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Isolation of Plastoquinones C and D from Spinach Chloroplasts 1, 2

M. D. Henninger and F. L. Crane

Department of Biological Sciences, Purdue University, Lafayette, Indiana

A total of 8 quinones have now been i(lentified in (lecrease in absorbancy represents re(luction of the
spinach chloroplast lipi(ls. These includle 4 plasto- quinone. WVe have use(l a v-alue of 15 for the inilli-
quinones Nwhlich have been designated by letter as mlolar extinction coefficient for the (lecrease in ab-
plasto(luinone A, B, C, ancl D. There are also 3 s,orbancy of plasto(quinones C and(l D since this is
tocopherylq(uinones a, 8, and -y as well as vitamin K1. characteristic of ty-pical isoprenoid( benzoquinones
In a previous note we reported the presence of plasto- (6). This value malay be revised when it is possible
quinone C in chloroplast lipids (7). We now find to obtain an accurate miiolecular wveight for these
that this plastoquinone C fraction contains another plastoquinones.
plastoquiinolne xvhich we halve referred to as plasto- Chlorophyll was (leterminie(l by the maetho(d of
quinone D (5). The latter represents about one third Arinoin ( 1).
of the total quinone in the plastoquinone C fraction.
In a previous paper wve halve reportecl on the differ- Extraction
ence in activity between plastoquinone C and D in
restoration of photosynthetic electron transport in ace- In contrast to plcastoquinones A and( B, plasto-
tone extracted spinach chloroplasts (5). In this quinone C and D are not easily releasecl from chloro-
paper we will describe the method used for the purifi- plasts by hydrocarboin solvents. For comiiplete ex-
,ation of the plastoquinone. traction acetone or mlixtures of a polar solvent suclh
as ethyl alcohol together with a nonipolar solvent
Materials such as heptane are necessary. The proce(lure which
we have generally- use(l involves conmplete extraction
Chloroplasts were prepared by differential centri- of the chloroplast lipids by a( mixture of propaniol
fugation of a spinach leaf honmogenate in 0.5 -m sucrose an(l n-lheptane. For this proce(lure 900 ml of chloro-
as previously describecl (2). Other miiaterials were plast suspension in 0.5 at sucrose, containing 1800 imlg
obtainedl as follows: n-heptane was fronm Phillips of chlorophyll, are (lilute(l with 1200 ml 0.2 M\ potas-
Petroleuimi Corp., Bartelsville, Oklahomla, and other siunm plhosphate buffer pH 7.5 andl 2500 ml of distille(d
solvents were reagent gradle obtained from commercial water. Then 1500 nil of n-pror-anol and 1500 nl
sources; decalso, Pernmutite Co., New York, New n-heptane are addled and the mlixture is shaken oIn a
York; silicic acid, Mallincro(dt, St. Louis, Mo.; ma- reciprocal shaker for 3 hours. The upper organic
terial for thin layer chromiiatography, Brinkmann, phase is renmovecl by mleans of a separatory funnel.
Great Neck, New York. Leuconlethylene blue reagent The aqueous residlue is honmogenizedl in a Wariing
for detecting quinones oIn thin layer chronmatography blenclor and then extracted by- stirring with petroleunii
plates was prepare(d as follows: 20 nml 10-3 m aqueous ether (30-60o). The upper petroleum ether layer is
methylene blue, 1 g zinc (lust and 3 ml of concentrated collectedl in separatory funnel and combined with the
sulfuric acid are mixecl and filtered through glass first extract. The aqueous residue is reextracte(d
wool to remove zinc pcarticles. When this colorless with petroleum etlher until all the cliolorphyll has beeii
solution is sprayed on thin layer plates quinones will extracte(d and a white pasty material remains in the
oxidize the reduced methylene blue to prodluce a blue aqlueous phase. The combine(d extracts are thein
spot. The strongly aci(d solution inhibits autoaxida- placedl under vacuum mlaintaine(l bY a water aspira-
tion of mlethylene blue on the rest of the p)late. tor until all the solvent is removeed. The remaininlg
Spectrophotometric (letermination of the plasto- green oil is theil taken up in 50 nil of heptane for
quinones is carried out by dissolving a sample to be chromiatography oil (lecalso.
teste(l in ethanol and measuring the absorbancy at 255
ni,a. A few grains of sodliunm borohydride (Metal Hy- Chromatographic Separation
drides, IInc., Beverly, Mass.) are then added to the
sample and after 2 nminutes to allow the borohydride to The extract is chromatographed on a decalso
settle, absorbancy is (letermiiined at 255 Imi,U again. The colunii to separate PQA, PQB., an(l epiphasic caro-
tenoidls fronm the PQC, PQD, chlorophyll, carotenoidIs,
' Received Oct. 24, 1963. the tocopherol quinones and a conmlyound with a 263
2 Supported in part under grant G567 from the Na- iillt absorbancy iiiaxiiiiuni.
tional Science Foundationi and GM 10741 from the Na-
tional Iinstitutes of Health. A dlecalso columnii 10 X 56 cml loosely packed
with 50/80 mesh decalso in the sodium form was chloroform in n-heptane removes a brown-yellow band
used. The column is washed with heptane and the containing PQC and PQD. The purified samples of
extract added in 50 ml of heptane. The column is PQC and PQD can now be prepared from this last
then eluted with 3000 ml heptane to remove an fraction by thin layer chromatography.
orange-yellow band which contains carotenes plus For preparation of thin layer plates a slurry of
PQA and PQB. This is followed by elution with 30 g of silica gel G in 60 ml of water stirred
