Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 72

C 0 1 C 0 2 C 0 3

(3.24)

Proto ð E400 F633 Þ¼ð E400 F633 Þ ð E400 F622 Þ ð E440 F640 Þ=

where (E440 F640) is calculated from Eqs. ( 3.11 )or( 3.12 ), and where

C 0 1 ¼ k 0 2 K 0 2 : K 0 4 = K 0 3 1 =

C 0 2 ¼ K 0 2 = K 0 3 (3.25)

C 0 3 ¼ K 0 1 þ K 0 2 : K 0 5 = K 0 3

and

K 0 1 ¼ 1 k 0 1 = k 0 2

K 0 2 ¼ k 0 3 = k 0 2

1

K 0 3 ¼ k 0 6 k 0 5 : k 0 3 = k 0 2

(3.26)

K 0 4 ¼ k 0 5 = k 0 2

K 0 5 ¼ k 0 5 : k 0 1 = k 0 2 k 0 4

The values of the k 00 constants were calculated according to Eq. ( 3.23 ). The
fluorescence amplitudes used in Eq. ( 3.23 ) were derived from the emission
spectra of standard Proto, Copro, and Pchlide dissolved separately in acetone/
H 2 O/0.1 N NH 4 OH (9:2:1, vv) that was previously extracted twice with hexane
(Rebeiz et al. 1975b ). The emission spectra of the standards were elicited by two
successive excitations at 400 and 440 nm. The k 00 values used for the calculations
of the K 00 and C 00 constants were the mean of at least five determinations
performed on five different concentrations of standard Proto, Copro, and Pchlide.
The K 00 constants were calculated from Eq. ( 3.26 )andtheC 00 constants from
Eq. ( 3.25 ). Under our instrumental conditions the values obtained were 0.25
for C 1 00 , 0.24 for C 2 00 , and 0.95 for C 3 00 .

3.3 Spectrofluorometric Determination of
Mg-Protoporphyrin Monoester and Longer
Wavelength Metalloporphyrins in the Presence
of Zn-Protoporphyrin IX at Room Temperature

When incubation for the biosynthesis of Mg-porphyrins are carried out in organello
or in vitro without adding enough ATP to the incubation mixture, the biosynthesis
of Mg-porphyrins is accompanied by the formation of Zn-Proto. Spectrofluoromet-
ric equations were developed for the calculation of Mg-porphyrins in the presence
of Zn-Proto as described below (Smith and Rebeiz 1977b ).