BioSystems, 25 (1991) 131--140
Elsevier Scientific Publishers Ireland Ltd.
Surfactant micelles containing protoporphyrin IX as models of primitive photocatalytic systems: a spectral study A . A . S i v a s h a, Z. Masinovsk:~ b a n d G.I. L o z o v a y a a 'W.G. Kholodny Institute of Botany, Ukr. SSR Academy of Sciences, Repin str. 2, Kiev (USSR) and bLaboratory of Evolutionary Biology, CzechoslovakAcademy of Sciences, Na Folimance 11, Prague (Czechoslovakia) (Received January 14th, 1991) (Revision received April 2nd, 1991)
Micelles of various surfactants containing protoporphyrin IX (PPIX) have been studied as models of primitive membrane-like photocatalytic systems. Spectral characteristics (absorption spectra, fluorescence emission and excitation spectra, fluorescence quantum yields and lifetimes) have been measured to indicate specific interactions of PPIX molecules with the micelles. Two types of PPIX aggregates are probably formed in water: non-fluorescent clusters corresponding to the absorption peak at 642--648 nm and fluorescent friable dimers with strong solute-solvent interactions corresponding to the absorption peak at 618 nm. The aggregates are solubilized by the micelles, which results in an increase of the fluorescence quantum yield (and thus in the increase of the PP]:X sensitizing ability). Solubilization of PPIX molecules by SDS-micelles is enhanced by the partial neutralization of the negative surface charge of the micelles. Neutral Triton X-100 micelles solubilize PPIX much better than SDS particles; however, ~ome of the clusters formed in the bulk aqueous phase of the detergent-water system remain aggregated in the crown of the micelle. The positively charged CTAB micelles have been shown to provide the best solubilization of PPIX accompanied by ~he highest increase of its emitting activity. The results are discussed in terms of the possible role of PPIX-containing membraneous systems in primitive photosynthesis.
Keywords: Surfactant micelles; Protoporphyrin IX; Primitive photosynthesis.
The membraneous arrangement of lightharvesting pigments, sensitizers, electron carriers and several enzymes within the membrane is of primary importance in the structural and functional organization of the contemporary photosynthetic machinery (Govindjee, 1982; Morris and Meisel, 191~9). Therefore, incorporation of porphyrin molecules into primitive membraneous structures may have beeen one of the most important steps of the early evolution of photosyntheses. In regard to the self-assembly of prebiotic systems, complexes of relatively simple amphi-
Correspondence to: Z. Masinavsk~.
philic molecules are suggested as predecessors of contemporary cytoplasmic membranes. The formation of amphiphiles by the photooxidation of linear hydrocarbons and their association into membraneous structures was reported by Deamer et al. (1989). The formation of micelles in aqueous solutions of various surfactants represents another model of self-assembly of amphiphiles. The interior of the surfactant vesicle contains hydrophobic hydrocarbon chains of amphiphilic molecules, while hydrophilic head groups are located at the surface. The interface of ionic micelles is charged and thus an electric double layer is formed. Surfactant micelles are one of the most exploited membrane-mimetic agents, especially in the study of the primary processes of photosynthesis (Turro et al., 1980; Fendler, 1981).
0303-2647/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Protoporphyrin IX (PPIX), the key compound of contemporary biosynthetic pathways of chlorophylls and heme, has been suggested as a possible photosensitizer of primitive photosynthetic reaction centers (Olson and Pierson, 1987; Masinovsk~ et al., 1989; Lozovaya et al., 1990). In the work reported here PPIX-containing micelles of several surfactants have been studied as models of primitive membrane-like photocatalytic structures. Materials and methods
The following substances were used: protoporphyrin IX, disodium salt (Sigma), sodium dodecylsulfate (SDS) (Sigma), p-(1,1,3,3-tetramethylbutyl) phenoxypolyoxyethylene glycols (Triton X-100) (Merck) and hexadecyltrimethylammonium bromide (CTAB) (Schuchardt, Mfinchen). PPIX was dissolved in 50 ~l of 25% NHaOH within a few seconds and made up rapidly to the required volume with water. PPIX concentration was calculated from the extinction value of the mixture of the PPIX solution (3 ml) and 36% HC1 (1 ml); the value of the molar extinction coefficient used was 2.75 × 105 M -1 (Gibson, 1964). Micelles of three types, negatively charged (SDS), neutral (Triton X-100) and positively charged (CTAB), have been prepared by mixing the aqueous solution of the corresponding surfactant with the PPIX solution. Parameters of the micelles studied in this work are summarized
in Table 1. Molar concentration of micelles, c,~ was calculated by the equation: c - CMC Cm
where c is the total surfactant concentration, CMC is the critical micelle concentration and n is the aggregation number. The %/c~ ratio (z), where cp is PPIX molar concentration, have been calculated. Absorption spectra were measured using a Specord M-40 spectrophotometer (Carl Zeiss, Jena). Fluorescence emission and excitation spectra were carried out on a Hitachi 850 spectrofluorimeter with automatic correction of the spectra. The fluorescence quantum yields were calculated after Friefelder (1976) using chlorophyll a as a standard according to the equation: Qx = I Q Asn2
where Q is quantum yield, I, integral intensity of fluorescence, A, absorbance at the excitation wavelength, n, refractive index, x, sample, s, standard. The following chlorophyll a standard values have been used: Qs = 0.32, As = 1.35. nx = n i l 2 0 ffi 1.33. Fluorescence lifetimes were detected by the method of time-correlated single-photon counting. A photochemical Research Associates Model 3000 ns lifetime fluorometer was used.
