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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