C-phycocyanin incorporated into reverse micelles: a fluorescence study

Colloids and Surfaces B: Biointerfaces 18 (2000) 51 – 59 www.elsevier.nl/locate/colsurfb

C-phycocyanin incorporated into reverse micelles: a fluorescence study Ruperto Bermejo a, Eva M. Talavera b, Carmen delValle b, Jose M. Alvarez-Pez b,* a

Department of Physical and Analytical Chemistry, Uni6ersity of Jaen, EUP of Linares, Linares 23700, Spain b Department of Physical Chemistry, Uni6ersity of Granada, Cartuja Campus, Granada 18071, Spain Accepted 15 September 1999

Abstract The solubilization of C-PC into aerosol-OT(AOT, sodium bis(2-ethylhexyl)sulphosuccinate)/water/i-octane reverse micelles has been investigated by fluorescence spectroscopy by following its behaviour as a function of the water-to-surfactant molar ratio, w0. The maximum wavelength emission decreased steadily with an increase in water content, together with a concomitant increase in steady-state anisotropy. These effects saturated at a w0 of around 30 and remained practically constant at w0 \30. These results are explained in terms of an inclusion into the inner core of the reverse AOT micelles. Strong interactions also took place between amino-acid residues neighbouring bilin chromophores and the AOT sulfonate headgroups, which resulted in the chromophores being located in the structured interfacial micellar water layer. The stabilization rate of the spectral parameters from micelles of protein solutions at pH 5.0 followed pseudo-first-order kinetics and afterwards remained stable for some days. The fluorescence intensity decay of C-PC in reverse micelles is described by a triple exponential function, showing a similar complex pattern to that in an aqueous solution. The fluorescence lifetime values of micelled C-PC indicate that the chromophores are shielded against solvent quenching by the structured interfacial water layer. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Aerosol-OT; Micelled C-PC; Chromophores; Interfacial water layer

1. Introduction In recent years there has been a general tendency towards the use of natural rather than synthetic dyes in foods and cosmetics, many synthetic pigments having been excluded from use in * Corresponding author. Tel.: +34-958-243831; fax: + 34958-244090.

food and cosmetics on the grounds that they are toxic, carcinogenic or otherwise unsafe. The consequent search for natural colorants might be resolved with natural dyes such as biliproteins from antenna complexes of algae. In general, algal pigments have not yet been approved for use in the food and cosmetics industries, although they have shown no signs of being toxic. In Japan, however, where algal cultivation is a well-devel-

0927-7765/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 1 2 9 - 0

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oped industry, these pigments have already been patented. Japanese patents on the uses of algal colorants in foods include the coloring of fermented milk products such as yoghurt [1]. Colorants prepared from red and blue-green algae are also suitable for use in cosmetics. Thus, a heatand pH-stable, low-molecular-weight phycocyanin produced from thermophilic blue-green algae has already been formulated as an eye shadow [2] and Arad and Yaron [3] have prepared pink and purple cosmetics from various red microalgae. Biliproteins are red or blue pigments with openchain tetrapyrrole prosthetic groups (bilin), which in their functional states are covalently linked via one or two thioether links to the specific cysteine residues in the apoproteins. They form light-harvesting antenna complexes (phycobilisomes) of cyanobacteria (blue-green algae) and of two groups of eukaryotic algae, the red algae and the cryptomonads. The biliproteins are broadly classified into four groups based on their spectroscopic properties: phycoerythrins (PE), the major prosthetic group of which is the red chromophore phycoerythrobilin; phycocyanins (PC), which contain either a mixture of phycocyanobilin (PCB) and phycoerythrobilin chromophores or phycocyanobilin alone, depending upon the species of origin; allophycocyanins (APC), with phycocyanobilin as their prosthetic group; and phycoerythrocyanins (PEC), which have phycocyanobilin and phycocryptoviolin chromophores. The apoproteins are built of equimolar quantities of two dissimilar subunits, a and b, with molecular masses in the range of 17 000– 20 000 Da. PE also contains a heavier (30 000 Da) g subunit. Each subunit is linked to one or more bilins. An ab unit is known as a monomer. The antenna systems of some cyanobacteria, such as Spirulina platensis for example, are composed of APC and C-phycocyanin (C-PC). The latter carries one PCB on the a subunit, the so-called a84 chromophore, and two PCB’s on the b subunit, the so-called b84 and b155 chromophores [4]. MacColl and colleagues have determined the aggregation states of phycocyanin. Monomers; trimers and hexamers of C-PC exist in equilibrium but the protein is primarily in a monomeric form at moderate ionic strength and concentrations of

