J. Phys. Chem. 1995,99, 10708-10715
10708
ARTICLES Dynamics of Porphyrin Molecules in Micelles. Picosecond Time-Resolved Fluorescence Anisotropy Studies Nakul C. Maiti, Shyamalava Mazumdar,* and N. Periasamy* Chemical Physics Group, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Bombay 400 005, India Received: February 13, 1995; In Final Form: April 24, 1 9 9 p
The fluorescence dynamics of protoporphyrin IX (PPIX) and protoporphyrin IX dimethyl ester (PPDME) and their zinc complexes in anionic (SDS), cationic (CTAB), and neutral (Triton X-100) micelles have been studied by picosecond time-resolved fluorescence and anisotropy decay measurements using a time-correlated single photon counting technique. The absorption and fluorescence emission spectra of the porphyrins (0.5- 1 pM) in the micelles (0.5-1 mM) are typical of the monomeric porphyrin. The absence of aggregation and homogeneity of the solubilization sites are confirmed by the fluorescence decay which is a single exponential for all the four porphyrins in the neutral and cationic micelles, Triton X-100 and CTAB. However, the presence of small concentrations of aggregates resulted in biexponential fluorescence decay in the anionic SDS micelles. The fluorescence anisotropy decay of the monomer-micelle systems is best fitted to a twoexponential function. The values of the rotational decay parameters are consistent with the model that the fluorescence depolarization occurs by both rotational and translational diffusion of the porphyrin inside the micelle and the tumbling of the micelle. The order parameter for the porphyrin intercalation and rotational and translational diffusion coefficients have been calculated. The model is consistent with the NMR results which show that the equilibrium distribution of porphyrin is confined to a region near the surface.
I. Introduction Micelles are nanometer scale aggregates formed as a dynamic unilaminar phase in aqueous solutions of surfactants. Structure, dynamics, and other phase properties of the micellar phase are subjects of extensive investigations in recent years.' Aqueous micelles are widely used to solubilize substances which are sparingly soluble in water. Interaction of various solutes with the micellar phase and encapsulation of molecules inside micelles are studied as models for the biomembrane* or a protein-molecule complex3 formation. The reactivity of the molecule in a micellar environment is either enhanced or becomes more specific? presumably due to the location or orientation of the molecule with respect to the interface in the micelle. The physical characterization of the micellar environment, namely, the core, the interface, and the immediate layers of water on the interface, has been the subject of numerous investigations by several experimental techniques5 and theoretical modek6 It is fairly well established now that practically all kinds of molecules have a surface-seeking tendency in the micelle and that the interfacial region is the preferred site for solubilization, even for hydrophobic molecule^.^ The fluorescence property of suitable molecules has also been used to probe the micellar environment near the region where it is solubilized.* An important advantage in using fluorescent molecular probes is the real time intensity decay measurements in the picosecond to nanosecond time scale which is comparable to the time scale of the dynamics of the excited fluorophore in micelles. The @
Abstract published in Advance ACS Abstracts, June 15, 1995.
0022-365419512099- 10708$09.0010
molecular dynamics of a fluorescent probe in the micelle consist of translational and rotational diffusion processes. Both these processes contribute to fluorescence depolarization of the probe. Thus, fluorescence anisotropy decay in the picosecond to nanosecond time scale is one of the best experimental data available for investigating both translational and rotational dynamics of the probe in the micellar environment. The interpretation of fluorescence anisotropy decay of molecules is essentially based on some assumed models for the molecular dynamics of the probe in the micellar environment. In order to have as simple a model as possible, it is essential that the probe is ideally distributed in the micelle such that the spectroscopic and molecular dynamical parameters of all probe molecules are identical. This would require (i) a sufficiently high partition coefficient for the probe for micellization so that there is a negligible amount of the probe in the aqueous phase, (ii) a single site of solubilization for the probe in the micelle, and (iii) absence of alternative structures (dimer, aggregates, acid-base forms, etc., which have different fluorescent and dynamical properties) for the probe. In practice, one would insist that the fluorescence decay of the probe in the micellar environment is a single exponential and attributable to a single species whose molecular dynamics is being modeled. Porphyrins, metalloporphyrins, and their analogs play a diverse role in biological systems. Various metalloproteins contain porphyrin complexes at their active site surrounded by peptide chains of the protein cavity. The dynamics of this prosthetic group inside the protein cavity has been proposed9 to be significant in the biochemical function of redox and 0 1995 American Chemical Society
Dynamics of Porphyrin Molecules in Micelles
R I = R 2 = R 3 * H , R , = Ph
>
TPP
, Zn T T P
Figure 1. Structures of porphyrins (left) and their zinc complexes (right).
