Bálint, Erzsébet

Hevesi, János

Vass, I.

English

Bálint Erzsébet

Hevesi János

Vass I.

University of Szeged

D E G R E E O F P O L A R I Z A T I O N I N D Y E - D E T E R G E N T \ M O D E L S Y S T E M S By E. BÁLINT, J. HEVESI and I. VASS Institute of Biophysics, Attila József University, Szeged The degree of fluorescence polarization and the absorption spectra of sodium-lauril-sulphate t (SLS) and thionine (Th) micellar solutions were measured. A simple consideration shows that the local concentration of dyes present in the micelles is proportional to the absorption coefficient. According to measurements the degree of polarization and the absorption characteristics of the systems are determined by the local concentration in the micelles. These dye concentrations (cal-culated from the concentration depolarization in micellar systems on the base of the FÔRSTER-ORE'S theory) are higher by 2 to 3 orders of magnitude than the bulk dye concentration and it seems to be similar to the pigment concentration present in the chloroplasts. The energy transfer between dye molecules can be adequately described by FORSTER'S theory. Introduction Dye-detergent systems are suitable for modelling energy transfer processes oc-curing during the photosynthesis [1]—[2]. In aqueous solution of detergents a g-, regates of colloidal dimensions — micelles — are formed above the critical-micelle-concentration (c.m.c.) [3]. If àppropiate dyes are added to the micellar solution, the dye molecules will be incorporated into micelles [4], with local concentration ex-ceeding the bulk concentration of the solution [5]. The average distance of the dye molecules, commensurable with those of in vivo systems (in chloroplast) can be reached. In the present paper the degree of fluorescence polarization and the absorption spectra of sodium-lauryl-sulphate (SLS) — thionine (Th) micellar solutions have been investigated in order to obtain informktion about the local dye concentration and the migration of electronic excitation energy within the micelles. Materials and methods The solutions examined were prepared as descriued in an earlier paper [6]. The concentrations of SLS and Th were varied between 0—8 • 10 - 3 M and 1 • 10~8—2-10-4 M, respectively. More details about the determination of the absorption spectra are given in [2]. The degree of fluorescence polarization was de-termined by using a photoelectric apparatus, constructed in the Institute of Medical Chemistry on the base of the description of KETSKEMETY et al. [7]. The calculations were performed by a minicomputer, type Wang—600. 124 E. BÁLINT Results and discussion The absorption spectra of the aqueous solutions of the systems (&(/.)) have two absorption bands with maxima at 600 nm (a-band) and 565 nm (/?-band) attri-buted to the monomer and to the dimer forms of the dye dissolved in the solution, respectively. In detergent solutions — above the c.m.c., found to be 3.5 • 10~3 M concentration of the detergent — new absorption bands appear at 460 nm (y-band) and at 640 nm (¿-band) attributed to dye-detergent complexes and to higher dye-aggregates, respectively (see Fig. 1). Dimer and aggregated form of the dye appear in consequence of high local concentration present in the micelles. Fig.l. The A"(/)max values of the absorption bands are proportional to the local con-centration of the corresponding dye form, provided no overlap of the bands exists [8]. In solutions examined only the monomer form of dye are fluorescent [9], therefore the fluorescence polarization depends on the local concentration of the monomers. The bands of the absorption spectra have significant overlap, thus the true values of &(/.)max in the different bands,can not be found directly from the measured spectra. Therefore the measured spectra were analysed by the method of ALJENCEV [10]. The true k(?.)max values of the different band — proportional to the local dye concentration — are plotted in Fig. 2 as a function of the dye concentration weigthed in at 4.5 • 10~3 M detergent concentration. Fig. 2 shows that the curves can be approximated by two straight lines having different slopes. With respect to the local dye concentration it can be stated that after reaching a certain dye "concentration (shown in Fig. 2 by a break-point in the straights) the micelles become saturated — DEGREE OF POLARIZATION IN DYE-DETERGENT SYSTEMS 125 Fig. 2. the ratio of the monomers and the dimers decreases — and simultaneously aggregates and dye-detergent complexes are formed. The break-point of the a-band maximum appears at higher dye concentration. Namely, in solutions containing more detergent, the number of micelles increases thus more and more dye molecules are necessary for obtaining the same local concen-tration in the micelles. It can be shown by measuring the degree of fluorescence polarization that the absorption and the polarization characteristics of the system examined are determined by the local concentration. lg k O J m a x ' * lg k ( \ . ) m a ) f Fig. 3. 