bilayer lipid packing density decrease).

References

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РАМН Арчакова А.И.//М.: Изд. ГУ НИИ биомедицинской химии РАМН – 2005 – 318 с.
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