7 2 6 PRELIMINARY NOTES

A new chlorophyll from green bacteria

The green photosynthetic bacteria are characterized by the presence of either chloro- bium chlorophyll (bacterioviridin)-65o or chlorobium chlorophyll-66o (numerical designation based on the position of the red-absorption peak in ether) z. Most strains of Chlorobium contain chlorophyll-66o which has its major red peak between 74 ° and 75 ° m/~ in FiFo. KATZ AND WASSINK z first reported an additional minor absorption peak at 81o mF for Chlorobium limicola in vivo and a corresponding minor peak at 780 m F in ethanol extracts. LARSEN ( F i g s . 6-11 in ref. 3) also observed this 8Io-mF absorption band in another Chlorobium strain and a corresponding peak at 77 ° m/~ in acetone extracts. In strain L of C. thiosulfatophilum the minor red peak appears at 800 mF, clearly resolved from the main peak of chlorophyll-65o at about 725 m/~ (see refs. I, 4). We have observed the minor red peak once again in Chloropseudomonas ethylicum, strain 2K (refs. 5, 6), as a shoulder at about 81o mF on the side of the 75o-mF peak of chlorophyll-66o (ref. 7). The evidence presented in this paper indicates that the absorption peak appearing between 800 and 81o m/, in vivo does not belong to chlorophyll-65 o (or -66o), but instead belongs to another chlorophyll species very similar to bacteriochlorophyll.

By the application of gradient differential centrifugation to a crude extract of C. thiosulfatophilum L, BERGERON AI~D FULLER i obtained a "yellow" zone in which the ratio of the 8oo-m/~ peak to the 725-m/~ peak was 1." 4 compared to 1:16 for the original extract. We have obtained from C. thiosulfatophilum L and Cps. ethylicum 2K a fraction with the major red-absorption peak at 805 or 81o m/~ (Fig. I) by following the general procedure of GIBSON s for extracting cytochromes from green bacteria.

L 6 - - r p - ~ i r J ~ - I r

\ '.~ / \ i

]

J ^ \ ,~ e, !

oe \ ,' I

+o, I

~" 1.6 +

0.8 ' L /

~ \ " , .- 1 , ~ / " i ./

0 I 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0

W A V E L E N G T H (mu. )

Fig. I. Absorp t ion spec t ra of Frac t ion-2 ex t r ac t s f r o m (A) C. tkiosulfatopkil**m L a n d (B) Cps. etkylicum 2 K in (I) o.oo5 M Tris buf fe r (pH 7.5) . . . . . . , (2) 8 o % m e t h a n o l (warm extract ion) . . . . , and (3) wet e the r - - . T he he i gh t of t he red-absorp t ion m a x i m u m is a rb i t ra r i ly set

equa l to I.O for each curve.

Biochim. Biophys. Aaa, 59 (1962) 726-728

PRELIMINARY NOTES 727

Washed bacteria in borate buffer were frozen and thawed three times. The crude extract was centrifuged 9 ° rain at 14 o, ooo × g. The pellet (Fraction I) contained the bulk of the chlorophyll-65o or -660. To the supernatant was added 20 % (w/v) (NH4),SO,. The precipitate (Fraction 2) contained the pigment with the absorption band at 805 or 81o m/,. The supernatant (Fraction 3) contained essentially all of the cytochrome material.

Fraction 2 was extracted first with 8o % methanol at 4 ° and subsequently with 80 % methanol at 4 o°. The cold-methanol extract contained most of the chlorophyll- 650 or -660 which had been present in Fraction 2. The warm-methanol extracts showed major peaks at 339 4- I, 364 + 2, 606 :[: 4, and 770 + I m/~ (Fig. I). The pigments were transferred to ether by mixing an equal volume of ether to the methanol extract and slowly adding IO % (w/v) NaC1 until two phases separated. The wet ether extract showed major peaks at 358 + I, 391 ~: I, 578 + 2, and 77 ° 4- I m/~ (Fig. I). (In the case of C. thiosulfatophilum L, additional peaks due to chlorophyll-65o were observed at about 427 and 658 mt~ in the warm-methanol extracts and at about 427 and 650 m/~ in ether (Fig. I).)

Spectroscopically the main pigment from Fraction 2 closely resembles bacterio- chlorophyll (see Table I). (SMITH AND BENITE# previously pointed out that the 780 - and 77o-ml~ bands in the extracts of KATZ AND WASSINK and LARSEN correspond to bacteriochlorophyU.) However, we shall use the designation "chlorophyU-77o" based on the position of the red-absorption maximum in ether, until further experi- ments are carried out to test whether or not this new chlorophyll from green bacteria is identical to bacteriochlorophyll from purple bacteria.

