Energy transfer from C-phycocyanin to chlorophyll a in Triton X-100 reverse micelles

Colloids and Surfaces B: Biointerfaces 11 (1998) 9–14

Energy transfer from C-phycocyanin to chlorophyll a in Triton X-100 reverse micelles Zhao Jiquan, Zhao Jingquan, Xie Jie, Zhang Jianping, Jiang Lijin * Institute of Photographic Chemistry, Academia Scnica, Beijing 100101, People’s Republic of China Received 2 July 1997; accepted 2 March 1998

Abstract The reverse micellar system of Triton X-100/pentanol/cyclohexane was used to imitate native thylakoid membrane and study the energy transfer from C-phycocyanin (C-PC ) to chlorophyll a. C-PC is completely solubilized in water and stable due to the protection of the water layer. When chlorophyll a is added into the system, the chlorophyll a molecules locate in the hydrophillic region. Energy transfer from C-PC to chlorophyll a takes place with much lower efficiency than in native cells. The efficiency of energy transfer increases with the concentration of chlorophyll a while it decreases with the increase of the molar ratio (R ) of water to Triton X-100. When the concentration of chlorophyll w a is 4.2×10−7 M and that of C-PC is 1.2×10−7 M in the reverse micelles with R values of 2.5 and 7.5, the efficiencies w of energy transfer are only 33 and 13%, respectively. The reason of the low efficiencies of energy transfer may be that the energy donor and acceptor are not coupled so well as in the native cells in which allophycocyanin (APC ) plays a role of the bridge for energy transfer. © 1998 Elsevier Science B.V. Keywords: C-phycocyanin; Chlorophyll a; Energy transfer; Reverse micelles; Triton X-100

1. Introduction Phycobiliproteins are photosynthetic antenna pigments found in cyanbacteria, red algae and the cryptomonads. In green algae and higher plants, the antenna pigments are represented by membrane-bound protein complexes of chlorophyll a and b, while only chlorophyll a is present in cyanbacteria and red algae. Early studies showed that energy absorbed by the biliproteins is utilized in photosynthesis with high efficiency and the major path of energy transfer is phycoerythrin phycocyaninallophycocyaninchlorophyll a [1]. But the mechanism of energy transfer from * Corresponding author. Tel: 86 010 64888164; Fax: 86 010 62029375. 0927-7765/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0 9 2 7 -7 7 6 5 ( 9 8 ) 0 0 02 8 - 9

the phycobiliproteins to chlorophyll a is still a challenging and interesting problem. Therefore, it may be helpful to study the mechanism of energy transfer from phycobiliproteins to chlorophyll a using a model system. In recent years, reverse micelles have been used as model systems to study biological events and some useful results have been obtained [2–4]. So it is possible to utilize the reverse micellar system to imitate the thylakoid membrane and study the energy transfer from phycobiliproteins to chlorophyll a. C-Phycocyanin (C-PC ) was chosen because of its stability in the Triton X-100 reverse micelles and its absorbance in the visible range from 560 to 640 nm [5]. Furthermore, the fluorescence spectrum of C-PC partially overlaps with the absorption spectrum of chlorophyll a which make the energy transfer possible. In order to simulate the natural environ-

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ment of the pigments, non-ionic surfactant Triton X-100 was used in the experiment, because the micellar and reverse micellar systems of Triton X-100 have been extensively studied in recent years [6–8]. Triton X-100 is the trade name of the liquid, nonionic surfactant poly(oxyethylene) (tetramethylbutyl )phenyl ether, with the formula CH C(CH ) CH C(CH ) C H (OCH CH ) OH. 3 32 2 32 6 4 2 2 9–10 When water is added into the dry nonionic reverse micellar solution, the water molecules first hydrate the oxyethylene (OE) groups of the polar chain of the surfactant. After hydration reaches saturation, subsequently added water molecules accumulated in the core of the reverse micelles to form water pools [9]. The system of Triton X-100/cyclohexane/pentanol can solubilize much more water than others.

