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Purification of photosystem I reaction centers from spinach stroma lamellae
Vol. 41, No. 1, 1970
PURIFICATION
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF PHOTOSYSTEM I REACTION CENTERS FROM SPINACH STROMA LAMELLAE' P. V. Sane and R. B. Park Botany
Department, Berkeley,
University California
of California, 94720
Received August 31, 1970 SUMMARY A procedure for the preparation of partially purified photosystem I reaction centers is d scribed. This procedure gives a preparation with a very high chl a/chl b 5 ratio which contains 1 P-700 per 16 chlorophylls. Starting material is a stroma lamella fraction obtained from spinach chloroplasts. Kok (1) taining
first
P-700.
cedure
for
using
Vernon
partial
acetone
chloroplasts further
reported
partial
and his
coworkers
purification
and Triton contains
of these
X-100 1 P-700
characterized
the
purification (2,3)
reaction
have
reaction
extraction. per
of a pigment
con-
described from
preparation
28 chlorophylls. centers
recently
centers
Their
complex
a pro-
plant
tissues
from spinach
Ogawa and Vernon
obtained
from
Anabaena
(4)
have
variabilis
and spinach. We recently chloroplasts
(5).
content
and is
present
in
close
showed
that
One is
physically
grana
obtaining
partially
fractions
without
lSupported by grant cine, U. S. Public
with
It system
purified the
located separated
regions.
association
there
are two kinds in the from
stroma
In this
photosystem
lamellae.
photosystem
has lcw P-700 2.
of photosystem
content paper
2.
It
has high
The second
and is
we describe
I reaction
I in spinach
centers
P-700
is
physically
in
a procedure from
for
these
use of detergents.
GM-13943-05 from the National Institute of General MediHealth Service, and the U. S. Atomic Energy Commission,
2The abbreviations used are: EDTA, ethylenediaminetetra-acetate; chlorophyll a; chl b, chlorophyll b. 206
chl
a,
Vol.41,No.
BIOCHEMICAL
1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS Chloroplasts sage through
a French
homogenate (grana
isolated
pressure
from
10,000
tions
were
used
tions
were
diluted
precipitates.
soluble
proteins.
preparing
with
a teflon
obtain acetone
material
two more times obtained
with
at the
taining volume
washed
M KCl.
oxidized (2)
using
difference
99.5%.
The precipitates
spectra
RC 160, preparations
estimated Electrophoresis
was determined by a standard
was conducted
obtained
with
vacuum
10 min.
The
(pH 7.4)
con-
in a small
from
procedure
using
to the method
from
160K,
reduced
a scattering
method
207
extracted
or homogeni-
of Vernon
to Arnon's
according
to
40K,
and RC 10 respectively.
equipped
microkjeldahl
under
resuspended
to the procedure
according
were
buffer
was determined
according
stirred
and other
x g for
M phosphate
thus
con-
were
sonication
finally
dropped
centrifuge
dried
at 30,000
RC 40,
to
The precipitates
were
by brief
were
a Cary 14 spectrophotometer
Chlorophyll
each time.
The preparations
of these
suspensions
chlorophylls
extraction
0.05
frac-
The final
The precipitates
centrifuged
These
EDTA wash were
to -20°C.
containing
acetone
centrifuga-
and centrifuged
on a clinical
discarded.
x g, and obtained
centers.
last
These
centrifuged
x g
once more to remove
was precooled
once with
and 10K have been designated content
the
mM EDTA (pH 8.0)
of the same buffer.
P-700
after
acetone
were
The FP
x g (160K)
mM EDTA (pH 8.0) was repeated
10 ml of cold
in 0.75
were 0.01
then
were
The suspensions
precipitates
0.75
which
end of third
and resuspended zation.
I reaction
The supernatants
soluble
(5).
The precipitates
photosystem
was more than
precipitates.
by a pas-
x g, 30 min at 40,000
and 160,000
The precipitates
rod and were
earlier
fraction).
The procedure
of acetone
fractionated
x g (40K),
with
10 ml of 100% acetone
centration
as described
lamella
40,000
50-fold
were
10 min at 3000 x g, 30 min at 10,000
x g (stroma
for
spinach
30 min at 30,000
x g (lOK),
obtain
cell
for
fraction),
60 min at 160,000
into
from market
was centrifuged
lamella
AND METHODS
(6). urea
minus
et al -A attachment.
Nitrogen
was
as a standard.
of Clarke
(7).
Prior
Vol.41,No.1,1970
BIOCHEMICALANDBIOPHYSICAL
to electrophoresis
the
material
RESEARCH COMMUNICATIONS
was solubilized
in 0.2% sodium
dodecyl
sulfate. RESULTS Table
I shows
preparations
balance
10% of the the total
per
(3).
a/chl
very
accurately
than
per
tion
is
than
(5),
the
the P-700
32 chlorophylls. the
RC 160.
