Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 104

and ( 3.25 ) are replaced by (E436 F618), (E450 F618), and (E424 F618) Soret excitation
amplitudes. This in turn transforms Eqs. ( 3.59 ) and ( 3.60 ) into:

MV Pchl ð ide Þð E436 F618 Þ

¼ 1 : 046 ð E436 F618 Þ 0 : 829 ð E450F = 618 Þ 0 : 099 ð E424 F618 Þ ð 3 : 62 Þ

ð Þð Þ

DV Pchl ide E450 F618

¼ : ð Þ : ð Þ : ð

E424 F 618

Þ ð : Þ

1 033 E450 F618 0 01 E436 F618 0 079 3 63

The calibration curve which relates authentic MV/DV Pchlide concentration
ratios (abscissa) to the calculated MV/DV Pchlide ratios of the net Soret excitation
amplitudes (ordinate) was a straight line that passed through the origin. It exhibited
a slope of 2.100 and a correlation coefficient of 0.990.

3.6.6 Sample Calculation of the Amount of Monovinyl and
Divinyl Protochlorophyllides in a Tetrapyrrole Mixture
Containing Divinyl Mg-Protoporphyrin Monoester

1. Authentic MV Pchlide, DV Pchlide, and DV Mpe were dissolved together in
hexane-extracted acetone. The total amount of MV + DV Pchlide and of Mpe
in the solution was determined by spectrofluorometry at 293 K(seeSects. 3.2
and 3.3 ) and amounted to 40 pmol of MV + DV Pchlides and 40 pmol of DV
Mpe/ml of solution.
2. The tetrapyrrole mixture was transferred to diethyl ether, and one Soret excita-
tion spectrum was recorded on the mixture at77 K, at an emission wavelength of
625 nm.
3. The non-deconvoluted Soret excitation amplitudes at (E451), (E437), and
(E424) nm were the next determined, in relative fluorescence units from the
F 625 nm excitation spectrum. The Soret excitation amplitudes at 451, 437, and
424 nm of the tetrapyrrole mixture amounted to 3.53, 8.02, and 4.03 relative
fluorescence units, respectively.
4. The deconvoluted net Soret excitation amplitudes of MV and DV Pchlide were
calculated from the aforementioned (E451), (E437), and (E424) Soret excitation
amplitudes with the help of Eqs. ( 3.60 ) and ( 3.61 ). The deconvoluted MV Pchlide
( E 437) net Soret excitation amplitude amounted to 5.69 relative fluorescence
units. That of DV Pchlide ( E 451) amounted to 2.96 relative fluorescence units.
5. The apparent ratio of the MV (E437)to DV (E451) net Soret excitation
amplitudes amounted to 5.69/2.96

1.92/1.
6. The true ratio of the MV Pchlide to DV Pchlide concentrations, R, was calculated
from the apparent MV (E437)/DV (E451)ratio of 1.92 and from the inverse of the

¼