2. Experimental Triton X-100 was purchased from Sigma (St. Louis, MO 63178, U.S.A.). Cyclohexane (>99.5%) was obtained from Beijing Chemical Factory (Chemical Road, Chaoyang district, Beijing 100022, P.R. China). Pentanol was purchased from The Third Reagent Factory (Chemical Road, Chaoyang district, Beijing 100022, P.R. China) and redistilled before use. Chlorophyll a was prepared from spinach leaves according to Ref. [10]. C-PC was prepared from Anabaena variabilis [11]. The ratio of Triton X-100 to pentanol in the used reverse micelles is 4:1 (w/w). The overall concentration of Triton X-100 and pentanol in the cyclohexane is 20% (w/v). To prepare the reverse micelles, a small amount of water was added into the system to hydrate the OE groups, then the C-PC stock solution was added, and at this step some more water had to be added by injection to maintain the [water]/[ Triton X-100] value (R ). In this way, a w series of reverse micelles were produced with a C-PC concentration of 1.2×10−7 M and R values w of 2.5 and 7.5, respectively. The chlorophyll stock solution of 10−3 M, prepared by dissolving chlorophyll a in the dry reverse micelles, was added into the C-PC reverse micelles and completely mixed by oscillation. The spectra were measured after an equilibration time of 30 min at 18±1°C.

Fig. 1. The absorption spectra of C-PC in water and reverse micelles of Triton X-100/penatol/cyclohexane. 1, Water; 2, R =2.5; 3, R =7.5; 4, R =1.0. w w w

The fluorescence quantum yield of C-PC in water was measured relative to Rhodamine B, in methanol, used as the reference; the quantum yield of Rhodamine B was 0.94. The concentration of the standard and C-PC were adjusted so that their absorption at the excitation wavelength 590 nm was 0.100. The results were calculated according to the equation

A B

A I W = 1 2 W 1 2 A I 2 1 The quantum yield of chlorophyll a was obtained similarly relative to C-PC. The absorption spectra were measured on a Hewlett Packard 8511 A spectrophotometer. Fluorescence spectra were recorded on a Hitachi 850 fluorescence spectrometer. The slit opening was 6 or 8 nm and the excitation wavelength was at 590 nm. For determining the energy transfer efficiencies, all the instrumental parameters were not changed in the experiment. The solutions were not out-gassed before measurements.

3. Results The absorption spectra of C-PC in water and reverse micelles (R =1.0, 2.5 and 7.5) are shown w

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Fig. 2. The fluorescence emission spectra of C-PC in water and reverse micelles. 1, Water; 2, R =2.5; 3, R =7.5. w w

in Fig. 1. It can be seen that the spectral properties of C-PC in water are maintained in the reverse micelles with R values ≥2.5 for 12 h. This indiw cates that the characteristic properties of C-PC are maintained when it is solubilized in the reverse micelles. However, when the R values are equal w to 1.0, the absorbance remarkably increases at the wavelengths <400 nm, indicating denaturation of C-PC. Fig. 2 shows the fluorescence spectra of C-PC in water and in reverse micelles with different R values. The fluorescence intensities in w reverse micelles are lower than those in water, while their emission maxima at 644 nm are invariable. Both absorption and emission spectra suggested that C-PC is stable in the Triton X-100/pentanol/cyclohexane reverse micelles and this system can be used to study the process of energy transfer from phycobiliproteins to chlorophyll a. The energy transfer from C-PC to chlorophyll a can be observed directly from Fig. 3Fig. 4. When chlorophyll a is added into the reverse micelles, the fluorescence intensity of C-PC decreases. The more chlorophyll a is added, the lower intensity of fluorescence emission of C-PC is observed.

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Fig. 3. Fluorescence spectra of the studied system at different chlorophyll a concentrations. The decrease of the fluorescence intensity of C-PC indicates the occurrence of energy transfer from C-PC to chlorophyll a. The fluorescence spectra of pure chlorophyll a at the same experimental conditions are given on the bottom of the figure. R =2.5 [ Triton X-100]=0.25 M, w Triton X-100; pentanol=4:1(w/w), [C-PC ]=1.2×10−7, [chlorophyll a]: (a) 2.1×10−7 M; (b) 4.2×10−7 M; (c) 1.1× 10−6 M.