TABLE 1 Some parameters of the surfactant micelles (adapted from Helenius and Simons, 1975). Detergent
Critical micelle concentration, CMC × 10 `3 (M)
Aggregation number, n
Triton X-100 SDS SDS CTAB CTAB
--0.5 M NaCl -0.02 M KBr
0,240 8.2 0.520 0.920 0.225
140 62 126 72 220
Results and Discussion
t.o Spectral and photochemical properties of protoporphyrin IX are influenced by pigmentpigment interactions as well as by the microenvironment of the pigment molecules. Absorption and fluorescence characteristics of PPIX in surfactant micelles have been studied in this work by comparison with those in the aqueous environment. PPIX aggregates readily in water (Brown et al., 1976; Lozovaya et al., 1990), this may be detected by the changes of its spectral characteristics. The absorption spectrum of PPIX in water differs from that in organic solvents by the following parameters (Fig. 1): • the broadening of the Soret band accompanied by the blue shift at higher PPIX concentrations which increases with time; • appearance of ~the new absorbing band at 455--475 nm; • considerable changes in the Q-band (500--630 nm); the characteristic maximum at 503 nm cannot be detected which may result from overlapping by the new band at 455--475 nm; the Q-band is shifted to the longer wavelength (red shift), the shift of the longest peak from 626 nm to 646 nm being the most dramatic. The presence of 1.0 M NaC1 brings about additional changes in Lhe absorption spectrum of PPIX, especially in the Q--band (Fig. 1). The influence of NaC1 is even more apparent when the fluorescence emission spectra are analyzed (Fig. 2). The increase in the ionic strength is accompanied by a sl:rong decrease in the intensity of fluorescence at 618 nm reflecting the enhanced pigment-pigment interactions which may result from the decrease of the effective concentration (activity) of water as well as from the screening of the negatively charged COOgroups of PPIX. Spectral characteristics of PPIX are considerably changed in the presence of surfactant micelles when compared to the aqueous environment; the differences depending on the detergent used have als~ been detected. No substantial changes in absorption spec-
¢ \ ! 564
WlIVELENaTH (rim) Fig. 1. Absorption spectra of protoporphyrin IX. (A) in methanol; (B) in water: (1) 2 × 10-5 M; (2) 4 x 10 -6 M; (C) in 1 M NaCl: (1) 2 x 10-r' M; (2) 4 x 10 -~ M.
trum were detected in the presence of the negatively charged SDS-micelles at the high pigment/micelles ratio (z = 0.1) when compared to
~t0 GJO 650 6?0 690 ?tO ?30 WAVELENGTH (rim)
Fig. 2. Fluorescence spectra of protoporphyrin IX. Excited at 400 nm: (a) in methanol; (1) 4 x 10-6 M PPIX in 0.25 M NaC1; (2) 4 x 10-6 M PPIX in 2.0 M NaCl; (3) 2 x 10-5 M PPIX in 0.25 M NaCl; (4) 2 × 10-5 M PPIX in 2.0 M NaC1.
the aqueous environment (Figs. 3A, 1B) indicating weak incorporation of PPIX molecules negatively charged by CO0- groups. With the decrease in the pigment/micelles ratio from 0.1 to 0.05 the absorption spectrum became more similar to that in methanol (Figs• 3A, 1A) reflecting probably the increased ratio between the solubilized molecules and the bulk of PPIX in the water phase. A similar effect was observed after 0.5 M NaC1 was added to the mixture with a high pigment/micelles ratio (z -- 0.2) indicating the higher degree of PPIX solubilization which might be a result of the partial neutralization of the negative surface charge of the micelles (Figs. 3B, 1). NaC1 was demonstrated to diminish the electric double layer, to influence the parameters of polarity of the micelles, etc. (Kryukova et al., 1973; Robson and Dennis, 1977). Some of these changes might also influence solubilization of PPIX. The results obtained from the absorption spectra are consistent with the corresponding values of the fluorescence quantum yield of PPIX (Table 2).
