Szabad, János

English

Szabad János

University of Szeged

TRANSFER OF ELECTRONIC EXCITATION ENERGY FROM /?-CAROTENE TO CHLOROPHYLL-a IN SOLUTION By J. SZABAD Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged (Received 15 June, 1972) From the sensitization of chlorophyll fluorescence by ^-carotene the critical distance of energy transfer R0 was found to be dependent on the wavelength of exciting light in the region 400—440 nm, ranging from 18 A to 22 A in the Soret band of chlorophyll-a. In the region 450—500 nm (where the absorption is mainly due to yS-carotene) Rc = 25 A. The value i?0 = 8,5 A was obtained from the absorption and fluorescence spectra. The superposition of a "fas t" transfer of energy involving the second excited singlet state of chlorophyll-a is offered as explanation. Introduction It is known that the electronic excitation energy of carotenoids is transferred to chlorophyll-a (Chl-a) and is useful in inducing photosynthesis [1—3]. In mixed solutions containing detergent, in monolayers containing carotene and Chl-a and in thin films, certain carotenoids are active in exciting Chl-a fluorescence [4—6]. In mixed solutions of /¿-carotene and Chl-a in castor oil the energy transfer was-observable only at low temperature [3]. SINGHAL and co-workers [7] supposed that J?-carotene in benzene and in 3-methoxy-l-butanol was ineffective in exciting fluo-rescence of Chl-a, but they did not exclude the possibility of energy transfer from /J-carotene to Chl-a in other solvents. DUYSENS [1] and RABINOWITCH [8] assume that the transfer of energy from carotenoids to chlorophylls takes place according to an inductive resonance mecha-nism obeying Forster's formulas [9]. LOSEV and GURINOVICH [3] and SINESHCHEKOV et al. [6] suppose that the mechanism of carotenoid — Chi energy transfer is not in-ductive resonance but something different. According to LOSEV and GURINOVICH [3] an effective carotenoid — Chi energy .transfer occurs if the system contains some type of "solid support surface", as in detergent solutions and in monolayers. There are cases in which the carotenoid—Chi energy transfer can not be explained by Forster's theory, since in a certain wavelength region of excitation the efficiency of transfer depends on the wavelength [10]. If Forster's theory is formally applied to the transfer at different exciting wavelengths, this dependence leads to different transfer frequencies and critical distances not predicted by this theory. A similar effect was observed by WEBER [11] and BAUER et al. [12]. In this paper ^-carotene — Chl-a energy transfer will be discussed in terms of the critical distances of transfer from sensitization of Chl-a fluorescence, and of absorption and fluorescence spectra. 134 J. SZABAD Material and Methods Chl-a was prepared from fresh spinach leaves according to JACOBS et al. [13] . The Chl-a solutions in diethyl ether were considered pure if the intensity ratios of the blue and red absorption maxima and those of red absorption maxima to the absorption at 5 0 5 nm were 1 ,32 and 4 5 — 5 0 respectively, /¿-carotene was a Merck product. The absorption spectrum of /¿-carotene showed that the preparation con-sisted only of the trans compound [14]. The mixed cyclohexane solutions contained 10-5 M Chl-a and 4 • lO"6, 6 • lO"6, 8 • lO"6, 10"5, 2 • 10"5, 4 • 10"5, 6 • 10"5, 8 • 10~5 and 10~4 M /¿-carotene. All measurements were carried out at room temperature. The critical distance (the average distance of donor and acceptor when the probability of transfer is equal to that of emission), is given by Eq. (1) [9] 6 9(lnl0)k2c*<Pd f , , , ^dv • * • = m n > n * N > J - 0 ) o Here A;2 = 2/3 (for random distribution of the molecules), c is the velocity of light, <Pd the fluorescence yield of the donor, n is the refractive index of the medium, N' is the number of molecules per mmole, / (v) the normalized fluorescence spectrum of the donor, e(v) is thé molar decadic extinction coefficient of the acceptor and v the frequency. The fluorescence spectrum of /¿-carotene was taken from the literature [15]. <Pd was taken as 10 4 [1]. According to CHERRY et al. [15], the yield of fluorescence of /¿-carotene is 1/500 of that of anthracene. The fluorescence spectra were normalized under the condition Jf(v)dv= 1. 