Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 66

protochlorophyll (Pchl) and tetrapyrrole intermediates in organello. In order to
achieve this goal it became mandatory to develop sensitive spectroscopic techniques
to monitor the products of in organello incubations at room temperature. These
techniques are described below.

3.2.1 Calculation of Protochlorophyllide Ester
by Fluorescence Spectroscopy at Room Temperature

Upon extraction of the chlorophyll (Chl) in hexane, protochlorophyllide a ester
(Pchlide a E) is extracted along with the Chl (see later sections below, dealing with
preparative techniques). Its determination by fluorescence spectroscopy at room
temperature is described below.
Let E refer to the Soret excitation maximum of a fluorescent compound and F to
the fluorescence amplitude of the short-wavelength emission maximum of that
same compound. Then let (E440F631) and (E440 F670) represent the respective
fluorescence amplitudes of a mixture of Pchlide a E and Chl a + b at 631 and
670 nm respectively the fluorescence being elicited by a 440-nm excitation of the
hexane extract. In hexane, a mixture of Chl a+b, where the ratio of Chla/Chl b is
about 2.6-3.0, usually exhibits an emission maximum at about 670 nm.
The following simultaneous equations can be written for the Pchlide E, Chl
mixture

ð E440 F670 Þ¼ Pchlide a E ð E440 F631 Þþ Chl ð E440 F633 Þ

þ Pchlide ð E400 F631 Þ; (3.1)

and,

ð E440 F670 Þ¼ k 1 Pchlide a E ð E440 F631 Þþ k 2 Chl ð E440 F631 Þ (3.2)

where

k 1 ¼ Pchlide a E ð E440 F631 Þ= Proto ð E400 F670 Þ

(3.3)

k 2 ¼ Chl ð E440 F670 Þ= Chl ð E440 F631 Þ

According to the above terminology, Pchlide E (E440 F631) fluorescence ampli-

tude of Pchlide E at 631 nm, which is elicited by a 440-nm excitation of the hexane
extract. Chl (E440 F631)

¼

fluorescence emission amplitude of Chl a + b at 631 nm,
which is elicited by a 440-nm excitation of the hexane extract; etc. By solving
Eqs. ( 3.1 )and( 3.2 ) for Pchlide E (E440 F631) the following equation is obtained

¼

Pchlide a E ð E440 F631 Þ¼½ð E440 F631 Þ ð ð E440 F670 Þ= k 2 Þ ð 1 = K Þ (3.4)