Fig. 3. Absorption spectra of protoporphyrin IX in SDS micellar system. (A) (1) z = 0.1; (2) z = 0.05. (B) z = 0.2: (1) in water; (2) in 0.5 M NaCl.
Neutral Triton X-100 micelles solubilize protoporphyrin IX much better than the negatively charged SDS particles. This is indicated by the similarity of the absorption spectra in the detergent-water system and in methanol (Figs. 4, 1A) as well as by the values of PPIX fluorescence quantum yield (Table 2). The best solubilization of PPIX was obtained by CTAB micelles (Fig. 5): the Soret band is sharp, the peak at 646 nm characteristic for the aqueous environment disappears and the whole
135 TABLE 2 Fluorescence quantum )ields of PPIX at different micro° environments. Micro-environment
Fluorescence • quantum yield
Methanol Water • with SDS • with SDS + 0.5 M NaC1 • with Triton X-100 • with CTAB • with CTAB + 0.02 M KBr
0.155 0.009 0.008 0.030 0.109 0.145 0.135
Q-band is similar to that in the absorption spectrum of PPIX in methanol (Fig. 1A). Electrostatic interactions between the positively charged surface of CTAB micelles and COOgroups of PPIX probably support incorporation of PPIX molecules by the particles. This is consistent with the comparison of the fluorescence quantum yield of :PPIX in the CTAB-water system with that of the methanol system (Table 2). When KBr was added to the CTAB system the value of the quantum yield decreased indicating the decreased solubilization of the negatively charged PPIX molecules due to the
decrease of the positive surface charge of the micelles. Solubilization of PPIX by various surfactants as well as its spectral properties in the micellar system depend on the physicochemical properties of the particles: surface charge, polarity, viscosity, water content, etc. In the case of the negatively or positively charged micelles formed by the ionic detergents such as SDS or CTAB (Fig. 6A) it is strongly influenced by electrostatic interactions sensitive to the ionic strength of solution. The specific properties of non-charged Triton X-100 micelles (Fig. 6B) are given by the chemical structure of this detergent: a relatively small non-polar component (tetramethylbutylphenol) and a long hydrophilic part (10 oxyethylene units) which determines the formation of a highly hydrated friable crown of the micelle. Such particles are not able to solubilize PPIX completely since a part of the clusters formed in the bulk aqueous phase of the Triton X-100/water system remains aggregated in the crown of the micelle. Information which comes from absorption O.D. 2.0
I ~ID. 2;
qSo w~vecEN~'r. Cnm)
Fig. 4. Absorption spectrum of protoporphyrin IX in Triton X-100 micellar system.
qSo w~vELeNeT~ (nm)
Fig. 5. Absorption spectrum of PPIX in CTAB micellar system.
, , . . . ' ~ ' " ~ ... ,.~ O o o ;'..
:o c ~ ,:~.~-~l'o~
Fig. 6. Localization of protoporphyrin IX molecules in charged (A) and uncharged (B) micelles. (A) (1) hydrophobic core; (2) charged surface (Stern layer); (3) diffuse double layer (Gouy-Chapman layer); (4) aqueous bulk phase. (B) (1) hydrophobic core; (2) hydrated crown; (3) aqueous bulk phase.
spectra is not sufficient to permit an understanding of the behaviour of PPIX molecules in a complex water-detergent system. A more detailed understanding comes from measurements of PPIX fluorescence. By comparing absorption and fluorescence excitation and emission spectra in water (Figs. 1, 2, 7), it becomes evident that the absorption maximum at 642--648 nm belongs to non-fluorescent (and thus photochemically inactive) PPIX clusters. However, the explanation of the properties of PPIX molecules which still emit in water is more complicated. PPIX fluorescence in methanol (dielectric constant, e -- 32) has a maximum at 633 nm (Fig. 2a); it is emitted from the excited state corresponding to the absorption peak at 626 nm (Fig. 1). In water (e -- 80) the maximum of PPIX fluorescence was found at 618 nm; it is emitted from the excited state corresponding to 610 nm (Figs. 2, 7). The absorption spectrum of PPIX in water (Fig. 1B) lacks a peak at 610 nm; therefore, the amount of the centres emitting at 618 nm is probably very small and is detectable only by the fluorescence spectroscopy. In the presence of micelles they disappeared completely. Fluorescence emission spectra of PPIX in the
Fig. 7. Fluorescence excitation spectrum of protoporphyrin IX in water (detected at 620 nm).
