Pheophytinization kinetics of chlorophyll c under weakly acidic conditions: Effects of acrylic acid residue at the 17-position

Article abstract of DOI:10.1016/j.bmc.2013.09.032

Pheophytinization of chlorophyll (Chl) c₁, which was isolated from the diatom Chaetoceros gracilis, was kinetically analyzed under weakly acidic conditions, and was compared with that of protochlorophyllide (PChlide) a and chlorophyllide (Chlide) a. Chl c₁ possessing a trans-acrylic acid residue at the 17-position exhibited slower pheophytinization kinetics than PChlide a and Chlide a, both of which possessed a propionic acid residue at the same position. The difference in pheophytinization properties between Chl c₁ and (P)Chlide a was ascribable to the electronegativity of the 17-substituent in Chl c₁ larger than that of (P)Chlide a due to the C17¹-C17² double bond with the conjugated 17 ²-carboxy group in Chl c₁. Demetalation kinetics of PChlide a was slower than that of Chlide a, which originated from the effect of the π-macrocyclic structures.

Full text of DOI:10.1016/j.bmc.2013.09.032

Bioorganic & Medicinal Chemistry 21 (2013) 6915–6919
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Pheophytinization kinetics of chlorophyll c under weakly acidic
conditions: Effects of acrylic acid residue at the 17-position
a
b
a
b,c
b
b
Kana Sadaoka , Sunao Shoji , Keiya Hirota , Yusuke Tsukatani , Taichi Yoshitomi , Hitoshi Tamiaki ,
a
a,
⇑
Shigenori Kashimura , Yoshitaka Saga
a
Department of Chemistry, Faculty of Science and Engineering, Kinki University, Higashi-Osaka, Osaka 577-8502, Japan
Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Pheophytinization of chlorophyll (Chl) c , which was isolated from the diatom Chaetoceros gracilis, was
1
Received 2 August 2013
Revised 10 September 2013
Accepted 11 September 2013
Available online 19 September 2013
kinetically analyzed under weakly acidic conditions, and was compared with that of protochlorophyllide
(
PChlide) a and chlorophyllide (Chlide) a. Chl c
exhibited slower pheophytinization kinetics than PChlide a and Chlide a, both of which possessed a pro-
pionic acid residue at the same position. The difference in pheophytinization properties between Chl c
and (P)Chlide a was ascribable to the electronegativity of the 17-substituent in Chl c larger than that of
P)Chlide a due to the C17 –C17 double bond with the conjugated 17 -carboxy group in Chl c . Demeta-
1
possessing a trans-acrylic acid residue at the 17-position
1
1
Keywords:
1
2
2
(
1
Chlorophyll c
Chlorophyllide
Demetalation
Photosynthesis
Protochlorophyllide
lation kinetics of PChlide a was slower than that of Chlide a, which originated from the effect of the p-
macrocyclic structures.
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
Physicochemical properties of Chls c are a clue to understanding
the metabolisms concerning on Chls c. We report herein kinetic
Chlorophyll (Chl) molecules play important roles in photosyn-
1
properties of pheophytinization of Chl c , which is isolated from
thesis. The molecular structures of Chls are widely diversified in
terms of their macrocyclic structures and peripheral substitu-
the diatom Chaetoceros gracilis.
Removal of the central metal from the cyclic tetrapyrroles of Chl
pigments, which is called pheophytinization, is a crucial reaction in
photosynthetic organisms: it produces the primary electron accep-
1
,2
ents. Most Chl pigments in oxygenic photosynthetic organisms
have the chlorin macrocycle, which is the 17,18-dihydroporphyrin.
The exception is Chls c in chromophyte algae and some photosyn-
tors in photosystem II reaction centers and proceeds Chl degrada-
1
–5
8–10
thetic prokaryotes.
Chl c , which is one of the major Chl c categories. Chls c are char-
acterized by the porphyrin-type -macrocycle instead of the chlo-
Figure 1 depicts the molecular structure of
tion.
In the early stage of Chl a degradation, the hydrolysis of
1
the phytyl ester and removal of the central magnesium occur
although the order of two reactions in photosynthetic organisms
p
1
1,12
rin macrocycle in other photosynthetically active Chls. In addition,
has been a little ambiguous.
