Biology Reference
In-Depth Information
The purity of the
14
C-Chl
a
fraction was tested further. A hexane extract from the
light incubation of the green homogenate (2.5-h irradiated cotyledons) was mixed
with carrier standard Chl
a,
and the specific radioactivity was determined at several
stages of purification. It appeared, that the
14
C-Chl
a
reached a constant specific
radioactivity after the Silica Gel H purification (Rebeiz and Castelfranco
1971b
).
These results strongly indicated that the green homogenate prepared from
cotyledons exposed to light for 2.5 h was able to synthesize
14
C-Chl
a
but not
14
C-Chl
b
(Rebeiz
1967
).
4.3.2 Biosynthesis of
14
C-Chlorophyll a and b by Green
Homogenates Prepared from Etiolated Cucumber
Cotyledons Pre-irradiated for 4.5 h
It is well known that Chl
b
biosynthesis and accumulation becomes noticeable after
etiolated tissues are partially greened. In etiolated cucumber cotyledons, that takes
place after about 4 h of greening under white light (Rebeiz
1967
). Thus when
etiolated, excised cucumber cotyledons were irradiated with white fluorescent light
for 4.5 h, they became partially green and capable of substantial Chl
b
biosynthesis
in addition to Chl
a
(Rebeiz
1967
). In order to find out whether homogenates
prepared from such greening cotyledons were capable of Chl
b
biosynthesis, they
were incubated in the light with
14
C-ALA. The crude Chl
a
and Chl
b
fractions were
both highly radioactive (Rebeiz and Castelfranco
1971b
). The
14
C-Chl
a
fraction
was subsequently purified to constant specific radioactivity. Chromatography on
Silica Gel H separated the
14
C-Chl
a
from other
14
C-porphyrins. This was accom-
panied by a strong decrease in specific radioactivity. Chromatography on cellulose
MN 300 separated the
14
C-Chl
a
efficiently from minor contamination by
l4
C-Chl
b
(Rebeiz and Castelfranco
1971b
). Spectrophotometric measurements indicated a
negligible Chl
b
contamination (about 2 %). Upon conversion into
14
C-pheophytin
a
and rechromatography on cellulose MN 300, the specific radioactivity remained
unchanged indicating that after the cellulose purification step, the
14
C-Chl
a
was
free of significant
14
C-porphyrin, phorbin, or colorless radioactive contaminants
(Jeffrey and Wright
1987
; Perkins and Roberts
1962
; Wickliff and Aronoff
1963
).
The
14
C-pheophytin
a
fraction was subsequently degraded to pheophorbide
a
according to Perkins and Roberts (
1960
), and an aliquot was chromatographed
on Silica Gel H in benzene: ethyl acetate:ethanol (8:2:5, v/v). As reported by Perkins
and Roberts (
1960
), this procedure degraded
14
C-pheophytin
a
extensively into
14
C-pheophorbide
a
and two slow moving red fluorescent radioactive products,
one of which was probably
14
C-pyropheophorbide
a
(Perkins and Roberts
1960
;
Wickliff and Aronoff
1963
).
The mixture of
14
C-pheophorbide
a
and its
14
C-tetrapyrrole derivatives was
degraded further to derivatives of the individual pyrroles, that is to maleimides
(Rebeiz and Castelfranco
1971b
). The Maleimides quenched short wavelength
Search WWH ::
Custom Search