Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 131

4.2 Total Protochlorophyll(ide) Biosynthesis in Organello
4.2.1 Radioactive Products of 14 C-ALA Incubation
Greening etiolated cucumber cotyledons were rapidly homogenized with mortar
and pestle at 4 C and the homogenate was squeezed through cheese cloth.
Incubation of 14 C-ALA with the crude homogenate was performed for 16 h at
28 C in the dark, with an array of cofactors listed below (Rebeiz and Castelfranco
1971a ). The incubation produced a highly radioactive ether extract. Upon chroma-
tography of the latter on thin layers of Silica Gel H, the radioactivity separated into
four bands. The band at the origin consisted probably of free porphyrins and was
not investigated any further. The remaining 14 C-bands moved with the same
chromatographic mobility of standard protochlorophyllide, Mg protoporphyrin
monoester, and protochlorophyllide ester (Rebeiz and Castelfranco 1971a ).
Upon elution of these bands and rechromatography on Silica Gel H, they moved
again with the same mobility as the standards. The 14 C-Mg protoporphyrin mono-
ester band was subsequently submitted to detailed chromatographic analysis.
It coincided in every respect with standard Mg-protoporphyrin monoester.
In order to confirm the identity of the radioactive components in the two Protochl
(ide) bands, the latter were submitted to further chromatographic analysis as
described below.
4.2.2 Confirmation of the Nature of 14 C-Protochlorophyllide
The 14 C-protochlorophyllide band was eluted from Silica Gel H and rechroma-
tographed as such on paper, in a variety of solvents, and after acidification on paper
and on Silica Gel H. In toluene the 14 C-protochlorophyllide band remained at the
origin with standard protochlorophyllide while standard Mg-protoporphyrin mono-
ester moved slightly from the origin, and standard Pchlide ester moved a little
further (Rebeiz and Castelfranco 1971a ). In this solvent the carotenoids move near
the front. Upon acidification and chromatography in toluene, the 14 C-Pchlide band
cochromatographed with standard protopheophytin.
In 2,6-lutidine:0.05 N NH4OH (5:3.5 v/v) the I4 C-Pchlide band cochroma-
tographed with standard Pchlide. Upon acidification its chromatographic mobility
decreased as expected for the Mg-free base in this solvent (Granick 1961 ), and it
cochromatographed with standard protopheophytin.
In acetone: petroleum ether: acetic acid (3:7:0.01 v/v), spectroscopically pure,
standard 14 C-Pchlide and 14 C-protopheophytin (Rebeiz 1967 , 1968 ) gave rise to
two major bands and one minor band (Rebeiz and Castelfranco 1971a ). In this case
too in vitro-biosynthesized 14 C-Pchlide chromatographed in this solvent, before
and after acidification, as standard 14 C-Pchlide and 1 protopheophytin respectively.
In this solvent Mg-protoporphyrin monoester (Mpe), protochlorophyllide ester,