Plant Physiol. ( 1967) 42, 1246-1254

Comparative Studies on PlastoquinoneII. Analysis for Plastoquinones A, B, C and D'

R. Barr, M. D. Henninger, and F. L. Crane2Department of Biological Sciences, Purdue University, Lafayette, Indiana 47907

Received AMay 15, 1967.

Summiarv. Two different methods for the extraction and assav of plastoqtinonesA, B, C and D from chloroplasts of green 'plants have been described. The long 'procedureinvolves separation of aqueous and lipid phases of extract in a separatory funnel, columnchromatography, purification on thin-layer plates, and spectrophotometric assays forquantitative determination of the various plastoquinones. The short procedure is basedon spotting lipid extracts firom 'ch'loroplasts on thin layer plates and comnparing leuco-methylene blue spots of unknown quinones with a series of spots produced by knownamounts of the 4 standard plastoquinones on the same plate.

Reliability of the 2 procedures is shown by presenting recovery data (82 % recoveryfor PQ A by the long method and 64-100 % recovery by the short method). Varioussolvent systems for quinone purification are described. Separation of plastoquinonesB and C into 16 components each is demonstrated for spinach and a tomato mutant, highpigment (hp). Plastoquinone C is shown to be equivalent to C1-C4 while D correspondsto PQ C5 and C6 according to Grif'fiths, Wallwork and Pennock's designation. Tiheterm PQ D is therefore redundant and §hould be abandoned in favor of specific designa-tion of PQ C ty'pe.

Spinaclh chloroplasts have been treated with avariety of organic solvents to extract plastoquinonies.Kofler et al. (8) extracted p'lastoquinone A withpetroleum ether from dried plant materials whileCrane (2) used isooctane with 5 % ethanol forextraction and a decalso column for purification ofalfalfa and corn seedling PQ A. Kegel, Henningerand Crane (7) reported the isolationi of plasto-quinones A, B and C from chloroplasts treated with0.2 NI phosphate buffer, propanol, heptane and u'.ater(1 :1.2:3 :2.5 v/v). This chloroplast and solventmixture was shaken on a reci,procal shaker for 3hours. The organilc extract was ifirst purified on adecalso and later on a silicic acid-supercel column(1:3 w/-w). Henninger and Crane (5) found theadditional quinone PQ D by this procedure. Hen-ninger, Barr and Crane (6) purified PQ B by slightmodifications of this procedure.

Egiger (3) assayed the PQ A content of numerousplants by means of acetone and petroleum ether ex-tractions. Bulcke et al. (1) determined the PQ A.C and D content of algae and various higher plantsusing acetone and light petroleum ether extracts.

1 Supported under grant GB 02920 from the NationalScience Founidation.

2 F. L. Crane is supported bv Career Award K6-21,839 from the National Institute of Gelneral -MedicalScieince.

It can be concluded from the above reports thatplastoquinones can be extracted with various organicsolvents. However, the relative efficiency of thesemethods has not been reported. We propose todescribe our methods for the isolation and assay ofplastoquinones A, B and C in detail by a long anda short procedure. We will also showr recovery (latafor both procedures and describe methods for theseparation of PQ B and PQ C into 6 separate typeseach.

Materials and Methods

Spinach for chloroplast preparation was obtainedfrom the local market. Organic solvents used forextraction were reagent grade with the exception ofheptane which wve redistilled. The following specialchemicals were obtained from the sources indicated:aluminum oxide (alumina) acid washed, Metck andCompany; 2,2'-bipyridine. Eastman Organic Chem-icals; ferric chloride, Hyflo Super-Cel, silicic acid,meta-precipitated (H2 SiU3.nH2O), Fi'sher ScientificCompany; methylene blue chloride, Matheson, Cole-man and Bell; potassium borohydride, Sigma Chem-ical Comnpany; silica gel GHR, Brinkman Instruments.

Chloroplast Preparation. All procedures werecarried out in a dimly lighted room using 7 W nightlights as the only illumination. Fresh leacsesvereobtained either from outdoors, greenhouse-grown

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BARR AND CRANE-DISTRIBUTION OF PLASTOQULINONES

plants, or conixnercial sources. They were deveinedand washed. Two-hundred gram quantities of leaveswere blended in 1.51 isotonic isolation mediuli con-sisting of 0.5 M sucrose with 2 IfnM phosphate bufferadjusted to pH 7.5 and centrifugged twice (600 X gfor 15 mins and 1500 X g for 20 mins) to obtainchloroplasts. The chloroplast pellet was resuspendedin 90 ml suspension medium and homogenized. Afterhomogenation, two 1-ml aliquots 'were removed forchlorophylMl determination according to Arnon's metliod.The homogenized chloroplasts were immediately ex-tracted or frozen for later use. All procedures forchloroplast isolation wvere carried out at 0 to 4°.