0 The critical distance can also be determined from quenching curves given by the following equation [9] : ^o c0 I I c0 J. (2) where cM is the concentration in mole/1, c0 the critical concentration (at which the probability of energy transfer is equal to that of emission) and (p(x) is the Gaussian error integral. <p0 and <P represent the fluorescence yield when the fluorescence of the donor is not quenched and quenched, respectively. The relation between R0 and c0 is Ko = -A • (3 ) 2 /7T3 N'c0 W As the yield of fluorescence of /¿-carotene is very low, <Z>/<i>0 is not measurable for /¿-carotene. However, the sensitization of the fluorescence of Chl-a by /¿-carotene and the quenching of the fluorescence of /¿-carotene by Chl-a are different mani-festations of the same process, and therefore from Chl-a fluorescence sensitization "quenching curves" for /¿-carotene fluorescence can be determined. The sensitiza-tion of Chl-a fluorescence in mixed solutions of carotene and Chi is usually char-acterized by the ratio F'JFC, where F'm is the measured and F'c the calculated fluo-rescence intensity of Chi in the mixed solution [7], [10]. F'c/F'm may represent the "quenching of fluorescence" of /¿-carotene. TRANSFER OF ELECTRONIC EXCITATION ENERGY FROM /^-CAROTENE TO CHLOROPHYLL-a 135 Results Using Eq. (1) R0 was calculated for four pigment pairs (Table 1). The spectra shown in Fig. 1 were used for the calculation. The red absorption band of Chl-a was taken into account, the higher frequency side of which is shown by a dotted 7 . 0 6 , 0 5 , 0 4 , 0 - u .< v x 10 (sec ) Fig. 1. Absorption and fluorescence spectra of jS-carotene and chlorophyll-a in cyclohexane line. In Fig. 2 the "quenching curves" of /¡-carotene fluorescence are shown for four exciting wavelengths (actually the reciprocals of the intensities of the relative fluorescence of Chl-a). From these curves the critical concentrations, and with Eq. (3) the critical distances were calculated. For the /¡-carotene Chl-a system in Table I i?0 values in A from the spectra for four pigment pairs. Rf, = 25 A calculated from "quenching curves" in the wavelength region 440—500 nm ~~~ Acceptor Donor Chlorophyll-a 0-carotone Chlorophyll-a 4 3 , 6 0 ^-carotene 8 ,5 ( 2 5 , 0 ) 6 ,5 1 3 6 J. SZABAD Fig. 2. Reciprocal of relative fluorescence intensities of chlorophyll-a ^-carotene mixtures as function of the concentration of /J-carotene the region 450—500 nm, R0 was obtained as 25 Ä independently of the wavelength of excitation. At exciting wavelengths in the region where the absorption of Chl-a is significant, c0 and, consequently R0 depend on the wavelength of the excitation, ranging from 18—22 Ä. Discussion Table I shows that, in principle, according to Förster theory the transfer from ^-carotene to Chl-a in cyclohexane solution is possible (with i?0 = 8.5 A); its efficiency under the present conditions, however, is negligible. /?-carotene is not able to quench the fluorescence of Chl-a (R0=0Ä), i.e. the ß-carotene — Chl-a transfer is irrevers-ible. There is practically no transfer of energy involving /^-carotene molecules (/?0 = 6.5 A).The "fluorescence quenching curves", however, show that in solution ^-carotene transfers its electronic excitation energy to Chl-a (Ä 0 =25Ä). R0 calculated from absorption and fluorescence spectra is much less than that obtained from the "quenching". This difference can be explained in two ways: 1. The fluorescence yield of /^-carotene introduced into Eq. (1) should be much higher than 10~4. If this were the cause of the difference, an absolute yield of fluo-rescence of ß-carotene of about 1,6• 10 - 3 should be expected; this may be the case because the fluorescence of ^-carotene, also under these conditions, is not easily measurable. 