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ultraviolet light (254 nm) and appeared as blue spots on fluorescent thin layers
viewed under ultraviolet light (Ellsworth and Aronoff
1968
).
The crude
14
C-Chl
b
fraction was also purified to constant specific radioactivity
(Rebeiz and Castelfranco
1971b
). After the first purification on thin layers of
cellulose, the specific radioactivity dropped sharply. This was due to the separation
of
14
C-Chl
a
from
14
C-Chl
b.
Upon rechromatography of the
14
C-Chl
b
on cellulose
the specific radioactivity remained unchanged indicating that after the first cellulose
purification the
I4
C-Chl
b
fraction was free of significant amounts of
14
C-porphyrins
or
14
C-Chl
a.
Spectrophotometric analysis indicated a negligible Chl
a
contamination
(about 1.5 %) after the first cellulose purification.
After a second cellulose purification no contaminating Chl
a
could be detected
by spectrophotometry. The radioactive shoulder running ahead of the bulk of the
14
C-Chl
b
probably represented
14
C-pheophytin
b
contamination
.
4.4 Accumulation of Spectroscopically Detectable Amounts
of Protochlorophyllide and Chlorophyll in Organello
After the achievement of
14
C-Pchl(ide) and
14
C-Chl
a
and
b
biosynthesis in
organello, it was realized that progress in this field of research depended on the
development of analytical techniques that allowed the detection of tetrapyrroles and
the determination of their chemical structure by spectroscopic methods. Since
tetrapyrrole were fluorescent, the research effort concentrated on the development
of spectrofluorometric analytical techniques (Chap.
3
). Thus by 1975 the first
qualitative and quantitative spectrofluorometric techniques were developed (Rebeiz
et al.
1975a
). However the observed biosynthetic rates were still rather low. Then
Paul Castelfranco and colleagues determined that concentrations of ATP higher than
what was routinely used in previous cell-free systems (Rebeiz and Castelfranco
1971a
,
b
) were needed for high rates of Mg-Proto biosynthesis in organello and
demonstrated that ATP was a cofactor for Mg-Proto chelatase activity (Pardo
et al.
1980
). Building on this information the cell-free systems capable of Pchlide
biosynthesis and accumulation were improved considerably as described below.
4.4.1 Development of in Organello Systems Capable of High
Rates of Mg-Proto Monoester and Protochlorophyllide
Biosynthesis in Organello
By early 1982 the best in organello system capable of Mpe and Pchlide biosynthesis
and accumulation is described in Table
4.1
displayed below.
The plastids were prepared from 4-day-oldetiolated cucumber cotyledons that
had been pre-irradiated for 4 h with 320
W/cm
2
of cool white fluorescent light.
μ
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