Chloroplast Extraction. Extraction was carriedout by blending 100 ml fresh or thawed chloroplasts(containing approximately 200 nmg h6lorophyll) ina Waring blendor in 100 ml of 0.2 M phosphatebufiffer (pH 7.5) for 5 mninutes. Then a mixture of250 ml' heptane and 250 ml 1- or 2-4propanol wasadded to the ground chloropla'sts. Larger amounts ofsolvents -had to be used if chloroplasts contained morethan 250 mg chlorophvll. The suspension was shakenon a reciprocal shaker for 1 hour at slow speed atroom temperature (220).

Separation of Aquieous and Lipid Fractions ofthe Extract. The green lipid fraction containingplastoquinones in heptane was separated from theaqueous propanol fraction in a 2000 ml separatoryfunnel. After the initial separation of the 2 phases,the green epiphase was set aside while the brownishhypophase was reextracted with mixtures of heptane,acetone, and benzene (1:2:1). This procedure wasrepeated until all the green had been removed 'fromthe aqueouts hypophase. When all the green epiphaseshad been pooled, it was desirable to rewash themwith a 1:1 mixture of absolute ethanol and water.This removed chloroplast glvcolipids from chlorophylland plastoquinones. The washed epiplhase was thendried with sodium sulfate and evaporated to drynessunder vacuum in a rotary evaporator.

Fractionation of Extract. Alfter evaporation todryness the lipid extract of chloroplasts was suspendedin a known amount of heiptane for chromatographyon a silicic acid-Super-Cel column or in petroleumether for chromatography on an alumina column.The extract could also be fractionated directly bythin layer chromatography.

Chr-omatography of Extract on Silicic Acid-Super-Cel Columns. A column (65 X 2.5 cm) was madefrom a mixture of 10 g silicic acid with 30 g Super-Cel in 500 ml heptane and was paoked under nitrogen(3-4 lbs pressure/square inch) allowing about 1 hourfor the adsorlbent to settle while additional heptanewas run through it. The packing was done stepwiseby adding a little adsorbent from the 500 -ml flaskwith additional heptane. This allowed an even 'pack-ing with no air bubbles trapped inside the adsorbent.

The chloroplast extract was put on the column inheptane with a pipette. It was applied carefullv inthe least possilble volume, such as 10 ml, to preventbreaks in the column. A'fter the extract was on the

column it was allowed to soak in before more solventwas applied. When the sample had been adsorbedon top of the column, 100 to 200 ml aliquots ofsolvents were applied.

The 'silicic acid-Super-Cel columns adsorbed andheld chlorophyll and xanthophyll on the top half ofthe column while carotene, vitamin K, tocopherols,plastoquinones, and tocopherylquinones were elutedwith mixtures of increasing concentrations of chloro-form in heptane as orange to light yellow bands.T-he amount of solvent varied according to how fastthe various, bands moved down'wards on the columnn.Generally, it was sufficient to use 200 ml heptane toelute fl-carotene in fraction 1, 200 ;ml 2 % chloro-form in heptane to e!ute vitamin K in fraction 2,200 ml 4 % chloroform in heptane to elute plasto-quinones A and B in fraction 3, and 200 ml 10 %chloroform in heptane to elute plastoquinones C andD in fralction 4. As collection of fraction 4 wascompleted, the green material adsorbed on the toppart of the column began to move down rapidlv. Toavoid contamination with chlorophyll, it was some-times best to stop collecting fraction 4 at this pointand go on to fraction 5 using 25 % chloroform inheptane to bring down the remaining plastoquinonesC and D with tocopherylquinones and chlorophyll.This fraction was purified later on thin-layer chroma-tograms. It was found that most of the chloroplastquinones had been eluted in fractions 2 to 5 so thatthere was no need to continue collecting fraction 6with 50 % chloroform in hoptane; instead, the columnwas flushed with absolute ethanol to remove all othersubstances remaining on the column.

Chromatography of Extract on an Alumina Col-umn. When better PQ C and D recoveries weredesired, it was advisable to use an alumina 'column.The alumina had to be deactivated with 8 % deionizedwater. To make a chromatography column, 200 gacid-washed alumina was mixed with 16 ml water.When all the water had ibeen adsorbed after vigorousmixing with a glass rod, the alumina was suspendedin petroleum ether and poured into a column. Thesolvent flow was so ra'pid at first that care had to betaken to prevent the column from running dry. Analumina column was ready -for use nearly as soon asit was built up to desired 'height. The chiloroplastextract was put on the column in petroleum ether.Successive fractions were eluted with 0, 2, 3, 5, 8,12, 20, 25 and 30 % diethyl ether in petroleunm ether(B.P. 30-700). Undiluted diethyl ether broughtdown the green components. Absolute ethanol wasused at the end to clean the 'column of whatever hadbeen tightly adsorbed on the alumina. Carotene waseluted in fraction 1, PQ A and B in fractions 2 to 4.PQ C in fraction 7 with' 20 % diethyl ether inpetroleum ether. Tocophervlquinones were partiallymixed with PQ C while a certain amount stayed onthe coltimn to be eluted together with the greencomponents in 25 to 30 % diethyl ether in petroleumether.

