Biology Reference
In-Depth Information
Table 3.1 (continued)
Equation
Constant
Mean value
standard deviation
k
47
0.16
0.04
0.02
The constants were calculated from fluorescence excitation spectra recorded at 674 and 660 nm,
respectively, on purified samples of Chl
a
, Chl
b,
Pheo
a
and Pheo
b.
Every constant reported in the
Table is the mean of 15-25 different determinations (Adapted from Bazzaz and Rebeiz
1979
)
k
48
0.14
(Sx
1
)/100 refers to the standard deviation (Sx
1
) of the determinations expressed as a
percentage of the pigment content. The Sx
1
values are also listed in Tables
3.2
and
their derivation is discussed in the ensuing sections.
Because of the partial overlap of the Soret excitation bands of Chl(ide)
a,
Pheo
(bide) a, Chl(ide)
b
and Pheo(bide)
b
(Bazzaz and Rebeiz
1979
), it was expected
that the uncertainty in determining the Soret excitation amplitude contributed solely
by Chl(ide)
a
in a mixture of the four tetrapyrroles would depend on the relative
concentration of the pigments in the mixture. For example, if the relative concen-
tration of Chl(ide)
a
in the mixture was high, its determination would be much more
reliable than if its relative concentration was low. Thus, it was necessary to test the
reliability of Eq. (
3.33
0
) with mixtures of tetrapyrroles containing various
proportions of Chl(ide)
a.
However, due to the infinite number of mixtures having
different relative concentrations that may be tested, only three ranges of
concentrations were investigated. In every case it was made certain that the
pigments in the mixture responded linearly to different dilutions. In these mixtures,
the relative proportion of Chl(ide)
a
was about 25, 10, and 5 % of the total
tetrapyrrole content in the mixture. The relative concentration of the other three
tetrapyrroles in the mixtures were always adjusted to equal proportions.
Thus, the reliability of Eq. (
3.33
0
) in calculating A
1
i.e.in calculating the Soret
excitation amplitude at 433 nm, contributed solely by Chl(ide)
a
in a mixture of Chl
(ide)
a
, Pheo(bide)
a,
Chl(ide)
b,
and Pheo(bide)
b,
was tested as follows: 1 ml of
HEAR solution containing 198 pmol of Chl
a
was freshly prepared, by dilution
from a stock solution of Chl
a
which was monitored by absorption spectrophotom-
etry, and its Soret excitation amplitude in relative fluorescence units, was deter-
mined from the Soret excitation spectrum which was recorded at the emission
maximum of Chl
a
at 674 nm. These experimentally determined fluorescence
excitation amplitudes are reported in Table
3.2
(second row, column 6). A 1 ml
HEAR solution containing 27 % (198 pmol) of Chl
a,
26 %
0
(195 pmol), 24 %
(178 pmol) and 23 % (170 pmol) of Chl
b,
Pheo
a
and Pheo
b,
respectively, were
prepared as described elsewhere (Bazzaz and Rebeiz
1979
).Then two Soret excita-
tion spectra recorded on the mixture at an emission wavelength of 674 nm and
660 nm, respectively. Three to four more identical mixtures were prepared and their
Soret excitation spectra were also recorded at 674 and 660 nm, respectively. The
Soret excitation amplitudes contributed solely by the 198 pmol of Chl
a
in the Five
duplicate mixtures of the four tetrapyrroles was then calculated from the Soret
excitation spectra with the use of Eq. (
3.33
0
)
.
These values, in arbitrary fluorescence
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