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In another approach, the isotropic hyperi ne coupling constant (
A
0
in Figure 10.2) of 16-SASL or
16-PC can be measured for l uid-phase membranes. A decrease in the
A
0
value indicates an increase
in hydrophobicity at the 16-SASL position (Figure 10.5c). However, this constant rel ects only the
hydrophobicity of the membrane center.
Both methods of determining membrane hydrophobicity have advantages and disadvantages,
which are discussed in Wisniewska et al. (2006).
10.3.4 P
HASE
T
RANSITION
The main phase-transition temperature of the lipid bilayer membranes can be monitored by observ-
ing the amplitude of the central line of the EPR spectra of 16-SASL or 16-PC (
h
0
in Figure 10.2).
The decrease in signal amplitude at the phase transition can be as much as 50
. For phase-transition
measurements, the temperature should be regulated by passing nitrogen gas through a coil placed in
a water bath and monitored by a copper-constantan thermocouple placed in the sample just above
the active volume of the cavity (Wisniewska et al. 1996). This way temperature can be regulated
with accuracy better than 0.1°C. To avoid aggregation of carotenoids in the gel phase membrane,
cooling experiments (l uid-to-gel phase transition) are preferred. The temperature should be lowered
by the addition of a small amount of cold water to the water bath with rapid agitation, permitting a
very low rate of temperature change,
%
∼
2°C
/
h. To avoid small cooling
/
heating cycles, a temperature-
controlling unit should not be used.
The phase-transition temperature,
T
m
, and the width of transition,
T
1/2
, were operationally
dei ned based on EPR data, as shown in Figure 10.6a. As a rule, in the presence of polar caro-
tenoids the phase transition broadens and shifts to lower temperatures (Subczynski et al. 1993,
Wisniewska et al. 2006). The effects on
T
m
are the strongest for dipolar carotenoids, signii cantly
weaker for monopolar carotenoids, and negligible for nonpolar carotenoids. The effects decrease
with the increase of membrane thickness. Additionally, the difference between dipolar and monopo-
lar carotenoids decreases for thicker membranes (Subczynski and Wisniewska 1998, Wisniewska et
al. 2006). These effects for lutein,
Δ
β
-cryptoxanthin, and
β
-carotene are illustrated in Figure 10.6b
1.0
1
0
0.8
-1
a
0.6
-2
LUT
β-CXT
β-CAR
0.4
T
m
Δ
T
½
-3
b
0.2
-4
DLPC
(C12)
DMPC
(C14)
DPPC
(C16)
DSPC
(C18)
DBPC
(C22)
20
21
22
Temperature (°C)
23
24
25
(a)
(b)
FIGURE 10.6
(a) Normalized amplitude of the central peak of the EPR spectra of 16-SASL plotted as a
function of temperature (cooling experiments) in the DMPC bilayer containing 0 (○) and 10 mol% lutein (
●
).
Dei nitions of
T
m
and Δ
T
1/2
are shown.
T
m
is the midpoint temperature at which the normalized EPR signal
amplitude equals (
a
+
b
)/2, where
a
and
b
are, respectively, intensities at given temperatures in the extended
linear portions of the upper and lower ends of the transition curve. As the sharpness of the transition, the
width Δ
T
1/2
is employed, which is dei ned by two temperatures at which the EPR signal amplitude is
(
a
+ 3
b
)/4 and (3
a
+
b
)/4. (b) Shifts of the main phase-transition temperature,
T
m
, of phosphatidylcholine (PC)
membranes (dilauroyl-PC (DLPC), DMPC, dipalmitoyl-PC (DPPC), distearoyl-PC (DSPC), dibehenoyl-PC
(DBPC) ) induced by the addition of 10 mol% carotenoid to the sample. Negative values indicate a decrease of
T
m
. Notice that the
x
-axis indicates the lipid as well as the number of carbon atoms in the alkyl chains. (From
Wisniewska, A. et al.,
Acta Biochim. Pol
., 53, 475, 2006. With permission.)
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