2. There exists some interaction between Chl-a and j?-carotene molecules pro-moting the carotenoid Chi energy transfer, e.g. the presence of mixed dimers or TRANSFER OF ELECTRONIC EXCITATION ENERGY FROM $-CAROTENE TO CHLOROPHYLL-a 137 higher aggregates. However there is no sign of these aggregates in the absorption and fluorescence spectra of mixed solutions.* The R0 values calculated from the "quenching curves" show the following characteristics. In the wavelength region where mainly /¿-carotene absorbs, R0 does not depend on the wavelength of excitation. In the region, where Chl-a also absorbs (400—440 nm), however, R0 depends on the wavelength. R0 in this region is a formal quantity, since this dependence is not predicted by the theory. This phenomenon should mean that the /¿-carotene — Chl-a energy transfer is complex: a "fast" transfer (to some extent uphill-type) is superimposed upon the Förster slow type transfer, involving the participation of the second excited state of Chl-a. *- -X- . • The author is indebted to Prof. I. KETSKEMETY, Head of the Luminescence and Semiconductor Research Group for permitting the use of the facilities of the Research Group. References [1] Duysens, L. N. M.: Thesis, Utrecht (1952). [2] Goedheer, J. C.: Biochim. Biophys. Acta 172, 252 (1969). [3] Losev, A. P., G. P. Gurinovich: Biofizika 14, 110 (1969). [4] Teale, F. W. J.: Nature, 181, 415 (1958). [5] Litvin, F. F., В. A. Gulyayev: Dokl. A. N. SSSR., 162, 1184 (1965). [6] Sineshchekov. V. A., F. F. Litvin, M. Das: Photochem. Phötobiol. 15, 187 (1972). 17] Singhai, G. S„ J. Hevesi, E. Rabinowitch: J. Chem. Phys. 49, 5206 (1968). [8] Rabinowitch, £., Govindiee: Photosynthesis, J. Wiley and Sons, New York—London—Sydney— Toronto (1969). [9] Förster, Th.: Fluoreszenz organischer Verbindungen. Vandenhoeck und Ruprecht, Göttingen (1951). [10] Szabad, J., L. Szalay: Fourth Int. Biophys. Congress, Moscow (1972). [11] Weber, G.: Biochem. J. 76, 335 (1960). [12] Bauer, R. К., E. Rabinowitch, L. Szalay, E. Tombdcz: Biophys. J., in press. [13] Jacobs, E. E., A. E. Vatter, A. S. Holt: Arch. Biochem. Biophys. 53, 228 (1954). [14] Zechmeister, L.: Isomeric carotenoid pigments, in: Fortschritte d. Chem. org. Natursf. 18, 45 (1961). [15] Cherry, R. J., D. Chapman, J. Langelaar: Trans. Farad. Soc. 44, 2304 (1968). П Е Р Е Д А Ч А Э Н Е Р Г И И ЭЛЕКТРОННОГО ВОЗБУЖДЕНИЯ В РАСТВОРЕ О Т /¿-КАРОТИНОИДА К Х Л О Р О Ф И Л Л У Я. Сабад Из спектров флуоресценции хлорофилла сенсибилизированных /i-каротиноидом опре-деляли «о, критическое расстояние передачи энергии. В полосе поглощения Соре, в области 400—440 нм это значение зависит от длины волн возбуждающего света й величина R018—22 А В области 450—500 нм (где в основном поглощает /?-каротиноид) Ä0 = 25 А. На основе спект-ров поглощения и флуоресценции i?0 = 8,5 А. Предполагается, что в переносе энергии прини-мает участие через второй синглетный уровень возбуждения хлорофилла одна „быстрая" передача. * An alternative explanation of this difference may be the incorrect choice of the upper fre-quency limit of the overlap integral (shown by the dotted line in Fig. 1) in Eq. (1). A higher limit would result in a higher R0 value. 3 

Transfer of electronic excitation energy from β-carotene to clorophyll-α : J.

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Transfer of electronic excitation energy from β-carotene to clorophyll-α : J.

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