All fractions from columns were evaporated to

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PLANT PHYSIOLOGY

dryness and suspended in a known amount of heptanieor absolute ethanol. Since most fractions containedseveral components, they had to be further purifiedlby thin layer chromatography.

TIhin-layer Chroni atographv of Extract. Frac-

t.ons from silicic acid-Super-Cel or alumina columns

could be purified by thin-layer chromatograplhy on

silica gel GHR 'plates. Silica gel GHR was preferredto silica gel G because GHR did not contain as much1free iron which colored the background bluish wlhenmethv'lene iblue spray was used on silica gel G.

To make 5 silica gel GHR plates (20 X 20 cim),30 g silica gel GHR were comibined with 60 mldeionized water in a 250 ml Erlenmever flask, shakenvigorously for 45 seconds to 1 minute and plateswere coated with a layer 250 microns thick by mean-s

of an appliicator. Plates 'vere air-dried for 5 minutes,sitacked in a raek, and incubated in a drying oven at

120° for 30 minutes. Overheating at higher tem-

peratures or for longer periods of time caused changes

in the silica gel which lead to spectrophotometrically-detectable imipurities in isolated fractions. Plateswere stored in a dessicating cabinet after they cooled.Only fresh plates wNere used for streakin'g. Platesmore than a day old were objectionable because theyencouraged tailing. Thiis was more noticeable ifnon-polar solvents were used to develop the chroma-tograms.

Extra-long thin-layer plates which measure 40 X

20 cm could also be used. The above amount ofs'ilica gel GHR coated 2 long and 1 regular plate in1 application. These longer plates permnitted betterresolution of closely a's'sociated compounds.

For qualitative identification of plastoquinones a

concentrated sample in ethanol or heptane obtainedfrom a column was applied with a p'ipette as a streak1.5 cm above the origin of a plate. Tihe same samplewas used to make a single spot in the left-hand corner

of the plate to identify the various, bands with a spray.

Anotlher spot could be added in the right corner if 2diifferent s.praa's were used for identification. Over-loading of the plates could be avoided if a samplewhich contained no more than 3 timig of chlorophyllwas applied to plate. Better separation resultedduring development if the applied bandl was thin andeven. Heptane was a better solvent than ethanolfor sample application because 'it evaporated fasterand did not allow the sample to spread over the silicagel.

After application of sam'ples, the silica gel plateswere developed in chromatography tankis lined withfilter paper to keep the atmospliere saturated witlsolvent.

Standard developing sol,vents for plastoquinonesincluded chloroform, benzene, or mixtures of heptane-benzene and heptane-chloroform or benzene-isooctaneand chloroform-isooctane. Heptane and isooctanekept all chloroplast comiponents at the origin of theplate wlhile chloroform and benzene tended to crowdcomponents into the upper 50 % of the plate. 'PIhusin chloroform it wx'as liard to separate PQ A and B

from 8l-carotenel, vitanlin K1, and PQ A 20. Purechlorofornm was sensitive to overloading witlh theresult that streaks of fl-carotene tailed over the upperth,ird of the plate. This couild lie amended by adding3 to 20 % heptane or isooctanie to the chloroform.Such mixtures gave excellent separation of plasto-quinones C aind tocopheryl,quinones from green conm-ponents of chloroplast extracts. Addition of fairlylarge anmounts of heptane or isooctane to chloroformll(30-50 %) slowed the movemlent of the upper bandsso that a better separation of plastoquinones A. A 20,B and vitaimin K1 wvas achieved at the sacrifice of noseparat.on in the lc;wer half of the plate where thetocopherylquinones stayed hiddlen in the chlbrophy 11.

Benzene was a good -general solvent for plasto-quinone separation. By adding varying amounts ofheptane or isooctane, plastoquinone A could have anRF value fromn 0.8 to 0.2. Ai mixture of 15 % hep-tane or isooctane in benzene was found to give goodseparation of fl-carotene. plastoquinone A 45, A 20,A 15 and A 10, as well as the various types of plasto-quinone B, a-tocopherol and reduced A 45. Plasto-quinone C stayed near the origin in these systems.T'hey could not he estimated quantitatively becausesome PQ C was masked by chlorophyll. Vitamin K,ran so close to PQ A in this systeim that it was hardto separate tllem1. In general, it couldl be said thatmixtures of heptane-benzene or isooctane-benzenewere best for separating fl-carotene frcml plasto-qulinones.

A general conclusioin about using solvents forchloroplast quinone separation by thin layer chroma-tography was that no single solvent was equally goo(dfor the separat'on of all 9. However, by addingheptane or isooctane to chloroform or benzene, solventsvstems could( he deveeloped xvhich favored the putrifi-cation of a single quinone.

flhe thin-layer plates, were developed to a iheighltof at least 15 Cmil. After removal from the tank, aplate wNas allowed to dry for 5 minutes. Thlen itsmajor part was covered with a gla,sis plate t(--o protectit during sprayinag.

