Coenzyme Q. XX. Isolation of coenzymes Q9 and Q10 from two Basidiomycetes

ARCEIIVES

OF

BIOCHEXISTRY

Coenzyme

ROBERT

From

AND

BIOPHYSICS

Q. XX. Isolation of Coenzymes from Two Basidiomycetes

E. ERICKSON,

the Merck

314-317 (1960)

90,

Sharp

Qs and Qlo

KEITH 8. BItOWn-, JR., DOh-ALD .~ND KARL E’OLKERS & Dohme

Research

Received

Laboratories,

Rahwa!l,

Yew

E. WOLF

Jersey

June 15, 1060

Prior examination of four Hasicliou~~gcetes did not, reveal the presence of any mem ber of the coenzyme Q group. However, further isolation studies have nox- )-ielded coCoenzymes Qg and Q~u were identified from enzyme Q’s from two Hasidiomycefes. Csfilago zea, and coenzgme Q9 from =Lgaricus ranpesfris. Evidence for the possible presence of plastoyuinone and bnsidioquinone was not, formd. INTRODUCTION

The members of the coenzyme & group which are now generally recognized (I) as having been isolated from nat)ural source materials are coenzymes Q,-Q,, (I). A closely related quinone, pln&oquinone (II), has been identified only from white and green tissue of higher plants (2). The coenzyme Q group is involved in mitochondrial

n = 10 = Q,a n = 6, etc. = Qa, etc.

eleckron transport,, while plastoquinone appears to be involved in photosynbhetic electron transport (2, 3). In their extensive study of t)he natural 314

distribut,ion of the coenzyme Q group, Lester and Crane (4) did not observe the presence of any member of the coenzyme Q group in their prelimiuary survey of four members of the Basidiomycetes; sporophores of Psalliota campestris, Coprinus atrammtarius, lago zea, and Polyporus sp. (bract).

C’sti-

This noteworthy absence of coenzyme Q was contrasted to the apparent presence of another quinone which was designated basidioquiuone. This latter quinone was indicated primarily by spect,ral observation. We have examined two Kasidiomgcetes and isolated coenzymes Q9 and Qlc from Ustilago xea and coenzyme Q9 from Agaricus campestris. The compounds were identified by comparison of their physical propert’ies with t)hose of aut,hentic coenzyme Qlo from beef heart, and coenzymc Q9 from Torzda yeast. The amounts are est’imated at approximately 0.05 pmole Qs/g. dry weight in Agaricrts campestris and 0.2 pmole Qlu and 0.02 pmole Q9jg. dry weight’ in C’stilago xea. r\;o evidence for the presence of plastoquinone was found, although one might’ not expect, to find this quinone in tissue which is not, associated with photosyllthesis. Furt.her, t,here was no evidence for the presence of another quinone which might correspond to basidioyuinone, ill t’hese isolations. The discrepancy could be due to differences in solwe material and/or isolation procedures.

EXPERIMENTAL ISOLATIOX

AiYD

RESULTS

QY Agaricus campestris OF

COESZPME

FI~OB~

To a mixture of i600 g. of common edible mushrooms (dgaricus campestris) and 7.2 1. of 1)5cb ethanol, blended into a paste in a Waring hlendor, was added 260 g. pyrogallol and the mixture was heated to reflux temperature. 9 solution of 000 g. potassium hydroxide in 1800 ml. water was slowI) addetl, and the cnt)ire mixture was hcatcd under reflus with stirring for 30 min. After cooling to room tcmperatjure, the mixtrtrP was extracted successivrly with 5.&, 1.8., 1%, and 1.8-l. portions of Skrllysolve B. The Pxtracts were cnn~ binrd and evaporated to dryness under vacuum t,o give 12.4 g. of nonsaponifinlrle lipid residue. This residue was dissolvPd in 200 ml. of warm Skrllysolve B and was kept at 5’C. for 10 hr. The precilritnte Lvhich formed MXS centrifuged, washed n-ith small portions of cold Skellysolve B until frrr of color. and discarded. The coml)inPd supernatant and washings were evaporated 1.0 dryness untlPr vacrtum, and the residtte was dissolved in $0 ml. of warm Skellysolve B. The precipitation at 5°C. \v:ts rPpPatrd. the supernntants and washings again bring comlrincd and takrn to dryness under “:,c,,,,,n.

