Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 67

where

K ¼ 1 ð k 1 = k 2 Þ (3.5)

Constants k 1 and k 2 were calculated according to Eq. ( 3.3 ). The fluorescence
amplitudes used in Eq. ( 3.3 ) were derived from the emission spectra of standard
Pchlide E and of a mixture of standard Chl a+b dissolved separately in acetone-
extracted hexane (see below, the sections on preparatory techniques). The ratio of
Chl a to Chi b in the mixture was about 2.6. Under our instrumental conditions the
mean of five determinations performed on different concentrations of standard
Pchlide E and Chl were 0.144 for k 1 and 145 for k 2 . The average value for
K calculated according to Eq. ( 3.5 ) was 0.999 (Rebeiz et al. 1975a ).

3.2.2 Calculation of Protochlorophyllide a by Fluorescence
Spectroscopy at Room Temperature

In developing analytical preparatory techniques for monitoring tetrapyrrole metab-
olism in organello and in vitro it was realized that the best way to monitor Pchlide
a was in the hexane-extracted acetone fraction (HEAR). In Addition to Pchlide
a that fraction contained chlorophyllide a (Chlide a ), Chlide b , protoporphyrin IX
(Proto) as well as coproporphyrin (Copro).
The spectrofluorometric correction for these tetrapyrroles in the HEAR fraction
while measuring Pchlide a is described below.

3.2.2.1 Correction for the Chlorophyllide a + b Fluorescence at 640 nm

Let E and F be defined as above and let (E 0 440 F 0 676) and (E 0 440 F 0 640) represent
the fluorescence amplitudes at 676 and 640 nm, respectively, of a mixture of several
pigments containing Chl and Chlide [Chl(ide)] a+b and which are elicited by a
440-nm excitation.
Then let Chl(ide) (E440 F676) and Chl(ide) (E440 F640) represent the Chl
(ide) a + b fluorescence amplitudes at 676 and 640 nm, respectively, upon excita-
tion at 440 nm. Finally let (E440 F640) represent the Chl(ide)-free fluorescence
amplitude at 640 nm of the pigment mixture containing Chl(ide) upon excitation at
440 nm.
The following equation can then be written:

E 0 440 F640

ð Þ¼ð E440 F640 Þþ k ð E440 F676 Þ (3.6)

where

k ¼ Chl ð ide Þð E440 F640 Þ= Chl ð ide Þð E440 F676 Þ¼ 0 : 015 (3.7)