The overlap of the 78o-m/z fluorescence band of chlorophyU-66o (ref. IO) and the 8io-m/~ absorption band of chlorophyU-77o in vivo makes it highly probable that excitation energy is transferred from the former to the latter. Light absorbed by either chlorophyll-65o (or -66o) or chlorophyll-77o in vivo causes the oxidation of c-type cytochrome(s) in both C. thiosulfatophilum and Cps. ethylicura 11. We suggest that chlorophyU-65o (or -660) may serve as an accessory pigment for collection of light in green bacteria, while chlorophyll-77o serves as the terminal excitation-energy acceptor which couples light absorption to photosynthetic electron transfer.

T A B L E I

ABSORPTION MAXIMA OF CHLOROPHYLL-770 AND BACTERIOCHLOROPHYLL

Solvent Chlovophyll-77o* Bacteviochlorophyll

(C. thiossdf. L) (Cps. ethylicum gK) (R. vub~m °) (Chromatium xt) (wtO (mU) (mU) (mU)

Methanol

E t h e r

77 ° 770 4- I 772 772 6o6 ~z4 604 i 3 608 6o9 364 zL 2 364 zL 2 365 366 338 339 ! I -- 34 °

77 ° 771 773 773 580 577 577 575 390 392 391.5 39 I 360 357 358-5 359

* In 80 % m e t h a n o l a nd w e t e ther .

Biochim. Biophys . A a a , 59 (I962) 726-728

728 PRELIMINARY NOTES

We thank Miss E. N. KONDRAT'EVA of Moscow University for giving us a culture of Chloropseudomonas ethylicum.

Research was carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission.

Biology Department, Brookhaven National Laboratory, Upton, N.Y. (U.S.A.)

JOHN M. 0LSON CAROL A. ROMANO

t R. Y. STANIER AND J. H. C. SMITH, Biochim. Biophys. Acta, 41 (196o) 478. 2 E. KATZ AND E. C. WASSINK, Enzymologia, 7 (1939) 97. 3 C. S. FRENCH AND V. M. K. YOUNG, in A. HOLLAENDER, Radiation Biology, Vol. 3, McGraw-Hill,

New York, 1956, p. 343. 4 j . A. BERGERON AND R. C. FULLER, in T. W. GOODWlN AND O. LINDBERG, Biological Structure

and Function, Vol. 2, Academic Press, Inc., L o n d o n - N e w York, 1961, p. 307. 5 V. N, SHAPOSHNIKOV, E. N. KONDRAT'EVA, E. N. KRASIL'NIKOVA AND A. A. RAMENSKAYA,

Dohlady Ahad. Nauk S.S.S.R., 129 (1959) 1424. ¢ V. V. SHAPOSI-INIKOV, E. N. KONDRAT'EVA AND V. D. FEDOROV, Nature, 187 (196o) I67. 7 E. N. KONDRAT'EVA AND L. V. MOSHENTSEVA, Doklady Akad. Nauk S.S.S.R., 135 (196o) 460. s j . GIBSON, Biochem. J., 79 (1961) 151. 9 j . H. C. SMITH AND A. BENITEZ, in K. PAECH AND M. V. TRACEY, Modern Methods of Plant

Analysis, Vol. 5, Springer-Verlag, Berlin, 1955, p. 142. 10 A. A. KRASNOVSKII, YU. E. EROKHIN AND I. B. FEDOROVICH, Dohlady Ahad. Nauk S.S.S.R.,

134 (196o) 1232. Xl j . M. OLSON, Federation Proc. (Abstracts), 21 (1962) 46. 12 I. R. KAPLAN AND H. SILBERMAN, Arch. Biochem. Biophys., 8o (1959) 114.

Received April I6th, 1962 Biochim. Biophys. Acta, 59 (1962) 726-728

A new dinucleotide peptide from rat liver

Previous studies from this laboratory I have shown the presence of adenine nucleotide peptides in cold-HClO 4 extracts of rat liver. One of these compounds (compound III) is similar to the nucleotide peptide described by WILKEN AND HANSEN 2 from bovine liver.

After a general screening 3 of all the ultraviolet-absorbing compounds present in this type of extract, it was possible to isolate a hitherto undescribed dinucleotide peptide formed by adenylic and cytidylic units.

The main steps for the isolation and purification of this material are the following: (a) cold-HClO 4 extraction 1, (b) separation in the cold by ion-exchange column chromatography 4, (c) concentration by an adsorption-elution procedure 5, (d) paper chromatography with the ethanol-ammonium acetate solvent e and (e) paper iono- phoresis with citrate buffer (pH 3.2) at 4-6 °.

An extract from 60 g of fresh rat liver was passed through a Dowex-I-chloride column (1.2 × 20 cm) in the cold and the third solvent (o.oi N HCl-o.o3 M NaC1) collected in bulk (79 ° ml with an absorbancy at 260 mt~ of 0.820). After treatment by the adsorption-elution technique 5 the material was rechromatographed in a longer column (o.77 × 40 cm) at 4-5 °. The elution diagram appears in Fig. I.

Peak B (see Fig. I) was adsorbed 5 and separated by descending paper chromatog- raphy*; in such chromatogram a nucleotide peptide (compound IV) with a smaller

Biochim. Biophys. Acta, 59 (I962) 728-730