Successive precipitations were t,hen carried out from 25, 10, 5, and 2.5 ml. acetone, respectively. l)y heating to dissolve the solublP residue from the pwvious prPcipitation and then cooling to O”C., thr precipitate bring washrd each time with rold awtone until colorless and the comhined supcrnatants arid washings being taken to dryness under vacttum. The weight of the solid from the evaporntiori of the tinnl supern:rt,ant and washings bvas 3ti:! mg. The :~cetonr-insolrtble rPsidues did not giw a positive LMB test,’ and probnl)lJwere quinonr frPc. The 3(i> mg. of impure quinone was dissolved in 3 ml. Skellyaolve B and applied to 15 g. Decalso in Skcllysolvc B in a 20.mm. glass column. Elution with 150 ml. Skcllgsolve B displaced only a white solid. A 200.ml. volume of 2(,;, cth,vl ethPr in Skcllgsolvc B elutetl a small amount of yellow matrrial, positive to t’he LMI3 test, md an adB ditional 200 ml. of 5( 0 ether in Skellysolve PlutPtl t,he main yellow band from the column, yictding -11’ mg. of yellow viscous oil. The ultra1 T,cwconwthylene blue is prepared by redtrcirrg of methylenr blue in 590 acetic a 0.1:; solution acid in Pthanol with excPss zinc dust. The matcrial to be tested is applied to filter paper and sprayed with the reagent,. The presence of quinones is indicated by the appearance of a blue spot.

violet spectrum of this oil in absolute ethanol. brfore and nftPr rrduction wit)11 sodium t)orohydride, was characteristic of PoenzJ-me Q and gavP 11” evidence for the presence of l,lastocl~~inone. 2 demonstrated the prcsl’aper chromatography ence of coenzyme Qy and thr alrs~ncc of plastoquinonP. ThP impure coerrzymP Q9 \vas dissolved in :L minimum volume of SkPllysolvc B and applied lo 2.5 g. E’lorisil in Skrllysolvr B in a T-nlrn. colrtmn. The Polumn was washed with Sketlq-solvr B (150 ml.) and elutrd with incrP:tsing nmourlts of cthrt in Skcllysolve 1~; 2’.; (80 ml.), 5’,; (100 ml.), 8’!;. (60 ml.), and 10’ ; (100 ml.). I~~lutiori of the, main yellow hand was :rchiPved with 35 ml. of 20’; ether and 60 ml. of 33’1, ether. Thr cPntjral fractions (1-l of thr 30 making ,111 the crntral band) were combined and evaporat Pd to dryness imdrt vacutun to give 11.2 mg. of orange solid. The solid was recr,vstallizPd from 1 ml. of at)solutP Pt~hanol t.0 give orangr rrystnlline clumps. m.p. 13.2-1-1.0” c. (con.), K: I:,,,. = 173 at 275 nip in isooct arIP. ‘I’hr~ pttre rompotmd was indisti,lguish:tl)le from coPnz\-mr QY in mPlting point, ultraviolP1, spcct rimi. and Iwhavior on paper chromatograma. The xllou~~t of coenzyme Qq in t,his species of mushroom, based on a yield of 10 mg. from the sample studied, a i0: 5 recovrrq-” and an 85’,; wat,er CoIltPnt, is estim:rtPd at :~pl~rosimntrl~~ 0.05 pmole/g. dr>- n-right. TsoLalYov

