1. Pigments extracted from reaction center particles of Rhodopseudomonas spheroides were separated and identified by means of thin-layer chromatography.\ud
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2. The results strongly suggest that bacteriochlorophyll a is the only bacteriochlorophyll pigment present in reaction centers.\ud
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3. Other compounds identified are bacteriopheophytin a, ubiquinone-10 and spheroidene

Barelds-van de Beek, P.G.M.M.

Beugeling, T.

Slooten, L.

English

1. Pigments extracted from reaction center particles of Rhodopseudomonas spheroides were separated and identified by means of thin-layer chromatography. 2. The results strongly suggest that bacteriochlorophyll a is the only bacteriochlorophyll pigment present in reaction centers. 3. Other compounds identified are bacteriopheophytin a, ubiquinone-10 and spheroidene

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Thin-layer chromatography of pigments from reaction center particles of Rhodopseudomonas spheroides

University of Twente Research Information

328 BIOCHIMICA ET BIOPHYSICA ACTA BBA 46405 THIN-LAYER CHROMATOGRAPHY OF PIGMENTS FROM REACTION CENTER PARTICLES OF RHODOPSEUDOMONAS SPHEROIDES T. B E U G E L I N G ,  L. S L O O T E N  AND P. G. M. M. B A R E L D S - V A N  D E  B E E K  Twente University of Technology, P.O. Box 217, Enschede (The Netherlands) and Biophysical Laboratory of the State University, Schelpenkade I4 A, Leiden (The Netherlands) (Received J u n e  5th, 1972) SUMMARY I. Pigments extracted from reaction center particles of Rhodopseudomonas spheroides were separated and identified by means of thin-layer chromatography. 2. The results strongly suggest that  bacteriochlorophyll a is the only bacterio- chlorophyll pigment present in reaction centers. 3. Other compounds identified are bacteriopheophytin a, ubiquinone-Io and spheroidene. INTRODUCTION During the past few years a number of papers have been published, dealing with the isolation of photochemically active reaction center particles from photo- synthetic bacteria (e.g. refs 1-6). In these particles the light-harvesting bacterio- chlorophyll and most of the other accessory pigments such as carotenoids, have been removed. The particles still contain specialized bactefiochlorophylls, P8oo and P87 o (ref. 7), of which the latter is reversibly photooxidizable ~. A pigment absorbing at about 756 nm, which was assumed to be bacteriophosphytin ~,~, and ubiquinone4,6, 9 are also present in the preparations. I t  has recently been concluded that K3Fe(CN)¢treated chromatophores from Rhodospirillum rubrum and K~IrCle-treated chromatophores from R. rubrum and Rhodopseudomonas slbheroides, in which the light-harvesting bacteriochlorophyll had been destroyed, contained bacteriochlorophyll a as the only type of bacterio- chlorophyll pigment 1°. In that  s tudy extracts of the particles were analyzed by means of thin-layer chromatography and spectra were recorded of the isolated pigments. However, the visible spectrum of reaction center bacteriochlorophyll showed, in addition to the characteristic absorption maxima of bacteriochlorophyll a, prominent peaks at 433 and 675 nm. It was assumed that the isolated reaction center bacterio- chlorophyll was still contaminated with one or more degradation products from the bulk bacteriochlorophyll, present in the rather impure particles. The present chroma- tographic investigations were carried out with purified reaction centers of Rps. * A l t h o u g h  t he  absorp t ion  peaks  nea r  8o0 and  865 n m  axe c o m m o n l y  called P8oo and  P87o, t h e y  poss ib ly  are more  p roper ly  descr ibed as  t he  jo in t  p rope r ty  of a se t  of closely in t e rac t ing  molecules  in t he  reac t ion  center,  and  no t  as t he  sepa ra te  man i f e s t a t i ons  of i n d e p e n d e n t  molecules  (see ref.  8). Biochim. Biophys. Acta, 283 (1972) 328-333 PIGMENTS FROM REACTION CENTERS 3 2 9  spheroides in order to elucidate this problem and in order to identify other pigments which are present in