1000 ml 10 % ethyl ether in heptane to elute a yellow vigorously for 1 minute is applied in a 250 u thick
band which contains more carotene plus PQA plus layer to five 20 X 20 cm plates. The plates are dried
PQB. These first 2 eluates may be combined for in an oven at 1000 for 30 minutes and stored at 800
isolation of PQA and PQB on thin layer chromatog- until used. Brinkmann Instrument company applica-
raphy. The decalso column is next eluted with 500 tor model SI1 is used for preparation of the plates.
ml of ethanol to elute a green band which contains Chromatography jars 10 X 23 cm base are filled
PQC, PQD, R263, a, ,6, and y tocopherol quinones to a height of 1 cm with developing solvent and lined
together with carotenes and chlorophyll. The solvent with Whatman 3 MM filter paper to keep the atmos-
is evaporated under vacuum at room temperature from phere saturated with solvent. For development the
this eluate and the oily residue is taken up in 50 ml solvent is allowed to ascend to a height of 15 cm.
of heptane for chromatography on silicic acid. Al- Development time with chloroform should be about
though greater resolution of the compounds in this 20 minutes and the 15 % ethyl acetate in benzene
fraction could be obtained by a more gradual increase about 35 minutes. Samples are applied in ethanol
of ethanol during elution of the decalso column, we as a strip 2.5 cm from the base of the plate with a
do not recommend this because PQC and PQD show single spot at one side. This spot is sprayed with
increasing tendency to decompose as they are sep- leucomethylene blue reagent after the plate has been
arated. It is best to separate PQC and PQD from developed and dried for 3 minutes. The major part
each other only at the last stage of purification. of the sample in the strip on the plate is protected
For the first silicic acid column 30 g of silicic acid from spraying by covering that part of the plate with
and 90 g of Hyflo Super-Cel are mixed as a thick filter paper. A strip of adsorbent corresponding to
suspension in n-heptane and packed in a 2 X 61 cm the developed quinone spots is scraped off the plate
chromatography column so that the column is about and suspended in 5 ml of ethanol. This suspension
75 % full. The column is packed and later eluted is centrifuged for 3 minutes at approximately 1000
under nitrogen gas at a pressure of 4 lb/in2. The X g to remove adsorbent and the ethanolic solution
pressure should always be removed to stop flow from of quinone is decanted. Extraction of the adsorbent
the column before the solvent goes below the top of is repeated by resuspension in 5 ml of ethanol and
the silicic acid to prevent formation of cracks. After may be repeated until extraction of quinone is com-
addition of the sample in a small volume of heptane plete as determined by spectrophotometric assay of
the column is eluted with 1000 ml n-heptane under the extract. Two or three extractions are usually
nitrogen pressure which removes a yellow band con- sufficient to remove the quinones.
taining PQA, PQB, and carotene not removed on the In the separation on the thin layer plates using
decalso column. Elution with 500 ml of a mixture chloroform as developing solvent PQC will run at
containing increasing amounts of chloroform (2- RF about 0.49 while PQD will be slightly lower at
25 %) in n-heptane will separate a brownish-yellow RF about 0.40. The fact that the material isolated is
band from the overlapping chlorophyll. This frac- the proper compound should be immediately con-
tion contains PQC, PQD, R263, and the tocopherol firnied by examining the UV spectrum of the sample
quinones. The exact elution mixture should be deter- in ethanol. Another spot moving at a higher RF will
mined by the progress of the brownish-yellow band often be encountered which in some cases will be
on the column. Recovery of all the plastoquinones traces of R263 and in other samples will be R253.
in this elution can be checked by further elution with Both of these compounds can be recognized by their
500 ml of 50 % chloroform in n-heptane. characteristic spectra (fig 4, 5). In some cases a
The solvent is evaporated from the fractions con- quinone mnoving at a lower RF than PQD may be
taining the brownish-yellow band under vacuum at detected. This will be a tocopherylquinone and
room temperature. The oil is taken up in 20 ml of can be recognized by its characteristic spectrum (fig
n-heptane for chromatography on the small silicic 6). A typical yield of plastoquinone C will be 0.021
acid column. To prepare this column 5 g of silicic umole per mg chlorophyll in the extracted chloro-
acid and 15 g Super-Cel are suspended in heptane. plasts. The yield of plastoquinone D will be 0.007
The second silicic acid column is packed as before pmole per mg chlorophyll. These quinones will de-
under 4 lb/in2 nitrogen in a 1.5 X 34 cm column. compose in 2 to 3 days either in ethanol solution or
After the sample is added to the column in a small as oil when stored at -15°.
volume the colunin is eluted under pressure with 60
ml of heptane which will remove a yellow band con- Properties
taining tocopherol quinones. Elution with 50 ml of
2 to 25 % chlorofo-rni in n-heptane removes a red The spectral and chromatographic properties of
band containing R263 and elution with 50 ml 50 % PQC and PQD are outlined in table I in comparison

