Identification of Flavonoids and Cinnamic Acid Derivatives from

F L A V O N O I D S A N D CINNAMIC A C I D D E R I V A T I V E S F R O M SPINACH
CHLOROPLASTS
177
Identification of Flavonoids and Cinnamic Acid Derivatives
from Spinach Chloroplast Preparations
W A L T E R OETTMEIER a n d ADELHEID HEUPEL
Abteilung für Biologie, Ruhr-Universität Bochum
( Z . Naturforsdi. 2 7 b , 177—183 [1972] ; eingegangen am 24. November 1971)
By immunochemical methods we had presented evidence for a primary acceptor complex for
photosystem I of photosynthetic electron transport. During purification of this complex, obtained
as a water soluble fraction from ether treated lyophilized chloroplasts, two low molecular fractions
are also obtained, which may or may not have relation to the primary acceptor. The purification
and identification of these low molecular fractions is reported. One, absorbing at 275 and 335 nm,
consists of several flavonoid glycosides. The aglycon of the main component is tentatively identified
as a 3-methyl-6,7-methylenedioxy-quercetagetin. The chromophore of the other low molecular
fraction, absorbing at 312 nm, contains two cinnamic acid derivatives, which on alkaline hydrolysis
yield p-coumaric acid. The relation of the two low molecular fractions to other fractions of various
activity isolated from chloroplasts is discussed.
In photosynthetic electron transport of chloroplasts on illumination water is oxidized and a terminal electron acceptor is reduced. In this reaction
two photosystems are involved. We have recently
reported on evidence for the existence of an endogenous primary acceptor of photosystem I in chloroplasts 2 . We had obtained an antibody against this
acceptor and had isolated a fraction from ether
treated chloroplasts, which would neutralize this
antibody 2 ' 3 . This fraction, called SL-eth ? should
therefore contain the antigen, i. e. the presumed
primary acceptor. We have reported on the purification of this fraction into a high molecular weight
protein, possessing the antigen activity and twro low
molecular fractions 3 . This paper presents results
on the isolation, separation and identification of the
later compounds.
Materials and Methods
1. Isolation and Separation
The antigen fraction, called SL-eth, was prepared
from spinach chloroplasts according to REGITZ et al. 2 ,
but the heat inactivation step was omitted. Crude SL-eth
(from broken chloroplasts of about 200 mg chlorophyll
content) was layered on top of a Sephadex G-75 column (2.5x30 cm). The volumetrically collected water
eluate yielded — as already described3 — a green
fraction, a yellow high molecular fraction (2max
275 nm), a blue fraction of plastocyanin and two low
molecular yellow fractions, which came down close
Requests for reprints should be sent to Dr. W. OETTMEIER,
Ruhr-Univ. Bochum, Abt. f. Biologie, D-4630 Bochum-Querenburg, Postfach 2148.
together. The low molecular fractions of five consecutive runs of the Sephadex G-75 column were concentrated in vacuo to about 3 ml, centrifugated and chromatographed on a Sephadex G-25 column (2 x 80 cm)
with water as eluant. Fractions with absorption maxima
at 260 and 310 nm (yielding low molecular fraction I
— the cinnamic acid fraction) and at 275 and 335 nm
(yielding low molecular fraction II — the flavonoid
fraction) were collected.
Low molecular fraction I was chromatographed on
a diethyl-aminoethyl cellulose column ( 3 x 6 cm, DE 52,
Whatman). The chromophoric group with an absorption maximum at 310 nm is adsorbed, whereas a substance with an absorption maximum at 260 nm comes
through the column without retardation. The column
is then washed with water and then with NaCl solutions of increasing NaCl content. The chromophoric
group, absorbing at 310 nm, is eluted from the column
at a concentration of 0.3 M NaCl. These fractions with
an absorption maximum at 310 nm are passed through
a Dowex 50 W X 8 column ( 3 x 6 cm) for removal of
Na® ions. The resulting acidic solution is lyophilized.
Further purification is achieved by dissolving the residue after lyophilisation in diethyl ether, extracting the
cinnamic acid derivatives into 5% NaHC0 3 , and recovering them from the acidified water phase by repeated extraction with diethyl ether.