The mechanism of in vivo pheo-
the 17-substituent of Chls c is different from that of other natural
phytinization has not been unraveled yet. Pheophytinization prop-
erties of both Chl pigments possessing a long ester chain and
chlorophyllide (Chlide) lacking the moiety will be helpful for bio-
chemical research of Chl degradation, but there is little information
on pheophytinization properties of Chlide-type pigments possess-
1
2
Chls: Chls c generally have a free acrylic acid (C17 @C17 ) at the
7-position, whereas other Chls possess a propionate residue
1
esterified with long hydrocarbon chains at this position.
Chls c function as light-harvesting pigments together with Chl a
and carotenoids in protein scaffolds in marine photosynthetic
2
13
ing a free carboxy group at the 17 -position while kinetic analy-
sis on pheophytinization of chlorophyllous pigments possessing an
6
,7
organisms, which contribute to a major part of global carbon cy-
cles. The metabolisms of Chls c as well as biogenesis and degrada-
tion of Chls a/c-type antenna proteins, therefore, have attracted
considerable attentions. However, little is known for such biologi-
cal phenomena in Chls c-containing photosynthetic organisms.
1
3–24
ester group has been extensively studied.
on pheophytinization kinetics of three Chlide-type pigments,
namely Chl c , protochlorophyllide (PChlide) a and Chlide a
Therefore, we focus
1
(Fig. 1). It is worth noting that PChlide a and Chlide a are important
intermediates in biosynthesis of naturally occurring Chls.2
Some of these previous reports on pheophytinization of natural
Chls and synthetic metallochlorins demonstrated the effects of
5,26
⇑
Corresponding author. Tel.: +81 6 6730 5880; fax: +81 6 6723 2721.
E-mail address: saga@chem.kindai.ac.jp (Y. Saga).
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.032

6
916
K. Sadaoka et al. / Bioorg. Med. Chem. 21 (2013) 6915–6919
with that of PChlide a and Chlide a, both of which have a propionic
acid at the 17-position.
A
D
B
C
N
N
N
N
Mg
2
. Results and discussion
1
8
1
7
1
Spectral changes of Chl c , PChlide a, and Chlide a through their
1
7
1
1
7
2
O
pheophytinization under acidic conditions are depicted in Figure 2.
Chl c had Soret and Q bands at 444 and 631 nm, respectively, as
shown in Figure 2A. The absorbance of these bands decreased
through the pheophytinization process, and new Soret and Q bands
appeared at 428 and 577/591 nm, respectively, accompanying the
isosbestic points at 437, 483, and 611 nm.
COOCH3
OH
O
Pigment
1
y
C171-C172
double bond double bond
C17-C18
Chl c1
PChlide a single bond double bond
Chlide a single bond single bond
PChlide a and Chlide a exhibited essentially the same spectral
1
Figure 1. Molecular structures of Chl c , PChlide a, and Chlide a.
changes as Chl c
C, respectively. The Soret and Q
1
under acidic conditions as shown in Figure 2B and
absorption bands of PChlide a at
y
peripheral substituents linked to the A-, B-, and C-rings of the cyc-
lic tetrapyrroles on pheophytinization kinetics of chlorophyllous
434 and 627 nm, respectively, decreased, accompanying appearance
of a new Soret band at 417 nm as well as Q bands at 568 and
588 nm. The isosbestic points could be observed at 425, 469, and
604 nm in this spectral change. In the case of Chlide a, the 432-nm
1
3–16,18–24
pigments.
However, no information is available on the ef-
fects of substituents in the D-ring on pheophytinization properties,
since it is difficult to change the 17- and/or 18-moieties directly
Soret and the 665-nm Q bands gradually lost their absorbance with
y
linked to the D-ring. Chl c
because of the presence of the 17-acrylic acid (C17 @C17 ). From
the viewpoints, we compare pheophytinization kinetics of Chl c
1
allows us to overcome such a problem
increase of a new 410-nm Soret band of pheophorbide (Pheide) a.
Theisosbesticpointscouldbeobservedat420,459,and551 nminthis
spectral change.
