Biology Reference
In-Depth Information
3.8.2 Determination of the Amount of 2-MV Pchl(ide) b in
the Presence of 2-MV Chl(ide) a and b, Using Room
Temperature and 77
K Spectrofluorometric Analysis:
Overall Strategy
The procedure involved:
(a) determination of the amount of 2-MV Chl(ide)
b
in the mixture by spectrofluo-
rometry, at room temperature as described in Sect.
3.4
and elsewhere (Bazzaz
and Rebeiz
1979
),
(b) selection of appropriate fluorescence wavelengths for the best discrimination
between the 2-MV Pchl(ide)
b
and 2-MV Chl(ide)
b
fluorescence signals,
(c) adaptation of the previously derived, general purpose simultaneous equations,
for the calculation of the net fluorescence signal generated by 2-MV Pchl(ide)
b
and 2-MV Chl(ide)
b
in diethyl ether at 77
K,
(d) calculation of the molar ratio of 2-MV Pchl(ide)
b
/2-MV Chl(ide)
b
from the
net fluorescence signals,
(e) calculation of the amount of 2-MV Pchl(ide)
b
from the total amount of 2-MV
Chl(ide)
b,
which is determined by spectrofluorometry at 293
K and from the
2-MV Pchl(ide)
b
/2-MV Chl(ide)
b
molar ratio.
3.8.2.1 Selection of Appropriate Wavelengths for the Calculation
of the Net 2-MV Pchl(ide) b Fluorescence Signal in the Presence
of 2-MV Chl(ide) a and b
Fluorescence excitation wavelengths at 463 and 455 nm, were selected from
excitation spectra recorded at emission wavelengths of 643 and 660 nm respec-
tively, in diethyl ether at 77
K, for the calculation of the net 2-MV Pchl(ide)
b
and
2-MV Chl(ide)
b
fluorescence excitation signals.
At these wavelengths, in diethyl ether at 77
K, contribution of the 2-MV Chl(ide)
a
fluorescence excitation is nil, since at emission wavelengths of 643 and 660 nm, Chl
(ide)
a
fluorescence emission is negligible (Belanger et al.
1982
). In other words,
under these conditions, a mixture of 2-MV Pchl(ide)
b
and 2-MV Chl(ide)
a
and
b
,
behaves like a dual mixture of 2-MV Pchl(ide)
b
and 2-MV Chl(ide)
b
. In excitation
spectra recorded at an emission wavelength of 643 nm in diethyl ether at 77
K[the
emission maximum of 2-MV Pchl(ide)
b
in ether at 77
K], the 2-MV Pchl(ide)
b
component exhibits a pronounced Soret excitation band with a maximum at
463 nm, while 2-MV Chl(ide)
b
exhibits a weaker excitation signal (Ioannides
et al.
1997
). On the other hand, in excitation spectra recorded at an emission
wavelength of 660 nm in diethyl ether at 77
K [the emission maximum of 2-MV
Chl(ide)
b
in diethyl ether at 77
K], the 2-MV Chl
b
component exhibits a Soret
excitation maximum at 475 nm and a measurable Soret excitation signal at 455 nm
(Ioannides et al.
1997
).
Search WWH ::
Custom Search