For the preparation of greater amounts of cinnamic
acid derivatives and flavonoids the following procedure
with whole spinach leaves was used. 500 g of spinach
leaves were cut to pieces and boiled for 1 hour with 11
of water. After passing the mixture through cheesecloth, the resultant filtrate was evaporated to dry in
vacuo. The residue was extracted with methanol in a
Soxhlet apparatus, the methanol evaporated in
vacuo and the residue dissolved in 15 ml of water and
acidified to pH 4. This solution was layered on top of
a /V-acetyl polyamide column (3.5 x 8 cm, MN Polyamid 6-AC, Macherey & Nagel) and eluted with water.
A brown eluate, appearing first, is discarded. The elu-
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178
W . O E T T M E I E R A N D A. HEUPEL
tion of cinnamic acid derivatives is monitored at
310 nm and about 700 ml are collected. This fraction
is further purified as described above.
Flavonoid compounds are then eluted with a water/
methanol gradient with increasing amounts of methanol up to 100 per cent. About 2 1 of yellow flavonoid
solution are obtained. The eluate of flavonoids is evaporated to dry in vacuo and dissolved in a minimum
amount of water. 2 ml of this solution are chromatographed on a cellulose column (3 x 90 cm, Avicell
Merck) with 15% acetic acid. Elution is monitored at
335 nm. Four fractions are obtained. Corresponding
fractions of several consecutive runs are combined and
lyophilized.
Flavonoids were sprayed with B e n e d i c t's reagent
and cinnamic acids with 2% diazotized sulfanilic acid
in 10% Na 2 C0 3 .
4. Spectroscopy
UV-spectra were determined in a Cary 15 spectrophotometer. The procedure of MABRY et al. 5 was used
to obtain diagnostic shifts of flavonoid spectra on addition of various reagents. Mass spectra were determined in a Atlas CH 7 mass spectrometer. Trimethylsilylation was achieved by adding a few ju\ of bis(trimethylsilyl) acetamide to the sample, prepared for
mass spectrometry, and heating to 100° for a few
minutes.
2. Hydrolysis and Chemical Procedures
Acid hydrolysis of flavonoids was carried out by
boiling 2 hours with 6% HCl and extracting the aglycon with ethyl acetate.
Cleavage of aromatic ether linkages was achieved
by boiling 1 hour with equal amounts of freshly destilled 67% hydroiodic acid and acetic anhydride. After
evaporation of the acetic anhydride in vacuo and dilution with water the flavone was extracted into ethyl
acetate. Elementary iodine in the ethyl acetate phase
was reduced by shaking with a dilute solution of
sodium thiosulphate and thus removed from the ethyl
acetate phase.
Quercetagetin was obtained from the hexamethyl
ether * in the same way.
Alkaline hydrolysis of cinnamic acid derivatives was
brought about by stirring with 2 N NaOH at room temperature for 4 hours under nitrogen atmosphere. After
acidification to pH 3, the cinnamic acid was extracted
with diethyl ether, re-extracted into 5% NaHC03 and
recovered from the NaHC03 phase by repeated extraction with diethyl ether prior to acidification.
p-coumaric acid methyl ester was synthesized according to HARBORNE and CORNER 4 .
Catalytic hydrogenation was achieved with palladium/charcoal as catalyst.
3. Thin Layer Chromatography
The following solvent systems were employed (all
mixtures by volume, if not indicated otherwise) :
Cellulose (DC-Fertigplatten, Merck) : a: tert. butanol-acetic acid-water 3:1:1; b: 15% acetic acid; c:
n-butanol-acetic acid-water 4:1:5 (upper phase); d:
water; e: ra-butanol-2 N NH3 1:1 (upper phase); f:
n-butanol-ethanol-water 4:1:2.2; g: benzene-acetic
acid-water 6:7:3 (upper phase); h: sodium formiateformic acid-water (10 g : 1 ml : 200 ml) ; j : 2% acetic
acid.
Polyamide (Macherey & Nagel, Polygram Fertigfolien Polyamid-6) k: methanol-acetic acid-water 90:
5:5.