The lmer
preparation
may be due to residual
not
in the
As expected,
P-700/chlorophyll
may be more useful
whole
for
chloroplasts characteristics absorption
than
characterization of Yamamoto
and one would
expect
of our most maximum of our
abundant
--et al.
and Vernon
(3)
fraction,
preparation,
like
208
namely, that
of spinach.
1 P-700
per
prepara-
between
b ratios
it
4, a
of Sane -2 et al RC 160
of the which
appears
I reaction
was prepared
such a preparation
is
for
of this
RC 10 or preparations
of photosystem
that
(2)
2 chlorophylls, reason
procedure. (3)
from
the work
a/chl
For this
as
We were
content
intermediate
photosystem
RC 160 preparation.
The P-700
b ratio
from
and chl
RC 160 preparation
The HP 700 preparation
a/chl
I).
and Vernon
of RC 10 ranges
The chl
the
to 1 P-700
10 by our
of Vernon
has characteristics
and RC 10.
chloroplasts
beyond
RC 160.
content
90%
two times
(Table
by Yamamoto for
90% of
of Yamamoto and Vernon
high
b ratio
to HP700 preparation
RC 40 preparation
present
a/chl
than
we are observing
approximately
very
less
about
12 chlorophylls
is
RC 160 is
the one observed
Based on 4 experiments, 28 to 1 P-700
chl
per
that
on 10 experiments,
HP700 preparation
of HP700 observed
similar
lower
for
that
Based
1 P-700
center
even though
indicates
preparation
as the
b ratio
b ratio
much less
This
from
showed
extraction
present.
this
content
a/chl
to measure
RC 10 is
by acetone
initially
Thus
P-700
The chl
The chl
RC 160 preparation
on the
of RC 160 ranged
for
unable
studies
was removed.
18 chlorophylls.
I reaction
fractions.
centers
content
of photosystem
from different
was destroyed
chlorophyll
purified
value
P-700
reaction
P-700
characteristics
obtained
Material
of the
the
would RC 10.
RC 10 are
that
the
made from centers. from whole have The red
of Yamamoto and Vernon(S),
BIOCHEMICAL
Vol. 41, No. 1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I CHLOROPHYLL, P-700, The figures acetone extraction. Preparation
AND NITROGEN CONTENT OF RC PREPARATIONS
in brackets
Chl a/Chl
show Chl a/Chl
b
Moles
> 10 (5-6)
RC 160*
RC 40
8-9
RC 10
7
b and P-700
content
of Chl a + Chl b Moles of P-700
16**
(100
to 130)
(3-4)
20
(200
to 250)
(2.2-2.4)
32
(500 to 650)
160 380
676 nm.
occurs
at 676 nm though
maximum is
toward
the blue If
the
compared
material
RC preparations prior
of lower
EDTA washing
The nitrogen nitrogen is
phoresis
evident of all
There
thoroughly
with
a/chl
b ratio,
this
material
kind
of reaction
will
with
a/chl
EDTA prior
b ratio
a more complete
of different generally
leered
other
protein
to acetone
of detergent
only
1-2 nm
It
extraction, appears
that
of chlorophyll is
given
with
EDTA.
b.
I.
in Table This
preparations.
in material
The
purifi-
The electro-
one protein-chlorophyll
bands
com-
not washed
extraction.
treatment,
the
and ease of resuspension be extremely
extraction
by washing
gives
to acetone
obtained.
of these
RC preparations
hmever,
are
RC preparations
from electrophoresis
EDTA prior
The absence chl
is
the
are,
chl
shifted
the HP 700 preparation.
not washed
permits
content
content
cation
plex.
is
with
spectrum
to
ug of N mumole of P-700
*Red absorption maximum for all preparations, **Difference spectrum maximum, 696-697 nm.
our difference
prior
useful
for
center.
209
high
P-700
content,
of the RC 160 suggest further
characterization
very
high
that of this
VoL41,No.
1, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES :: 3. 4. 5. 6. 7.
Biophys. Acta 48, 527 (1961). Kok, B., Biochim. Vernon, L. P., Yamamoto, H. Y., and Ogawa, T., Proc. Nat. Acad. Sci. U.S. 63, 911 (1969). Yamamoto, H. Y., and Vernon, L. P., Biochemistry 8, 4131 (1969). Ogawa, T., and Vernon, L. P., Biochim. Biophys. Acta 197, 332 (1970). Sane, P. V., Goodchild, D. J., and Park, R. B., Biochim. Biophys. Acta (1970), in press. Arnon, D. I., Plant Physiol. 24, 1 (1949). Clarke, J. T., Ann. N. Y. AcaT Sci. 121, 428 (1964).