less than 8 mg/ml [5]. Saito et al. [6] have also undertaken an extensive series of experiments on phycocyanin aggregation. At pH 5.4, their data could be fitted to a monomer-trimer equilibrium. Sodium perchlorate (1.0 M) and sodium thiocyanate dissociates C-PC to a homogeneous solution of monomers but does not dissociate the monomers into their two subunits [5,7]. In a previous paper, we described the visible absorption, fluorescence emission and steady-state excitation fluorescence anisotropy spectra of diluted C-PC solutions from S. platensis in 20 mM sodium phosphate buffer, pH 7.0, and in the same buffer supplemented with 1 M sodium perchlorate. The absorption and fluorescence spectra of C-PC from S. platensis in trimeric and monomeric forms show that the spectral shapes are affected only slightly by aggregation changes in the protein, although when its aggregation state changes from trimer to monomer [8] there is a loss in visible absorption, a decrease in fluorescence efficiency and a considerable increase in the excitation fluorescence anisotropy spectrum. The three-dimensional structure of the C-PC trimer has been determined from Fremyella diplosiphon [9–11] and Agmenellum quadruplicatum [12]. The crystal unit cell of C-PC from these cyanobacteria contains three (ab)6 hexamers and each hexamer is made up of two trimers superimposed head-to-head. The unit-cell dimensions of C-PC crystals from S. platensis are between the values obtained for C-PC from F. diplosiphon and A. quadruplicatum [13]. In the trimer, the a84 chromophore in one monomer and the b84 chromophore in an adjacent one are in close contact and energy is transferred from the a84 chromophore to the b84. Nevertheless, the close proximity of the chromophores is not necessarily sufficient for complete energy transfer and other structural features are required in this process. The chromophores’ microenvironment plays an important role in the native trimer, and the chromophore configuration and polypeptide conformation affect each other to give the most favourable structure for efficient energy transfer. Upon disaggregation and denaturation there is a dramatic increase in the excitation fluorescence anisotropy spectra due to a decrease

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in energy transfer between chromophores. In the presence of strong protein denaturing agents the two subunits of the phycocyanin monomer can be completely separated from each other. Denaturation of the native C-PC structure is accompanied by a loss in visible red absorption and an increase in absorbance in the near-ultraviolet band (decrease of Rred/UV). Biliproteins are highly soluble in water, but some applications, as indicated above, require their being dissolved in apolar solvents. Moreover, the advantageous fluorescence properties of biliproteins could provide a versatile model when included into reverse micellar media, a microheterogeneous system which mimics the watermembrane interface well. For these reasons we have studied the possibility of using reverse micelles of sodium bis (2-ethylhexyl) sulphosuccinate (AOT)/water/i-octane to dissolve C-PC from S. platensis in apolar media and we have made use of the fluorescence properties of this biliprotein to locate the relative position of its chromophores when incorporated into reverse micelles of AOT. Our results indicate that electrostatic interaction of the charged amino-acid side chains with surfactant polar-head groups determine the location and conformational adaptability of C-PC in AOT micelles.

2. Materials and methods

2.1. Reagents Highly pure C-PC was obtained from cells of S. platensis, generously provided by IMADE S.L. (Granada, Spain), by several initial stages and two chromatographic steps, as described elsewhere [8,13]. Blue single crystals of C-PC were taken from the capillary tube where they were obtained [13] and carefully cleaned with diluted solutions of PEG. The crystals were then dissolved in 20 mM phosphate buffer at different pH values between 5.0 and 7.0, to provide moderately concentrated stock solutions. Isooctane and n-heptane (uvasol grade) were from Merck (FRG). All other chemicals were from Sigma Chemical (St Louis, MO). All chemi-

53

cals were used as received. The purity of AOT was confirmed by the close agreement of its UV-absorption spectrum with that found in the literature [14].