Plastoquinone bands were identified by spray ing1 of the 2 developed spots in the corners of the platewith reduced methylene blue spray. A stock solutionof 1 ni- metllene blue chloride in d&ionized wvatercould be kept indefinitely at roomn temperature. T"ento 15 minutes before use, 30 mil stock solution wvasput into a 50 ml Erlenmieyer flask and 1 to 2 g zincdust alnd 2 to 3 ml concentrated sulifuric acid were

added. Trhe spray mnixture was shaken for 1 minuteor uintil blue color (lisappeared. This meaint tllat themethyleine blue chllor'ide hlad been suffi cientlyv reduced.The spray was noxw ready for use btut such freshlyprepared reduced metllylene blue was unstable andwas likely to stain the background of the plate. Itwas allowXed to age with the zin,c dust and sulfuricacid for 5 to 10 minutes. The leucomethylene bluesoluto was filtered into a spray bottle through glasswool to remove zinc (lust particles. Such aged sprayrenlainedi useabhe for 30 minutes. After this tirme,

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BARR AND CRANE-DISTRIBUTION OF PLASTOQUINONES

the reduced methylene blue gradually changed backto its blue oxidized formm. It was useless for plasto-quinone identification at this stage because it againstained the background of the plate blue.

Properly prepared and used, reduced methyleneblue spray gave blue spots on a white backgroundwith all plastoquinones and tocopherylquinones.Vitamin K gave a yellow spot after spraying whichgradually turned blue in 2 to 10 hours. Othersubstances such as a-tocopherol could sometimes giveblue spots with reduced methylene blue so that plasto-quinone standards were applied alongside the samplesto aid in their identification.

After a tentative identification of plastoquinoneswith reduced methylene blue spray, the various bandscorresponding to standard blue spots were scrapedoff the plate, suspended in 3 to 5 ml absolute ethanol,and, centrifuged in a clinical table model centrifugeat 200 X g for 5 minutes to remove the silica gel.

Extraction with ethanol gave best quinone re-covery from silica gel. To be sure that all of thequinone had been eluted, the silica gel could be re-washed by suspending it in 5 ml more of absoluteethanol and recentrifuging. The recovered ethanolicfractions could be pooled, concentrated by evaporation,and red'issolved in a known amount of absoluteethanol for spectrophotometric assays of plasto-quinones.

Oxidation of Redulced Plastoquiinonzes on Columns.If hydroquinones wvere pre-sent, they were oxidizedon the alumina or silica gel columns by the longmethod. On thin layer chromatography they werenot oxidized in the 30 to 40 minutes require(d for thedevelopment of a plate. If they were not oxidized,then values for plastoquinone A were low. Possiblyrelatively high values for iPQ C in such a case couldbe derived from contamination of PQ C with thehydroquinone of PQ A. Therefore a separate platewas run with an aliquot of sample and sprayed withferric ehloride-dipyridyl reagent (equal amounts of0.2 % ferric chloride in absolute ethanol an(d 0.5 %2,2'4bi-pyridine in the region where PQ C is found.Olf course, a-tocopherol was detected by this pro-cedure as a pink spot between PQ B and PQ C inmost solvent systems.

Spectrophotometric Assay of Plastoquinones. Aspectrophotcmetric assav as previously described(7, 8) was used for quantitative determination ofplastoquinones. Quinone impurities could often bedetected by variations in the spectra. The absorptionmaxima of the various plastoquinones are at 255 m,uand of tocopheryl,quinones between 260 to 270 mnu.The various plastoquinones all have the same spec-trum. a-Hydroxy plastoquinone A has a spectrumlike y-tocopherylquinone.A single assay in a Beckman DK-Rezording

Spectrophotometer was done as follows: to 0.5 mlof ethanolic solution fronm a blue band from a silicagel plate, 0.5 ml absolute ethanol was adided, and itsspectrum taken in the 220 to 340 mi region againstan absolute ethanol blank. A pinch of potassium

borohydride, KBH4 was added, the suspension wasshaken 5 seconds, and its spectrum recorded againover the same UV region. Aifter 2 minutes, thespectrum was retaken without adding more boro-hydride to make sure that all of the quinone had beenreduced. If isosbestic points of plastoquinones werenot at 232 and 276 mu one of the following reasonscould account for it: A) too much or too littleborohydride added, B) sample not concentratedenough, C) impurities present in sample to changeisobestics or give additional peaks. In the lattercase, the sample had to be restreaked on a silica gelplate.

The changes in absorbancy induced by the addi-tion of borohydride at 255 m/u could be used as aquantitative test to calculate the amount of plasto-quinone present in the extract. The d4iference inabsorbance between' oxidized and reduced forms at255 mp was divided by the millimolar extinctioncoefficient (e) to obtain micromoles of quinone perml of sample in the cuvette (or nmoles per liter).

umoles PQ = AA oxid-red 255 m,u

AeThe Ae (255 m,u, oxidized minus reduced) for anyplastoquinone was 15.

The procedure for extraction outlined to thispoint was referred to as "the long method'. It took5 days to extract and purify quinones from 1000 mlchloroplasts containing 2500 mg chlorophyll.

