Role of Functional Groups and Surfactant Charge in Regulating

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J. Phys. Chem. B 2002, 106, 1446-1454

Role of Functional Groups and Surfactant Charge in Regulating Chlorophyll Aggregation in Micellar Solutions Angela Agostiano,*,†,‡ Lucia Catucci,†,‡ Giuseppe Colafemmina,† and Hugo Scheer§ Dipartimento di Chimica, UniVersita` degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy, CNR Centro Studi Chimico Fisici Sulla Interazione Luce-Materia, and Botanisches Institut der UniVersita¨ t, Menzinger Str. 67, 80638 Mu¨ nchen, Germany ReceiVed: May 7, 2001; In Final Form: October 24, 2001

A series of modified chlorophylls, namely, pyrochlorophyll a, Zn-pheophytin a, Zn-pheophorbide a, chlorophyllide a, [3-acetyl]-chlorophyll a, and bacteriochlorophyll a, have been investigated in micellar solutions. The study is aimed at establishing the role played by the different functional groups of the chlorophyll in the molecular organization of chlorophylls in a microheterogeneous environment. The surfactants (AOT, CTAB, and Triton X-100) have been chosen mainly on the basis of the different charges carried by their polar heads, to study the effect of a point charge on the spectral characteristics of the pigments. Besides optical techniques used to investigate the spectral properties of the pigments, the state of the micellar system was studied by resonance light scattering (RLS) and NMR (self-diffusion and relaxation time measurements). The results of the UV-vis measurements evidence the role played by the functional groups of the chlorophylls in the formation of the different species in solution. In view of the few cases reported in the literature on blue-shifted chlorophyll species, special attention has been devoted, to the behavior of the modified chlorophylls in CTAB where a species absorbing around 642 nm is always formed. CD, RLS, and NMR data identify this species as a pigment-surfactant aggregate, in which the positive charge of the surfactant interacts with the C-132 ketoester group of a monomeric chlorophyll.

Introduction Chlorophylls (Chls) are the universal photosynthetic pigments, involved in the biological light driven processes of almost all photosynthetic organisms.1 Their organization in specific proteinpigment complexes is responsible for their functional duality. In antennas, these pigments are efficiently collecting and funneling light energy,2-6 and in the reaction centers, they act as electron carriers during energy transduction.7-9 The different roles played in any such organism are often not performed by different kinds of Chls but by the same molecules in differently organized forms and environments.10,11 This is due to specific interactions of the pigments with the functional groups of the protein moiety in which they are imbedded, to interactions with neighboring pigments, and to more global influences of the native protein on the pigment’s electronic states, e.g., through the formation of electrical macrodipoles.12-15 The presence of point charges on the periphery of the macrocycle also seems to play a role in determining the in vivo shift of the absorption bands compared to the spectra of the molecules in solution.15,16 A characteristic feature of Chls, relevant for their molecular organization processes, is the central metal of the tetrapyrrole macrocycle. It provides the site of binding to nucleophilic electron donors and is relevant for the pigment’s molecular organization.17 This central metal is almost always Mg, but recently Zn has been recognized to replace it in the major pigment of certain bacteria.18 In the periphery of the macrocycle, * To whom correspondence should be addressed. e-mail: agostiano@ area.ba.cnr.it. Phone: 0039-080-5442060. Fax: 0039-080-5442128. † Universita ` degli Studi di Bari. ‡ CNR Centro Studi Chimico Fisici Sulla Interazione Luce-Materia. § Botanisches Institut der Universita ¨ t.