these preparations. The results strongly suggest that  the pigments of both P8oo and P87o are identical with bacteriochlorophyll a. Further, it was found that  the ubiquinone present in the reaction center particles is ubiquinone-io. Bacteriopheophytin a and spheroidene were also isolated with the help of thin-layer chromatography. MATERIALS AND METHODS Reaction center particles were prepared from chromatophores of Rps. spheroides as previously described by one of us 6. Briefly, chromatophores were incubated with 0.3 % sodium dodecyl sulphate and layered on a 0.5 M sucrose solution. After centri- fugation at 200 ooo × g, the reaction center particles were isolated from the super- natant  layer. They were further purified by treatment with Triton X-Ioo and urea at pH IO, followed by a sucrose gradient (o.I-I  M) centrifugation at 20o ooo x g. The reaction center particles obtained in this way were called AUT-RC particles. The solvents used for extraction and chromatography were of analytical grade (Merck and BDH). Ubiquinone-6 and ubiquinone-Io were purchased from Sigma. Methanol extracts for chromatography were made from freeze-dried particles which were kept in the dark. The extracts were prepared just before chromatography. The adsorbent used for thin-layer chromatography was Kieselgel G, obtained from Merck. Thin-layer plates were developed in the dark. The spotting of the samples and elution of the spots from the scraped off adsorbent layer was carried out in dim light. Spectra were recorded with a Cary Model 14 R spectrophotometer equiped with a scattered light transmission assembly. RESULTS AND DISCUSSION The absorption spectrum of an AUT-RC preparation from Rps. spheroides is shown in Fig. IA of ref. 6. The spectrum shows a low absorption around 685 nm, proving that  the reaction center particles contain only small amounts of degradation products from the bulk bacteriochlorophyll, as compared to the preparations used before 10. A good separation of the pigments from AUT-RC particles was obtained with thin-layer chromatography in which a mixture of light petroleum, acetone and n-propanol (see the text  of Fig. I) was used as the mobile phase. This solvent mixture is a variation of a solvent system given by Lynn Co and Schanderl n for the separation of plant pigments. Fig. I shows the result of a separation. A bright blue spot is indicated with R. In a separate experiment, extracts of both AUT-RC particles and whole cells of Rps. spheroides were chromatographed on the same plate. In this way it was estab- lished that  the RF value of spot R was the same as that of bacteriochlorophyll a. I t  must be noted here that  in the experiments with KsFe(CN)e- and K2IrC1 e- treated chromatophores mentioned above 1°, three different combinations of mobile Biochim. Biophys. Acta, 283 (1972) 328-333 330 T. BEUGELING et al. y 0 0 o y o r ~ BPh b ,  ~ R  g , °l o 4  t , i .2 0.1 4 0 0  5 0 0  c o o  7 0 0  h ( n m )  4 i J 8 0 0  Fig. I. Thin-layer chromatography of a methanol extract from AUT-RC particles of Rps. spheroides. Adsorbent: Kieselgel G (Merck), not activated, 0. 3 mm thickness. Developing solvent: light petroleum (boiling range 60-80 °C) -acetone-n-propanol (80 : IO : 5, v/v/v). Developing time: 30 min. Indicator: none. The methanol extract was spotted on the place of the lowest spot; the solvent front is shown at the top of the drawing. R, reaction center bacteriochlorophyll. BPh, bacteriopheophytin a. The spots of R and BPh are blue and purple, respectively. Yellow spots are indicated with y. An orange spot and an orange band are indicated with o. A green spot, a blue spot and a reddish spot are indicated with g, b and r, respectively. Fig. 2. Absorption spectrum of reaction center bacteriochlorophyll in acetone, isolated alter preparative thin-layer chromatography of a methanol extract from AUT-RC particles of Rps. spheroides. and  s ta t ionary  phases were used. Also in these cases the RE value of the blue spot was the same as tha t  of bacteriochlorophyll  a. The absorpt ion spect rum of the isolated blue p igment  is given in Fig. 2 and  peak max ima  and  relat ive in tensi t ies  are l is ted in Table I, together with peak char- acteristics of bacteriochlorophyll  a. The most  plausible in te rpre ta t ion  of our da ta  is tha t  the blue spot on the th in- layer  chromatogram was bacteriochlorophyll  a, and  tha t  this compound is responsible for the peaks near  80o and  865 n m  in the reaction center  preparations.  