Where it was not necessary to obtain largeamounts of purified quinones but only assay theamount present in an extract, a time-saving variantof the long method 'was done as follow,s: A) 1 or 2ml of original extract suspended in heptane wasstreaked on the middle section of a long :chroma-tography plate (40 X 20 cm) coated with silica gelGHR, B) 0.1 to 0.2 ml additional extract was spottedin corners of plate, C) chromatogram was developedin an appropriate solvent system, D) plate vasallowed to dry, E) the middle section was coveredwith a glass plate, F) 1 corner spot was sprayed withmethylene blue s1pray to identify oxidized plasto-quinones and tocopheryl,quinones, G) the spot in theother corner was sprayed'with ferric ichloride-dipyridylspray (equal amounts of 0.2 % iferric chloride inabsol-ute ethaniol and 0.5 % 2,2'-bi-pyridine in abso-lute ethanol mix'ed just before use) to identifya-tocopherol and reduced quinones, H) all bands ofthe covered section corresponding to blue or pinkspots of the sprayed areas were scraped, I) quinoneswere eluted with absolute ethanol and samples cen-trifuged to remove silica gel, J) spectrophotometricassays on each sample were run, K) micromolesquinone per 1 or 2 ml of original extra'ct were calcu-lated, L) total was multiplied by dilution factor, 25,for example, if 1 of 25 ml of original extract wasused. This shortcut procedure was more accuratethan the long method with chromatography columnsbut the amounts of purified quinones obtained weresmall.

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PLANT PHYSIOLOGY

\VNhen it wTCas desirable to coimipare the 1relativeamotunit of quinones in many similar samiples. sucli asclloroplast extracts from various plants, the shortor spot method was the best to use. A) Pure samplesof staindard plastoquinones A, B and C wN-ere preparedand suspended in a known amount of sol-ent, P)junioles PQ per ml were determined by spec-trophoto-mletric assay, C) 2 dilfferent concentrations of eaclhstandiard were spotted on a long chromatographyplate with a known aamount of several tinknlkiowlextracts, D) after plates had been developed andsprayed wx ith imethylene blue reagent, sizes of spotswere conipared visually or w-ith a densitometer. Withpractice it was possil)le to tel' whether the uinknowncontailned as mucih, lmore, or less of each plasto-qunAlone as the standard spot, E) amiount of eachquinonie fromi the unknowiin wxas calculated by com-parison ag-ainst standards. The short method allowedqtuanititative detemiination of 4 iplastoquinones in anunstr-eaked sam-ple. It reduced total handling tirmeper sample to 4 to 8 hours.

Special Procedtres for Fractioniationt of PQ Band PQ C. Griffiths et al. (4) showed that PQ Banid PQ C could each be resolved into 6 separateentities designated B1-B, and C1-C, by 2-dimensionalthini layer chromatography, A simple procedure forthis wvas as follows: A) lipid extract from spinaclhor any other green plant was streaked on a thin layerplate. B) the plate 'was developed in heptane-benizene

(40 :60) or chloroformi-heptane (60 :40), C) thequinone hand belcx\v PQ A xw hich had been identifiedby leco-methvlene lbltue spray as paler than the spotfor PQ A was scraped and eltted. D) eluate wasconcentrated( bx evaporation, E ) PQ B extract wasapplied as a streak on another tlin layer plate anddeveloped in 15:85 helptanie-l,enzeiie, F) ipart of theband or a separate spot was !spraved witlh leuco-methylene blue spray to locate the 3 blue PQ Bband; xx-hiclh separated in this solvent system, G) thesePO B bands were scraped separately, eluted withetihanol and concentrated, H1) each PQ B fractionwas spottedl on a thlird thin layer plate with standard'sof PQ A anid C anid developed in diiso,propyl ether-petroleum ether (12:88). Wihen developed this plateshowed 2 blue spots from each of the 3 samples oraltog,etiher 6 formiis of PQ B.

To fractionate PQ C, the third and fourth bluebands below PQ A fromii a thin layer plate developedin ichloroform-heptane ('97 :3) or the PQ C fractionfrom an alumiina column xvere taken. The presenceo,f chloropyhyll directly below PQ C in chloroform-heptane (97 :3) made it easy to locate the PQ Cbands. PQ C extracts were aipplied as a single spotto another thin laver plate and developed in diiso-propyl ether-benzenie (15:85) in both directions asany 2-dimensionlal chromatogram. A series of 6 bluespots diagonally aCross the plate could be seen afterspraying wvith leticomethylene blue spray.

Table I. Con 4parison betwecn Plhisoquinbone Values by the Lonig anid th/ Shlort ProceduresThe 3 samples shown in each column represent separate extractions and determinlations from January spinach

containinig 347 mg total chlorophyll each.