the carbonyl groups at C-131, C-133, and C-173 behave as nucleophilic electron donors. In Chl-proteins, they can form hydrogen bonds, and in solution, they can interact with metals19 including the central one of a neighboring chlorophyll.20 The alkyl-groups in the positions 3, 7, and 8 of the macrocycle can result, too, in relevant functional differences.21 Last but not least is the 173-esterifying group (mostly phytol), not only relevant for the hydrophobicity of the molecule but also for specific interactions in aggregates and in chlorophyll proteins and their assembly. Recently, the presence of the phytyl chain has been shown as a factor determining the ordered insertion of the pigment into lamellar phases.22 To investigate the interplay between the molecular organization of chlorophylls and their photophysical properties, the behavior of Chl and its derivates in polar and nonpolar solvents and in a series of membrane mimetic systems has been studied.22-30 In extending previous studies of Chl in micellar systems of different surfactants, we have investigated a series of modified chlorophylls in which the central metal, some of the peripheral groups, or the macrocyclic conjugation system were varied (Figure 1). Pyrochlorophyll a (PyroChl), which lacks the 132carbomethoxy-group, Zn-pheophytin a (Zn-Phe), in which the central Mg is replaced by Zn, Zn-pheophorbide a (Zn-Pheid), which in addition lacks the 173-phytyl chain, chlorophyllide a (Chlide), which lacks the 173-phytyl chain but retains Mg as central metal, [3-acetyl]-chlorophyll a (Acetyl-Chl), which contains a 3-acetyl instead of the vinyl group, and bacteriochlorophyll a (Bchl) have been investigated. The present study reports on the role played by the different functional groups of the chlorophyll in the pigment molecular organization into microheterogeneous systems. Surfactants bear-

10.1021/jp011718j CCC: $22.00 © 2002 American Chemical Society Published on Web 01/15/2002

Chlorophyll Aggregation in Micellar Solutions

J. Phys. Chem. B, Vol. 106, No. 6, 2002 1447

Figure 2. Scheme of Stejskal-Tanner sequence for NMR self-diffusion coefficient measurements.

Figure 1. Stuctures of chlorophyll a and the modified pigments.

ing different charges on their polar heads have been used in order to study the effect of the presence of a point charge on the spectral characteristics of the pigments. Optical techniques were used to investigate the spectral properties of the pigments, whereas the state of the micellar system was studied by resonance light scattering (RLS) and NMR (self-diffusion and relaxation time measurements).

synthesized by standard procedures,36,37 whereas Bchl was purchased from Sigma. All pigments were stored in the dark at 243 K under N2 atmosphere. Micellar solutions containing the different pigments were generally prepared by drying appropriate amounts of stock solutions in a stream of Ar and redissolving the pigments in the detergent solutions. Alternatively, the appropriate amounts of alcoholic stock solution of pigment and aqueous surfactant were mixed together, dried in a stream or Ar, and redissolved in water. B. Methods. Visible absorption spectra were recorded using a JASCO 8700 spectrophotometer. Fluorescence and resonance light scattering measurements were carried out using a PerkinElmer model LS-5 spectrofluorimeter. The CD spectra were recorded using a JASCO J810 spectropolarimeter. Self-diffusion coefficients of CTAB were determined by NMR using a FT-PGSE pulse sequence on a Tesla BS 587A 80 MHz spectrometer equipped with a pulsed-field gradient unit (Stelar snc) and controlled by the HROCH software. A classical spin-echo sequence (90°-τ-180°-τ), modified with two rectangular field gradient pulses of δ duration separated by a time interval ∆, was used38 (Figure 2). In our measurements, a gradient field strength G ) 0.04 T m-1, a separation time of ∆ ) 0.1 s, and a gradient time δ, ranged from 0.001 to 0.06 s, were used. The echo amplitude at 2τ is given by

A(2τ) ) A(0) exp[-γ2G2Dδ 2(∆ - δ/3)]

(1)

where γ is the proton gyromagnetic ratio and D is the selfdiffusion coefficient. Echo amplitudes vs [δ2(∆ - δ/3)] were fitted with an exponential function. The temperature of the sample was maintained at 306 K. NMR relaxation times, T1 and T2, were obtained on a Varian XL 200 spectrometer using inversion recovery and CPMG sequences, respectively.

