Biology Reference
In-Depth Information
The value of k was the mean of eight different determinations performed on
eight different concentrations of Chl a+b dissolved in hexane-extracted acetone.
Upon substituting for k in Eq. ( 3.6 ), Eq. ( 3.8 ) is obtained:
E 0 440 F 0 640
ð
Þ¼ð
E440 F640
Þþ
0
:
015 Chl
ð
ide
Þð
E440 F676
Þ
(3.8)
In a mixture containing several pigments in addition to Chl(ide) and where the
contribution of the other pigment components to the Chl(ide) fluorescence at
676 nm is negligible, the following equation can be written
E 0 440 F 0 676
Chl
ð
ide
Þð
E440 F676
Þ¼ð
Þ
(3.9)
Substituting Eq. ( 3.9 ) into Eq. ( 3.8 ) gives:
E 0 440F 0 640
E 0 440 F 0 676
ð
Þ¼ð
Þþ
:
ð
Þ
E440F640
0
015
(3.10)
and
E 0 440 F 0 640
E 0 440 F 0 676
ð
E440 F640
Þ¼ð
Þ
0
:
015
ð
Þ
(3.11)
In the extracts of irradiated etioplasts that are still in the lag phase of Chl
accumulation, the contribution of Chl fluorescence to Pchlide fluorescence at
640 nm is negligible, therefore:
k
¼
ð
Þð
Þ=
ð
Þð
Þ¼
Chl
ide
E440 F6640
Chl
ide
E440 F676
0
and Eq. ( 3.11 ) becomes
E 0 440 F 0 640
ð
E440 F640
Þ¼
Þ
(3.12)
In the hexane-extracted acetone fractions of developing chloroplasts excited at
440 nm, the contribution of Copro, Proto and Pchlide to Chli(de) fluorescence at
676 nm is also negligible. This is caused by the small amounts of Copro + Proto +
Pchlide encountered in the extracts as well as by the very low fluorescence yield
of these tetrapyrroles at 676 nm. This was checked further by preparing mixtures
of Copro + Proto + Pchlide in hexane-extracted acetone that contained the same
concentration of tetrapyrroles as the extracts of the plastids. Excitation at 440 nm
did not elicit any fluorescence at 676 nm. It was therefore concluded that the use
of Eq. ( 3.12 ) for calculating the Chl(ide)free fluorescence at 640 nm (E440 F640)
of the hexane-extracted acetone fractions of developing chloroplasts is valid.
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