1
2
1
Pheophytinization kinetics can be quantitatively analyzed by
absorbance changes at the Soret peaks of the three pigments. Fig-
ure 3 depicts time courses of Soret peak absorbances of Chl c , PCh-
1
lide a, and Chlide a through pheophytinization in aqueous acetone
at the proton concentration of 7.1 ꢀ 10 M, which corresponds to
0
0
0
0
.4
.3
.2
.1
(A)
ꢁ4
pH 3.1. The pheophytinization reactions were regarded as pseudo-
first-order reactions because of linear time-dependency of the log-
arithm of their Soret absorbance changes. This is consistent with
the reaction conditions in which the proton concentrations were
much higher than the pigment concentrations. The reaction rate
constants of pheophytinization, ks, could be estimated by fitting
the time courses of the Soret absorbances of the chlorophyllous
pigments to the following kinetic equation:
0
.0
.5
ln½ðA ꢁ A
where A
1
Þ=ðA
, A, and A
0
ꢁ A
1
Þꢂ ¼ ꢁkt
0
(
B)
0
1
are their Soret absorbance at onset of the mea-
surement, at time t, and at the complete pheophytinization,
respectively.
0.4
0.3
0.2
0.1
1
The pheophytinization rate constants of Chl c were estimated to
ꢁ
3
ꢁ2
ꢁ1
ꢁ1
be 6.0 ꢀ 10 , 2.6 ꢀ 10 , and 1.3 ꢀ 10 min , at the proton con-
ꢁ4
ꢁ4
ꢁ3
centrations of 3.6 ꢀ 10 , 7.1 ꢀ 10 , and 1.4 ꢀ 10 M (=pH 3.4, 3.1,
and 2.9), respectively. These k-values were smaller than those of
PChlide a (2.2 ꢀ 10 , 9.6 ꢀ 10 , and 4.9 ꢀ 10 min ) and Chlide
ꢁ2
ꢁ2
ꢁ1
ꢁ1
0
.0
.5
0.0
0
(
C)
0.4
0.3
0.2
0.1
0.0
-
-
-
0.5
1.0
1.5
0
2
4
6
8
10 12 14
400
500
600
700
Time / min
Wavelength / nm
1
Figure 3. Kinetic plots for pheophytinization of Chl c (open circle), PChlide a (open
Figure 2. Spectral changes of Chl c
1
(A), PChlide a (B), and Chlide a (C) in acetone/
triangle), and Chlide a (open square) in acetone/water (3/1, vol/vol) at the proton
ꢁ
4
ꢁ4
ꢁ4
water (3/1, vol/vol) at the proton concentration of 7.1 ꢀ 10 , 3.6 ꢀ 10 , and
concentration of 7.1 ꢀ 10 M. Absorbance changes were monitored at 445, 433,
ꢁ
4
1
.4 ꢀ 10 M, respectively. (A): spectra from 0 to 240 min at a 10-min interval. (B):
spectra from 0 to 360 min at a 30-min interval. (C): spectra from 0 to 300 min at a
0-min interval. The arrows show the direction of the absorbance changes.
1 0 1
and 432 nm for Chl c , PChlide a, and Chlide a, respectively. A , A, and A are Soret
absorbances of the three pigments at the onset of measurements, at time t, and at
the complete pheophytinization, respectively.
1

K. Sadaoka et al. / Bioorg. Med. Chem. 21 (2013) 6915–6919
6917
ꢁ1
ꢁ
3
ꢁ2
ꢁ1
ꢁ1
10
as follows. Chl a: 6.0 ꢀ 10 , 2.1 ꢀ 10 , and 5.1 ꢀ 10 min , 8-
ꢁ3
ꢁ3
ꢁ1
vinyl Chl a: 2.7 ꢀ ꢀ 10 , 8.9 ꢀ 10 , and 2.0 ꢀ 10 min , 3-ethyl
ꢁ3
ꢁ2
ꢁ1 22
Chl a 9.6 ꢀ 10 , 4.0 ꢀ 10 , and 1.1 min
.
The difference in the
relative ratios between Chl c and Chl a analogs would originate
1
1
from the rather stronger electronegative effect of an acrylic acid
residue than a sole vinyl group due to the conjugation of a C@C
bond with COOH. The difference in positions of substituents on
the chlorin macrocycle might also result in the difference in the ra-
tios of pheophytinization rate constants.
-
1
1
0
Pheophytinization properties of Chl c
features of the other categories of Chls c, namely Chls c
Chls c and c possess a vinyl group at the 8-position instead of
an ethyl group in Chl c . The 7-substituent is further replaced to
a methoxycarbonyl group in Chl c . The replacements of the sub-
stituents in the B-ring of Chls c and c would make them more sta-
ble to the removal of the central magnesium than Chl c because of
1
allow us to presume the
2
and c
3
.