Materials and Methods A. Materials. Sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and p-(1,1,3,3-tetramethylbutyl)phenoxypoly(oxyethylene)glycol (Triton X-100) were obtained from Sigma and used without further purification. Hexadecyltrimethylammonium chloride (CTAC) and hexadecyltrimethylammonium bromide (CTAB) were purchased from Fluka and purified by crystallization from ethanol. Hexadecylpyridinium bromide (CPB) and 2H2O (99.9% 2H) were from Aldrich. Chl was isolated from fresh spinach leaves as previously described31-33 and stored, in the dark, in n-pentane at 243 K under an N2 atmosphere. Purity and concentration were routinely checked using the criteria described elsewhere.34 Chlide was synthesized and purified according to Fiedor et al.35 PyroChl, Zn-Phe, Zn-Pheid, and Acetyl-Chl were also

Results and Discussion A. Visible Absorption Spectra of Pigments in Micellar Systems. In all surfactants examined, the absorption spectra of Chl (recorded immediately after the sample preparation) show the presence of a long wavelength band with a maximum around 745 nm. Its spectral characteristics are similar to those recorded for Chl in water saturated pentane39 and are indicative of pigment aggregation. In the nonionic surfactant Triton X-100 (Figure 3A), an increasing of the surfactant concentration results in the conversion of the 744 nm band into one with a maximum at 668 nm, characteristic of the monomeric pigment.40,41 At a constant Chl concentration, the intensity change of the aggregate band observed varying the surfactant concentration can be fit well by assuming a Poisson distribution of the pigment in the

1448 J. Phys. Chem. B, Vol. 106, No. 6, 2002

Agostiano et al.

Figure 4. Dependence of the Chl absorption ratio between 745 and 668 nm on surfactant concentration. [Chl] ) 1 × 10-5 M in all micellar system reported.

Figure 3. Absorption spectra of 1 × 10-5 M Chl in micellar solution of (A) 1 × 10-3 M Triton X-100, (B) 1% (wt %) AOT, and (C) 1 × 10-3M CTAB. The dotted line shows the spectrum recorded one month after sample preparation, under otherwise identical conditions.

micelles.42 Assuming the concentration of the free surfactant is equal to its cmc (0.24 mM) and an aggregation number of 143,43 at [Chl] ) 1 × 10-5 M and [surfactant] ) 5 × 10-4 M, the fit gives an average number of seven pigments per micelle. At a surfactant concentration of 5 × 10-2 M, conversely, 97% of the micelles are empty, thus accounting for the observed increased amount of monomeric Chl. The presence of a charge on the surfactant polar head promotes Chl aggregation (Figure 3B,C), irrespective of the charge being negative (AOT) or positive (CTAB). The band around 745 nm is now present at all surfactant concentrations examined. Moreover, in contrast to the Triton X-100 data, the aggregate/monomer ratio even increases slightly by increasing the surfactant concentration (Figure 4). The presence of a net charge therefore profoundly alters aggregation, which can no longer be described by a Poisson distribution of the chlorophyll in micelles of a constant size. The electrostatic interactions between the charge of the surfactant and Chl can account for the inadequacy of the distribution model and for the increase of the aggregate formation with increasing ionic surfactant concentration. The formation of the Chl aggregate can be ascribed to the increased ionic strength and dielectric constant of the solution, as previously reported for Chl in water-organic solvent mixture.44 In CTAB, the spectra change with time: the absorption band at 740 nm is gradually replaced by one with maxima at 642

Figure 5. Absorption spectra of 1 × 10-5 M of (A) Chl, (B) PyroChl, (C) Zn-Phe, and (D) Zn-Pheide in micellar solution of 1 × 10-3 M Triton X-100.