Chromatograms were examined in UV-light of
350 nm, in the presence or absence of ammonia vapors.
* Quercetagetin hexamethyl ether was a generous gift from
Prof. Dr. H. WAGNER, Munich.
Results
As has been reported already, the crude antigen
fraction SL-eth» which neutralizes the antibody inhibition of the presumed primary acceptor of photosystem I, can be separated on a Sephadex G-75
column into several fractions 3 . The two low molecular fractions thus obtained may or may not be
related to this primary acceptor. Their further purification is described in methods. As will be documented in the following tables and figures, we have
identified the compounds in low molecular fraction I as cinnamic acid derivatives and in the low
molecular fraction II as a mixture of flavonoid
compounds.
1. Low molecular fraction II — flavonoids
Two dimensional thin layer chromatography of
the purified low molecular fraction II revealed the
presence of nine flavonoid compounds. /?/-values are
given in Table 1. Compounds, only present in traces,
are set into paranthesis.
Designation o f
the c o m p o u n d
i?/-values in solvent systems
a
b
A
(BI)
B 2
CI
C 2
(DI)
(D2)
(D 3)
(D4)
0.29
0.29
0.39
0.24
0.40
0.24
0.34
0.37
0.47
0.26
0.34
0.38
0.46
0.48
0.71
0.64
0.78
0.68
Table 1. /?/-values of flavonoids from low molecular fraction
II in thin layer chromatography (solvent systems are described
in methods).
The final column chromatography on the cellulose column yields four fractions. They correspond
F L A V O N O I D S A N D CINNAMIC ACID DERIVATIVES F R O M SPINACH
to the following compounds in thin layer chromatography (in the order of elution) : D 1 — D 4, C 1
and C 2, B 1 and B 2 and finally A. Compound A
proved to be chromatographically pure and idenfification was started with this compound. As rational
for the identification procedure it was assumed that
compound A (^ max (methanol) 250, 276 and 338
nm) is a glycosidated flavonoid. Acid hydrolysis
yielded 1, whose absorption spectrum could not be
referred to a common flavone. Additional substituents, especially ether linkages, were assumed.
Therefore further cleavage with hydroiodic acid
was carried out, yielding the basic flavonol 2. Mass
spectrometric analysis, done by Dr. D. MÜLLER
(Abt. Chemie, Ruhr-Universität Bochum), of the
trimethylsilyl derivative of 2, showed it to be a
hexahydroxy flavone. By comparison with an
authentic sample, the structure of quercetagetin
could be established for 2 (Rf-values in solvent systems a: 0.17; b : 0.02 and k: 0.17). Amax (methanol) 257, 273 and 356 nm are in good agreement
with the data Amax (ethanol) 259, 272 and 364
n m , r e p o r t e d b y HARBORNE
6.
OR4
1:
2:
3:
4:
R 1 , R 2 - C H , - bridge, R 3 = C H 3 , R 4
R1 = R 2 = R3 = R4 = H
=
R 2 = CH 3 , R 1 = R 3 = R 4 = H
=
R2 = R4 = CH 3 , R 1 = R 3 = H
=
= H
quercetagetin
patuletin
spinacetin
The presence of a methyl and a methylenedioxy
group was established in 1 by mass spectrometric
analysis, again done by Dr. D. MÜLLER. The mass
spectrum of the tris (trimethylsilyl) derivative of 1
is shown in Fig. 1; only peaks > 3 5 0 m/e are indicated.
—O'^y^oc^
OH
B
0
545
SeoSölimpuityl
A00
450
500
550
m
/e
Fig. 1. Mass spectrum of (trimethylsilyl) derivative of compound 1 (3-methyl-6,7-methylenedioxy-quercetagetin).
CHLOROPLASTS
179
Neglecting an impurity at 563 m/e, 560 m/e proved to be the molecular ion. As known from the
mass spectroscopy of trimethylsilyl compounds, a
methyl group is split off very easily, yielding the
fragment ion 545 m/e. The high intensity of 545
m/e, compared to the molecular ion, is due to the
formation of a six membered silyl ring and a very
stable aromatic system.