OF

Coslvzvnle

Q

FROM

Iktilago zca C~slilago zen (corn smut) w-35 grown in submerged culture, and the cells were harvested h> centrifugation. ThP wet cell rake was dried I)> slurr>-ing OIICP wit,h acetone, centrifuging, and then dying the rentrifugnte in a vacu~~m own. The acetone washings7 conceritrat,ed t 0 :I fr\\ litjets, and the dried cells \vPrP thrn PsaminPd separately for cwrrzyme Q. The dried cell cake (4560 g.) was ground to 60 mesh and extracte(l by stirring with successive lo.%, i.2, 3.&, and 3.61. portions of Skellysolve B at rcflus trmpPrature, cooling, and separating the solution. TIIP extracts werP comlrined and evaporated to dr,vnPss under vacuum to give 13.4 g. of tarry solid. The ~~11s w’re thrn cstractcd three times witjh 1-l. portions of boiling ahsolrite * Circular chromatography on Whatman No. 1 papPr imprrgnnted with Vaseline by dipping in 5’:C Vaaelinr solution in hexane and air drying; din~ethylformamidP as mot)ile phase; see page rt al. (5). 3 This recovery is tensed on that observed by Etgr

Pt

al.

(5).

ethanol. The ethanol extracts were combined and evapor:tW under vacuum to give lo%.9 g. of tarry residue. The Skrllysolve 13 extract was purified 1)J precipitation of impurities and chrom~togr:tph) as described above to give 219 mg. of yellow oil in which paper chromatography showed the presence of coenzyme Qlo plus a small amount of coenzyme Q!, (about 5P10’b; ). Spectrophotornrtric assuy (lb) of thr oil supported a cocnzyme Q ront,ent of (Xi’,,;, :tnd the amount of coenzyme Q in the Skellysolvc B eAract, assuming a TO”;, recover\-, is estimated at, 200 Ill#. Similar purification of the ethanol extract gave GO0 mg. material in which paper chromatography showed the prcscnce of mostly coenzyme QIe plus a trace (5-107;) of coenzyme Qy Spectrophoto+ metric LIL ~rwy, as above, supported a 20!‘;, roenzgme Q content, ant1 the total amount of rocnzyme Q in the dried acetone-washed cells, assl~ming a iO’,~j recovery, is estimat,etl at 100 mg. Sinre paper chromatography indicated that the quinoidal material in the et,hano1 extract was cssentially ident,ical t)o that, in the Skellysolve 13 extract, both materials were combined. A small amount, of the combined extract was placed on a paper chromatogram in a continuous circle anti developed until the coenzyme Q10 and coenzymc Qg were well resolved. The cocnzymes Q were lorated l)y ultraviolet :thsorpt.ion, and the entire coenzymc Q10 hand, the entire coenzymc Qy I~md, and some Vaselined paper from i he edge of the rircle, earh of npprosimatrl>the same Lveight, were cut. out and extensively elut,etl with boiling isooctnne. The eluates were taken to dryness under vacuum and dissolved in 3.5 ml. isooctnne, and the optical densit,ics at 107 mr wcrc rampared. The relst)ive opt,ical densitirs indicated that, of the total coenzyme Q which had been spotted on the paper, about 10Cf; xvas coenzymc Q3 and OO’jb was coenzyme QlO. Bilanized silica gel ((i) was prepared and dried under vacuum at 115”. To 150 g. of the adsorbent was added 30 ml. of the upper phase of I mistluc dimeth~lformurnidc~w:ttrr-isooct,anr (100:3:30 by volume). The mixture was vigorously shaken and slurried into a 30~mm. diameter gIass c~olumn in 200 ml. of t.he lower phase. The column volume was measllred as approximately 135 ml. A portion (158 mg.) of t,hc combined rxtract was added to this column in C, ml. of the lowe phase, and elut,ion of t hc column was carried out at the rate of 12.5 ml. (one fraction) per 15 min. .4 yellow color was notirr:tt,le in fractions SE. These frnct,ions were combined, an eqrull volume of water was added, and an extraction was made wit,h petroleum et her (3OMiO”). This extract, WRY