nm (Qy-region) and 416 nm (Soret region; Figure 3C, dotted line). A similar behavior of Chl is observed in micellar solutions of hexadecylpyridinium bromide (CPB), indicating the need of a positive charge for the formation of the “642” species (data not shown). To relate the different behavior of Chl in different surfactants to structural features of the pigment, the study was extended to a series of modified chlorophylls. The processes of selfaggregation and interaction with the different host systems were investigated by UV-vis spectroscopy (Figures 5-7). In micellar solutions of Triton X-100, the modified pigments PyroChl, Zn-Phe, and Zn-Pheid are present, contrary to Chl, predominantly as monomers at all surfactant concentrations studied (see Figure 5). The lack of aggregation of Zn-Phe can be related to the coordination capability of the central metal. Magnesium is an alkaline-earth metal with the 3d orbitals empty, which can be easily employed in coordination. The Mg ion is most stable in its octahedric form. In Mg-tetrapyrroles, it is therefore coordinatively unsaturated, which has been recognized as a dominant factor for chlorophyll aggregation in organic solvents.45 The tetracoordinate Mg in chlorophyll a can therefore further coordinate molecules to form aggregates. The Zn ion,



Figure 6. Absorption spectra of 1 × 10-5 M of (A) Chl, (B) PyroChl, (C) Zn-Phe, and (D) Zn-Pheide in micellar solution of 1% (wt %) AOT. Dotted lines show spectra recorded one month after sample preparation, under otherwise identical conditions.

Figure 7. Absorption spectra of 1 × 10-5 M of (A) Chl, (B) PyroChl, (C) Zn-Phe, and (D) Zn-Pheide in micellar solution of 1 × 10-3 M CTAB. Dotted lines show spectra recorded one month after sample preparation, under otherwise identical conditions.

conversely, generally utilizes the 4s0 and 4p0 orbitals to coordinate four ligands in a tetraedric structure, and therefore, the Zn tends to remain tetracoordinated. The pronounced difference in micellar solution is nonetheless surprising in view of the ready aggregation of BPhe, which lacks a central metal,46,47 and points to more subtle differences among the Znand Mg-tetrapyrroles, e.g., in their electronic structure of the macrocycle. A striking example on such a change is the solvent dependence of the cation radical of the Zn-tetraphenylporphyrin.48 The lack of a polymer band in the spectrum of PyroChl at low concentrations of the surfactant points to a function of the ring E β-ketoester group in the forming of the long-wavelength absorbing aggregates. This is again different from aggregates in organic solutions, where PyroChl forms tighter aggregates.19,49-51 The aggregation behavior is profoundly influenced, however, by the detergent used. In AOT micellar solution, both Zn-Phe and PyroChl aggregate (λmax ≈ 730 nm, Figure 6). PyroChl has an absorption maximum at 730 nm which shifts in time toward shorter wavelengths (690 nm). The existence of a band peaking at 690 nm has been already reported in the literature for Chl in water/methanol solution and attributed to the formation of an eso-dimer in which one water molecule is coordinated to the Mg of one chlorophyll and linked by hydrogen bonding to the keto group of the second one.52 Such dimers are more readily formed when the C-132 ketoester group is blocked or absent, as is the case of PyroChl. We therefore attribute the band at 730 nm to oligomers whose basic unit is the monohydrated dimer of PyroChl. Zn-Phe also forms aggregates in AOT, but the stronger monomer absorption points to less stable aggregates as compared to Chl or PyroChl. Obviously, the negative charges of the surfactant polar head promote aggregation, e.g., by varying the orbital energies.48 Zn-Pheide is present as a monomer at all surfactant concentration examined, thereby evidencing the role of the phytyl chain in aggregation.46 Aggregates are also formed by all pigments examined, again with the exception of Zn-Pheid, in micelles of the positively charged CTAB (Figure 7). Interestingly, the long-wavelength absorbing forms of Chl and Zn-Phe convert in time to blue shifted forms, with a maximum around 640 nm and an intense

