Biology Reference
In-Depth Information
The means of 12 determinations amounted to
k
1
¼
0.07902
0.005;
k
2
¼
1.915
0.111;
k
3
¼
0.524
0.032;
k
4
¼
12.72
0.727;
K
1
¼
0.959;
K
2
¼
0.959. By substitution of the above constants into Eqs. (
3.74
) and (
3.75
),
the latter transform into:
2-MV Pchl
ð
ide
Þ
b
ð
E463 F643
Þ¼
1
:
04
ð
E463 F643
Þ
0
:
54
ð
E440 F635
Þ
(3.76)
2-MV Pchl
ð
ide
Þ
a
ð
E440 F635
Þ¼
1
:
04
ð
E440 F635
Þ
0
:
08
ð
E463 F643
Þ
(3.77)
The calculated ratio of the net fluorescence amplitudes of 2-MV Pchl(ide)
b
(E463 F643)/2-MV Pchl(ide)
a
(E440 F635) was next converted to an authentic
molar ratio of 2-MV Pchl(ide)
b
/2-MV Pchl(ide)
a
prior to the calculation of the
actual amount of 2-MV Pchl(ide)
b
. This was achieved by reference to a standard
calibration curve that was constructed as follows:
(a) mixing authentic 2-MV Pchl(ide)
b
and 2-MV Pchl(ide)
a
in known
proportions,
(b) recording the required 77
K emission spectra in diethyl ether for every mixture
by excitation at E440 and E463 nm,
(c) calculating the net fluorescence amplitudes of 2-MV Pchl(ide)
b
(E463 F6443)
and 2-MV Pchl(ide)
a
(E440 F635) with Eqs. (
3.76
) and (
3.77
),
(d) calculating the apparent 2-MV Pchl(ide)
b
(E463 F6443)/2-MV Pchl(ide)
a
(E440 F635) emission ratio, and
(e) plotting the 2-MV Pchl(ide)
a
/2-MV Pchl(ide)
b
molar ratios, on the abscissa,
against the apparent 2-MV Pchl(ide)
a
(E440 F635)/Pchl(ide)
b
(E463 F6443)
emission ratio on the ordinate, or
vice versa
. The linear relationship between the
2-MV Pchl(ide)
a
/2-MV Pchl(ide)
b
molar ratios (Y), and the apparent Pchl
(ide)
a
(E440 F635)/Pchl(ide)
b
(E463 F6443) emission ratio (X) obeyed the
following equation:
R
2
Y
¼
1
:
827 X
þ
0
:
55
ð
n
¼
12
;
¼
0
:
976
Þ
(3.78)
3.8.1.3 Determination of the Amount of 2-MV Pchl(ide) b in a Mixture
of 2-MV Pchl(ide) b and 2-MV Pchl(ide) a
The amount of 2-MV Pchl(ide)
b
in the presence of 2-MV Pchl(ide)
a
was deter-
mined from the total amount of Pchl(ide)
a
which was calculated from 293
K
fluorescence emission spectra as described elsewhere in Sect.
3.2
and from the
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