Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 95

the MV to DV Soret excitation amplitudes which can be readily computed for any
given sample, from the relative amplitudes of the net MV and DV Soret excitation
signals (vide supra), is in turn independent of the frozen condition of the sample.
The ratio of the MV net Soret excitation amplitude at 417 nm to the DV net Soret
excitation amplitude at 424 nm is not necessarily identical, to the authentic ratio of
the amounts of MV to DV Mg-Protos in the mixture. This is because the MV and
DV Soret excitation magnitudes of the MV and DV components depend (a) on the
fluorescence quantum yield of the MV and DV MgProto components, which may
not be necessarily identical, and (b) on the fluorescence emission wavelength at
which the MV and DV excitation spectra are recorded.
However, once the MV to DV Mg-Proto net Soret excitation ratio has been
determined it can be readily converted to an authentic ratio of MV to DV Mg-Proto
concentrations, by reference to a standard calibration curve. The latter is
constructed (a) by mixing authentic MV Mg-Proto and DV Mg-Proto in known
proportions, (b) by recording the two required 77 K excitation spectra on every
mixture, (c) by calculating the ratio of the net MV to the net DV Soret excitation
amplitudes for every mixture with the help of Eqs. ( 3.44 ) and ( 3.45 )or( 3.46 ) and
( 3.47 ), and finally (d) by plotting the authentic MV/DV Mg-Proto concentration
ratios, on the abscissa for example, against the calculated MV/DV ratio of the net
Soret excitation amplitudes on the ordinate. Under our instrumental conditions,
such a plot of authentic concentration ratios of MV/DV MPE against the
corresponding net Soret excitation ratios of MV MPE (E417/587)/DV MPE
(E424/591), yielded a straight curve that passed through the origin and that
exhibited a slope of 1.12 and a coefficient of correlation, r, of 0.998. The concen-
tration of MV MPE and DV MPE in the mixture was then calculated from the total
amount of MV + DV MPE, which can be readily determined at 293 K for any
MV + DV MPE mixture (see Sect. 3.5.2.1 ), and from the slope of the calibration
curve (vide infra).
The reliability of Eqs. ( 3.44 ) and ( 3.45 ) in determining the proportions of MV
and DV Mg Proto Monoester in mixtures of the two tetrapyrroles amounted to
0.8 6 % and to 1.85 4.8 %, respectively (Table 3.6 ).
3.5.2.5 Sample Calculation of the Amounts of MV Mg-Proto
Monoester and DV Mg-Proto Monoester Present
in a Mixture of the Two Tetrapyrrole
1. The sample of MV and DV MPE, under consideration, was free of Zn-Proto
contamination. The total amount of MV + DV Mg-Proto monoester in the
sample was determined at 293 K in hexane-extracted acetone from the area
under the fluorescence emission band between 580 and 610 nm, as previously
described (Rebeiz et al. 1975a ; Smith and Rebeiz 1977a ). It amounted to
20 pmol of MV + DV Mpe/ml of solution.