Chemistry Reference
In-Depth Information
of these carotenoids to the samples during preparation
are plotted as a function of the membrane thickness.
where shifts of
T
m
induced by adding 10 mol
%
10.4 SATURATION-RECOVERY EPR
The saturation-recovery EPR method of measuring spin-lattice relaxation time (
T
1
) is a pulse tech-
nique in which recovery of the EPR signal is measured at a weak-observing microwave power
after the end of the saturating microwave pulse. The time scale of this recovery is characterized
by the spin-lattice relaxation time,
T
1
(Eaton and Eaton 2005), which for lipid spin labels can be as
long as 1-10
s. To obtain the correct spin-lattice relaxation time, the sample should be thoroughly
deoxygenated, which can be achieved by equilibrating the sample in a gas-permeable capillary with
nitrogen gas, which is also used for temperature control (Hyde and Subczynski 1989, Subczynski
et al. 2005). Presently, Bruker produces EPR spectrometers capable of saturation-recovery measure-
ments at X-band.
μ
10.4.1 O
XYGEN
T
RANSPORT
P
ARAMETER
The bimolecular collision of molecular oxygen (a fast-relaxing species) and a nitroxide (a slow-
relaxing species) induces spin exchange, which leads to a faster spin-lattice relaxation of the nitrox-
ide. This effect is measured using the saturation-recovery EPR technique. An oxygen transport
parameter,
W
(x), was introduced as a conventional quantitative measure of the collision rate between
the spin label and molecular oxygen (Kusumi et al. 1982b):
()
(
)
(
)
−
1
−
1
Wx
=
T
Air,
x T
−
N ,
x
(10.4)
1
1
2
T
1
(Air,
x
) and
T
1
(N
2
,
x
) are spin-lattice relaxation times of nitroxides in samples equilibrated with
atmospheric air and nitrogen, respectively. Note that
W
(
x
) is normalized to the sample equilibrated
with the atmospheric air.
W
(
x
) is proportional to the product of the local translational diffusion
coefi cient
D
(
x
) and the local concentration
C
(
x
) of oxygen at a depth x in the membrane, which is
in equilibrium with the atmospheric air:
()
() ()
Wx
=
ADxCx
,
A
=
8
π
pr
(10.5)
0
where
r
0
is the interaction distance between oxygen and the nitroxide radical spin label (about 4.5 Å)
(Windrem and Plachy 1980)
p
is the probability that an observable event occurs when a collision does occur and is very close to
1 (Hyde and Subczynski 1984, Subczynski and Hyde 1984, Subczynski and Swartz 2005)
A
is remarkably independent of the solvent viscosity, hydrophobicity, temperature, and spin-label spe-
cies (Hyde and Subczynski 1984, Subczynski and Hyde 1984, Subczynski and Swartz 2005)
Figure 10.7a shows typical saturation-recovery curves for 14-PC in the DMPC bilayer containing
10 mol
9-
cis
zeaxanthin in the presence and absence of oxygen. The recovery curves are i tted by
single exponentials, and decay time constants (
T
1
's) are determined. To obtain the oxygen transport
parameter, in principle, two saturation-recovery measurements should be performed, one for the
sample equilibrated with nitrogen and the other for the sample equilibrated with air (see Equation
10.4). However, to increase accuracy, saturation-recovery measurements are carried out systemati-
cally as a function of oxygen concentration (
%
air) in the equilibrating gas mixture. Figure 10.7b,
in which the
T
1
−1
values for 14-PC in the DMPC bilayer containing 10 mol
%
%
9-
cis
zeaxanthin are
plotted as a function of oxygen concentration (
air) in equilibrating gas mixture, shows the method
of calculating the oxygen transport parameter. Experimental points show a linear dependence up
%
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