Triplet-triplet transfer as a mechanism of a photodynamic reaction

53 ~

SHORT COMMUNICATIONS

The two-dimensional chromatograms (2o cm ~ 2o cm) were developed first with 80 % (w/v) aq. phenol for 6 h in an atmosphere of NH a provided by keeping a beaker containing aq. NH 3 (sp. gr. 0.88). The second development was with n-butanolacetic acid-water (4o:1o:5o, v/v/v) for 4 h. This solvent was prepared by mixing the ingredients thoroughly in a separating funnel and discarding the lower immiscible aqueous portion. After complete evaporation of the solvents from the chromatograms they were uniformly sprayed with the Sakaguchi reagents as described by PANT9. Appearance of pink spots characterized the position of the guanidine derivatives. Creatine was spotted by the method described by ROCHE et al. 11. This consisted in spraying the chromatogram with a freshly prepared solution containing o.i ml diacetyl (added just before spraying) in 15 ml 1 % (w/v) a-naphthol in 6 o~, (w/v) NaOH. The appearance of a purple spot characterized creatine. Table I shows the Ry's of different guanidine derivatives in the five solvent systems employed. Fig. I shows their comparative movements in the various solvents while Fig. 2 shows their position in two-dimensional chromatograms when 80 ?'o phenol-NH a and n-butanol-acetic acid-water are employed as irrigating solvents. This work was supported by grant and a research assistantship to S. S. DUBEY by the Council of Scientific and Industrial Research, New Delhi, India, to whom our thanks are due. RADHA PANT

Biochemistry Section, The University, Allahabad (India)

S.S. DUBEY

1 R. CONSD~:N, A. H. GORDON AND A. J. P. MARTIN, Biochem. J., 38 (1944) 244. 2 p. K o N m , Actas e trabalhose do terceiro Congresso Sud-Americano de Chimica, Rio de Janeiro e Sao Paulo, Vol. 2, 1937, p. 234. 8 A. TISELIUS, Trans. Faraday Soc., 33 (1937) 524. 4 S. LlSSlTZKY, ISABELLA GARCIA AND J. ROCHE, Experienlia, X/9 (1954) 379. 5 E. SCHUTTE, Z. physiol. Chem., Hoppe Seyler's, 279 (1943) 52. s N. VON THOAI AND Y. ROBIN, Biochim. Biophys. Acta, 14 (1954) 76. v R. PANT AND S. S. DUBE¥, Biochem. J., 74 (196°) 491. 8 j . p. MORRISON, A. I t . ENNOR AND D. E. GRIFFITHS, Biochem. J., 68 (1958) 447. 9 R. PANT, Biochem. J., 73 (1959) 3 o. 10 S. P. DATTA, C. E. DIgNT AND 1-I. HARRIS, Science, 112 (195 o) 621. 11 j . ROCHE, N. VoN THOAI AND J. L. HATT, Biochim Biophys. Acla, 14 (1954) 71.

Received February 25th, 196o Biochim. Biophys. ,4cta, 41 (196o) 536-538

Triplet-triplet

t r a n s f e r as a mechanism of a p h o t o d y n a m i c reaction

In previous papers we have reported the photodynamic destruction of the fungicide pimaricin by visible light if riboflavin (or lumichrome) is present1, 2. Pimaricin belongs to a group of polyene antibiotics 3. Though its structure is rather complicated, the part of the molecule which is responsible for the u.v. spectrum (maxima at 281, 292, 3o4 and 319 m/z) is practically limited to a tetraen-grouping. On irradiation in the presence of riboflavin with light of a wavelength of 445 m/z, the peaks at 3o4 and 319 m/z disappear rapidly, though pimaricin itself cannot absorb these quanta. So the light must be absorbed by riboflavin, whic h has maxima at 223, 268, 373, 445 m/,, and then transferred to pimaricin. Biochim. Biophys. Acla, 41 (196o) 538-541