2
3
-
2
3
1
1
0
0
3
2
3
1
-
the additional electron-withdrawing effects. Hence, all Chl c pig-
ments are tolerant to pheophytinization compared with Chl a
coexisting in Chls a/c-type antenna proteins in marine photosyn-
thetic organisms. Previous reports have demonstrated that Chl b,
which functions as light-harvesting pigments in higher plants
and green algae, is also more stable against pheophytinization than
1
-
4
-3
-3
2
× 10
10
3× 10
+
[
H ] / M
1
Figure 4. Pheophytinization rate constants of Chl c (open circle), PChlide a (open
triangle), and Chlide a (open square) in acetone/water (3/1, vol/vol) dependent on
examined proton concentrations.
1
3,14,16,19
Chl a.
Chls b/c found in light-harvesting antenna systems,
ꢁ
2
ꢁ1
ꢁ1
therefore, are rather tough against pheophytinization in oxygenic
photosynthetic organisms whereas Chl a, which is also utilized in
reaction centers, seems to be apt to release the central magnesium.
Such features of these antenna Chls might be advantageous to
metabolisms of photosynthetic light-harvesting proteins and adap-
tation to environmental changes.
a (8.8 ꢀ 10 , 3.7 ꢀ 10 , and 1.7 min ) under the same conditions.
These k-values are the average of three independent measurements,
where the standard deviations were less than 11%, 5%, and 5% of the
averaged values for Chl c , PChlide a, and Chlide a, respectively. The
1
k-values obtained from the kinetic analysis are summarized in Fig-
2
ure 4. It should be noted that the 17 -carboxy groups in the three
The slower demetalation kinetics of PChlide a than Chlide a can be
ascribed to the effects of p-macrocyclic structures, namely the por-
pigments are mainly neutral (COOH form) under the present acidic
conditions where the pH is estimated to be distributed between 2.9
and 3.4 based on the assumption of complete dissociation of hydro-
chloric acid in acetone/water (3/1), as was ensured in previous
phyrin-type and chlorin-type macrocycles for PChlide a and Chlide
a, respectively. The relative ratios of pheophytinization rate constants
between the two pigments, k (PChlide a)/k (Chlide a), were ranged
from 0.25 to 0.30 in the proton concentration range between
1
5,17
reports,
which correspond to the acrylic and propionic acid residues in Chl
and (P)Chlide a, are 4.80 and 4.76, respectively (data from
SciFinder).
Figure 4 indicates that Chl c
a
since the pK of 2-butenoic acid and butanoic acid,
ꢁ
4
ꢁ3
3
.6 ꢀ 10 and 1.4 ꢀ 10 M (the pH range between 3.4 and 2.9).
c
1
Thesevalues were not largelydifferentfromk (PChl a)/k (Chla), which
were estimatedto bewithin0.16and0.27inthe proton concentration
1
possessing a trans-acrylic acid res-
ꢁ
4
ꢁ3
range between 1.3 ꢀ 10 and 1.3 ꢀ 10 M (the pH range between
idue at the 17-position is more tolerant to removal of the central
metal than PChlide a and Chlide a possessing a propionic acid res-
idue at the same position. The kinetic stability of Chl c
the other two pigments against pheophytinization can be ascribed
to the presence of the double bond between C17 and C17 atoms
1
with the conjugated 17 -carboxy group in Chl c . It was reported
that vinyl groups directly linked to the A- and B-rings of the chlorin
macrocycle provided higher resistance to pheophytinization than
ethyl groups. The effect of the double bond in the 17-acrylic acid
residue of Chl c on pheophytinization properties demonstrated in
2
2
3
.9 and 2.9). It isnotedthat thereportedk-valuesof PChlawereesti-
ꢁ4
ꢁ3
ꢁ1
ꢁ1
matedtobe9.7 ꢀ 10 , 4.2 ꢀ ꢀ 10 , and1.4 ꢀ 10 min atthepro-
1
relative to
ꢁ4
ꢁ4
ꢁ3
ton concentrations of 1.3 ꢀ 10 , 2.5 ꢀ 10 , and 1.3 ꢀ 10 M (=pH
22
1
2
3.9,3.6,and2.9),respectively. Therefore, thedifferenceofpheophyt-
inization kinetics between PChlide a and Chlide a is quantitatively in
line with the previous report concerning (P)Chl a possessing a phytyl
2
2
2
ester at the 17-propionate residue. Comparison of demetalation
2
2
kinetics between (P)Chlide a in this study and (P)Chl a esterified with
2
2
phytol indicates that the demetalation rate constants of (P)Chlide a
are about twice larger than those of the corresponding (P)Chl a under
similar acidic conditions. The slightly faster demetalation of Chlide-
type pigments relative to Chl-type pigments would be ascribable to
differences in steric hindrance over the chlorin macrocycles by the
1
this study is in line with the effects of vinyl groups in the A- and B-
rings. Such effects can be rationalized by large electronegativity of
C@C moieties relative to C–C.22 The substituents possessing high
electron-withdrawing abilities can decrease the electron densities
of core nitrogen atoms in the chlorin macrocycle, providing higher
activation energy in proton attack to the nitrogen atoms and the
17-substituents and/or microenvironmental structures including sol-
ventmoleculesaroundpigments.Thepresentresultsonkineticstabil-
ities of chlorophyllous pigments lacking an ester group at the 17-
position should help to understand the early process of Chl degrada-
tion including the steps of pheophytinization and ester cleavage.