unstructured Soret band at 418 nm. By contrast, the spectrum of the PyroChl aggregates remains basically unchanged, indicating a stable equilibrium between monomeric and oligomeric forms. The four pigments discussed so far all have polar side-chains only on rings C and D. They are compared in Figure 8 to two pigments bearing and additional polar side chain at ring A, viz., BChl and Acetyl-Chl. BChl is present as a monomer in Triton X-100 and aggregates in the charged surfactants. The aggregation is less pronounced for Acetyl-Chl. It forms aggregates only in AOT, whereas it remains monomeric in CTAB. Interestingly, the latter solution develops in time a blue shift of the Qy band and a sharp, intense Soret band indicative of the formation of similar species as discussed above for the other pigments. The results of the UV-vis measurements evidence the role played by the functional groups of the chlorophylls in the formation of the different species in solution. We will focus on the blue-shifted Chl species, discussing more in detail the behavior of the modified chlorophylls in CTAB, the surfactant that promotes the formation of the 640 nm absorbing species. Although the formation of blue shifted Chl aggregates has seldom been documented in the literature,53,54 excitonic theory predicts that both blue and red absorption band shifts can occur in dimers depending on the relative orientation of the two pigment molecules. A reversible blue shift in the red absorption maximum of the chlorophyll can also be caused by a point charge on the macrocycle periphery, which again depends critically on the position of this charge. Because PyroChl never forms the 640 nm species, the ring E β-ketoester group seems to be involved in its formation, possibly by interaction with the positively charged polar head of the CTAB. The conjugation of the ketoester group with the aromatic system through the C-131 keto group and the C-131-C-132 double bond in the enol has been reported in several papers describing either enols or chelates of this system.19,55-57 In these complexes, a red shift of the Qy band was observed, indicative of the influence of the state of the system on the exited state energy, together with a broadening of the signal that states for heterogeneity and distribution of states with different energies. Therefore, if properly positioned, the charge can induce red-shift, and this possibility cannot be


Agostiano et al.

Figure 8. Absorption spectra of 1 × 10-5 M of (A) Acetyl-Chl and (B) Bchl, in micellar solution of different surfactants. Dotted lines show spectra recorded after one month from sample preparation, under otherwise identical conditions.

Figure 9. Fluorescence spectra of different chlorophyll a forms (1 × 10-5 M) in 1 × 10-3 M CTAB and 1 × 10-2 M CPB solution. (A and B) Fluorescence of monomeric and “642” Chl species in CTAB, respectively. (C and D) Fluorescence of monomeric and “642” Chl species in CPB, respectively. The excitation wavelength was 416 nm, and the cell path length was 1 cm.

rouled out for shift in the blue region. Stabilization of porphyrin aggregates with CTAB by both Coulombic and hydrophobic interactions with surfactants has recently been reported in the

literature.57 The larger amount of this species with Zn-Phe, as compared to Zn-Pheid, points again to the importance of the phytyl chain in interactions of Chls with their environment.



TABLE 1: Relative Fluorescence Yields, OFrel, and Fluorescence and Absorption Maxima (λflu and λab) of the Different Species of the Pigments in Et2O and in a 1 × 10-3 M CTAB Solutiona φFrel [%]

λflu [nm]

λabs [nm]

Chl 100 7.8 12.3

665 670 645

660 668 641 745

PyroChl 100 7.5

663 673

658 671 733

monomer in Et2O monomer in CTAB “642” in CTAB

Zn-Phe 72 1.1 4.3

657 670 641

653 665 638

monomer in Et2O monomer in CTAB “642” in CTAB

Zn-Pheid 72 9.7 24.7

654 670 643

653 666 641

monomer in Et2O monomer in CTAB

BChl 1.1 7.3

770 770

770 774

monomer in Et2O monomer in CTAB

Acetyl-Chl 88 36

682 692

674 686

monomer in Et2O monomer in CTAB “642” in CTAB aggregate in CTAB monomer in Et2O monomer in CTAB aggregate in CTAB

a Fluorescence yields are relative to that of the Chl momomer in diethyl ether, at the same absorption and at the excitation wavelength, to which the value of 100% has been assigned

