Development of Cell-Free Systems - Chlorophyll Biosynthesis and Technological Applications - page 160

purification of 4VCR was achieved in the ammonium sulfate eluate of the
DEAE-Sephacel column.
Further purification of the DEAE-Sephacel 4VCR eluate was achieved by
chromatography on Cibacron Blue 3GA-1000 Agarose.
Cibacron Blue 3GA-1000 agarose is a non-specific affinity chromatography
medium. Upon chromatography of the concentrated DEAE-Sephacel eluate
on Cibacron Blue 3GA-100 agarose, about 65 % of the proteins were not retained
by Cibacron and passed through the column. Elution of adsorbed 4VCR in column
buffer containing 5 mM CHAPS resulted in an overall purification of about 20-21-
folds (Kolossov and Rebeiz 2001 ). 4VCR yields ranged from 11 to 17 % of the total
original activity. 4VCR activity of the Cibacron eluate was stable for several
months at 81 C.

The electrophoretic profiles of the membrane and solubilized fractions as well
as that of the Cibacron eluate are depicted in Kolossov and Rebeiz ( 2001 ).

Demonstration of 4VCR Activity in Barley Chloroplast Membranes

It was previously assumed that 4VCR activity disappeared or decreased to unde-
tectable levels in photoperiodically grown plants (Abd-El-Mageed et al. 1997 ).
That conclusion was based on experimentation involving isolated chloroplasts
having a full complement of Chl. It has now become apparent that in addition to
4VR inhibition by the plastid stroma, the high concentration of Chl interfered with
the 4VR spectrofluorometric assays. Upon solubilization of the 4VR activities
from chloroplast membranes, the stromal inhibition was relieved, most of the Chl
was left behind in the membranes, and the 4VCR activities became unmasked.
It amounted to 7.6 nmoles of MV Chlide a formed per mg of purified protein after
5 min of incubation (Kolossov and Rebeiz 2010 ).

4.6.6 Development of a Cell-Free System Capable
of the Conversion of Chlorophyllide a
to Chlorophyll a

4.6.6.1 Preparatory Techniques

Etiolated cucumber cotyledons were excised without hypocotyl hooks, and were
placed in a large Petri dish, 15 cm in diameter prior to light treatment. One 100 g of
etiolated tissues were harvested in darkness under a safe green light. The tissue was
homogenized using a blender (Waring blender 7010, model 31BL91), in 250 ml of
homogenization buffer. Homogenization consisted of two bursts of 3 s each.
The homogenization buffer consisted of 500 mM HEPES, 1 mM MgCl 2 ,1mM
EDTA, 30 mM TES, 5 mM cysteine, and 0.2 % BSA (w/w), adjusted with KOH to