Long method Slhort method80:20* 15 :85** 80:20*

Plastoquinone ,Linoles PQ/mg chlorophyll tmoles PQ/mg chlorophyll

A 0.03 0.03 0.03 0.04 0.03 0.04 0.12*** 0.04 0.03 0.04 0.12***B <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01C (C1 to C4) 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.02D (C, + C6) 0.003 0.003 0.002 0.004 0.005 0.003 <0.01 0.004 0.004 0.003 0.01

* 80:20 designates solvent system chloroform-isooctane.*1 15:85 designates solvent system heptane-benzene.*8* Sample from a separate extraction of late February spinach containing 217 lmg Lotal chlorophyll.

Table II. Recovery of Added Plastoquinone A by the Short lIssay P-oh-edurzeSolvenit system was 97 % chloroform-3 %, heptane for development of tllin layer plate.

Volumeapplied to

plateSample

Amount ofPQ A found

,umoles

Amount ofPQ A adde

ymoles

f Difference Percentd from amounit recovery

in extract of added PQ A

Extract 0.01 0.0255 ... ... ...Extract plus 0.005 0.018 0.019 0.006 100 %12 ,umolec PQ A 0.01 0.037 0.038 0.012 100 %

0.02 0.084 0.076 0.032 133 %0.03 0.121 0.114 0.043 120 %0.05 0.255 0.185 0.125 208 %*

* At excessively high levels of quinionie the plate becomes overloaded and spreading of spot gives high values. Totalvoltlimie of sample vas 10 ml.

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BARR AND CRANE-DISTRIBUTION OF PLASTOQUINONES

Table III. R. Values of Plastoquinones A, B, C and D, Vitamint Kj, Cocetzymiies Q10 and Q6, and a-Tocopheryl-quinone in Various Solventt Systems

No. Solvent systemDielectricconstant PQ A PQ B PQ C

RF ValuesVit.

PQ D K1 Co. Q1l, Co. Q6 a-TQ PQ A 20*

1. Heptane-benzene (15 :85)2. Chloroform-heptane (97:3) ...

3. Chloroform-isooctane (80:20) ...

4. Diisopropylether-benzene (20 :80)5. Pentane-diethvlether-acetic acid 2.4

(70:30:2)6. Pentane-ethanol (100 :0.5) ...

7. Pentane-ethanol (100 :0.8)8. Peintane-ethanol (100:2) ...

9. Chloroform-cyclohexane-methanol ...

(90:10:5)10. Chloroform-cyclohexane (80:20)11. Methanol 32.612. Methanol-pentane (90:10)13. Methanol-cyclohexane (60:40)14. N-amyl acetate ...

15. Diisopropyl ether16. Pentane-diisopropylether (90:10) ...

17. Petroleum ether-benzene-ethanol(80 :30:7.2)

18. i\Iethanol-pentane-acetic acid(80:20:1)

19. Pentane 1.820. Ethyl acetate-chloroform (90:10) ...

21. Chloroform-ethyl acetate (90:10)22. Tertiary butyl alcohol 11.723. Isopropanol 18.124. Ethanol 24.325. Heptane-etlhanol (90:10) ...

26. Pentane-tertiary amyl alcohol 3(85 :15)

27. Benzz!ne-methanol (95 :5) 328. Methanol-dichloroethane-water ...

(100:18:20)29. Pentane-ethanol (50:50) ...30. Toluene-ethvl formate-formic acid 10

(50:40:10)31. Pentane-ethanol (100:1)32. Cyclohexane 233. Cyclohexane-chloroform (60:40) ...

34. Chloroform-cyclohexane (60:40) ...

35. Benzene--

0.770.920.910.950.98

0.680.940.830.970.98

0.190.570.410.810.78

0.15 0.72 0.350.48 0.83 0.690.35 0.88 0.570.71 0.88 0.850.72 0.95 0.85

0.20 0.08 0.33 0.00 0.29 0.330.57 0.58 0.47 0.00 0.73 0.240.71 0.81 0.36 0.27 0.79 0.550.92 0.91 0.87 0.74 0.87 0.92

0.770.610.690.620.850.870.850.93

0.770.640.600.530.92

0.870.89

0.350.770.820.670.890.790.210.63

0.27 0.71 0.52... 0.71 0.73

0.73 0.67 0.63... 0.60 0.61... 0.84 0.88

0.73 0.81 0.770.13 0.77 0.440.57 0.85 0.76

0.59 0.54 0.63 0.71 0.57 0.59

0.070.890.900.730.650.800.950.99

0.470.920.950.710.630.810.950.98

0.270.890.87

0.640.810.950.97

0.00 0.09 0.00.-. 0.85 0.89

0.82 0.85 0.89... 0.65 0.70... 0.57 0.63... 0.74 0.79... 0.91 0.95... 0.97 0.99

0.95 0.93 0.87 ... 0.66 0.920.78 0.77 0.66 ... 0.79 0.73

0.99 0.99 0.96 ... 0.97 0.990.92 0.91 0.87 ... 0.81 0.91

0.780.070.370.57IA

0.700.000.210.50

-1

0.080.000.100.18n

0.47 0.85 0.210.00 0.47 0.000.07 0.31 0.150.15 0.51 0.28Ali Ai6 M

0.270.670.540.790.78

0.050.290.230.340.45

0.70

0.89.. .

