Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 75

3.3.2 Validation of Equation ( 3.32 )

In order to determine the reliability of Eq. ( 3.32 ), the fluorescence emission spectra
of various concentrations of standard Mg-Porphyrins, and Zn-Proto were recorded
before and after mixing with one another. The various mixtures were prepared
in the same proportions as encountered in organello experiments (i.e. the
mixtures contained 20-100 % Mg-Porphyrins). The Mg-porphyrins fluorescence
integral between 592 and 620 nm, i.e. Mg-Porphyrins E420 R 620

592

was
determined by planimetry from the emission spectrum of the Mg-porphyrins before
mixing with Zn-Proto. After mixing with Zn-Proto the fluorescence integral of
the Mg-Porphyrins between 592 and 620 nm was calculated from the emission
spectrum of the mixture according to Eq. ( 3.32 ). The actual and calculated
Mg-Porphyrins Mg-Porphyrins

ð f ðλÞ d λÞ

E420 R 620
592

values were compared and
both the percent error and the discrepancy were determined (Smith and Rebeiz
1977a ). In addition, the mean percent error

ð f ðλÞ d λÞ

the standard deviation of the mean

percent error and the mean discrepancy the standard error of the mean discrep-

ancy were also determined for the following single component and double compo-
nent solutions: (a) Mg-Porphyrins, (b) Zn-Proto and (c) Mg-Porphyrins + Zn-Proto
(Smith and Rebeiz 1977a ).
The mean discrepancies for all errors ranged from 0 to -16 % (Smith and
Rebeiz 1977a ).

3.4 Determination of Chlorophyll and Chlorophyllide
a [Chl(ide a)] in the Presence of Chl(ide) b and
Pheophytin and Pheophorbide [Pheo(bide)] a and b
Spectrofluorometry at Room Temperature

The use of absorption spectrophotometry to monitor the amount of Chl a and b,
which is extractable in organic solvents, is satisfactory when the ratio Chl a to Chl
b is less than 6.0 (Bazzaz and Rebeiz 1979 ). This technique is not suitable, however,
for detecting and determining small amounts of Chl degradation products in the
presence of large amounts of Chl. Since it now appears that the formation of small
amounts of chlorophyllides (Chlides) (Chl that has lost its phytol) and pheo-
phorbides (Pheobides) (Chlides hat have lost their Mg) signals the beginning of
chloroplast degradation, before the disappearance of Chl becomes evident, (Bazzaz
and Rebeiz 1978 , 1979 ) it has become essential to develop sensitive techniques for
the early detection of these degradation products with minimum processing of
the chloroplast extract. This precaution was dictated by the lability of the Chl
extracted in organic solvents. Indeed, it has been our experience that Chl can easily
generate chemical artifacts during chromatography, which interfere with the detec-
tion of small amounts of Chlides and Pheobides. As a consequence sensitives