Table I. Properties of Chloroplast Quinones and Related Compountds

Absorbance Isosbestic* RF
in ethanol E1 z RF 15 % ethyl
Compound maximum shoulder
El1cma points 1 %7o ether in acetate
maximum m,u chloroform
m,u n benzene
PQA 255 262 246 276,233 0.74 0.81
PQB 255 262 218 276,233 0.78 0.88
PQC 262 255 120 283,232 0.49 0.0
PQD 262 255 75 290,226 0.40 0.0
aTQ 261 269 414 282,232 0.37 0.21
fTQ 261 430 280,229 0.33 0.19
yTQ 258 430 279,230 0.25 0.16
R253 from PQD 253 15 .. 0.69 0.53
R263 263 43 .. 0.72 0.70
* Isosbestic poinits determined by reduction with potassium borohydride.

gO 3



230 2 25 2020 370 200 2_0 00 * 0 20 220 230 240 no 240 270 200 no Zoo No no0 330


07 "ss
os \


m 5\


220 230 200

25 0 2?0 290 a1 2- - |-,

FIG. 1. Spectrum of plastoquinone C in ethanol. (-) oxidized form ---) after reduction with borohydride.
0.15 mg dry weight in 3 ml ethanol.
FIG. 2. Spectrum of plastoquinone D in ethaniol (-) oxidized form, (---) after reduction with borohydride.
0.17 mg dry weight in 3 ml ethanol.
FIG. 3. Spectrum of plastoquinone B in ethanol (-) oxidized form, ---) reduced with borohydride. 0.10
mg dry weight in 3 ml ethanol.
FIG. 4. Spectrum of R263 in ethanol. (-) oxidized form, --- -) reduced with borohydride. 0.29 mg dry
weight in 3 ml ethanol.
FIG. 5. Spectrum of R253 in ethanol. This sample was formed by aginig a sample of 0.03 ,umole/ml of PQD in
ethanol at -15° for 4 months. 0.78 mg dry wt in 3 ml ethanol.
FIG. 6. Spectrum of a tocopherol quinone. (-) oxidized form, --- -) reduced with borohydride.

to the other quinones and quinone-like compounds the oxidation of reduce(d methylene blue on thin
from spinach chloroplasts. Both of these compounds layer chromatogranis, but the spectruml of the com-
appear as pale yellow oils when purified. Both com- pound does not show increased absorbancy at 230 nlu
pounds are unstable in the purified form and are on reduction. The ral)idl degradlation of plastoqjui-
converted on standing at -15° for 2 or 3 days into nones C and D is in marked contrast to the excellent
materials which no longer show the typical quinone stability of plastoquinones A andl B.
spectrum. We have referred to these degradation Since benzoquinones in general have nmolar ex-
products as R253 because they show a broad absorp- tinction coefficients (E) of about 15,000 the F II
tion maximum at 253 mu which decreases on addition found for l)lastoqluinones C and( D would suggest a
of borohydri(le as s6own in figure 5. R253 causes mlolecular weight in the region of 1.500 if the coIll-
Extract from chloroplasts
o800 mg chlorophyll
decalso column

Eluate I Eluate II Eluate III

3000 ml n-heptane 1000 ml 10% ether 500 ml ethanol
yellow in heptane PQC + PQD + R263
carotene, PQA and PQB yellow + tocopherol
(separate by thin layer PQA + PQB quinohes
chromatography) (separate by thin layer
first silicic
acid column