The spectrum of Fig. 2 does not  show addi t ional  absorpt ion peaks at  675 and 433 nm. This proves tha t  the peaks at these wavelenghts  found in earlier experi- ments  1° in fact or iginated from degradat ion products  of the bulk  bacteriochlorophyll ,  present  in the crude s ta r t ing  material .  A quan t i t a t ive  analysis  of reaction center  bacteriochlorophyll  could not  be made since the yield of recovery of bacter iochlolophyll  from cells, chromatophores and  react ion centers was low and  rather  variable (about 30 %). A small  blue spot on the chromatogram, shown in Fig. I, which has a lower RE value t h a n  react ion center  bacteriochlorophyll,  is marked  b. Such a spot was also Biochim. Biophys. Acta, 283 (1972) 328-333 PIGMENTS FROM REACTION CENTERS 3 3 1  TABLE I P R I N C I P A L  L I G H T  A B S O R P T I O N  M A X I M A  A N D  R E L A T I V E  I N T E N S I T I E S  I N  A C E T O N E  F O R  I S O L A T E D  REACTION CENTER BACTERIOCHLOROPHYLL AND BACTERIOPHEOPHYTIN a Values from the literature for both bacteriochlorophyll a and bacteriopheophytin a are given for comparison. Maxima of bacteriochlorophyll a and bacteriopheophytin a (literature) measured in ether, are also given (BChl, bacteriochlorophyll a; BPh, bacteriopheophytin a). Pigment ~rnax (nm) and relative intensities (in parentheses) Reaction center bacteriochloro- phyll from AUT-RC particles 359 (99) 392 (67) 57 ° (25) BChl (ref. 19) 358 (97) 390 (58) 577 (28) BChl from Rps. spheroides 358 (98) 390 (sh) 578 (27) (ref. 20) BChl from R. rubrum 36o (76) 392 (56) 57 ° (23) (in ether) BPh from AUT-RC particles 357 (IOO) 382 (58) 52o (27) BPh from Rps. spheroides 357 (ioo) 385 (52) 523 (27) (refs 19, 20) BPh from R. rubrum 357 (IOO) 387 (51) 528 (23) (in ether, ref. 21) 705 (17) 77 ° (IOO) 773 (IOO) 771 (IOO) 71o (II) 773 (IOO) 616 (8) 676 (15) 747 (59) 675 (II) 745 (48 ) 616 (4) 680 (14) 75 ° (61) found after  chromatography  of extracts  f rom whole cells and crude reaction centers 1°. Only  small amounts  of this pigment  were observed. This seems to  exclude the possi- bil i ty tha t  it has an impor tan t  role in photosynthesis .  I t  is conceivable tha t  the compound is a degradat ion product  of bacteriochlorophyll  a. The green p igment  g in Fig. I m a y  also be such a degradat ion product.  According to Clayton and co-workers s purified reaction centers of Rps. sphe- roides contain fixed amounts  of bacter iopheophytin.  The absorption band  at 756 nm in our AUT-RC particles also indicates the presence of bacteriopheophytin.  This was confirmed wi th  thin-layer  chromatography.  A purple spot was observed on the chromatogram of Fig. I ,  which is marked BPh. The spectrum of the pigment  is given in Fig. 3 and peak characteristics are listed in Table I, together with values for bacter iopheophyt in  a f rom the literature. F rom these da ta  it is evident tha t  the isolated compound is bacter iopheophyt in  a. Whether  this pigment  is involved in pr imary  reactions of bacterial  photosynthesis  remains open to speculation. I t  is well known tha t  reaction center particles f rom purple bacteria contain ubiquinone4,6, 9. In  AUT-RC particles from Rps. spheroides 6 and in KsFe(CN)6- t rea ted  chromatophores f rom R. rubrum 4 the molecular ratio of ubiquinone and P87o was found to be approximate ly  r : i .  Measurements of E P R  spectra n and of the difference spect rum and kinetics of l ight-induced ultraviolet absorbance changes is in reaction centers of Rps. spheroides indicate tha t  ubiquinone acts as the pr imary electron acceptor in a one electron reaction. Therefore, it is impor tant  to characterize it completely.  