The absorption blue shift in the Soret region of the spectrum is reminiscent of the H-type aggregates of porphyrins in surfactants investigated by Maiti et al.58 However, these aggregates show a concomitant red shift of the Qy band which is opposite to the situation in the complexes investigated here. B. Fluorescence. Table 1 summarizes the fluorescence results for the different pigments in ethyl ether and in a micellar solution of CTAB. The fluorescence is reported as the fluorescence yield relative to that of the momomer in diethyl ether, at the same absorption at the excitation wavelength, to which the value of 100 has been assigned. The red-shifted aggregates of all pigments studied did not show any detectable fluorescence. This agrees with a number of studies on the Chls aggregates.25 By contrast, the low fluorescence of monomers in CTAB is surprising, compared to that of the respective monomer in ether. The same low fluorescence is, however, not only seen in another cationic detergent, CPB, with the same counterion but also when the counterion is changed to Cl-. We therefore conclude that the presence of a positive charge near the tetrapyrrole might be responsible for the fluorescence quenching through the formation of charge-transfer complexes between the pigments and the polar headgroup of the surfactant, which is capable to dissipate the excitation energy. Unfortunately, no fluorescence data have been reported on the Chl bearing a positive point charge studied by Pearlstein et al.15 Significantly, the fluorescence of the “642” species has a comparable intensity as the monomer. The fluorescence spectra of monomeric Chl and of the “642” form in CTAB and in CPB are shown in Figure 9. The fluorescence of monomeric Chl in CPB is somewhat lower than that in CTAB (Figure 9C). The opposite behavior is found for the “642” species. (Figure 9D). Under the assumption that the “642” species is a pigment-

Figure 10. RLS Spectra of different chlorophyll a forms (1 × 10-5 M) in 1 × 10-3 M CTAB solution. (A) RLS spectrum of CTAB solution, (B) RLS spectrum of “642” Chl species in CTAB solution, and (C) RLS spectrum of aggregated Chl species in CTAB solution.

surfactant aggregate, in which the positive charge of the surfactant interacts with the ring E β-ketoester, the lower fluorescence value obtained in CPB (1% compared to 12% in CTAB) could be explained in terms of a higher charge delocalization from the pyridinium polar headgroup of CPB. Fluorescence quenching via H transfer, because of the formation of H bonding to enolic forms of the β-ketoester system, could on the other hand also explain the above results. The fluorescence quenching effect of charges present on the surfactant are supported by the intense fluorescence of the monomer in Triton X-100 and the lack of a detectable fluorescence of monomers in the AOT micellar solution. C. Resonance Light Scattering. RLS is a sensitive and selective probe to chromophore aggregation.59 In particular, Pasternack et al..60,61 utilized RLS to study porphyrin aggregation. They observed very strong light scattering in the absorption region of porphyrin aggregates, whereas it was absent in monomers and small oligomers. The amount of light scattering increases with a “good” electronic coupling among the chromophores. Figure 10 shows the RLS profiles of the different species of chlorophyll a in micellar solution of CTAB. The Chl polymer species absorbing at λ higher than 700 nm show a scattering peak at 458 nm (Figure 10C) with an intensity typical of a large,


Agostiano et al.

Figure 11. (A) Circular dichroism spectra of different Chl forms (1 × 10-5 M) in 1 × 10-3 M CTAB solution: monomer (dotted line) and “642” species (continuous line). Inset: CD spectrum of aggregated Chl species. (B) Circular dichroism spectra of “642” species of Chlide in micellar solution of CTAB (1 × 10-3 M) at two different concentrations: 2 × 10-5 M (dotted line) and 1 × 10-5M (continuous line). Inset: absorption spectra of the same samples.