Besides, the energy transfer efficiencies are also related to the R values, that is, the energy transfer w efficiency is higher in reverse micelles of lower R w value. The energy transfer efficiency can be calculated according to: F (1) E=1− da F d where F and F are the fluorescence intensities da d in the presence and absence of the acceptor, respectively. For a given system, the overall fluorescence intensity is the sum of those of all components, so the F can be written as follows: da F =F−F −(F −F )W (2) da chl d da chl where F is the overall fluorescence intensity of the system; F is the fluorescence intensity of chlorochl phyll a excited directly at 590 nm. F −F is the d da

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Z. Jiquan et al. / Colloids Surfaces B: Biointerfaces 11 (1998) 9–14 Table 1 The efficiency of energy transfer from C-PC to chlorophyll a [chlorophyll a] (M )

Efficiency (%)

2.5

2.1×10−7 4.2×10−7 1.1×10−6

26 33 37

7.5

2.1×10−7 4.2×10−7 1.1×10−6

0.91 13 24

R

w

[C-PC ]=1.2×10−7 M.

0.128, 2.2 and 0.45, respectively, in reverse micelles with R value of 7.5; while those as 0.136, 2.2 and w 0.43, respectively, with R of 2.5. The same W w chl value of 0.13 was obtained in both cases. Therefore,

Fig. 4. Fluorescence spectra of the studied system at different chlorophyll a concentrations. The decrease of the fluorescence intensity of C-PC indicates the occurrence of energy transfer from C-PC to chlorophyll a. The fluorescence spectra of pure chlorophyll a at the same experimental conditions are not given. R =7.5, [ Triton X-100]=0.25 M, Triton X-100: w pentanol=4:1(w/w), [chlorophyll a]=(a) 2.1×10−7 M; (b) 4.2×10−7 M; (c) 1.1×10−6 M; (d ) 1.6×10−6 M.

decrease of fluorescence intensity of C-PC caused by energy transfer to chlorophyll a. Therefore, (F −F )W should be the fluorescence intensity d da chl of chlorophyll a excited by the accepted energy from C-PC, W is the quantum yield of chlorochl phyll a. The value of W can be determined chl experimentally by the following equation: F A W chl = chl PC (3) W F A PC PC chl F A chl PC W =W (4) chl PC F A PC chl where A and A are the absorbances of C-PC PC chl and chlorophyll a at the wavelength of 590 nm. F , F , A and A were directly obtained from chl PC PC chl experimentally measured spectral data, while W PC was measured as 0.65 in water, therefore F /F , A /A and W were determined as chl PC PC chl PC

F =1.2F−1.2F −0.15F da chl d Using Eqs. (1) and (5) one can find that:

(5)

(F−F ) chl (6) F d The values of F, F and F can be obtained from chl d Figs. 3 and 4. Thus, the energy transfer efficiencies at different chlorophyll a concentrations can be calculated using Eq. (6). The results are shown in Table 1, from which it can be seen that the energy transfer efficiency is related to R . w E=1.15−1.2

4. Discussion Although the reverse micelles of Triton X-100 have been previously studied by several authors [7–9], a model of the reverse micelles has not yet been proposed. Fortunately, the model of the micelles of Triton X-100 was proposed by Robson and Dennis [12] and Chauvet et al. [13]. Based on the model of the micelles of Triton X-100 and a knowledge of the fundamental properties of reverse micelles, a model is proposed for the reverse micelles of Triton X-100. It is assumed that the reverse micelles are spherical and composed of three parts—the water pool in the core, the hydrophillic region in the middle and the peripheral hydrophobic region. The hydrophillic region con-

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Fig. 5. Possible situations for a biopolymer solubilized in a micellar hydrocarbon solution, (a) water-shell model, (b) concerted-micelles mechanism, (c) protein partially exposed to the hydrocarbon solvent and (d) ion-pair interactions between the biopolymer and the ionized surfactant heads.

