Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 132

protoporphyrin monoester, and protopheophytin ester exhibited chromatographic
mobilities strikingly different than 14 C-Pchlide and 14 C-protopheophytin. No
efforts were made to determine whether the segregation of Pchlide and its
Mg-free base into multiple bands was due to pigment degradation or to a separation
of closely related, spectroscopically identical, compounds. A similar case was
reported for radioisotopically and spectroscopically pure 14 C-pheophorbide a and
b chromatographed on icing sugar (Perkins and Roberts 1962 ). After acidification
the 14 C-Pchlide band cochromatographed on Silica Gel H in benzene:ethyl acetate:
ethanol (8:2.5:5 v/v) with standard protopheophytin.
The foregoing results strongly suggested that the cell-free system did indeed
synthesize 14 C-Pchlide.
4.2.3 Confirmation of the Nature
of 14 C-Protoehlorophyllide Ester
The 14 C-Pchlide ester band was eluted in ether from Silica Gel H and rechroma-
tographed as such and after acidification on paper. It was also chromatographed on
Silica Gel H after partial acid hydrolysis.
In 2,6-lutidine:0.05N NH4OH (5:3.5 v/v) it moved differently than standard
Pchlide and Mpe. It moved with the same mobility as standard Pchlide ester (Rebeiz
and Castelfranco 1971a ). In this solvent some standard Pchlide ester and
14 C-Pchlide ester remained at the origin together with some carotene. It was
conjectured that this may be due to interference by excess carotene in this solvent.
After acidification, the 14 C-Pchlide ester band moved with standard protopheo-
phytin ester ahead of Mpe and protopheophytin. In acetone:petroleum ether:acetic
acid (3:7:0.01 v/v) the 14 C-Pchlide ester band moved with standard Pchlide ester,
ahead of Mpe. After acidification it cochromatographed with standard protopheo-
phytin ester ahead of Mpe and the multiple bands of protopheophytin. Upon partial
hydrolysis of the 14 C-Pchlide ester band in 12 N HC1 and chromatography on Silica
Gel H in benzene:ethyl acetate:ethanol (8:2:5 v/v), the radioactivity exhibited
the same mobility as standard protopheophytin ester and its hydrolysis product
protopheophytin. In this case too, the results strongly suggested that the cell-free
system was indeed synthesizing 14 C-Pchlide ester.
4.2.4 Minimal Cofactor Requirement of the Tissue
Homogenate Biosynthetic System
The minimal cofactor requirement for the incorporation of 14 C-ALA into
14 C-Pchlide and 14 C-Pchlide ester by the crude homogenate consisted of: CoA
plus GSH, potassium phosphate, methyl alcohol, and Mg 2+ . The absolute requirement
for oxygen was also evident (Rebeiz and Castelfranco 1971a ).