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Biophys J, September 2001, p. 1511-1520, Vol. 81, No. 3
and
*Department of Biochemistry, McMaster University, Hamilton, ON L8N
3Z5, Canada; Department of Biological Chemistry, Weizmann
Institute of Science, Rehovot 58102, Israel; Center for
Technological Education, Holon 58102, Israel; and
§Chemical Services Unit, Weizmann Institute of Science,
Rehovot 76100, Israel
Phosphatidylserine and cholesterol are two major
components of the cytoplasmic leaflet of the plasma membrane. The
arrangement of cholesterol is markedly affected by the presence of
phosphatidylserine in model membranes. At relatively low mol fractions
of cholesterol in phosphatidylserine, compared with other
phospholipids, cholesterol crystallites are formed that exhibit both
thermotropic phase transitions as well as diffraction of x-rays. In the
present study we have observed and characterized a novel thermotropic
transition occurring in mixtures of phosphatidylserine and cholesterol.
This new transition is observed at 96°C by differential scanning
calorimetry (DSC), using a heating scan rate of 2°C/min. Observation
of the transition requires that the hydrated lipid mixture be incubated
for several days, depending on the temperature of incubation. The rate
of formation of the material exhibiting a transition at 96°C is more rapid at higher incubation temperatures. At 37°C the half-time of
conversion is ~7 days. Concomitant with the appearance of the 96°C
peak the previously known transitions of cholesterol, occurring at
~38°C and 75°C on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrous cholesterol and to the dehydration of cholesterol monohydrate, respectively. The loss of the 75°C peak takes a longer time than that of the 38°C peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75°C and subsequently is converted by
additional time of incubation to an altered form of the monohydrate, showing a phase transition at 96°C. After several weeks of incubation at 37°C, only the form with a phase transition at 96°C remains. If
such a sample undergoes several successive heating and cooling cycles,
the 96°C peak disappears and the 38°C transition reappears on
heating. For samples of 1-palmitoyl-2-oleoyl phosphatidylserine or of
1-stearoyl-2-oleoyl phosphatidylserine having mol fractions of
cholesterol between 0.4 and 0.7, the 38°C transition that reappears after the melting of the 96°C component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at
least for these samples, the amount of anhydrous cholesterol crystallites formed is indeed a property of the lipid mixture. We have
also examined variations in the method of preparation of the sample and
find similar behavior in all cases, although there are quantitative
differences. The 96°C transition is partially reversible on cooling
and reheating. This transition is also scan rate dependent, indicating
that it is, at least in part, kinetically determined. The enthalpy of
the 96°C transition, after incubation of the sample for 3 weeks at
37°C is dependent on the ratio of cholesterol to 1-palmitoyl-2-oleoyl
phosphatidylserine or to 1-stearoyl-2-oleoyl phosphatidylserine, with
the enthalpy per mole cholesterol increasing between cholesterol mol
fractions of 0.2 and 0.5. Dimyristoyl phosphatidylserine at a 1:1 molar
ratio with cholesterol, after incubation at 37°C, exhibits a
transition at 95°C that reverses on cooling at 44°C, instead of
60°C, as observed with either 1-palmitoyl-2-oleoyl phosphatidylserine
or 1-stearoyl-2-oleoyl phosphatidylserine. These findings along with
the essential absence of the 96°C transition in pure cholesterol or
in cholesterol/phosphatidylcholine mixtures, indicates that the
phospholipid affects the characteristics of the transition, and
therefore the cholesterol crystallites must be in direct contact with
the phospholipid and are not simply in the form of pure crystals of
cholesterol. These observations are particularly important in view of
recent observations of the presence of cholesterol crystals in
biological systems.
Biophys J, September 2001, p. 1511-1520, Vol. 81, No. 3
© 2001 by the Biophysical Society 0006-3495/01/09/1511/10 $2.00
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