2.2. Experimental procedures The anionic surfactant AOT can solubilize variable amounts of water in apolar solvents, the concentrations of which are generally expressed as w0 = [H2O]/[AOT], the molar ratio of water to surfactant. Reverse micelles were prepared with varying w0 values and biliproteins were incorporated into them from stock solutions prepared at pH 5.0–7.0 in 0.02 M phosphate buffer. The [protein]/[micelle] ratios were always constant at 0, 1 in order to render the probability of multiple occupation of the micelles negligible [15]. To incorporate the biliprotein into the micelles a few microliters of the appropriate C-PC solution was injected into the AOT solution in iso-octane and shaken till clear [16]. For the experiments, w0 was increased from 2 to 60 by adding the appropriate quantities of biliprotein stock solution, AOT-isooctane solution and water. The absorption spectra were recorded with a Perkin–Elmer lambda 16 spectrophotometer. Fluorescence emission spectra were measured with a Shimadzu RF 50001 spectrofluorometer with slit widths of 3 nm for both the excitation and emission monochromators. Typical excitation and emission bandwidths of 5 nm were used to measure steady-state anisotropy. The cuvette holders were thermostatically controlled to 25°C throughout the experiments. The kinetic incorporation of C-PC into AOT reverse micelles was followed by measuring maximum wavelength emission versus time through a system comprising w0 = 35 and phosphate buffer to different pH values. Stability was assessed by recording the fluorescence spectra versus time. Time-resolved fluorescence intensity was measured in the single-photon counting mode with an Edinburgh Instrument (UK.) spectrometer, model FL900. The data were obtained with a free running H2 discharge flashlamp operating at 7.0 kV, at 0.45 bar and a frequency of 40 kHz. The lamp pulse was recorded at the same wavelength as that

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of the excitation. The bandwidth of the monochromators was 10 nm. Cumulation was stopped when 5000 counts had been stored in the peak channel for the total fluorescence intensity decay and five decay profiles were collected, each at fixed excitation and emission wavelengths. The simultaneous analysis of multiple fluorescence decay experiments is frequently referred to as ‘global analysis’ and has proven useful for time-domain data. Each term in the analysis (t, a) may be fixed or floating in the fitting and may also be a linked parameter in the analysis. Any parameter that is linked will be held in common between each data file in the fitting, allowing related terms between the decays to be analysed over the whole experiment. We have linked ti and ai over all experiments of a family of five decay data from the same solution. The quality of fittings was judged by the reduced x 2 method, and the weighted residuals were checked for random distribution. The standard deviations of t1 and a1 listed in the table are purely statistical in origin and cannot be directly equated to parameter errors because they take no account of parameter correlation’s or systematic errors. They are calculated on the basis of the mean error. The mean fluorescence life-

Fig. 1. Variation in maximum emission ( ) and anisotropy () of C-PC as a function of water-to-surfactant molar ratio (w0) in reverse micelles; ( ) lexcitation = 600 nm; () lexcitation = 600 nm, lemission = 635 nm.

times B t\ are calculated by means of the expression: %i ait 2i B t\ =

(1) %i aiti

3. Results and discussion

3.1. Steady-state measurements Visible absorption, steady-state fluorescence and anisotropy spectra of C-PC in micellar solutions with w0 in the range of 5–60 have been recorded. Visible absorption and steady-state emission spectra of the protein when incorporated into solutions of reverse micelles of various w0 values exhibit blue shifts. The maximum wavelength emission of C-PC decreased steadily in concomitance with an increase in micellar water content until reaching saturation at a w0 value of around 30 (Fig. 1). In the same figure it can be seen that anisotropy increased with water content to remain practically constant at w0 ] 30. Similar variations in the absorption, emission and anisotropy parameters have been reported for a number of proteins and their fragments solubilized in reverse micelles [17–19]. The visible absorption, fluorescence emission and steady-state excitation fluorescence anisotropy spectra of C-PC trimers, monomers and micellar solution with a water content of w0 =40 are shown in Figs. 2–4. The spectra of the micelled C-PC are substantially different from the spectra pertaining to trimeric and monomeric CPC in aqueous solutions. Nevertheless, they are clearly different from the absorption spectra corresponding to fully denatured subunits of C-PC (absorption characteristics of both subunits are similar to each other with absorption maxima at 280, 350 and 553 nm, and the 553 nm absorbance of the C-PC b-subunit is twice that of the a-subunit [8]). The anisotropy values in Fig. 4 are higher than those corresponding to monomeric C-PC and the spectrum shows a pronounced step at a higher wavelength, as might be expected for

