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Jun 1, 1993 - Guang Li, Linda B. McGown. J. Phys. Chem. , 1993, 97 (25), ... Laurie L. Amundson , Rui Li and Cornelia Bohne. Langmuir 2008 24 (16), 84...
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J. Phys. Chem. 1993,97, 6745-6752

6745

A New Approach to Polydispersity Studies of Sodium Taurocholate and Sodium Taurodeoxycholate Aggregates Using Dynamic Fluorescence Anisotropy Cuang Li and Linda B. McCown’ Department of Chemistry, P. M.Gross Chemical Laboratory, Duke University, Box 90344, Durham, North Carolina 27708-0344 Received: October 8, 1992; In Final Form: April 1, 1993

Sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC) bile salt micelles in aqueous solution were investigated using perylene as a fluorescenceanisotropy probe. By the introduction of a “reduced” correlation time in the anisotropy decay theory, it was possible to resolve the rotational correlation time of the smallest aggregate from these polydisperse systems. For both bile salts, comparison of the resolved correlation times with calculated values identified the smallest aggregate as the dimer. Temperature studies confirmed this result. The hydrodynamic radii of the bile salt dimers, calculated from the anisotropy results, were 7.2 f 0.1 A for NaTC and 7.3 f 0.1 A for NaTDC. The NaTC dimer as a percentage of all aggregates decreased from 68% to 56% a t 20 OC and from 82% to 67% at 40 OC as the total monomer concentration of NaTC was increased from 20.0 to 100.0 mM. For NaTDC a t 20 OC, a decrease from 43% to 35% was observed for the same increase in total concentration. The average aggregate size was found to be larger for NaTDC than NaTC. In the presence of 3.0 M NaCl, the percent dimer in NaTC solution remained relatively constant a t approximately 33% as NaTC concentration was increased, although the average aggregate size increased. Addition of Tb3+ was found to significantly decrease the percent dimer in NaTC solution. A primary-secondary aggregation model is presented for NaTC in which the primary micellar unit is a dimer “sandwich” with perylene located between two bile salt monomers. The effects of dimer shape and the relationships between internal rotation of the probe and overall rotation of the probs-dimer complex on the experimental results are discussed.

htroduction Bile salts, which are naturally occurring,biological detergents, exhibit many interesting and unique properties with respect to aggregation and sol~bilization.~-~ Thedevelopment of an accurate model of bile salt aggregation is important both for fundamental studies of their function in biological systems and for guiding their application in chemical separations and analysis. Despite numerous studies, however, an aggregation model has yet to be agreed upon. At least five different models have been proposed.24 Moreover, fundamental properties such as aggregation number, size, and size distribution (polydispersity) of bile salt micelles remain uncertain. Techniques for determining particle size and polydispersity, such as quasi-elastic light scattering (QELS)and small-angle X-ray scattering (SAXS),are of limited value in studying bile salt aggregates because the dimension of the primary bile salt micelle is on the edge of the size limits of these methods. In contrast, since the micellar rotational diffusion correlation time is proportionalto the third power of the radius of a particle rather than first power dependence on its translational diffusion, techniques based on fluorescence rotational anisotropy should be more powerful for recovering the size and size distribution of small bile salt aggregates. It is well known that the trihydroxy bile salts form smaller aggregates than the dihydroxy It was reported in an early paper that the trihydroxysalt sodium taurocholate (NaTC) exists in aqueous solution as a tetramer, while two other trihydroxy salts, sodium cholate (NaC) and sodium glycocholate (NaGC), preferentially form dimers.’ A later 2H-NMR study confirmed that NaC forms dimers and tetramers at total monomer concentrationsup to 100 mM,5 while a NMR NOE experiment showed that the mean size of NaC aggregates is no larger than a tetramer at concentrations less than 50 mM.6 For thedihydroxy salt sodium deoxycholate (NaDC), a mean radius of 12.2 A was found by 2H-NMR.7 Mean radii of -18.5 and -10 A were reported for the dihydroxy salt sodium taurodeoxycholate (NaTDC) and NaTC, respectively, in 0.15 mM NaCl at 20 OC, based 0022-365419312097-6745$04.00/0