210
PURIFICATION
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OF PHOTOSYSTEM I REACTION CENTERS FROM SPINACH STROMA LAMELLAE' P. V. Sane and R. B. Park Botany
Department, Berkeley,
University California
of California, 94720
Received August 31, 1970 SUMMARY A procedure for the preparation of partially purified photosystem I reaction centers is d scribed. This procedure gives a preparation with a very high chl a/chl b 5 ratio which contains 1 P-700 per 16 chlorophylls. Starting material is a stroma lamella fraction obtained from spinach chloroplasts. Kok (1) taining
first
P-700.
cedure
for
using
Vernon
partial
acetone
chloroplasts further
reported
partial
and his
coworkers
purification
and Triton contains
of these
X-100 1 P-700
characterized
the
purification (2,3)
reaction
have
reaction
extraction. per
of a pigment
con-
described from
preparation
28 chlorophylls. centers
recently
centers
Their
complex
a pro-
plant
tissues
from spinach
Ogawa and Vernon
obtained
from
Anabaena
(4)
have
variabilis
and spinach. We recently chloroplasts
(5).
content
and is
present
in
close
showed
that
One is
physically
grana
obtaining
partially
fractions
without
lSupported by grant cine, U. S. Public
with
It system
purified the
located separated
regions.
association
there
are two kinds in the from
stroma
In this
photosystem
lamellae.
photosystem
has lcw P-700 2.
of photosystem
content paper
2.
It
has high
The second
and is
we describe
I reaction
I in spinach
centers
P-700
is
physically
in
a procedure from
for
these
use of detergents.
GM-13943-05 from the National Institute of General MediHealth Service, and the U. S. Atomic Energy Commission,
2The abbreviations used are: EDTA, ethylenediaminetetra-acetate; chlorophyll a; chl b, chlorophyll b. 206
chl
a,
Vol.41,No.
BIOCHEMICAL
1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS Chloroplasts sage through
a French
homogenate (grana
isolated
pressure
from
10,000
tions
were
used
tions
were
diluted
precipitates.
soluble
proteins.
preparing
with
a teflon
obtain acetone
material
two more times obtained
with
at the
taining volume
washed
M KCl.
oxidized (2)
using
difference
99.5%.
The precipitates
spectra
RC 160, preparations
estimated Electrophoresis
was determined by a standard
was conducted
obtained
with
vacuum
10 min.
The
(pH 7.4)
con-
in a small
from
procedure
using
to the method
from
160K,
reduced
a scattering
method
207
extracted
or homogeni-
of Vernon
to Arnon's
according
to
40K,
and RC 10 respectively.
equipped
microkjeldahl
under
resuspended
to the procedure
according
were
buffer
was determined
according
stirred
and other
x g for
M phosphate
thus
con-
were
sonication
finally
dropped
centrifuge
dried
at 30,000
RC 40,
to
The precipitates
were
by brief
were
a Cary 14 spectrophotometer
Chlorophyll
each time.
The preparations
of these
suspensions
chlorophylls
extraction
0.05
frac-
The final
The precipitates
centrifuged
These
EDTA wash were
to -20°C.
containing
acetone
centrifuga-
and centrifuged
on a clinical
discarded.
x g, and obtained
centers.
last
These
centrifuged
x g
once more to remove
was precooled
once with
and 10K have been designated content
the
mM EDTA (pH 8.0)
of the same buffer.
P-700
after
acetone
were
The FP
x g (160K)
mM EDTA (pH 8.0) was repeated
10 ml of cold
in 0.75
were 0.01
then
were
The suspensions
precipitates
0.75
which
end of third
and resuspended zation.
I reaction
The supernatants
soluble
(5).
The precipitates
photosystem
was more than
precipitates.
by a pas-
x g, 30 min at 40,000
and 160,000
The precipitates
rod and were
earlier
fraction).
The procedure
of acetone
fractionated
x g (40K),
with
10 ml of 100% acetone
centration
as described
lamella
40,000
50-fold
were
10 min at 3000 x g, 30 min at 10,000
x g (stroma
for
spinach
30 min at 30,000
x g (lOK),
obtain
cell
for
fraction),
60 min at 160,000
into
from market
was centrifuged
lamella
AND METHODS
(6). urea
minus
et al -A attachment.
Nitrogen
was
as a standard.
of Clarke
(7).
Prior
Vol.41,No.1,1970
BIOCHEMICALANDBIOPHYSICAL
to electrophoresis
the
material
RESEARCH COMMUNICATIONS
was solubilized
in 0.2% sodium
dodecyl
sulfate. RESULTS Table
I shows
preparations
balance
10% of the the total
per
(3).
a/chl
very
accurately
than
per
tion
is
than
(5),
the
the P-700
32 chlorophylls. the
RC 160.