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haviour as a function of the water-to-surfactant molar ratio (w0). This parameter also determines the degree of deviation of the properties of the entrapped water from those of normal bulk water. Within certain ranges of water content and AOT concentration (e.g. w0 from 5 to 50) the reverse micelles form pseudo-stable microemulsions. The radius of their water pool may be between 10 and 80 A, and in fact the inner-core radius of reverse micelles in the AOT-iso-octane system at room temperature is of 50 A, for w0 = 30 [20]. For the AOT system an approximate relationship between w0 and this inner-core radius, r (A, ), is r: 1.75w0 [21]. Preliminary X-ray diffraction studies of C-PC from S. platensis give unit-cell constants of a= b= 182.38 A, , c =60.87 A, [13]. These unit-cell dimensions are between the values obtained for C-PC from F. diplosiphon [10,11] and A. quadruplicatum [12]. Thus it is reasonable to assume that

Fig. 2. Absorption spectra of C-PC. (—) C-PC in 20 mM sodium phosphate buffer, pH 5.0; ( — ) C-PC in 1 M sodium perchlorate, 20 mM sodium phosphate buffer, pH 5.0; and ( ) C-PC in reverse micelles (w0 = 40) from a stock solution of 20 mM sodium phosphate buffer, pH 5.0.

two or more non-equivalent chromophores coupled by energy transfer. In view of the heterogeneity and aggregation of the chromophores we have not attempted to evaluate an angle between the excitation and emission transition dipole moments from these data. Before discussing the structural aspects of C-PC solubilized in reverse micelles, it might be worthwhile commenting on micellar and C-PC dimensions. Besides the high optical transparency of the AOT/i-octane/water system, another important feature of this ternary system is that the distribution of the AOT micelles is quite narrow and their size depends upon the water/amphiphile ratio. Macromolecules dissolved in the aqueous core of reverse micelles have been studied on the whole by spectroscopic methods, following their be-

Fig. 3. Fluorescence emission spectra of C-PC (lexcitation =580 nm). (- - - -) C-PC in 20 mM sodium phosphate buffer, pH 5.0; (· · · ·) C-PC in 1 M sodium perchlorate, 20 mM sodium phosphate buffer, pH 5.0; and ( – – –) C-PC in reverse micelles (w0 =40) from a stock solution of 20 mM sodium phosphate buffer, pH 5.0.

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Fig. 4. Excitation anisotropy spectra of C-PC. The anisotropy values shown in the figure are the means of 5 values and were calculated for each 5 nm. ( ) C-PC in 20 mM sodium phosphate buffer, pH 5 0 (lemission = 650 nm) () C-PC in 1 M sodium perchlorate, 20 mM sodium phosphate buffer, pH 5.0 (lemission = 640 nm). () C-PC in reverse micelles (w0 =40) (lemission =635 nm).

the 3D structure of crystalline C-PC from S. platensis is 6ery similar to that of C-PC from these cyanobacteria and that the crystal unit cell of C-PC from S. platensis contains three (ab)6 hexamers. Rough calculations provide an anhydrous sphere equivalent radius for the trimeric C-PC from S. platensis of 43.2 A, . The effects described in the preceding paragraphs can be explained in the following way: the structure of the C-PC molecule is amphipathic, which also explains its self-association in solution. Monomers, trimers and hexamers of C-PC exist in equilibrium at a pH value close to neutral but at the stock concentration and ionic strength of the buffer used the proportion of trimers was greater than monomers and hexamers [8]. Since the C-PC trimer is longer than the micelles when the water pool is relatively small (w0 B30) it is reasonable to assume that the protein remains partially exposed to the hydro-

