Synopsis - Chlorophyll Biosynthesis and Technological Applications - page 44

permitted the viewing and determination of the actual chemical structures of the
generated end-products. It also required the development of cell-free systems that
produced enough end-products to allow rigorous chemical structure determinations.
2.9.1.1 Choice of Analytical Spectroscopic Technique for Room
Temperature Spectroscopy
After a few trials using absorption spectroscopy, it became obvious that absorption
spectrophotometry was not suitable as a major tool for achieving our goals. The
method lacked the required sensitivity and was not rigorous enough for the task on
hands. Then I remembered that while at UC Davis, I was impressed by the fluores-
cence spectroscopic techniques used by Eloise Tappel to study the formation of
malonylaldehydes in his Vitamin E metabolic studies. I therefore investigated the
market for the availability of recording fluorescence spectrofluorometers and was very
impressed by what I learned about their sensitivity and their two-window functio-
nality. Indeed, the great sensitivity of these instruments and the possibility of varying
the excitation wavelength (one window) or the emission wavelength (second window)
imparted a considerable flexibility and usefulness to this technique. Thus when my
first tetrapyrrole proposal submitted to the UI Research Board in 1972 was funded,
I immediately purchased a Perkin-Elmer MPF-3 recording spectrofluorometer, which
at that time was a state-of-the-art instrument. Consequently, I started using fluores-
cence spectrofluorometry in my Chl biosynthesis studies.
Usually at the end of an incubation period, I made an 80 %Acetone extract of the
incubation mixture, subjected it to fluorescence analysis, and looked for fluorescing
metabolic intermediates. However there was so much Chl in the extract that its
fluorescence masked everything else. I then started extracting the 80 % acetone
extract with hexane and started monitoring the fluorescence of the hexane extract
for possible metabolic intermediates. That effort also failed because the Chl passed
into the hexane and its fluorescence masked everything else.
Then one day I noticed that after extraction with hexane, the residue what was
left behind was clear, and slightly yellowish. That residue was usually dumped.
I felt that this hexane-extracted acetone residue (HEAR) contained so few
metabolites that it was not worth looking at. Then one day I decided for the fun
of it, to look at the fluorescence of the HEAR. To my great surprise, the fluores-
cence spectrofluorometer kept on recording large peak after peak, the identity of
which was unknown. I then realized that (a) all the metabolic products of the
incubation were hydrophilic enough to pass in the HEAR that we usually dumped,
and (b) that although the HEAR looked devoid of any metabolites, I had greatly
underestimated the sensitivity of fluorescence spectroscopy. After this unexpected
discovery, I purchased nearly every available tetrapyrrole I could find on the market
and ran their emission and excitation spectra in order to build a database of
porphyrin fluorescence that would help me identify the fluorescence peaks being
generated during in vitro incubations.