SHORT COMMUNICATIONS

539

Compounds with a tetraenstructure are usually very sensitive to free radicals and we originally supposed, therefore, that the photodynamic destruction was due to free radicals, which could have their origin in the decomposition of riboflavin by ligh0. From further work on this subject we have to conclude that neither an oxidation, as described in the literature on other photodynamic destructions4, 5 in which riboflavin is involved, nor a free-radical mechanism 6-8 is responsible for our reaction. Though the structure of pimaricin is very complex we have nevertheless continued to use this compound, for a change in this molecule under influence of light m a y be easily detected spectrophotometrically. Moreover simple tetraens are rare or very labile compounds, whereas pimaricin is rather stable and commercially available*. In this paper arguments will be given that a direct transfer of excitation energy from riboflavin or lumichrome to pimaricin by collision may be involved. It is not likely that this transfer proceeds via a riboflavin-pimaricin or lumichrome-pimaricin complex because we were not able to detect these complexes by absorption and fluorescence measurements. SZENT_GY~RGYI 9,10 has recently given some examples of charge-transfer complexes, but the concentrations used in his experiments (io -a M) were much higher than those in ours ( < lO -5 M). However, we cannot completely exclude intermediate complex formation. Further observations of the reaction mentioned have brought us to the conclusion that the sensitizers riboflavin and lumichrome react in their triplet states. These triplet states have a rather long lifetime compared with the corresponding singlets and their importance in many photosensitized reactions has been recognized in the last decade TM12. These long-lived excited states can be demonstrated by phosphorescence of the frozen solutions. In our reaction the average time for collision between riboflavin and pimaricin in a IO-'~ M solution will be about IO-4 - lO -5 sec 1~. The lifetime of the singlet state being about io -s sec, it is evident that only relatively long-lived excited states like triplets have a reasonal chance to transfer their energy. We therefore propose the following reaction sequence as an explanation of the photodynamic destruction of pimaricin: Riboflavin + h v - - + Riboflavin* (singlet)--+ Riboflavin* (triplet) Riboflavin* (triplet) + Pimaricin ~

Riboflavin + Pimaricin* (triplet)

(I) (2)

In reaction (I) riboflavin is first excited by a 445 m/, light quantum to its singlet state and next a singlet-triplet transition takes place (conversion of the spin moment). From this state an energy transfer to a pimaricin molecule may be possible if the triplet of pimaricin is lower than the similar state of riboflavin, for reaction (2) is allowed by spin-conservation rules 14. A few examples of such transfers have been described recently 12,15. The overall result of this triplet-triplet transfer would be an excited pimaricin molecule. It is conceivable that a very sensitive compound like pimaricin with its reactive tetraen structure decomposes if it is excited to its triplet state. The lability of pimaricin is demonstrated by its sensitivity to u.v. light. * We wish to express our gratitude to t h e R o y a l D u t c h F e r m e n t a t i o n Industries, Delft, Netherlands, for supplying this fungicide.

Biochim. Biophys. Acta, 41 (196o) 538-541

54 °

SHORT

COMMUNICATIONS

The reaction mechanism first mentioned is supported by the effect shown by several substances upon the system riboflavin-pimaricin. A compound that quenches the phosphorescence of riboflavin is supposed to act in this way by facilitating the radiationless dissipation of excitation energy by collisions with molecules of the solvent. Consequently the lifetime of the triplet state is shorter. This means the triplet-triplet transfer is less probable. It appeared that the quenching effect on the phosphorescence of the compounds tested ran completely parallel to the degree of inhibition of the photodynamic destruction of pimaricin in the combination riboflavin-pimaricin. On the other hand, compounds without visually observable quenching showed no inhibition, but in many cases even accelerated the pimaricin destruction. For illumination a Kromayer high-pressure mercury lamp was used. The aqueous solutions containing 2.1o -5 M riboflavin and 9 . I o - ~ M riboflavin were buffered with a o.o6 M phosphate buffer at pH 6.8. The distance between lamp and solution was 3 cm. By using an interference filter, light with a wavelength of about 443 m/, was obtained. The destruction of pimaricin was traced by measuring the extinctions at 3o 4 and 319 m/, by means of a Unicam Spectrophotometer S.P. 5oo. The quenching effect of compounds on the phosphorescence of riboflavin was tested by adding them to a 2'1o 5 M aqueous solution of riboflavin and visually observing the phosphorescence in u.v. light after freezing (light source, Philips lamp H.P.W. 125) 16. Besides the compounds mentioned in Table I it was found that all paramagnetic ions tested, and also oxygen, diminish the photodynamic pimaricin decomposition. TABLE EFFECT

OF V A R I O U S

SUBSTANCES ON

ON

I

FHOTODY'NAMIC DESTRUCTION

RIBOFLAVIN

Phototynamic destruction of pimaricin

A dried substance"

Conch.

-Cystine Cysteine Methionine Thioglycolic acid Glutathione

-l o -a M io a ~I 1o-a M 1 o - a :'ll 1 o - a ~11 l o - a :11 i o -a M i o - a A:[ Jo -3:11 l o -4 M i o-a 31 ~o - a M IO -4 M

NasSO a

E t h y l iodide Monoiodoacetic acid KI KI

Potassium flmdanide Thiourea Thiouracil Ascorbic acid Hydroquinone Methanol Ethanol Plmarlcln

I j

5" l o s ~1I 5" l o 5 : 1 1 75 vol. ~'i) 96 vol. %

--

OF

P1MARICIN,

AND

PHOSPHORESCENCI~

% loss pimaricin

23 35 33 2t 79 58 93 2z 23 2 5 5 3 8 inhibition

Riboflavin phosphoresamce Comm.

