Synopsis - Chlorophyll Biosynthesis and Technological Applications - page 45

2.9.1.2 Development of Room Temperature Analytical
Fluorescence Techniques
After having built a voluminous data base of known tetrapyrroles fluorescence
emission and excitation peaks at room temperature, I was able to identify all the
Chl metabolic intermediates detected at room temperature during the Chl biosyn-
thetic studies. Usually the metabolic intermediates that were formed and passed into
the HEAR at room temperature depended upon the incubation conditions and the
plastid fraction that was used. In most cases the accumulated metabolites consisted
of uroporphyrin, (Uro) coproporphyrin (Copro), Proto, Mg-porphyrins, protochlor-
ophyllide (Pchlide) and chlorophyllide (Chlide). These metabolites were detected by
their emission and excitation peaks. However since several peaks occurred in every
recorded spectrum, there was a considerable fluorescence band overlap. In order to
quantitate the amount of every detected tetrapyrrole by reference to standard calibra-
tion curves, the various fluorescence band overlaps had to be deconvoluted and
computed out. Thus several simultaneous fluorescence equations were derived that
allowed the deconvolution of the various fluorescence bands with great precision
(Rebeiz et al. 1975 ). These techniques will be referred to throughout this monograph.
2.9.1.3 Development of Cell-Free Systems Capable of the Net Synthesis
of Chlorophyll Biosynthetic Metabolic Intermediates
Armed with sensitive fluorescence techniques and quantitative measuring
capabilities we proceeded with the development of cell-free systems that formed
net metabolic tetrapyrrole intermediates in vitro (Rebeiz et al. 1982 ). That effort
spanned many years of research and culminated with the development of cell-free
systems capable of very high rates of Pchlide and Chl biosynthesis in vitro (Daniell
and Rebeiz 1982 ). These cell-free systems will be referred to throughout this
monograph.
2.9.1.4 Demonstration of Precursor-Product Relationships
During Chlorophyll Biosynthesis
By 1975 we felt confident that with the newly developed techniques it would be
possible to start systematic investigations of possible precursor product relation-
ships during chlorophyll biosynthesis. It was thus possible to detect the insertion of
Mg into Proto (Smith and Rebeiz 1977 ) and the conversion of exogenous Proto,
Mg proto and its monoester into Pchlide (Mattheis and Rebeiz 1977b , c ). Although
these results confirmed the paper pathway proposed by Granick in 1950 (Granick
1950 ), we could not demonstrate the conversion of exogenous Pchlide into its
phytylated analog, Pchlide ester as was commonly believed (Mattheis and Rebeiz
1977a ). These precursor-product relationships will be discussed in various chapters
of this monograph.