micellar systems (E close to that of methanol) have been found to be almost identical to that in methanol (Fig. 2a). Such a strong shift (from 618 nm to 633 nm) cannot be explained only by the change of the solvent dielectric properties (Ae = 48). Actually, Kessel and Rossi (1982) have observed a very small (if any) blue-shift in PPIX fluorescence as the solvent dielectric constant decreased from 80 (dimethylformamide) to 32 (methanol). In our previous work (Lozovaya et al., 1990) we have proposed that the maximum at 618 nm corresponds to the clusters, in which the processes leading to the loss of excitation energy are not efficient enough to prevent the fluorescence. To obtain more information concerning this suggestion we have measured the lifetimes of PPIX fluorescence in different micro-environments. Our results together with some other references are summarized in Table 3. Single exponential fluorescence decay curves (Fig. 8) have been obtained for both aqueous and micellar systems indicating, together with the data of Table 3, the monomeric-type fluorescence for both cases. The differences between the lifetimes indicated here are negligible when compared to those reported by Andreoni et al. (1983) for hematoporphyrin dimers (4--5 ns) and monomers (13--1.5 ns). Our results argue against the strong intermolecular coupling within the PPIX diimers in water which emit at 618 nm. If the interaction of PPIX molecules within the dimer is entirely dipole-dipole it is given by
the following equation (FSrster, 1948): Vint =
where gin t is the interaction energy, n = 1.33, the refractive index, K -- 2, the orientation factor adopted for the dipoles aligned in one plane and parallel to each other, R -- 0.5--1.0 nm, distance between dipoles. The transition dipole moment (~) was obtained from the absorption spectrum of the Gaussian line shape after Colbow and Danyluk (1976): #2 = 1.955 x 10 -3s emVl/2P/1 where em is the extinction coefficient at the maximum, ~1/2 is the half width at half peak height and va is the absorption maximum. With these conditions the interaction energy is 60--25 cm-1. Since the shift of absorption maximum from 626 nm to 610 nm (Fig. 1) corresponds to 420 cm-1, even in the strongly coupled PPIX dimers it cannot be explained only by exciton coupling after theoretical considerations. Thus, both solvent dielectric properties and dipole-dipole interactions could contribute to the effects observed, but do not explain them completely. Some additional shift probably results from the different solute-solvent interactions of PPIX dimers and monomers. However, the possibility that the fluorescence at 618 nm is emitted by a small amount of monomers which are not clustered in the aqueous environment, but interact with water molecules by some very
TABLE 3 The lifetimes of PPIX fluorescence in different micro-environments. Micro-environment
(ns) Methanol Water * with CTAB • with Triton X-100
17.50 15.52 16.01 16.20
Dzhagarov and Gurinovitch (1982) This paper This paper Savitsky et al. (1979)
t 0 -"re.
tJU~fL N/.BIKA eO0 t,~O 4~ I
460 • k~ ZOO 220 2qO
i ee Qo
ee* ~ e
GO gO T/14E (~l~OSeCOel~)
Fig. 8. Fluorescence decay profile of protoporphyrin IX in water. Excitation: 400 nm. Emission: 620 nm. rf = 15.520 ± 0.287 ns.
effective but still unknown way, cannot be excluded. Such interactions have also been suggested for the aqueous solution of hematoporphyrin (Smith et al., 1989). Micelles strongly influence both pigmentpigment and pigment-water interactions by
solubilizing PPIX within their hydrophobic micro-environment which may be supported (e.g. with the CTAB micelles) by the strong electrostatic interactions. Solubilization of PPIX by the micelles increases its emitting and photosensitizing abilities.
the following parameters for the energy transfer between the PPIX molecules: the FSrster parameter of R0 = 37 A and the efficiency of transfer o f F = 2.1 x 109 s -1. When compared with the corresponding values of chlorophyll-chlorophyll transfer (R0 -- 65 ~, and F -- 0.44 x 1012 s -1) these parameters allow only for a limited transfer between the PPIX molecules. In spite of the fact that their assembly within the membrane-like structure might have facilitated this transfer, protoporphyrin IX still does not seem to be an adequate candidate for the light-harvesting function (cf. Olson and Pierson, 1987). On the other hand, our experiments with surfactant micelles have demonstrated that PPIX might have sufficiently increased its absorbing and emitting (and, thus, sensitizing) abilities by being incorporated in a membrane-like structure. Comparing the absorption Q-bands of PPIX in CTAB micelles and that of chlorophyll (Fig. 9) as well as the corresponding crosssections (1.6 x 10 -16 and 3.5 x 1 0 - 1 6 c m - 2 respectively) with the spectral distribution of the natural solar radiation, one may suggest that some simple PPIX-containing membraneous structure could efficiently absorb solar energy and utilize it for photochemical transformations in primitive photocatalytic systems. References
w,qve.Le No"r~'l (nm) Fig. 9. Spectral distribu~ion of solar radiation on the Earth's surface (A) compared to the absorption Q-bands of protoporphyrin IX (B) and chlorophyll a (C).
Two possible functions of PPIX-containing membrane-like structures should be discussed in the context of the early photosynthesis: lightharvesting and photosensitizing. Concerning the first point, we have calculated
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