The lmer
preparation
may be due to residual
not
in the
As expected,
P-700/chlorophyll
may be more useful
whole
for
chloroplasts characteristics absorption
than
characterization of Yamamoto
and one would
expect
of our most maximum of our
abundant
--et al.
and Vernon
(3)
fraction,
preparation,
like
208
namely, that
of spinach.
1 P-700
per
prepara-
between
b ratios
it
4, a
of Sane -2 et al RC 160
of the which
appears
I reaction
was prepared
such a preparation
is
for
of this
RC 10 or preparations
of photosystem
that
(2)
2 chlorophylls, reason
procedure. (3)
from
the work
a/chl
For this
as
We were
content
intermediate
photosystem
RC 160 preparation.
The P-700
b ratio
from
and chl
RC 160 preparation
The HP 700 preparation
a/chl
I).
and Vernon
of RC 10 ranges
The chl
the
to 1 P-700
10 by our
of Vernon
has characteristics
and RC 10.
chloroplasts
beyond
RC 160.
content
90%
two times
(Table
by Yamamoto for
90% of
of Yamamoto and Vernon
high
b ratio
to HP700 preparation
RC 40 preparation
present
a/chl
than
we are observing
approximately
very
less
about
12 chlorophylls
is
RC 160 is
the one observed
Based on 4 experiments, 28 to 1 P-700
chl
per
that
on 10 experiments,
HP700 preparation
of HP700 observed
similar
lower
for
that
Based
1 P-700
center
even though
indicates
preparation
as the
b ratio
b ratio
much less
This
from
showed
extraction
present.
this
content
a/chl
to measure
RC 10 is
by acetone
initially
Thus
P-700
The chl
The chl
RC 160 preparation
on the
of RC 160 ranged
for
unable
studies
was removed.
18 chlorophylls.
I reaction
fractions.
centers
content
of photosystem
from different
was destroyed
chlorophyll
purified
value
P-700
reaction
P-700
characteristics
obtained
Material
of the
the
would RC 10.
RC 10 are
that
the
made from centers. from whole have The red
of Yamamoto and Vernon(S),
BIOCHEMICAL
Vol. 41, No. 1,197O
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table I CHLOROPHYLL, P-700, The figures acetone extraction. Preparation
AND NITROGEN CONTENT OF RC PREPARATIONS
in brackets
Chl a/Chl
show Chl a/Chl
b
Moles
> 10 (5-6)
RC 160*
RC 40
8-9
RC 10
7
b and P-700
content
of Chl a + Chl b Moles of P-700
16**
(100
to 130)
(3-4)
20
(200
to 250)
(2.2-2.4)
32
(500 to 650)
160 380
676 nm.
occurs
at 676 nm though
maximum is
toward
the blue If
the
compared
material
RC preparations prior
of lower
EDTA washing
The nitrogen nitrogen is
phoresis
evident of all
There
thoroughly
with
a/chl
b ratio,
this
material
kind
of reaction
will
with
a/chl
EDTA prior
b ratio
a more complete
of different generally
leered
other
protein
to acetone
of detergent
only
1-2 nm
It
extraction, appears
that
of chlorophyll is
given
with
EDTA.
b.
I.
in Table This
preparations.
in material
The
purifi-
The electro-
one protein-chlorophyll
bands
com-
not washed
extraction.
treatment,
the
and ease of resuspension be extremely
extraction
by washing
gives
to acetone
obtained.
of these
RC preparations
hmever,
are
RC preparations
from electrophoresis
EDTA prior
The absence chl
is
the
are,
chl
shifted
the HP 700 preparation.
not washed
permits
content
content
cation
plex.
is
with
spectrum
to
ug of N mumole of P-700
*Red absorption maximum for all preparations, **Difference spectrum maximum, 696-697 nm.
our difference
prior
useful
for
center.
209
high
P-700
content,
of the RC 160 suggest further
characterization
very
high
that of this
VoL41,No.
1, 1970
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES :: 3. 4. 5. 6. 7.
Biophys. Acta 48, 527 (1961). Kok, B., Biochim. Vernon, L. P., Yamamoto, H. Y., and Ogawa, T., Proc. Nat. Acad. Sci. U.S. 63, 911 (1969). Yamamoto, H. Y., and Vernon, L. P., Biochemistry 8, 4131 (1969). Ogawa, T., and Vernon, L. P., Biochim. Biophys. Acta 197, 332 (1970). Sane, P. V., Goodchild, D. J., and Park, R. B., Biochim. Biophys. Acta (1970), in press. Arnon, D. I., Plant Physiol. 24, 1 (1949). Clarke, J. T., Ann. N. Y. AcaT Sci. 121, 428 (1964).
210