488 m/e, which is the base peak in this spectrum,
is not due to an additional fragment ion, but to the
bis (trimethylsilyl) derivative of 1 with one free
hydroxyl group. As is known from methylation, the
strongly hydrogen bonded hydroxyl group in the
6 position is only substituted with difficulties. The
corresponding fragment ion after loss of a methyl
group, 473 m/e, has only very low intensity, because a stabilizing effect by ring closure is impossible in this case. Additional proof for the presence
of a methyl and a methylenedioxy group in 1 was
given by high resolution mass spectrometry. The
molecular weight found was within the value of
0.009 mass units for the exact calculated molecular
weight of 1. Further information, concerning the
position of the above substituents, could not be obtained from this mass spectrum. No additional
characteristic fragment ions could be observed.
The substitution pattern is tentatively established
by UV-spectroscopy (^max of 1 in methanol: 258,
268, 275 sh and 350 nm). A shift of 17 nm of
band I, produced by boric acid/sodium acetate,
postulates the presence of two free ortho hydroxyl
groups in the 3' and 4' position. A shift of 22 nm
of band I, caused by aluminium chloride/hydrochloric acid, indicates the presence of a free hydroxyl group in the 3 or 5 position. Since 1 is stable
on addition of sodium methylate, a free hydroxyl
group in the 3 position is excluded. Thus, the
structure of 1 is tentatively established as 3-methyl6,7-methylenedioxy-quercetagetin.
Nature and position of the substituent, which is
split off on acid treatment from A, i. e. the original
compound in the low molecular fraction II, are not
yet known.
Information on the remaining compounds B 1
and B 2 and C 1 and C 2 (the compounds of the D
series were omitted, because these components were
only present in traces), which are not yet resolved
into single components, could be obtained by chromatographic comparison of their hydrolysis products with those of A, as shown in Table 2.
W . O E T T M E I E R A N D A. HEUPEL
180
Compounds
hydrolysis 6 % HCl
Rf-values in solvent
systems
a
b
k
A
B1/B2
C1/C2
0.65
0.66
0.77
0.78
0.84
0.04
0.04
0.11
0.11
0.39
0.39
0.49
0.46
cleavage hydroiodic
acid
i?/-values in solvent
systems
k
b
a
0.16
0.18
0.40
0.18
0.02
0.02
0.17
0.17
0.02
0.17
Table 2. Comparison of the hydrolysis products of compounds
from low molecular fraction II in thin layer chromatography
(solvent systems are described in methods).
1, as obtained from A, is also detected in the HCl
hydrolysate of the mixture of B 1 and B 2. Therefore, one of the B compounds differs from A only
in the nature of the substituent, which is split off
by acid hydrolysis, or in its point of attachment.
Finally, also C 1 and C 2 must have the basic quercetagetin structure, because 2 is found after cleavage with hydroiodic acid.
Therefore, in addition to compound A, at least
three other derivatives of quercetagetin are present
in low molecular fraction II. These findings are in
agreement with the results of Z A N E and W E N D E R 7 ,
who isolated patuletin 3 and spinacetin 4 from
spinach leaves, but as free aglycones and not as
glycosides.
2. Low molecular fraction I — cinnamic acid
derivatives
The chromophore of the purified low molecular
fraction I proved to consist of two quite similar
compounds, whose resolution into single components has not yet been achieved (/?/-values in solvent
systems c : 0.70, 0.81; d : 0.88; e: 0.02 and f :
0.26, 0.35; red colour reaction with diazotized sulfanilic acid). The UV-spectrum of the mixture of
these two compounds in methanol is shown in
Fig. 2.
Only one absorption maximum beyond 250 nm
at 312 nm is observed, which can be shifted to
358 nm on addition of sodium methylate. The maximum of fluorescence emission lies at 453 nm (on
excitation with 375 nm).
On alkaline hydrolysis, only one acidic hydrolysis product was obtained from the mixture of both
compounds, confirming again their close resemblance. By comparison with a commercial sample,
the structure of p-coumaric acid could be established
500 m[i.