carbon solvent. Interactions take place between entrapped water, hydrophilic amino-acid sidechains and the AOT sulfonate headgroups, whereas the hydrophobic parts of the protein are directly exposed to the hydrocarbon solvent or interact with the lipophilic chains of the surfactant molecules. All these interactions result in conformational changes in the protein structure and disturb the energy transfer between chromophores, all of which can lead to the unfolding of the trimeric structure and partial monomerization. The spectral parameters become similar to those of the protein monomer and both a decrease in fluorescence efficiency and large, although not maximum, anisotropy values are to be expected. Since conformational changes can be seen in the absorption spectra [a slight decrease of Rred/UV (data not shown)], we take the decreased energy transfer to be the main cause of the initial increase in anisotropy. Until w0 = 30 the maximum wavelength emissions decreased and the anisotropy values continued to increase. After this, however, further increases in w0 did nothing to modify the values of these parameters. These effects can be explained in the following way: at w0 \ 30 the water pool is larger that trimeric C-PC and thus the protein must be completely confined in the water pool. Thus, steady-state anisotropy increased to its maximum value and the maximum wavelength emission remained constant. Likewise, the absorption spectrum indicates pronounced conformational changes, represented by a considerable decrease in Rred/UV as shown in Fig. 2. After a relatively long time in the micellar water pool, amphiphilic interactions might partially unfold both trimers and monomers and perhaps lead to the dissociation of monomers into their subunits (possibly partially unfolded too). Moreover, the unchanging values of both fluorimetric parameters, anisotropy and lmax, when the system has a water content of w0 \30 implies that chromophore mobility within reverse micelles is limited by the very strong interaction of the chromophores with the AOT sulfonate groups and the structured interfacial water layer in which they are probably embedded.

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3.2. Kinetic measurements We also recorded the absorption, fluorescence and anisotropy spectra of the C-PC micelles versus time at w0 =40. When micellation was done with C-PC solutions at pH 5.0, all steady-state parameters remained practically constant for 1 week or more, even when a precipitate phase appeared at the bottom of the cuvette (on the third or fourth day). The spectral parameters with the protein stock solutions at pH 7.0 were less stable than those with solutions of pH 5.0. The results discussed above agree well with those found from stability measurements, since the phycocyanobilin chromophores are very labile when uncoupled from the protein [22] and bleaching processes take place rapidly. Our results show, however, that absorbance and steady-state fluorescence remain constant for at least a week (the maximum emulsion stability time). This would indicate that the chromophores are embedded into the structured interfacial water layer intimately adjacent to the AOT sulfonate head

groups, and may be supposed to protect the chromophores from the bleaching processes. We also made experiments to establish the kinetics of C-PC incorporation into reverse micelles at different pH values between 5.0 and 7.0. Steady-state fluorescence spectra were recorded versus time and their maximum wavelengths were measured. The spectral parameters stabilised most readily at pH 5.0, which is the isoelectric point of C-PC; at pH 6.0 the stability rate decreased and continued to do so as the pH increased to 7.0. If we assume that the emission quantum yield of free protein is the same as that for micelled protein then the apparent lmax at intermediate states of incorporation will reflect the fraction of these states. In our system at pH 5.0, the micellar state fluoresced only about 80% as much as did the free protein, therefore the latter will just slightly dominate the apparent lmax and this approach may be used as a rough first step to compute approximately the kinetic constants of micellation. In this way we can calculate the fraction of free protein using the expression:

fraction

Fig. 5. Pseudo-first-order kinetic plot for the incorporation of C-PC into AOT reverse micelles. The slope of the straight line obtained is −0.182 and the correlation coefficient is 1.00.

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of

free

C-PC= 1−

l max − l max 0 t max l 0 − l max

(2)

where l max is the initial maximum wavelength 0 emission of the aqueous C-PC (pH 5.0)/AOT/iis the maximum fluorescence octane system, l max wavelength when the system remains stable over time and l max are the maximum fluorescence t wavelengths at different incorporation times. Fig. 5 is a semilogarithmic plot of free protein percentages versus time made from fluorescence measurements for a typical experiment at pH 5.0. The linearity of the data shows that the incorporation process is one of pseudo-first order in terms of free C-PC concentration, which is acceptable in processes carried out in the presence of excess micellar phase. At higher pH values the difference between the fluorescence efficiency of free and micellar C-PC is greater and the quick method described cannot be used. The data in Fig. 5 allow us to calculate an apparent kinetic rate constant of 0.182/min.

