Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 97

3.6 Quantitative Determination of Monovinyl
(MV) and Divinyl (DV) Protochlorophyllides
(Pchlides) by Spectrofluorometry at 77 K

The determinations of DV and MV Pchlides in the absence and presence of other
tetrapyrroles are described below.

3.6.1 Generalized Equations for the Determination of the
Net Monovinyl and Divinyl Fluorescence Signals
of a Particular Tetrapyrrole Pair in the Presence
of a Third Interfering Tetrapyrrole

Such a situation arises when the determination of the amount of MV and DV Pchl
(ides) is attempted in the presence of large amounts of Mg-Protos. This condition is
encountered under certain in vivo (Rebeiz et al. 1984b ) and in vitro (Daniell and
Rebeiz 1984 ; Rebeiz et al. 1984a ) conditions. The problem arises because
Mg-Protos exhibit a pronounced and broad vibrational emission band, which in
diethyl ether at 77 K extends from about 618 to 660 nm (Belanger and Rebeiz
1982 ). As a consequence, when Pchl(ide) Soret excitation spectra are recorded at
the emission maximum of Pchlide, i.e., at 625 nm or at a shorter wavelength, at
618 nm for example, the Pchl(ide) Soret excitation bands overlap with the DV
Mg-Protos Soret excitation band between 430 and 450 nm(Belanger and Rebeiz
1982 ). Therefore in order to calculate the net Soret excitation amplitudes of MV
and DV Pchl(ide), the contribution of the DV Mg-Protos Soret excitation
amplitudes to the Pchlide Soret excitation bands must be eliminated. The reverse
is not true, however, since the Mg-Protos Soret excitation bands, which are
recorded at an emission wavelength between 591 and 584 nm, do not overlap
with those of MV and DV Pchl(ides). This is because MV and DV Pchl(ides) do
not exhibit any significant fluorescence between 584 and 591 nm.
The deconvolution of the net fluorescence signals Ea and Fb or Ec and Fd of two
different compounds X and Y, from the fluorescence signals, Eg and Fh, of a third
interfering compound, Z, has already been discussed above (see Sect. 3.2 ). It was
then pointed out that the net fluorescence signals generated by any one of the three
aforementioned compounds can be separated from the fluorescence signals
generated by the other two compounds with the use of three unknown simultaneous
equations. The latter are of the form

X ð E a F b Þ¼ð E a F b Þ Q 1 ð Ec F d Þ Q 2 ð E g F h Þ= Q 3 (3.48)