Fig. 2. Absorption spectrum of the chromophoric group of
low molecular fraction I, as obtained from crude SL-eth
(
methanol,
methanol + sodium methylate).
for the hydrolysis product (Table 3 ) . The absorption spectrum of the hydrolysate (in methanol / m a x :
228, 295, 305; after addition of sodium methylate:
305 sh, 325 nm) is identical with that of p-coumaric
acid.
Solvent
system
g
h
i
j
p-coumaric acid
hydrolysate
trans
CIS
trans
CIS
0.37
0.34
0.89
0.37
0.38
0.74
0.93
0.65
0.40
0.33
0.90
0.38
0.41
0.76
0.91
0.64
Table 3. Comparison of Rf-\alues of the alkaline hydrolysate
of the chromophoric group of low molecular fraction I with
that of p-coumaric acid in thin layer chromatography (thin
layer chromatography was carried out in different runs, combining g/h and i / j in two dimensional chromatogramm; solvent systems are described in methods).
Identity of p-coumaric acid in the alkaline hydrolysate of the chromophore from low molecular fraction I is confirmed by its catalytic hydrogenation to
phloretic acid and comparison with authentic phloretic acid, obtained from p-coumaric acid in the
same way (Rf-values in solvent systems g: 0.41 and
h: 0.73; purple violet colour reaction with diazotized
sulfanilic acid, Amax 277, 285 sh nm).
The substituent, which is split off by alkaline hydrolysis from both compounds, must be linked to
the carboxylic group of p-coumaric acid. The hydroxyl group must be free, otherwise the bathochromic shift of 46 nm on addition of alkali cannot
be explained. The methyl ester of p-coumaric acid
FLAVONOIDS A N D CINNAMIC ACID DERIVATIVES F R O M SPINACH
CHLOROPLASTS
181
shows quite the same UV-spectrum as the mixture
vatives of
of both cinnamic acid derivatives isolated, which
plasts, are related to or identical with the recently
supports the assumption that the oarboxylie group
described p-coumaryl-meso-tartaric acid and its ace-
of p-coumaric acid is esterified by a compound
tyl derivative, isolated f r o m spinach leaves
without a marked selfabsorption in both cases. The
chemical
nature
of these substituents is not
yet
p-coumaric acid isolated f r o m
9a.
At present, it is not possible to relate the two low
molecular fractions to the antigen moiety in our
SL-eth preparation.
known.
chloro-
Since the protein purified
by
REGITZ 3 f r o m SL-eth already neutralizes the antiDiscussion
body
The terminal electron acceptor of the photosynthetic electron transport chain in chloroplasts is the
water soluble ferredoxin, which connects
the re-
ducing power of photosystem I to NADP®-reduction.
Since
there
are numerous
Hill
reactions
independent of the addition of ferredoxin, and since
viologen dyes with much more negative redox potentials than ferredoxin are reduced by isolated chloroplasts, it was speculated that there is a primary
acceptor of photosystem I different f r o m ferredoxin.
Recently evidence has (been presented f o r the existence of such a c o m p o u n d between photosystem I
and
ferredoxin.
YOCUM
and
SAN PIETRO 8 ' 9
dis-
covered a substance, called FRS — ferredoxin reducing substance — which in addition to ferredoxin
is required to restore NADP®-reduction in highly
sonicated chloroplasts.
against the primary
acceptor,
we
have
at
present no means to show that one of the low molecular fractions had been dissociated f r o m the protein fraction during purification and therefore is
perhaps the prosthetic group of the primary acceptor. As has been pointed out b y REGITZ et al. 2 , the
antibody not necessarily must be directed against
the prosthetic group of the primary acceptor and
therefore the antigen may be the apoenzyme
or
even only a structural component of the primary
acceptor
complex.
As
already
discussed2'3,
the
designation of the antigen isolated f r o m SL-eth ?
as
the primary acceptor o f photosystem I and an electron carrier rests on its relation or part identity to
F R S o f Y O C U M a n d S A N PIETRO.