on QELS measurements;*a mean aggregationnumber for NaTC of 3 1 reported as well. The same NaTC radius was found by a later study using NMR.9 These mean values were found to be nearly independentof the total concentration of bile salt monomer. In contrast, a later paper reported apparent, mean aggregation numbers of -5 for NaTC and -20 for NaTDC at high concentrations” and suggested that intermicellar interactions may actually result in these “apparent” values being underestimated. Several studies have addressed the size distribution of bile salt aggregates.7.8J0J3 In a solution of NaDC that contained the maximum NaCl concentration (0.7 M) possible without precipitation, some 30% of the aggregates were reported to be tetramers? For both NaTC and NaTDC in 0.15 M NaCl solutions, polydispersity of approximately 20-4596 about the mean aggregate size was r e p ~ r t e d .The ~ coexistence of the dimer and larger aggregates of NaC was reported in a solubility study.1° Based on ESR measurements, at least two kinds of micelles were suggested to coexist in dihydroxy bile salts at 100 mM total concentration.13 In this paper, we present a theoretical approach to resolving the rotational correlation time of bile salt dimers using perylene as the anisotropy probe and apply it to NaTC and NaTDC in aqueous solution. The monomeric structures of these bile salts are shown in Figure 1. Perylene was chosen as the fluorescence anisotropy probe in this study because it is an established anisotropy that has relatively simple photophysical behavi0r.~~320Perylene is one of the polycyclic aromatic hydrocarbons that have been used in previous fluorescence probe studies of bile salt micellar system^.^^-*^ In addition to NaTC and NaTDC in simple aqueous solutions, results are also shown for NaTC at a higher temperature and in the presence of NaCl and Tb(N03)~.The proportion of dimer, e x p r e s d as a percentage of total aggregates, is compared for the different micellar solutions, and the results are interpreted in terms of a model in which a basic unit is a sandwich complex of perylene in an NaTC dimer. 0 1993 American Chemical Society

6746

The Journal of Physical Chemistry, Vol. 97, No. 25, 19'93

Li and McGown size distribution for the aggregates is a good approximation to this system if the aggregation number of the m i d e s is smll,1-3 which is usually the case for bile salt micelles. If the probability for a certain micellar volume VMJis BMJ, then a summation can be introduced into eq 2 to include all volumes of micelles in the system. Therefore, the new anisotropy expression for perylene in bile salt micellar system should be

R =OH (NaTC) = H (NaTDC)

Molecular structure of bile salts: (A) structure of the taurocholates NaTC and NaTDC; (B)stereochemicaldepiction of NaTC and NaTDC.

m

e 1.

-ry

Fluorescence anisotropy decay can be used to study reorientation by rotational diffusion of the transition dipole moments of a fluorescent probe (if nonrotational reorientation mechanisms are limited) during its excited-state lifetime. Since the reorientation process of a probe that is bound to a host structure is related to the rotation of both the probe and its host (in this case, the micellar aggregate), it is possible to obtain information about both the anisotropic microenvironment of the probe and the size and size distribution of the host. While the former possibility has been extensively applied in studies of restricted probe rotation,1418little work has been done on the use of fluorescence anisotropy decay to study the polydispersity of the host system. It is expected that the rotation of a probe (assumed in the following discussion to be perylene) will be restricted to some degree in a host binding site. Therefore, an approximate expression for the time-dependent anisotropy of the probe, [r(t)]p, can be given as1k16

where ro and rm( f0) are the limiting anisotropies at t = 0 and t = a,respectively; 4p.i is the ith rotational correlation time of the probe, and ap,i is the normalized preexponential factor of QpJ. For perylene, [r(t)]p can be approximated by two 4 p terms, of which the smaller refers to the in-plane rotation and the larger refers to the out-of-plane rotation. For a probe in a solution of monodisperse, spherical micelles in which the rotations of the probe and micelles are independent of each other, the total anisotropy expression for the probmicelle complex, [f(t)]pM, is simply the product of [r(t)]p from eq 1 and the factor exp(-t/4M), where 4~ is the rotational correlation time of the micelle:16 The hydrodynamic volume (V M )and the rotational correlation time ( 4 ~of) the micelle are related by the Debye-Stokes-Einstein equation:

where & is the rotational diffusion coefficient of the micelle, ke is the Boltzmann constant, Tis the absolute temperature, q is the bulk viscosity of the solvent, and VMis the hydrodynamicvolume of the micelle. In a polydisperse micellar solution there will be a distribution of ~ M S each , correspondingto a different sizeof micelle. A discrete

where4pMUis a new quantity, the 'reduced" rotationalcorrelation time, which is a characteristic of the combined probmicelle system and is equal to the measured or 4apparent" correlation time recovered from the experimental data:

(5) I/4PM,ij I l/b,t + ' / h i 4 Two basic relationshipsfollow from the definitionof the reduced correlation time. First is the general relationship that for any 4P.i and 4 M j Values, 4PM,ij

min(bP,t,4MJ)

(64

The second relationship is the following approximation: if max(~p,,,4Mj)/min(4p,i.4MJ) 2 10,

then h

, i j

= min(b,&MJ)

(6b)

The new preexponential factors in eqs 4a and 4b are determined not only by ro, rm,and UP but also by BM. For example, if BMJ = 0.2, ~ M =J 0.3 ns, and all 4 p > 1 0 4 =~ 3.0 ~ ns, then the apparent normalized preexponential factor (7,) of 0.2 will correspond to the exponential term with the reduced correlation time 9 P M j equal to -0.3 ns. That is to say that @MJ is nearly equal to the apparent preexponential factor in this case,i.e., 7, = B M j [ ( f O - r,)Ziarp,t + rm]/rot BMJ = 0.2. Further, b a d on the physical meaning of BMJ, the preexponential factor will represent the fraction of aggregates having ~ M 5J 0.3 ns in the micellar solution. It is assumed that the probe does not significantly perturb the composition of the micellar aggregates, as discussed in a later section.