tains the OE chain of Triton X-100, pentanol and water molecules which bind to oxygen atom in the OE chain by a hydrogen bond. The hydrophobic region consists of an octyphenyl group and cyclohexane. The volumes of water pools and reverse micelles increase with R value though not monow tonically. The water pools in the system are in dynamic equilibrium, in which water molecules can move from one water pool to the others, so do the solutes. For protein solubilization in reverse micelles, there are four situations [14] (Fig. 5). In the first situation, the protein is confined in the water pool and is protected by a layer of water molecules from the surfactant heads. In the second situation, the solubilization is accomplished by the concerted action of several small micelles. In the third situation, part of the protein is directly exposed to the hydrocarbon solvent. Finally, the solubilization might occur via ion-pair interactions between the charged head groups of the surfactant and the ionized side chain of the protein. For a nonionic surfactant, the last situation is absent. From Fig. 1, it is concluded that the solubilized C-PC is not denatured when the R value ≥2.5, since the w

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absorption spectrum of denatured C-PC exhibits an hypochromicity and a blue shift of the absorption maximum. Furthermore, the ratio of visible to near-ultraviolet for a denature state is almost up to 1 [15]. Based on the experimental results (not shown), C-PC would precipitate when mixed with hexane and pentanol, which indicates that C-PC is very easily denatured by pentanol and cyclohexane, therefore, it is only solubilized in the first situation in the experimental conditions (R ≥2.5), which means the dimension of the water w pool must be large enough to hold a C-PC trimer. The solubility of chlorophyll a in both organic and aqueous solutions implies that chlorophyll a is mainly located in the hydrophillic region with the large tetrapyrrole rings near the water pool [13], in which the bound water in the OE chain may coordinate as a ligand to the Mg ion in the chlorophyll a molecule. Therefore, the chlorophyll a molecules may act as an acceptor of energy from C-PC. Generally, the excitation energy can transfer from one molecule to others by collision or dipole–dipole interaction. In the reverse micellar system, C-PC is protected by the water layer and chlorophyll a locates in the hydrophillic region, therefore they cannot contact directly. The energy transfer can occur only by dipole–dipole interaction so-called Fo¨rster mechanism [16 ]. Based on this mechanism, the rate of energy transfer should depend on the coupling of the energy levels and the spatial separation as well as the orientation of the transition dipoles of the donor and the acceptor [17]. Besides, it also depends on the number of donor–acceptor pairs. The efficiency of energy transfer (E ) is E=

R6 0 R6 +R6 0

where R is the distance at which the efficiency of 0 energy transfer is 50%. In the reverse micelles of Triton X-100 each water pool could hold only one C-PC trimer due to limited space while the hydrophillic region can accommodate many more chlorophyll a molecules. That is why the efficiency of the energy transfer is proportional to the concentration of chlorophyll

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a (although not monotonically). From Table 1 it can be seen that the efficiency is only 37% when the chlorophyll a concentration is 1.1×10−6 and R is 2.5. The low efficiency of energy transfer is w because C-PC and chlorophyll a are not properly coupled in the reverse micelles. On the other hand, in the native cells, there exist allophycocyanin (APC ) between C-PC and chlorophyll a, which acts as the energy transfer bridge. In fact, the fluorescence spectrum of C-PC overlaps the absorption spectrum of APC fully, and in turn, the fluorescence spectrum of APC overlaps completely with the red band of the absorption spectrum of chlorophyll a. The distances and orientations between the chromophores are optimized in native cells. These make the light energy absorbed by the C-PC flow in an energy cascade to the chlorophyll a. Therefore, APC is necessary for high energy transfer efficiency in native cells. On the other hand, the dimensions of water pool, which determines the distance between C-PC and chlorophyll a in the reverse micelles of Triton X-100, generally increases with the water content [8]. The high R will produce a larger dimension w of water pool. Therefore, the efficiency of energy transfer is higher in the reverse micelles with lower R than that with higher R . That is why the same w w amount of chlorophyll a give more fluorescence of C-PC in Fig. 3 than in Fig. 4.

Acknowledgment This work was supported by Natural Science Foundation of China (NSFC ) with No. 29773049.

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