subsequent removal of the central metal.1
8–24
The 17-acrylic acid
residue in Chl c
zation. The relative ratios of pheophytinization rate constants of
Chl c over those of PChlide a, k (Chl c )/k (PChlide a), were esti-
mated to be 0.27 in the proton concentration range between
1
also has such substitution effects on pheophytini-
1
The logarithms of pheophytinization rate constants of Chl c ,
1
1
PChlide a, and Chlide a exhibited linear relationships with exam-
ined proton concentrations in Figure 4, in which the slopes were
determined to be 2.2, 2.2, and 2.1, respectively. These values were
consistent with those of Chl a analogs, which were reported to be
ꢁ
4
ꢁ3
3
.6 ꢀ 10 and 1.4 ꢀ 10 M (the pH range between 3.4 and 2.9).
The values were smaller than the reported ratios of k (8-vinyl Chl
a)/k (Chl a) and k (Chl a)/k (3-ethyl Chl a), which were distributed
2
2
between 0.39 and 0.63.22 It is worth noting the reported k-values of
1.9–2.2 under similar experimental conditions.
These suggest the
participation of two protons in the rate-limiting step of pheophyt-
inization of the three pigments under the acidic conditions.
Chl a, 8-vinyl Chl a, and 3-ethyl Chl a at the proton concentrations
of 1.3 ꢀ 10 , 2.5 ꢀ 10 , and 1.3 ꢀ 10 M (=pH 3.9, 3.6, and 2.9)
ꢁ
4
ꢁ4
ꢁ3

6
918
K. Sadaoka et al. / Bioorg. Med. Chem. 21 (2013) 6915–6919
To summarize, we first demonstrated that pheophytinization
1
2
(
A)
1
kinetics of Chl c possessing C17 @C17 was slower than that of
1
2
PChlide a and Chlide a possessing C17 ꢁC17 . These results were
ascribable to the difference in the inductive effects of the 17-sub-
stituents in the D-ring of the chlorin macrocycle, namely an acrylic
acid in Chl c
of Chl c shown in the present study will help to understand the
metabolisms and geochemical events concerning on Chls c.
1
and a propionic acid in (P)Chlide a. Kinetic stabilities
1
3
3
. Materials and methods
.1. Apparatus
(
B)
Visible absorption spectra were measured with a Shimadzu UV-
450 spectrophotometer, where the reaction temperature was reg-
2
ulated with a Shimadzu thermo-electric temperature-controlled
cell holder TCC-240A. HPLC was performed with a Shimadzu LC-
2
0AT pump and an SPD-M20A or SPD-20AV detector. LC-MS was
measured with a Shimadzu LCMS-2020 system equipped with an
electrospray ionization probe.
(
C)
3
.2. Pigment preparation
Chl c
ing to previous reports.
treatment with chlorophyllase. PChlide a was prepared by oxida-
tion of Chlide with 2,3-dichloro-5,6-dicyano-1,4-benzoqui-
These chlorophyllous pigments were purified by
1
was extracted from a diatom Chaetoceros gracilis accord-
2
7,28
Chlide a was obtained from Chl a by
2
9
a
2
8,30
none.
reverse-phase HPLC before measurements of demetalation
kinetics.
