Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 99

influences the values of the k2, k 4 , K 1 , and K 2 constants in Eqs. ( 3.37 ) and ( 3.38 ). As
expected, the choice of the appropriate Pchl(ide) a, c excitation wavelengths and of
the b, d emission wavelengths is dictated by the fluorescence emission and excita-
tion characteristics of MV and DV Pchl(ide) in ether at 77 K. At this temperature
(a) 90 % of the Pchl(ide) pool occurs in the pentacoordinated state, while the
remaining 10 % occurs in the hexacoordinated state (Belanger and Rebeiz 1984 ),
(b) the B (0-0) Soret excitation electronic transitions of the pentacoordinated MV
and DV Pchl(ide) are very clearly split into shorter wavelength By (0-0) and longer
wavelength Bx (0-0) components (Rebeiz and Lascelles 1982 ), (c) although
pentacoordinated MV Pchl(ide) (E437/F625) and pentacoordinated DV Pchl(ide)
(E443/F625) possess very similar fluorescence emission maxima at 624 and625 nm,
respectively, they exhibit different By (0-0) Soret excitation maxima, E, at 437and
443 nm respectively. The corresponding Bx (0-0) transitions of pentacoordinated
MV and DV Pchl(ide) exhibit excitation maxima at 443 and 451 nm respectively
(13). As a consequence of these observations and because of the forementioned
Soret excitation overlap between the pentacoordinated MV Pchl(ide) Bx (0-0) and
the DV By (0-0) transitions at 443 nm the following strategy was adopted. It was
decided to discriminate between the pentacoordinated MV and DV Pchl(ides) in
Eqs. ( 3.37 ) and ( 3.38 ), via the MV Pchlide By (0-0) Soret excitation maximum at
437 nm and via the DV Pchlide Bx (0-0) Soret excitation maximum at 451 nm,
respectively.
Therefore in Eqs. ( 3.37 ) and ( 3.38 ) X was considered to represent the
deconvoluted net Soret excitation amplitude of pentacoordinated MV Pchl(ide)
By (0-0) (E437/F625) at437 nm. Likewise Y was considered to represent the
Soret excitation amplitude of pentacoordinated DV Phclide Bx (0-0) ( E 451 F 625)
at 451 nm. Therefore in this context ( E a Fb ) refers to ( E 437 F 625) and represents the
Soret excitation amplitude at 437 nm, when the excitation spectrum of a MV + DV
Pchl(ide) mixture is recorded at an emission wavelength of 625 nm. Likewise
( Ec Fd ) refers to ( E 451 F 625) and represents the Soret excitation amplitude of
the MV + DV Pchl(ide) mixture at 451 nm, when the excitation spectrum of the
mixture is recorded at an emission wavelength of 625 nm.
With the forementioned assignment, Eqs. ( 3.37 ) and ( 3.38 ) transform into:

MV Pchl ð ide Þð E437 F625 Þ¼ ð ½ E437 F625 Þð E451 F625 Þ= k 6 ð 1 = K 3 Þ (3.53)

DV Pchl ð ide Þð E451F625 Þ¼ ð ½ E451 F625 Þð E437 F625 Þ= k 8 ð 1 = K 4 Þ (3.54)

and Eq. ( 3.40 ) becomes

k 5 ¼ MV Pchl ð ide Þð E451F625 Þ= MV Pchl ð ide Þð E437F625 Þ

k 6 ¼ DV Pchl ð ide Þð E451F625 Þ= DV Pchl ð ide Þð E437F625 Þ ð 3 : 55 Þ

k 7 ¼ DV Pchl ð ide Þð E437 F625 Þ= DV Pchl ð ide Þð E451 F625 Þ

k 8 ¼ MV Pchl ð ide Þð E437F625 Þ= MV Pchl ð ide Þð E451F625 Þ