In  this connection it is interesting to note tha t  the ubiquinone present in KsFe(CN)6-treated chromatophores from R. rubrum was found to be ubiquinone-Io (ref. 14). The position of ubiquinones on the chromatogram of Fig. I is just below the upper  yellow spot. They  can be detected if the chromatogram is sprayed with Rhodamine  B in ethanol and examined in ultraviolet light. Using the procedure outl ined before 14 we found tha t  the ubiquinone isolated from a thin-layer plate, Biochim. Biophys. Acta, 283 (1972) 328-333 332 T. B E U G E L I N G  et  al. 0.12 l 0.1 008 ¢ 0-06 / 0,04 0.02 0 ' "4;0 = 5;0 ' 6;0 = 7;0 ' 8()~0 Mnm) Fig. 3. Absorption spectrum of bacteriopheophytin a in acetone from an AUT-RC preparation of Rps. spheroides, after thin-layer chromatography. as shown in Fig. I, was ubiquinone-Io. This result is not surprising, because relatively large amounts of ubiquinone-Io were found in whole cells and chromatophores of Rps. spheroides. The latter data are in agreement with those of Maroc et al. 15, but Takamiya et al. le reported that ubiquinone- 9 is present in chromatophores of Rps. spheroides. The spots indicated with y, o and r presumably come from carotenoids. So far, carotenoids have not been removed completely from the AUT-RC particles. I t  is not clear, whether these pigments play an important role at all, since they are lacking in reaction centers prepared by Reed and Clayton 5 and, since blue green mutants show normal photosynthetic growth with no carotenoids. Carotenoids were extracted and purified according to a procedure which is generally used for the extraction of ubiquinone (ref. 17, except that no ferricyanide was added). The isolated material was thereafter spotted on a thin layer plate which was developed with a mixture of chloroform and benzene ( i : i ,  v/v). The pigment from the biggest spot (RF value 0.57) showed absorption maxima at 428, 454 and 484 nm and a shoulder at 4o4 nm. These spectral characteristics are indicative of spheroidene (Pigment Y of ref. 18). A C K N O W L E D G E M E N T S  One of us (L.S.) was supported by the Netherlands Foundation for Chemical Research (S.O.N.), financed by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). Thanks are due to Dr J. Amesz for stimulating discussions. REFERENCES i R. K. Clayton, Biochim. Biophys. Aaa, 75 (1963) 3 I2. 2 P. A. Loach, G. M. Androes, A. F. lViaksim and M. Calvin, Photochem. Photobiol., 2 (1963) 443. 3 R. K. Clayton and W. R. Sistrom, Photochem. Photobiol., 5 (1966) 66x. 4 T. Beugeling, Biochim. Biophys. Acta, 153 (1968) 143. Biochim. Biophys. Acta, 283 (1972) 328-333 PIGMENTS FROM REACTION CENTERS 333 5 D. W. Reed and R. K. Clayton, Bioehem. Biophys. Res. Commun., 3 o (i968) 471. 6 L. Slooten, Bioehim. Biophys. Acta, 256 (1972) 452. 7 R, K. Clayton, Photochem. Photobiol., 5 (1966) 669. 8 R. K. Clayton, H. Fleming and E. Z. Szuts, Biophys. J., 12 (i972) 46, 9 D. W. Reed, J. Biol. Chem,, 244 (1969) 4936. IO T. Beugcling, in G. Forti, M, Avron and A. Melandri, Proe. znd Int. Congr. Photosynth. Res., Stresa, I97I, Dr. W. Junk, The Hague, 1972, p. 1453. t i  D. Y. C. Lynn Co and S. t5. Schanderl, J. Chromatogr., 26 (1967) 442- 12 G, Feher, M. Y. Okamura and J. D. lV[cElroy, Bioehim. Biophys. Acta, 267 (I972) 222. 13 L. Slooten, Biochim. Biophys. Aeta, 275 (1972) 208. 14 T. Beugcling, in 15. Metzner, Progress in Photosynthesis Research, 15. Laupp, Jr, Tfibingen, I969, p. 11Ol. 15 J. Maroc, H. de I~lerk and iV[. D. Kamen, Biochim. Biophys. Acta, 162 (1968) 62I. 16 K. Takamiya, M. Nishimura and A. Takamiya, Plant Cell Physiol., 8 (1967) 79. 17 K. Takamiya and A. Takalniya, Plant Cell Physiol., IO (1969) 363. 18 T. W. Goodwin, D. G. Land and I~. E. Sissing, Bioehem. J., 64 (1956) 486. 19 A. Jensen and O. Aasmundrud, Aeta Chem. Stand., 17 (1963) 907. 2o A. Jensen, O. Aasmundrud and K. E, Eimhjellen, Biochim. Biophys. Acta, 88 (1964) 466. 21 W. S. 1Kim, Biochim. Biophys. Acta, i t2  (1966) 392. Biochim. Biophys. Acta, 283 (1972) 328-333 

Thin-layer chromatography of pigments from reaction center particles of Rhodopseudomonas spheroides

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