Biology Reference
In-Depth Information
The calculated ratio of the net fluorescence amplitudes of 2-MV Pchl(ide)
b
(E463 F643)/2-MV Chl(ide)
b
(E455 F660) was next converted to an authentic
molar ratio of 2-MV Pchl(ide)
b
/2-MV Chl(ide)
b
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 diethyl ether solutions of authentic 2-MV Pchl(ide)
b
and 2-MV Chl
(ide)
b
in known proportions,
(b) recording the required Soret excitation spectra at emission wavelength of
643 and 660 nm at 77
K for every mixture,
(c) calculating the net fluorescence excitation amplitudes of Pchl(ide)
b
(E463
F6443) at 463 nm, and Chl(ide)
b
(E455 F660) at 455 nm with Eqs. (
3.81
)
and (
3.82
),
(d) calculating the apparent Chl(ide)
b
(E455 F660)/Pchl(ide)
b
(E463 F6443)
excitation ratio, and
(e) plotting the authentic 2-MV Chl(ide)
b/
2-MV Pchl(ide)
b
molar ratios, on the
ordinate, against the apparent Chl(ide)
b
(E455 F660)/Pchl(ide)
b
(E463 F6443)
excitation ratio on the abscissa, or
vice versa
. The linear relationship between
the 2-MV Chl(ide)
b
/2-MV Pchl(ide)
b
molar ratios (Y), plotted on the ordinate,
against the apparent 2-MV Chl(ide)
a
(E455 F660)/2-MV Pchl(ide)
b
(E463
F6443) excitation ratio (X) plotted on the abscissa obeyed the following
equation:
R
2
Y
¼
1
:
493 X
þ
1
:
003
ð
n
¼
10
;
¼
0
:
9869
Þ
(3.83)
3.8.2.3 Determination of the Amount of 2-MV Pchl(ide) b
in a Mixture of 2-MV Pchl(ide) b and 2-MV Chl(ide) b
The amount of 2-MV Pchl(ide)
b
in the presence of 2-MV Chl(ide)
b
was deter-
mined from the total amount of Chl(ide)
b
which was calculated from 293
K
fluorescence excitation spectra as described in Sect.
3.4
and elsewhere (Bazzaz
and Rebeiz
1979
) (10), and from the 2-MV Chl(ide)
b
/2-MV Pchl(ide)
b
molar ratio
which was calculated as described above. The reliability of calculating the amount
of 2-MV Pchl(ide)
b
in the presence of 2-MV Chl(ide)
b
amounted to
2.7
10.3 % (Table
3.13
).
3.8.2.4 Sample Calculation of the Amount of 2-MV Pchl(ide) b
in a Mixture of 2-MV Pchl(ide) b and 2-MV Chl(ide)b
1. An ether solution containing 161.4 pmol of 2-MV Pchl(ide)
b
and 410.0 pmol of
Chl(ide)
b
per ml was prepared.
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