electron transfer proteins. Porphyrins and almost all fourcoordinated metalloporphyrins are highly planar and stable fluorescent probes which have been shown to have a strong tendency to get encapsulated inside micelles in monomeric forms.Io There has been no report so far on studies of dynamical properties of porphyrins inside micelles using time-resolved techniques. One difficulty in using the structurally simple and symmetric porphyrins such as TPP and ZnTPP as fluorescent probes is the readiness with which they aggregate in micelles even at moderate concentrations thereby leading to multiexponential fluorescence decays. The natural porphyrin (protoporphyrin E, Figure 1) and its substituted analogs have been shown to be readily solubilized as monomer in most micelles. The diameter of the porphyrin ring is (-13 A) much smaller than the dimensions of micelles, and the solubilization site of the porphyrin inside micelles has been established from NMR studies" to be -6 from the surface of the micelle. The natural porphyrin complexes encapsulated inside micelles have biomimetic significance as models for various heme proteins and other porphyrin-containingbiomolecules. We have chosen in this study some protoporphyrin M derivatives with the acid or diester forms which are solubilized as monomers inside aqueous micelles. The aqueous micelles chosen in this study are sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), and Triton X-100 (TX-100) with increasing size (SDS < CTAB < TX-100) of the micelles. The fluorescence decay is a single exponential, and the fluorescence anisotropy decay is found to be a two-exponential function. The results of the fluorescence and anisotropy decay parameters are discussed with reference to the molecular dynamics of the probe in the micelle.
II. Experimental Section Materials. Tetraphenylporphyrin (TPP) and ZnTpP were prepared by standard procedures. Protoporphyrin IX (A) and the dimethyl ester (B)were obtained from Sigma Chemicals, USA, and used without further purification. The zinc complexes of A and B were prepared by refluxing for 5 h the porphyrin solution in dimethylfomamide with excess zinc acetate. The reaction mixture was cooled to room temperature and diluted with water to precipitate the zinc complex. The zinc complex was finally purified by column chromatography. Sodium dodecyl sulfate (SDS) (Sigma), Triton X-100 (TX-100) (Sigma), and cetyltrimethylammonium bromide (CTAB) (Aldrich Chemicals, USA) were fluorescence-free and used as such. All solvents used in this study were of Anal@ or spectroscopy grade. Micellar samples were prepared" by stirring a 2% surfactant
J. Phys. Chem., Vol. 99,No. 27, 1995 10709 solution with the porphyrin for several hours at 30-40 "C. Finally, the solution was filtered through Millipore filters. Methods. Absorption and fluorescence emission spectra and steady state anisotropy data were obtained using commercia1 spectrometers (Shimadsu W-2100 and RF-540 models). NMR spectra were recorded on a Bruker 500 MHz (AMX 500) FTNMR instrument. NMR studies were carried out at 25 "C using D20 as the solvent. 'HNMR experiments were carried out at 500.137 MHz with 10 ps as the 90" pulse width, and I3C NMR experiments were done at 125.77 MHz with 8.3 ps as the 90" pules width. The picosecond laser-excited time-correlated single photon experimental setup has been described previou~ly.'~The light source is the rhodamine 6G dye laser (pulse width; 6-10 ps; tunability; 570-630 nm; cavity-dumped repetition rate, 800 kHz) derived from the CW mode-locked, frequency-doubled Nd:YAG laser. The excitation wavelength used for the porphyrins and their zinc complexes was either 580 or 630 nm. The fwhm of the instrument response function is typically 80100 ps using a microchannel plate photomultiplier (Hamamatsu R2809). Fluorescence decays were obtained at the magic angle (54.7"), parallel and perpendicular orientation of the emission polarizer with respect to excitation polarization. It is convenient to collect polarized fluorescence components which is corrected for the geometry factor (G-factor) of the spectrometer. The G-factor is defined as the ratio of the detected signal for parallel and perpendicular orientations of the polarizer when a depolarized emission source is kept in the place of the sample. The G-factor-corrected data sets imply a common scale factor for parallel and perpendicular decay components (thus eliminating one parameter when the decay data are fitted). The G-factor for the different emission wavelengths was determined by using ethanol solutions of cresyl violet as a standard for which the lifetime is 3.23 ns and the rotational correlation time was 280 ps. The G-factor is the ratio of the times taken to collect perpendicular and parallel polarized emissions to match the fluorescence tails exactly. The average of several trials is taken to determine the G-factor. In micellar solutions parallel polarized fluorescence was collected to give a peak of (1-2) x lo4 counts taking a time of, say, t s (typically 200-300 s). The perpendicular component was collected for a time t multiplied by the G-factor to give a G-factor-correcteddata set. Analysis of Decay Data. The experimental fluorescence decay is the convolution of the instrument response function and the intensity decay equation predicted by a kinetics/ dynamics model of the excited state. The decay data were fitted to the appropriate equations by the iterative nonlinear least squares method using Marquardt's algorithmI4 making use of Grinvald-Steinberg recursion relations.I5 The fluorescence decay obtained at the magic angle was fitted to the decay function which is either one or two exponentials given by eq 1.