taken to d~~yness under vacuum. The solid old tnined gave a positive LMB test, ran at t,he front of n paper rhromntogram iR( .!IO-.98), showetl aho1rt 20’; of a qrlinonr absorbing at 275 rnp, mid probably represented :t degradation product of roenzyme Q. A strong yellow cwlor was present in fractions 18-110. The material was retracted as above from frnrtions 50, .53, 5(i, GO, ti3, 00, 70, 73, 7ti, and X0. and each solid xvas spotted OIL a paper rhronxtogram. Frnrtjions 50 and 53 ront.ained wenzyme Qy only, while fraction 5ci ront,aintd a very .s~nall amount of roenzyme Qlll , which increased to ii Iarge amount in fraction X0, while the coenzymr Q!, drrrcasrtl to essentially none in fraction 80. Fractions -18, -10, 51, and 5’1 were comhincd arid extracted with petroleum ether as ahove. TOP pxtracts were taken to dryness and dissolved fol rwryst allization in 1 .O ml. hot, :tl-wolutje ethanol. The corrrzyme Qy was ind\lccd to crystallize after bring seeded wit,11 a very small crystal and krpt in an ice-salt bath for several hours. The compound was ol,taine(l as orange clum~~s of rrysta1.q. m.p. $J.&43.3” irorr.), Ei’j’,,, = 150 at 35 rnr in ethanol, AIS:,“;,. at 2i5 111~= 131, indistinguishable from p\lre roenzyme Q!, on paper rhromatograms. Fractions 85AO were similarl>- treated, the cofrom 2.0 ml. enzyme Q,,, being recrystallixcd absolute ethanol. The coenzyme Q10 crystallized quickly. giving yello\r--orange clumps of rrystnls? m.p. 19.5-50.3” Ccorr.) E:‘:,,, = l(i8 at, 275 111~ in rthano1, AEh::,Y,,at, 2i5 rnp = IJO, irltiistirlgrlish:~l,Ir from coenzymr QIO on paper chromatograms. Thr acetone wash liquid \vas diluted with an equal volume of water and extracted t,wice \vith 7.2 and 3.C I. Skellysolvc B, resl)ectivcly. The ertrac t,s \vrre caombined and evaporatPt1 lo tlryricss und(lr vacuum to yield a tarry solid. neighing 114.9 g. l’urific>ation of this material by chrom:ltograph\- gave 1770 mg. of yellow oil. Spectrophotomet,ric assay supportrd an 18”; content of roenzyme Q, and the mnorlnt of cocnzyme Q in the total acetone washes, assuming a recovery of 70(,b, is estimat,ed to he 500 mg. A semiquantitative determination of the relative amount of cwnzymw Q, and Q,,, was made I)>- paper chrom:ltographJ- and ~~ltraviolct comparison at 107 nip as described above. The relative amolmts of rornz,vmrs Q8 and Qlll were found IO he 7 and %‘,l, rrspwtively. The total u-eight of coenzymr Q in the tissue examined is estimat,ed at 000 my. On the basis of the \vcight of the dried aretone-washed cells, the amount of coenzymes Qy and 4,” in I’x!iluqo zen is estimated at 0.02 and 0. 2 pmole/g. tlr~. wright., respectively.

The 1~0 major components of the fcrmentnt,ion mcdirlm (corn steep liquor and :I prot,ein hydrolyz:ite) were extensively estr.:tcteti with prt roleurn ether. The extra& contained very little material, none of which cordd he assigned by paper chromatography, chromatography on I>ecslso, or dtrnviolet spedra t0 being coerizyme Q. It ma?thlts be assumed that all coenzyme Q fotmtt came from the growing organism. REFERE?;CES

et Niophys. .-1c/n 33, 169 (1959); (h) I,INN, H. O., I'AGE. A. c., *JR., 'C!'OSG, Ji:. I,., (;AI>E, 1'. H., SJI~-SK, c‘. H., ANI) FOLKERS, Ii., J. .lm.

U~ett/. Sot. 81, -1007 (1959). 2. CRANE, F. I,., Plan! Ph!/siol. 34, 1% (1959). 3. JSISHOP, 1. J., P,oC. :VUl/. .-1CCd. hi. I'. s. 45,

IliRG (1959). 4. I,~+;sTER, 1t. I,., .4NI) CRANK, E'. I,., J. Bid. Chenz. 234, 2169 (1959). 5. PAGE, A. C., ,JR., (;ALh, I’. H., J
85, 471 (1959). G. VANDEN~C~VEI,, Anal. Chew

F. A., ANI) 28, 838 (1956).

\'AT~:IIER,

I).

II.,