well coupled aggregate. The “642” species, by contrast, shows no anomalously high scattering (Figure 10B), again excluding the presence of H type aggregates. On the contrary, the scattering intensity at 416 nm is reduced, because of absorption in the Soret band. In summary, the characteristics give no evidence that the “642” species has Chl molecules close to each other and well coupled; rather, its RLS spectrum (Figure 10B) is quite similar to that of the monomer spectrum reported by Pasternack et al.59 D. Circular Dichroism. CD measurements have been performed in aqueous CTAB solution containing the different species of Chl (monomer, polymer, and “642”). Chl solutions containing the monomer (Figure 11A, dotted line) show CD spectra characterized by the presence of a nonconservative peak of low intensity in the red region at 668 nm, the wavelength corresponding to the monomer absorption. In the Soret band region, there are two bands of opposite sign at 422 and 390 nm

together with a series of bands located in the UV region, between 350 and 220 nm. Both series of signals are sensitive to the stereochemistry of C-13 and indicate the presence of the C-13(R) epimer of the chlorophyll.62 In the same figure, the CD spectrum of the “642” species is shown (solid line). Although it is similar to the monomer spectrum in the red and Soret region, it shows some differences in the UV part, reasonably related to the already mentioned interaction between the C-13 ketoester group with the surfactant. In the inset of the same figure, the spectrum of the polymer is shown, evidencing the presence of a strong conservative double band with a zero crossing at 745 nm. As it has already been reported for Chl in wet organic solvents,63 this is indicative of coupling between the dipole transitions among the molecules of an aggregate.34,64 In part B of Figure 11, the CD spectrum of the “642” species of Chlide in CTAB is reported (solid line). It is quite similar to the corresponding spectrum of chlorophyll in the short-



TABLE 2: Measured Self-Diffusion Coefficients and Relaxation Times T1 and T2 of CTAB Trimethylammonium Group in 2H2O without Pigment ([CTAB] ) 5 × 10-2, 1 × 10-3, and 5 × 10-4 M) and with 5 × 10-5 M Solutions of Different Chlorophyll a Species ([CTAB] ) 1 × 10-3 M) sample (CTAB solutions)

D (m2 s-1)

T2 (ms)

T1 (ms)

5 × 10-2 M 10-3 M 5 × 10-4 M in the presence of Chl monomer in the presence of Chl aggregate in the presence of Chl “642”

(0.65 ( 0.06) × 10-10 (3.3 ( 0.3) ×10-10 (5.9 ( 0.06) ×10-10 (3.7 ( 0.4) ×10-10

253 ( 12 323 ( 12 507 ( 25 180 ( 9

492 ( 25 678 ( 19 710 ( 35 509 ( 25

(3.1 ( 0.3) ×10-10

183 ( 9

690 ( 19

44 ( 2

132 ( 6

wavelength region but contrasts by a weak split signal in the red part of the spectrum. It is reasonable to suppose that the absence of the phytyl chain forces the macrocycle near the surface of the micelle, allowing for stronger interactions between the pigment molecules. This assumption is further supported by the presence of two conservative bands in the CD spectrum of a more concentrated solution of Chlide (dotted line). It is interesting to note the similarity of the absorption spectra of the two solutions (see inset of the figure), which, on the other hand, are similar to the corresponding absorption spectrum of the “642” species of the esterified pigment Chl, which never shows conservative CD bands. E. Self-Diffusion Coefficients and Relaxation Times T1 and T2. In isotropic systems and in the absence of thermal or concentration gradients, the mean square displacement in time of a molecule or aggregate is proportional to the self-diffusion coefficient D:

〈r2〉 ) 6Dt Self-diffusion coefficients of detergents in micelles generally range from 10-9 to 10-12 m2 s-1. Therefore, measurements carried out in the observation period of ∆ ) 0.1 s and D ) 10-9 m2 s-1 allow us to observe root mean-square displacements on the order of 10 µm. For a molecule confined in an aggregate of a size

Role of Functional Groups and Surfactant Charge in Regulating

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