0
10 20 30 40 50 60 70
Retention time / min
3
.3. Kinetic measurements
Figure 5. HPLC elution patterns of pheophytinization products (solid lines) of Chl c
1
(
1
A), PChlide a (B), and Chlide a (C) in aqueous acetone at the proton concentration of
.4 ꢀ 10 M for 60 min, 7.1 ꢀ 10 M for 150 min, and 3.6 ꢀ 10 M for 80 min,
A 1050
Soret absorbance = 0.5 at the 1-cm pathlength) was mixed with a
340 L distilled water, and a 10 L of aqueous hydrochloric acid for
lL acetone solution of purified chlorophyllous pigments
ꢁ
3
ꢁ4
ꢁ4
(
respectively, as well as the intact pigments before pheophytinization (dotted lines).
Pigments were eluted on an Inertsil ODS-P column with methanol/acetonitrile/
5
0
Chlide a were recorded at 422, 415, and 407 nm, respectively. Chromatograms of
intact Chl c , PChlide a, and Chlide a were recorded at 441, 432, and 430 nm,
respectively.
l
l
0 mM ammonium acetate (pH 5.2) (3/1/1, vol/vol/vol) at
a
flow rate of
volumetric analysis (Wako Chemical Industries, Ltd.) was added to
the solutions. Pheophytinization kinetics was examined by moni-
toring the absorbances at the Soret peak positions of the three pig-
ꢁ
1
.75 mL min . Chromatograms of reaction products of Chl c
1
, PChlide a, and
1
ꢁ
3
ꢁ4
ments at the proton concentration of 1.4 ꢀ 10 , 7.1 ꢀ 10 , and
ꢁ4
3
.6 ꢀ 10 M under the control of reaction temperature at 25 °C.
.4. HPLC analysis of reaction products
3
The reaction products of the three pigments by incubation in
acidic aqueous acetone were analyzed by reverse-phase HPLC as
shown in Figure 5. The products of Chl c exhibited the main frac-
1
tion at 50 min (Fig. 5A). This fraction had the Soret band at 422 nm
in the HPLC eluent, and gave a molecular ion peak at m/z 589.0,
Reaction products after measurements for pheophytinization
kinetics of the three pigments were extracted from reaction mix-
tures with dichloromethane, and were analyzed on a reverse-phase
HPLC column Inertsil ODS-P (3.0 mm i.d. ꢀ 150 mm, GL Science
Inc.) with methanol/acetonitrile/50 mM ammonium acetate (pH
+
which corresponded to the calculated value for [MH] , 589.2, in
the measurement of liquid chromatography–mass spectrometry
5
.2) (3/1/1, vol/vol/vol) at a flow rate of 0.75 mL/min.
(
LC-MS). Therefore, this product was ascribed to the demetalated
pigment of Chl c . Some minor products could be observed after
pheophytinization of Chl c under the present conditions. In the
1
Acknowledgments
1
case of pheophytinization reaction of PChlide a, a major product
was eluted at 45 min (Fig. 5B). This fraction, which gave a molecu-
lar ion peak at m/z 591.0, had the Soret band at 415 nm in the HPLC
eluent, indicating that the main product was protopheophorbide a.
Slight by-products were detected at 15 and 20 min. Pheophytiniza-
tion reaction of Chlide a gave a major product, which was eluted at
We thank Dr. Tadashi Mizoguchi, Ms. Yuki Kimura, and Ms. Har-
una Yokota of Ritsumeikan University as well as Dr. Jiro Harada of
Kurume University for their experimental assistance. This work
was partially supported by a Grant-in-Aid for Scientific Research
(
C) (No. 23550201 to YS) from the Japan Society for the Promotion
of Science. KS was supported by JSPS fellowship for Young
Scientists.
1
y
6 min (Fig. 5C). This fraction had the Soret and Q bands at 407
and 665 nm, respectively, in the HPLC eluent, and gave a molecular
ion peak at m/z 593.0. These analysis assigned this product to
Pheide a. Some minor products could also be observed after pheo-
phytinization of Chlide a. These results indicated that the pheo
phytinization reactions of the three pigments predominantly
occurred under the acidic condition with slight by-products.
References and notes
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