It
is interesting
to
note,
however,
that
SAN
PIETRO 10 in a recent report on purification of FRS
also obtained low molecular fractions, which he has
not yet identified, but which as judged f r o m their
W e have presented evidence that an antiserum
absorption spectra, seem to be identical with our
against the thylakoid system of chloroplasts con-
low molecular fractions. It seems to be m o r e than a
tains an antibody against the unknown primary ac-
coincidence
ceptor of photosystem 1 1 ' 2 . REGITZ 2' 3 obtained an
obtained f r o m chloroplasts in completely different
extract f r o m ether treated lyophilized chloroplasts,
preparations, but both containing the presumed pri-
called S^.eth, which would neutralize the inhibitory
mary acceptor.
effect of the antibody
preparation
and
therefore
that the low molecular fractions
The two low molecular fractions, we have
are
ob-
should contain the antigen, i. e. the presumed pri-
tained f r o m SL-eth ? show similarities to other com-
mary acceptor.
pounds reported in the literature.
REGITZ 3 has recently reported on the purification
CRS — cytochrome c reducing substance — iso-
of the protein fraction f r o m SL-eth » which carries the
lated
antigen property. In the crude SL-eth
MURANO
also two low
molecular fractions are present. The results in this
by
FUJITA
12
and
MYERS
11
and
FUJITA
and
shows great resemblance to FRS of SAN
PIETRO and to our SL-eth • It has been suggested by
paper have identified one as a mixture of several
the authors that CRS might be related to the pri-
flavonoid
mary acceptor of photosystem I
of
glycosides and the other as two derivatives
p-coumaric acid. The main compound in the
flavonoid
fraction is tentatively characterized as a
12.
REGITZ et al.
2
reported that SL-eth has CRS activity.
A number of naturally occurring cofactors
of
3-methyl-6,7-methylenedioxy-quercetagetin glycoside.
photosynthetic phosphorylation have been isolated
The two derivatives of p-coumaric acid are esterified
f r o m chloroplasts. The cofactors, isolated b y KROG-
with not yet identified compounds. Quinic acid or
MANN a n d STILLER
glucose, which are the most c o m m o n
compounds
to be of flavone type of unknown structure. They
esterified with p-coumaric acid in nature, do not
correspond to the quercetagetin derivatives of low
seem to be involved. It seems possible that our deri-
molecular fraction II f r o m Sy^-eth • " P h o s p h o d o x i n " ,
13
a n d GEE et al.
14,
are r e p o r t e d
182
W. OETTMEIER
A N D A. HEUPEL
a preparation by BLACK et a l . 1 5 , exhibits similar ab-
As stressed already, we have not yet shown that
sorption maxima in the regions of 2 6 0 — 2 8 0 and
the two low molecular fractions in S^eth are related
315 — 3 3 0 nm, as do both low molecular fractions
to the protein moiety with the antigen activity and
"phospho-
therefore to the primary acceptor. However, we can
d o x i n " is claimed to be a p t e r i d i n 1 6 ' 1 ' . No pteri-
rule out pteridines being related to the primary ac-
din, however, could be detected in low molecular
ceptor, because we find no evidence f o r a pteridine
in our preparation S^eth • Strong suggestions that
from SL-eth • The active compound
of
fractions of S^eth • A fluorescent protein factor, isolated by W u and MYERS 18 and its phosphorus containing
chromophoric
group
("P-compound")
19
a pteridin might act as the prosthetic group of the
primary acceptor of photosystem I had been made
of " P - c o m p o u n d " has not yet been achieved, but a
b y F U L L E R and N U G E N T 2 1 , based on the observations that the presence of pteridines in spinach had
number
been established 16
both stimulate photophosphorylation. Identification
of
characteristics of
dines is reported
19.
unconjugated
pteri-
UV-data, fluorescence behaviour
and chemical properties of " P - c o m p o u n d " , as reported by W u et a l . 1 9 , are quite similar to those of
the p-coumaric acid derivatives, we obtained from
low molecular fraction I f r o m SL-eth • W e think
it possible therefore that both are identical and
therefore " P - c o m p o u n d " should be considered as
a p-coumaric acid derivative.