where i is the number of discrete exponentials required to fit the emission profile. Ai are the amplitudes and z~ are the lifetimes. Polarized fluorescence decays (parallel and perpendicular components) were fitted simultaneouslyI6to eqs 2 and 3 to obtain a single set of parameters common to both the decays.
Maiti et al.
10710 J. Phys. Chem., Vol. 99, No. 27, 1995 400 500 600 700
TABLE 1: Fluorescence and Anisotropy Parameters of Various Porphyrins in THF Solution (T = 25 "C) porphyrin
L (nm)
ZnTPP ZnPPIX ZnPPIXDME TPP TPP PPIX PPIX PPIXDME PPIXDME
580 580 580 5 80 630 580 630 580 630
1em
(nm)
sr(ns)
ro
t,(ns)
1.78 2.07 2.02 9.54 9.29 11.69 11.50 11.78 11.47
0.11 0.10
0.13 0.18 0.15 0.16 0.17 0.15 0.17 0.17 0.18
640 640 640 640 650 640 640 640 650
0.10 0.11 0.32 0.11 0.35 0.10 0.32
tfis the lifetime of the excited state obtained for the single exponential decay at magic angle polarization. r(t) is the anisotropy decay function which is a single exponential (in pure solvents)"
r(t) = r, exp(-f/z,)
(4)
-0.1
1
,
,
,I
h (-) Figure 2. Absorption (solid line) and fluorescence (broken line, lex = 580 nm) spectra of 1 p M protoporphyrin dimethyl ester, PPDME
(B), in 2% TX-100 solution. The plot of steady state anisotropy ( i s i ) against excitation wavelength (,lex) of the sample is shown in the inset at the top of the figure (Aem = 640 nm).
or multiexponential (in micelles) 400
a
h
ro is the initial anisotropy and pi (E /3; = 1) and t r i are the preexponents and the anisotropy decay constants, respectively. Only the data of those samples for which the fluorescence decay shows a single exponential are processed for analysis of anisotropy decay. Goodness of fits were examined by the random distribution of the weighted residuals, the chi-square value,'* and a good fit of the calculated anisotropy function with the experimental data. The recovered parameters for anisotropy decay in micelles (tf,ro, pi, and t r i ) are used to calculate steady state anisotropy ( I s s ) using eq 6 and checked to be consistent with the independent experimental value.
,
500
600
700
0.07
0.04
120 5
0.6 a
e
4 9
0.4 0.2 0.0 400
500 600 A (nm)
700
Figure 3. Absorption (solid line) and fluorescence (broken line, ,lex A standard sample (cresyl violet or nile red in ethanol) was regularly used to test the performance of the instrument for reproducibility of lifetimes and random distribution of residuals.
111. Results Figure 1 shows the molecular structure of protoporphyrin IX (A), protoporphyrin dimethyl ester (B), and their zinc complexes (C and D, respectively). The fluorescence decay as well as fluorescence anisotropy of the two protoporphyrins (A and B) and their Zn complexes (C and D) is a single exponential in tetrahydrofuran (THF). The concentrations used in these experiments are in the range of 0.5-1 pM. The fluorescence lifetimes (zf),initial anisotropies (ro), and rotational correlation times (tr) measured in THF for all the porphyrins are given in Table 1. For the metal-free porphyrin, the maximum value of ro corresponds to excitation at the longest wavelength absorption band at -630 nm. The data for TPP and ZnTPP are also included in Table 1 for comparison with A-D. The lifetimes for TPP and ZnTPP are also in good agreement with those reported in the literature.I9 The concentration of the porphyrins used for the micellization studies are in the range of 0.5- 1 p M , and the concentration of the micelle (in aggregated form) is at least 100 times higher. Assuming Poisson statistics to be valid, the probability that two or more molecules occupy a single micelle is