1 o -a 114 lO a :lI i o -a M IO ~ 11I I o-a M l o a 3.1 io a M 1o - a M l o a :1,1 l o '* 3 1 ~ o - a ~,I l o a 3,1 ,o 4 31 l

' o --

5" 5" 5 5 2.

I o 5 ~'~I 1o -s ~ I

vol. °,o v o l . of, J o -5 ~'I

(;clout of frozen part o! the solution

()range ( )range ()range ( )range Yellow Yellow ()range

Orange Orange N o colour N o colour N o colour N o colour N o colour N o colour N o colour Yellow Yellow \ V e a k l y orange

* O n a c c o u n t o f t h e low s o l u b i l i t y of the p i m a r i c i n a 2 - 1o ~ 3 I solution of riboflavin was u s e d to observe the p h o s p h o r e s c e n c e quenching.

Biochim.

Biophys.

, l c l a , 41 ( 1 9 6 o ) 5 3 8 -541

SHORT COMMUNICATIONS

541

These observations are apparently in complete agreement with the paramagnetic quenching of the triplet state 14 (the oxygen molecule is also paramagnetic). The hypothesis of the triplet-triplet transfer as an explanation for the photodynamic reaction described is also strongly supported by the discovery of the quenching of the riboflavin phosphorescence by pimaricin itself. In summary, a triplet-triplet transfer is proposed as a mechanism of the photodynamic destruction of the polyene antibiotic pimaricin in the presence of riboflavin. The inhibiting effect of added substances on this destruction runs completely parallel to the quenching of the riboflavin phosphorescence by these compounds.

Biochemical Laboratory of the Technological University, 67 Julianalaan, Delft (The Netherlands)

J . POSTHUMA W . BERENDS

B. HENDRIKS AND "W. BERENI~S, Rec. tray. chim., 77 (I958) 145. 2 E. ZONDAG, J. POSTHUMA AND g . BERENDS, Biochim. Biophys. Acta, 39 (I96O) 178. a j. B. PATRICK, R. P. WILLIAMS AND J. S. WEBB, J. Am. Chem. Soc., 8o (1958) 6689. 4 p. A. KOLESNIKOW, Biokhimia, 22 (I958) 434. 5 A. W. GALSTO~, Science, i i i (195 O) 619. e H. R. MERKEL AND \V. J. NICKERSON, Biochim. Biophys. Aeta, 14 (1954) 3o3 . x~V. J. RUTTER, Acta Chem. Scan&, 12 (1958) 438. s L. P. VERNON, Biochim. Biophys. Acta, 36 (I959) 177. 9 I. ISENBERG AND A. SZENT-GYbRGYI, Proc. Natl. Acad. Sci. U.S., 44 (1958) 857:45 (1959) 1231. 10 G. I%ARREMAN, I. ISENBERG AND A. SZENT-GV6RG¥1, Science, 13o (i959~J 119i. 11 C. REID, Excited states in chemistry and biology, L o n d o n 1957, c h a p t e r 512 G. PORTER, Proc. Chem. Soc. London, (I959) 291. 13 G. OSTER AND A. H. ADELMAN, J. Am. Chem. Soc., 78 (1956) 913 . 14 G. PORTER AND M. t{.. WRIGHT,Symposia Faraday Soc. Nottingham, 1959. 15 A. TERENIN AND V. ERMOLAEV, Trans. Faraday Soc., 52 (1956) lO42. 16 A. SZENT-GYGRGYI, Bioenergetics, Acad. Press, New York, 1957. I

Received March 29th, 196o Biochim, Biophys. Acta, 41 (I96O) 538 541

Uptake of bacitracin by sporangia and its incorporation into the spores of bacillus licheniformis Previous studies1, ~ have suggested a possible relation between the production of the polypeptide antibiotic, bacitracin, and the spore-forming metabolism of Bacillus licheniformis. Under a number of different physiological conditions, it has been shown that bacitracin is produced only under cultural conditions that permit eventual spore formation. Thus addition of ethyl malonate to a growing culture inhibits both bacitracin production and spore formation. While investigating the incorporation of D-[3H~phenylalanine and DL-iaHl ornithine into bacitracin by whole cells of B. licheniformis, we observed that some of the label appeared in the spores. In these experiments the efficiency of incorporation of label into the spores was the same as that into bacitracin, e.g. about 1%. These observations prompted a consideration of the possibility that the bacitracin molecule is incorporated into the spore and thus comprises part of the spore coat 3. The possibility that bacitracin is incorporated into spores without prior degradaBiochim. Biophys. Acta, 41 (196o) 541-543