There also might b e some connection between the
primary acceptor of photosystem I and the light
activation factor of WLLDNER et a l . 2 0 . Ribulosediphosphate carboxylase from tomatoe plants shows
a stimulation of activity upon irradiation with light
of 325 nm. Responsible for light activation is a protein with a chromophore, absorbing at 325 nm and
identified as chlorogenic acid 20 . But the possibility
cannot be ruled out that another compound with
similar spectroscopic properties (i. e. a p-coumaric
acid derivative) but in low concentrations is masked
by chlorogenic acid, which is one of the most common phenolics in plants 2 0 .
1
2
3
4
5
6
7
8
9
9A
R . BERZBORN, W . MENKE, A . TREBST, and E. PISTORIUS,
Z . Naturforsch. 2 1 b , 1057 [ 1 9 6 6 ] .
G. REGITZ, R . BERZBORN, and A . TREBST, Planta Berlin
9 1 , 8 [1970].
G. REGITZ and W . OETTMEIER, P r o g r e s s in Photosynthesis
Research, Stresa 1 9 7 1 , in press.
J. B. HARBORNE and J. J. CORNER, B i o c h e m . J. 8 1 , 2 4 2
[1961].
T . J. MABRY, K . R . MARKHAM, and M . B . THOMAS, T h e
Systematic Identification of Flavonoids, p . 35, SpringerVerlag, B e r l i n - H e i d e l b e r g - N e w Y o r k 1 9 7 0 .
J. B. HARBORNE, P h y t o c h e m . 4, 647 [ 1 9 6 5 ] .
A . ZANE and S. H . WENDER, J. org. Chemistry 2 6 , 4 7 1 8
[1961].
C. F. YOCUM and A . SAN PIETRO, Biochem. b i o p h y s i c . R e s .
Commun. 36, 614 [ 1 9 6 9 ] .
C. F . YOCUM and A . SAN PIETRO, Arch. Biochem. B i o p h y sics 1 4 0 , 1 5 2 [ 1 9 7 0 ] ,
K . TADERA, Y . SUZUKI, F. KAWAI, and H . MITSUDA, A g r .
Biol. C h e m . 3 4 , 5 1 7 [ 1 9 7 0 ] ; K . TADERA and H. MITSUDA,
A g r . Biol. C h e m . 3 5 , 1431 [ 1 9 7 1 ] .
and that the redox
potentials
of tetrahydropterines as measured by polarographic
methods seemed to b e negative enough to reduce
ferredoxin 21 . However, recently it has been shown
that the hitherto reported redox potentials for the
quinonoid dihydropterin-tetrahydropterin couple of
— 0 . 7 V should be revised. A R C H E R and S C R I M G E O U R 22 reexamined the standard reduction potential and reported a ^g'-value of + 0 . 1 5 V. R E E D and
MAYNE 23 could not find any pterin in the fully
active reaction
spheroides.
centers
of
Rhodopseudomonas
Recently M A L K I N and B E A R D E N 24 suggested that
a bound ferredoxin may be the primary acceptor
of photosystem I and H I Y A M A and K E 2 5 reported
on a spectral change at 4 3 0 nm attributed to the
primary acceptor of photosystem I. At present it is
not possible to correlate their data with ours and
those of S A N P I E T R O .
This work was supported by the "Deutsche Forschungsgemeinschaft". We are indebted to Prof. Dr. A.
TREBST for many helpful discussions and to Dr. D.
MÜLLER for mass spectrometric analysis.
10
11
12
13
14
15
16
17
18
A . SAN PIETRO, Progress in Photosynthesis Research,
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19
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23
24
25
CHLORELLA
183
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[Amsterdam] 2 2 6 , 4 7 7 [ 1 9 7 1 ] .
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[1971].