Eluate 1 Eluate 2 Eluate 3
1000 ml n-heptane 500 ml 2-25% 500 ml 50%
light yellow chloroform in chloroform
n-heptane in n-heptane
brownish yellow green
PQC, PQD, R263 chlorophylls
to copherol quinones

second silicic
acid column

Eluate 1
Eluate 2
Eluate 3
60 ml n-heptane 50 ml 2-25% 50 ml 50%
yellow chloroform chloroform
to copherol in n-heptane in n-heptane
quinones red-yellow brown-yellow

thin layer thin layer thin layer

chromatography chromatography chromatography
4 plates 2 plates 2 plates

a, l and T R263 PQC PQD

tocopheryl -
FIG. 7. Flow sheet of isolation of plastoquiinones from chloroplasts.

pound(s which wve have isolated are pure (6). Since however, until mletlhodIs are foundl for stabilizing the
this is a much higher mlolecular weight than we have compoundls at the level of purification which we have
encountere(d with any of the other terpenoid benzo- achieved. The spectra of PQC and PQD as shown
qJuinones we suspect that further purification may be in figure 1 and 2 differ from that of PQA or PQB
possible. Further purification will not be possible, (fig 3) in that the absorption maxinmum is at 262 m,t

with a shoulder at 255 nmf insteadl of a maximum at lipids from which plastoquinones A and B, tocopheryl-
255 m,u. PQC and PQD also show a wider wave- quinones and an unknown lipid with oxidation-reduc-
length spread between the isosbestic points formed tion capability, R263, can also be isolated by prepara-
when the quinone is reduced with borohydride. These tive thin layer chromatography. The properties of
differences in the spectrum may indicate a different plastoquinones C and D are describecl as well as the
arrangement of substituants on the (quinone ring than conversion to an unknown derivative R253 which
is foundl in PQA. occurs after the plastoquinones C and D are separated
froml each other.
Plastoquinones C and D are quite simiiilar in their
\N'e thank Mr. Tom Arnett and Miss Pat Sollars for
spectral properties, stability, antl chronmatographic their assistance during the developmennt of these methods.
behavior. They do show considlerable differences in
abilitv to restore photoreductase activity in acetone- Literature Cited
extractecl chloroplasts. These (lifferences have been
dletailed in a previous communication (5). The most 1. ARNON, D. 1. 1949. Copper enzymiies in isolate(d
noteworthy difference is the restoration of NADP chloroplasts. Polyphenoloxidase in Bet(a vuilaris.
reduction in the presence of ascorbate, indophenol, Plant Physiol. 24: 1-15.
an(d chllrophenylmethyl urea by PQC, but not by 2. CRANE, F. L. 1959. Isolation of 2 quinionles with
PQD. On the other hand, the overall reduction of coenzyme Q activity from alfalfa. Plant Physiol.
34: 546-51.
NADP starting from water requires the addition 3. DIL LEY, R. A. AND F. L. CRANE. 1963. Liglht in-
of PQA, PQC, and PQD (4). Changes in the oxido- duced chaniges of a-tocopherylquinone and plasto-
redluction state of PQD in chloroplasts in response to quinonie D in spinach chloroplasts. Plant Physiol.
light have also been observed (3). 38: xxx.
An analog of plastoquinone A. namiiely PQA with 4. HENNINGER, MI. D. ANi) F. L. CRANE. 1963. Resto-
a 20 carbon side chain PQA (20) rather than a ration of tripllosphopyridine nucleotide photoreduc-
45 carbon side chain PQA(45) has been isolated by tion in acetone-extracted chloroplasts by plasto-
Eck and(t Trebst (8) from horse chestnut (Aesculus). quinonies. Plant Physiol. 38: xi-xii.
The chromatographic properties an(d spectra of this 5. HENNINGER, MI. D. AND F. L. CRANE. 1963. Resto-
ration of photoreductase activities in acetone-ex-
comlpoun(l do not correspond to the properties of tracted chloroplasts by plastoquinones and toco-
PQC or PQD. Eck and Trebst (8) have also re- pherylquinlones. Biochem. 2: 1168-72.
portecl the isolation of dimers of PQA(45) and PQA 6. ISLER, O., R. RiTEGG, A. LANGEMANN, P. SCHUDEL,
(20) from horse chestnut. These compounds showed G. RYSTER, AND J. WeRSCH. 1960. Chenmistry of
no activity in extracted chloroplasts. but further com- ubiqu;inone and related compounds. Ciba Founda-
parison should be madle between these compounds tion Symposium, Quinones in Electron Transport.
and PQC and PQD. Little Brown and Co., Boston, Mass. 79-99.
1962. Two new quinones from chloroplast. Bio-
Summary chem. Biophys. Res. Commun. 8: 294-98.
A method has been described for the isolation of die konstitutioni eines weiteren plastochinonis und
plastoquinones C and D fronm spiniach chloroplasts. seines dimereni aus kastanienblittern. Z. Natur-
The method also providles fractionis of the chloroplast forsch. 18b: 446-51.