Biology Reference
In-Depth Information
Chapter 3
Development of Analytical and Preparatory
Techniques
Integrity without knowledge is weak and useless, and knowledge
without integrity is dangerous and dreadful (Samuel Johnson)
3.1 Prologue
The successful investigations of the chlorophyll biosynthetic pathway would not
have been possible without the development of new analytical and preparatory
techniques. These techniques form the object of this chapter.
Prior to 1975 practically all investigations involving metabolic tetrapyrroles
used absorption spectrophotometric techniques. Then in the early 1970s it came
to our attention that metabolic tetrapyrroles were fluorescent. A comparison of
absorption and fluorescence techniques revealed that fluorescence techniques were
about a 100 fold more sensitive and were more flexible than absorption techniques.
Indeed while absorption spectroscopy is a one-window technique fluorescence is a
two windows technique. This means that with fluorescence techniques it is possible
to record sensitive fluorescence emission spectra by varying the emission wave-
length while keeping the excitation wavelength constant. Or one can record equally
sensitive excitation spectra by varying the excitation wavelength and keeping the
emission wavelength constant.
3.2 Determination of Metabolic Tetrapyrroles
by Room Temperature Spectrofluorometry
After the usage of
14
C-
-aminolevulinic acid (ALA) to achieve the total biosynthesis
of protochlorophyllide
a
(Pchlide
a
), and Chl
a
and
b
in organello (Rebeiz and
Castelfranco
1971a
,
b
). The need arose to achieve the net synthesis of Chl,
δ
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