Über Teilreaktionen der Photosynthetischen Sulfatreduktion
in zellfreien Systemen aus Spinatchloroplasten und Chlorella *
Enzyme Reactions Involved in Photosynthetic Sulfate-Reduction in Cell-free Systems
of Spinach Chloroplasts and Chlorella
AHLERT
SCHMIDT
Ruhr-Universität Bochum, Abteilung für Biologie
( Z . Naturforsch. 27 b , 1 8 3 — 1 9 2 [1972] ; eingegangen am 18. November 1971)
Sulfatreduktion, A P S , P A P S , S-Sulfoglutathion
Cellfree extracts f r o m Chlorella,
spinach leaves or spinach chloroplasts convert radioactive
sulfate into a S-sulfocompound already in the dark. The reaction requires A T P , M g 2 ® ions and
c o m p o u n d s with S H groups. The later are needed for activation of the enzym and as substrates and
acceptors for the sulfogroup. The c o m p o u n d formed if glutathion is used has been identified as
S-sulfoglutathion. Other products formed from sulfate and A T P in the dark b y spinach or
chlorella
extracts are A P S and P A P S . The formation of P A P S requires the presence of a SH-group. Both
cellfree extracts transfer the sulfate group from A P S and P A P S onto glutathion to form S-sulfoglutathion. N o A T P is needed in this reaction, neither with P A P S nor with A P S as substrate.
Über den Mechanismus der photosynthetischen
Sulfatreduktion in pflanzlichen Organismen liegen
bisher nur wenig Informationen v o r K ü r z l i c h
konnten wir zeigen, daß isolierte Chloroplasten 2 - 6
bei Belichtung Sulfat bis zur Stufe des Cysteins zu
reduzieren vermögen. Zell- und partikelfreie Systeme
sowohl aus Chloroplasten 2 - 4 als auch aus der Grünalge Chlorella7-9
können ebenfalls Sulfat reduzieren. Der Sulfatreduktion geht eine nicht lichtabhängige, aber dann A T P benötigende Sulfataktivierung
voraus. Über die Sulfataktivierung zu APS und
PAPS und den Umsatz der sulfathaltigen Nukleotide zu einer 5-Sulf overbindung soll hier berichtet
werden. Da die bisherigen eigenen Versuche mit
Enzymen aus Spinat auf Unterschiede zu dem zellfreien System der Arbeitsgruppe von SCHIFF aus
Chlorella hinzuweisen schienen, werden die Systeme
aus Spinat und aus Chlorella in ihren Eigenschaften und Cofaktorbedürfnissen miteinander verglichen.
Sonderdrudeanforderungen
an Dr. AHLERT SCHMIDT,
Ruhr-Universität Bochum, Institut für Biochemie der Pflanzen, D-4630
Bochum,
Postfach 2148.
Methodik
a) Chloroplastenextrakt:
Spinatchloroplasten, nach
der Methodik von JENSEN und BASSHAM 10 hergestellt,
wurden osmotisch aufgebrochen und der wäßrige
Überstand (entsprechend einer Chlorophyllkonzentration von 0,6 mg/ml) als Enzympräparation benutzt 4 .
b) Chlorella-Extrakt: Chlorella pyrenoidosa wurde
nach der Vorschrift von BÖGER 11 ohne Vitaminzusatz
angezogen und nach einem abgeänderten Verfahren
von S c h i f f in der French-Presse aufgebrochen. 61
Kulturlösung werden abzentrifugiert und in 0,02 M
Tris-HCl-Puffer pH 7,8 (pro 1 werden 200 mg GSH
zugegeben) so aufgenomen, daß zu 1 g Chlorella-Zellen (Naßgewicht) 5 ml Puffer gegeben werden. Diese
Lösung wird in der French-Presse (Sorvall Ribi RF I)
bei 20000 PSI aufgebrochen, wobei die Temperatur
zwischen 10 und 15 °C gehalten wird. Dieser so gewonnene Rohextrakt wird sofort in Portionen zu jeweils 10 ml eingefroren. Zum Versuch wird jeweils
eine Portion aufgetaut und für 10 Min. bei 10000 g
abzentrifugiert.
Durch
eine
Sephadex-G-50-Säule
(1,7-25 cm) werden alle niedermolekularen Verbindungen abgetrennt (0,02 M Tris-HCl-Puffer pH 7,8 mit
* Folgende Abkürzungen wurden verwendet: A P S = Adenosinphosphosulfat, P A P S = Phosphoadenosinphosphosulfat, P A P = 3'-5'-Adenosindiphosphat, G S H = reduziertes
Glutathion, GS-SG = oxidiertes Glutathion, G S - S 0 3 H =
S-Sulfoglutathion, Cys = Cystein.