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Table 1 Fluorescence intensity decay parameters of the C-PC solutions of trimers, monomers and micellesa Sample

t1

t2

t3

a1

a2

a3

x2

Bt\

C-PC trimer C-PC monomer C-PC micelle

0.069 0.02 0.259 0.07 0.309 0.04

1.109 0.09 0.959 0.07 1.33 9 0.14

2.30 90.19 2.18 90.18 3.25 9 0.38

0.69 9 0.21 0.52 90.10 0 789 0.07

0.25 9 0.02 0.43 9 0.09 0.19 9 0.02

0.06 90.02 0.05 9 0.01 0.03 90.01

1.06 1.05 1.03

1.38 90.13 1.01 9 0.09 1.25 90.13

a The excitation wavelenght was 600 nm and the emission was recorded at 630 nm: lifetimes are in nanosecond and pre-exponential factors have been normalized.

3.3. Time resol6ed fluorescence Time-resolved measurements of fluorescence intensity from the aqueous solutions of C-PC trimers and C-PC monomers showed complex decays. According to x 2-minimization and deviation function criteria a triple exponential model improves the fitting of the experimental fluorescence intensity decay data. The best-fit parameters (lifetime, ti, and normalized amplitudes, ai ) are listed in Table 1. The decay in fluorescence intensity of C-PC in reverse micelles reveals the same complex pattern as it does in aqueous solution and is best described by a triple exponential function. The instrumental response, decay data and the best-fitting together with its residuals are plotted in Fig. 6 and the fitting parameters are given in Table 1. The lifetime components of trimers and monomers in aqueous solutions lie at around 63 ps and 2.3 ns, which in general agrees with the values found for C-PC by several other research groups [7]. The observed decay traces are always dominated by a long-lived component with a lifetime of about 2 ns. In micelles a rise time is observed although its relative amplitude is very low and the mean fluorescence lifetime B t\ decreases slightly with regard to C-PC trimers in aqueous solution although it is longer than that of C-PC monomers. This is in accordance with the steady-state fluorescence efficiency of the micelle protein content. The complex decay kinetics of C-PC are in part a manifestation of heterogeneity’s in the chromophore-protein arrangements and this heterogeneity continues to be evident in the micellar inner core. At present it is not possible to assign the decay components to each chromophore. X-

ray analysis has shown, however, that the chromophore arrangements of b84 and a84 are rather similar, and that both are close to the protein surface in monomers and subunits [11]. b155 is much more twisted and may be the one to become uncoupled first from the protein; thus it is tempting to assume that its lifetime is significantly reduced compared with b84 and a84 given that conformational mobility is a cause for the rapid internal conversion in bile pigments [23]. A com-

Fig. 6. (A) Fluorescence intensity decay of C-PC in reverse micelles (w0 =40), curve fitting based on a triple exponential model (line), and lamp pulse at 600 nm. (B) Residuals lemission =600 nm; lemission =630 nm; spectral bandwidth 10 nm; x 2 =1.03. For further parameters see Table 1.

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parison of the fluorescence lifetime data of free C-PC monomers and C-PC-content micelles in Table 1 shows that in the latter system t2 and t3 are substantially larger whilst t1 remains practically constant. These results indicate that the structured interfacial water layer in which the chromophores are embedded shields them from solvent quenching, thus increasing their lifetime. It is reasonable to assume that these chromophores might be the nearest to the protein surface and therefore the above results can be useful to assign the longer lifetimes of C-PC monomers to the b84 and a84 chromophores.

4. Conclusions It may be concluded that C-PC has in fact been solubilized into reverse micelles of AOT/water/ioctane when w0 \30. The microemulsions had a nice blue color with absorption and emission maxima of 611 and 625 nm, respectively. The steady-state fluorescence data show that the mobility of the chromophores inside reverse micelles is limited by strong interactions of the chromophores with the AOT sulfonate groups and the structured interfacial water layer in which they are embedded. At pH 5.0, the isoelectric point of C-PC, the stabilization kinetic of the spectral parameters is one of pseudo first order and the incorporation rate is greater than when the pH is increased. The spectral parameters remain stable as long as the microemulsion is stable. The fluorescence intensity decay of C-PC in reverse micelles is described by a triple exponential function, showing a complex pattern similar to that in aqueous solutions. The fluorescence decay of micelled C-PC might indicate that the chromophores embedded into the structured interfacial water layer may well be b84 and a84 The solubilization of biliproteins in apolar solvents could lead to the use of natural rather than synthetic dyes in hydrophobic foods and cosmetics. .

Acknowledgements We thank our colleague Dr John Trout for his

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comments and revision of our English text.

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