126 E. BÁLINT Fig. 3 contains the degrees of fluorescence polarization measured at 4.5 • 10 - 3 M and at 5 • 10~3 M detergent concentration as a function of log &(A)max. The curves are typical conceritrational ones. The supposition that the concentrational depolari-zation in micellar systems can be described by the theory of Forster-Ore [11—12], permits to calculate the local concentration of the dye molecules present in the micelles. According to this theory the point at plp0— = 0.55 lies at a given c0 concentration pro-portional to the inverse of the cube of the energy transfer constant [13]; Table cTh from 1 • 10"e M to 2-10" M c s t s .10"»(M) cioC I0~3 (M) 2.5 4.100 t 22 3.5 . ^ 0.811 - 1 1 4.5 0.405 - 1 1 5.0 0.212 - 1 4 8.0 0.046 - 1 5 1 Cn 4 N'Rl 0) Calculating with i?0=46 A (obtained from the overlap of absorption and fluorescence spectra of the systems), c 0 =4 • 10 - 3 M. Knowing the values of k (A)max and that ^Wmax=Kcloc pertaining to c0=cloc the value of the K constant can be determined and the local dye concentration for another solution can be calculated. ; In the table the calculated values of the local concentration are given in the concentration range of 1 • 10® M—2-10 - 4 M. It can be seen from the data that the local concentrations are higher by 2 to 3 orders of magnitude than the bulk dye concentration. The local concentrations given in the table correspond to the average distances from 10 to 60 A between the dye molecules. These values are similar to the mean distances of the real pigments present in the chloroplasts. l 9 C l o c fig- 4. DEGREE OF POLARIZATION IN DYE-DETERGENT SYSTEMS 127 Fig. 4 shows the calculated (solid iine) and the measured (circles) values of the relative degree of fluorescence polarization (p/p 0) as a function of the logarithmic öf the local concentration. The calculation was carried out by the Förster-Ore theory. It can be seen from the figure that at lower dye concentrations the calculated values and the measured ones of pjp0 are in a good accordance. At higher concen-trations, from the point Bp — corresponding to the local concentration pertaining to the break points in the Fig. 2 — the deviations between the measured and the calcu-lated values are more significant. These deviations, however, can be interpreted by the formation of aggregates and their participation in the energy transfer. Conclusions \ The presented results of fluorescence polarization measurements prove that the structure and the dimensions of the chloroplasts are similar — in certain detergent concentration range — to those found in dye-detergent systems. The excitation energy transfer between the dye molecules present in the micelles can be described by the Förster's theory. Useful information concerning the photophysical processes óf the photosynthesis may be expected from the results of further investigations of the same kind of dye-detergent systems. * * The authors are indebted to Prof. L. SZALAY, Director of the Institute of Biophysics for the helpful discussions and for the valuable advices during the pre-v paration of present paper. References [1] Singhal, G. S., E. Rabinowitch, J. Hevesi: Photochem. Photobiol. 11, 531 (1970). [2] Hevesi, J., E. Lehoczki, E. Bálint: Zs. Prikl. Spectr. 13, 440 (1970). [3] McBain, J. W.: Colloid Science (Heath, Boston) (1950). [4] Corrin, M. L„ W. D. Harkins: J. Chem. Phys. 14, 641 (1946). [5] Hevesi J., E. Bálint, E. Lehoczki: Acta Phys. Polon. Sei. A38, 829 (1970). [6] Bálint, E„ E. Lehoczki, J. Hevesi: Acta Phys. et Chem. Szeged, 17, 16 (1971). [7] Ketskeméty, I., L. Gargya, E. Salkovits: Acta Phys. et Chem. Szeged. 3, 16 (1957). [8] Lehoczki, E., J. Hevesi: Acta Phys. et Chem. Szeged 18, 11 (1972). -[9] Lehoczki, E., E. Bálint, J. Hevesi: Zs. Prikl. Spectr. 16, 97 (1972). [10] Fok, M. V.: Trudi FIAN 59, 3 (1972). [11] Förster, Th.: Z. Naturfors. 4a, 321 (1949). [12] Ore, A.: J. Chem. Phys. 31, 442 (1959). [13] Lehoczki, E„ K. Csatorday: BBA, 396, 86 (1975). СТЕПЕНЬ ПОЛЯРИЗАЦИИ ЛЮМИНЕСЦЕНЦИИ МОДЕЛНОЙ СИСТЕМЫ КРАСИТЕЛЯ-ДЕТЕРГЕНТА Э. Балинт, Я. Хевеши и И. Ваш Установили, что спектры поглощения и степень поляризации люминесценции растворов тионинав натрийлаурил-сульфате зависят ,от локальной концентрации тионина в мицеллах. На основе полученных кривых концентрационой деполяризации по теории Ф ё р с т е р а -рассчитали, что локальная концентрация тионина в мицеллах на 2—3 порядка больше чем введенной в раствор концентрации красителя и достигает концентрации пигмента в ми-целлах, характерной для хлоропластов. Перенос энергии между молекулами тионина проис-ходит в соответствии с теорией Ф ё р с т е р а . . I 

Degree of polarization in dye-detergent model systems

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Degree of polarization in dye-detergent model systems

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