* *

0.00 0.000.17 0.200.47 0.230.&i9 0.73

0.450.780.820.630.830.690.330.69

0.250.770.810.690.750.480.150.48

0.72 0.73

0.000.850.830.670.610.780.930.98

0.000.820.550.680.570.790.890.98

0.89 0.730.69 0.41

0.97 0.970.83 0.74

0.170.000.130.25n9^

0.530.000.400.08nA

. ..

. . .

. . .

n<~~~~~~~~~~~~~~~.V V... .V _.I It vw.1V v*U.sV- v.Jv_ v."ov.vv v.U0

* Synthetic PQ A 20 standard.

Table IV. Relative Amounts of the Various Types of Plastoquinone C Found in Spinach and aTomato Mutant (hp)

Spinach Tomato (hp),umoles PQ C Percent of IAmoles PQ C Percent of

Plastoquinone C per mg chlorophyll total PQ C per mg chlorophyll total PQ C

typePQ C1 0.001 5 0.009 30PQ C2 0.004 20 0.0045 15PQ C3 0.008 40 0.003 10PQ C4 0.002 10 0.0045 15PQ C5 0.001 5 0.0015 5P0 c6 0.004 20 0.0075 25

1251

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PLANT PHYSIOLOGY

ResultsA comnparison of the amounts of plastoquinones

in a sample of spinach chloroplasts determined by thelong and short method is Shown in table I. Reli-ability of the assay procedures is indicated by theresults with triplicate anIples shown in table I.Recovery data for the short method are shown intable II. Results with 3 separate solvent systemswere similar to the example shown.

Table III -shows RI, values for plastoquinones A,B, C and D, a-tocopherylquinone, vitamin K, andcoenzymes Q10 and Q6 in 35 different solvent systems.For isolation of the 4 plastoquinones on a single thinlayer plate, solvent systems chloroform-heptane(97:3), chloroform-isooctane '(80:20), heptane-ben-zene (15:85), or heptane-ethanol (95:5) are recom-mended. Figure 1 shows a diagram of spinach lipidextract separated in chloroform-isooctane (80:20).

For special ppurposes, such as fractionation ofPQ B and PQ C into 6 components, solvent systemsused by Griffiths et al. (4) are best. Figures 2and 3 illustrate the separation of PQ B and PQ Cfrom spinach into 6 different types each. Figure 4shows the separation of 6 different types of PQ Cfrom a mutant tomato (hp). Table IV shows therelative concentrations of each type of PQ C inspinach and tomato chloroplasts. Figure 5 shows

Cq7 c3 CJ(Q'7c

0 90 i? 9

$-CAROTE

POA 45

p

P0B

88 8

ORIGIN * . *B B1 B3 BS

82 B4 B6FIG. 2. Various types of plastoquinone B separated

in diisopropylether and petroleum ether (12:88).cochromatographic separation of PQ C2 and C. fromspinach and the complete PQ C series !from high-pigment tomato. Filled circles designating overlapbetween PQ C2 and PQ C3 from the 2 plants indicategreater color density.

Discussion

Plastoquinones A, B and C found in spinachohloroplasts can hbe extracted with lipid solvents bya nuniber of different procedures. We have de-scribed a long and a short method for separation and

e-TOCOPHEROL (withterric chloride spray)

UNKNOWN OUINONE

ODOoPOC

49=:)<> =) 4=), POD

JI

a-TO

BREAKDOWN

PRODUCTS AND

CHLOROPHYLL b

O * *I

ORIGIN

FIG. 1. Chromatogram of lipid extract from spinachin chloroform-isooctane (80 :20).

I

C'

C2

C3

C4

C5

C6

0 ORIGIN

FIG. 3. Six forms of spinach plastoquinone C sepa-rated in diisopropylether-benzene (15 :85) in both di-

rections.

00

1252

* * .0

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BARR AND CRANE-DISTRIBUTION OF PLASTOQUINONES

Ci

C2C3

c4C5

C6

I ORIGIN

FIG. 4. Six forms of tomato plastoquinone C fromhigh pigment mutant (hp) separated in diisopropylether-benzene (15 :85) in both directions.

CI

C2C3

C4C5

C6

ORIGIN

TOMATO ANDSPINACH. PQC APPLIED

ORIGIN

SPINACHPQC APPLIED

FIG. 5. Cochromatographic separation of tomatoplastoquinone C1-C. in presence of spinach plastoqui-none C, anid C3, Solvent diisopropylether-benzene(15 :85) in both directions. Plastoquinones C2 and C.are the major compoinents in the preparation previouslydescribcd as plastoquinone C.

assay. The long procedure is ibest for 'handling largevolumes of chloroplast extracts (up to 2,500 mg

chlorophyll) when the purpose is to obtain largequantities of purified quinones. The short methodis better for assay purposes.

We tested the reliability of the long versus shortassay procedures on 3 replications of spinach chloro-plasts. The differences within replications did notexceed 25 % but there was a loss of u,p to 25 %PQ A on aluimina compared to data from thin layer

plates by the short method (table I). The Januaryspinach used for the 3 replications 'contained onlv a

third of the expected amount of PQ A normally found

in spinach (0.1 mole/mg chlorophyll). A laterpreiparation done by the short method showedl 0.12mole PQ A/mg chilorophyll. Recovery data with 12,Ixmoles PQ A added to chloroplas'ts (143 mg chloro-phyll) before extraction by the long method gavean 82 % recovery.

The amounts of PQ C found by the long methoddid not differ from amounts found by the shortmethod (table I) but recovery data withl 6 umolesadded PQ C to chl'oroplasts before extraction resultedin 39 % overall recovery. The added PQ C was notthere at the end of extraction. Its disappearancecannot 'be explained as a loss on alumina becausefrom 0.06 ,umole 'pure PQ C put on 80 g deactivatedalumina 53 % was recovered. It is possible that ailenzyme or oth-er substance in whole chloroplasts de-stroved the added PQ 'C while the ch'loroplasts werebeing extracted on a reciprocal shaker for 1 hour.

Thin layer chromatography of plastoquinones fallsinto 3 categories: A) recognition and isolation ofall 4 plastoquinones on a single plate, B) )urificationof a single quinone from the lipid extract of chloro-plasts, and C) special procedures for fractionationof PQ B and C into 6 components eaclh. For (A)w-e prefer chloroforim-heptane (97:3), heptane-ben-zene (15:85), pure benzene, or chloroform-isooctaiie(80 :20). Combinations of Iheptane and benzeneseparate the PQ B region into 2 or 3 (listinct bands,but PQ 'C mixtures stay close to the chlorophyllsnear the origin and must he alilowed sufficient t.meto resolve from each other and the chlorophylls.(B) is a matter of personal preference. Individualsolvent systems should be chosen to accomplish thedesired results (table IV). (C), the resolution ofPQ B and C into 6 different 'forms each, can beaccomplished 'by the methods of Griffiths et al. (4).Thev report the presence of plastoquinones B1-Bsand C1-C6 from Ficuis and 8 other species of plants.We find themn in -spinach and tomato. According tothese authors (4), their PQ 'C1-C3 is equivalent toHenninger and Crane's PQ C while PQ C.,-Cr, isPQ D.

Examination of PQ C and D prepared fromspinach in this laboratory by means of the solventsystem's employed by 'Griffiths et al. (4) revealedthat PQ C has 2 major components equivalent toPQ C., and C3. These are accompanied by 2 lesserquinones -which can be designated PQ C1 and C4.PQ D prepared 'by us from spinach consists mainlyof PQ C6 with traces of PQ C5. The nomenclatureof Griffiths et al. (4) thus appears to be more pre-cise and we propose to abandon the terim PQ D.PQ C from high pigment tomato, a mutant for higherthan normal amounts of ,8-carotene and chlorophyll,,gives more PQ C1 and C4 than are found in spinach.

Acknowledgment

We thank Dr. MI. L. Tomes for providing high-pigment tomato 1k aves and Mrs. M. Wetzel and Mr.J. Logan for assistance in preparation of plastoquinones.

1253

e""'%

.01% I...)

I :

f'. :

."'P",1-1

I

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PLANT PHYSIOLOGY

Literature Cited

1. BUCKE, C., R. M\. LEECH, M. HALLA\\AY, AND R.A. -MORTON. 1966. The taxonomic distributionof plastoquinone and tocopherolquinone and theirintracellular distribution in leaves of lVicia fabaL. Biochim. Biophys. Acta 112: 19-34.

2. CRANE, F. L. 1959. Isolation of two quinioneswith coenzvme Q activity from alfalfa. PlantPhv-siol. 34: 546-51.

3. EGGER, K. 1965. Die Verbreitunig v-oIn VitaminK1 and Plastochinon in Pflanzen. Planta 64:41-61.

4. GRIFFITHS, WV. T., J. C. \\ALLWORK. AND J. F.PE-NNOCK. 1966. Presence of a series of plasto-

quinones in plants. Nature 211: 1037-39.5. HENNINGER, IA. D. AND F. L. CRANE. 1964. lso-

lation of plastoquinones C and D fromii spinachchloroplasts. Plaint PhVsiol. 39: 598-6q2.

6. HENNINGER, AL. D., R. B,ARR, AND F. L. CRANE.1966. Plastoquinone B. Plant Phvsiol. 41: 696-700.

7. KEGEL, L. P., Al. D. HENNINGER,, ANXD F. L. CRANE.1962. TNxvo new quinoines fromii chloroplasts. Bio-chem. Biopliys. Res. CoIm1muin. 8: 294-98.

8. KOFLER, AL., A. LANGEMANIN, R. RUEGG. L. H.CHOPARD-DIT-JEAN, A. RAYROUD, AND 0. ISLER.1959. Die Structur eines pflanizlicheni Chinonsmit isoprenoider Seitenkette. Helv. Chliml. Acta42: 1283-92.

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