Experimental Section M.terials. Sodium taurocholate (NaTC) (ULTROL grade, >99%, Calbiochem), sodium taurodaoxycholate (NaTDC) (>98%, Sigma), perylene (Gold Label, >99%, Aldrich), sodium chloride (99.999%, Aldrich), and terbium(II1)nitrate (99.999%~~ Aldrich) were all used as received. All aqueous solutions were prepared with HPLC grade water. Perylene stock solution was prepared in absolute ethanol. Solutionsof 10.0MMperylenein aqueous bile salt were prepared as follows. The appropriatevolume of the perylene stocksolution was added to a volumetric flask, and the ethanol was evaporated with a gentle stream of Nz(*). Aqueous NaTC or NaTDC was then added, and the solution was sealed and sonicated for 1 h. For the relevant NaTC solutions, NaCl or Tb(NO3)s was added after sonication and the volume of the solution was adjusted to 5.0 mL. All of the samples were equilibrated overnight before measurement. Merememente. The dynamic anisotropy experiment was performed on a 4850 multiharmonic Fourier transform (MHF) phase-modulation fluorometer (SLM Instruments, Inc.) in the T-format, in which a 470-nm. 10-"bandwidth band-pass interference filter (Oriel) was set in each of the two emission

The Journal of Physical Chemistry, Vol. 97, No. 25, I993 6747

Polydispersity Studies of NaTC and NaTDC

TABLE I: Estimated Radii and Rotatiod Correlation

Times ($M) Calculated for NaTC and NaTDC Micellar Aggregates in AQWOWSolution aggregate aggregation no. radius,' A dimer 2 7-7.5 10.0 trimer 3 4 11.0 tetramer 6 12.5 hexamer 14.0 octamer 8 15.0 decamer 10 20.0 multimer >2oc 25.0 30.0 40.0

h , b ns T = 20.0 O C T = 40.0 O C 0.36-0.44 1.04 1.38 2.03 2.85 3.50 8.30 16.2 28.0 66.4

0.224.27 0.63 0.84 1.24 1.74 2.14 5.06

9.89 17.1 40.5

The radii were obtained using molecular modeling calculations. The first hydrate shell is included in the hydrodynamicradii. The correlation time is calculated from DebyeStokes-Einstein equation based on the estimated radius. The bulk viscosity of the NaTC solution is assumed to be that of pure water. These radii do not correspond to particular aggregation numbers and are intended only to show the dependence of rotational correlation time on the micellar radius.

channels. A HeCd laser (Liconix) was used to provide exciting light at 442 nm. In the T-format experiment, the excitation polarizer is set to pass vertically polarized light, and one of the two emission polarizers is alternated between horizontal and vertical polarization settings while the other is set at horizontal to serve as a reference. The dynamic measurements were made using a base modulation frequency of 5.0 MHz. Data were collected at 30 frequencies in the range 5-1 50 MHz. The base cross-correlation frequency for detection was 4.167 Hz. The fluorescence phase angle differences (PADS) and modulation amplitude ratios (MARS) were calculated as the averages of 15 replicate measurements, in which each measurement was, in most cases, the internal average of 100 samplings taken over a 24-s period. For the NaC1-containing solutions, an average of 50 internal samplings was used for each measurement. The temperature of the sample compartment was maintained at 20.0 i 0.1 or 40.0 i 0.1 OC by a circulating water bath. Samples were placed in the sample compartment for at least 15 min prior to measurement to allow for thermal equilibration. Fluorescence lifetimes were measured on the SLM 4850 instrument in the L-format and using scattered light as the lifetime reference. The emission polarizer was set to the magic angle to eliminate photoselection effects. Fifteen modulation frequencies in the range 5-75 MHz were used. Other conditions were the same as in the anisotropy measurements. Data Analysis and Computation. Heterogeneity analysis software from Globals Unlimited (Version 3) and SLM Instruments, Inc. (Version 1.60), were used to analyze the lifetime data and dynamic anisotropy data, respectively. In both cases, the acquired standard deviations of the measured values were input as the "errors" in the nonlinear least-squares fits. SYBYL molecular modeling software (Version 5.4, TRIPOS) was used to estimate the radii of some small bile salt aggregates. Goodness of fit for fluorescence lifetime and rotational correlation time analyses was judged for various models (one-, two-, and three-component models, using both discrete and distributed components) by the reduced xzvalues ( x z ~as) well as the randomness of the distribution of residuals across the modulation frequency range. Results and Discussion

Molecular Modeling Results. Table I shows the estimated radii for NaTC and NaTDC aggregates over a range of aggregation numbers, including a peryleneNaTC dimer sandwich, that were obtained using molecular modeling software and assuming the aggregates to be spherical. The estimated radii include 7-7.5 A for a dimer, 10 A for a trimer, 11 A for a tetramer, 12.5 A for

a hexamer, 14 A for an octamer, and 15 A for a decamer. These values are in general agreement with values reported elsewhere.7JJ Perylene was incorporated into the models for the above calculations. It was found that inclusion of the relatively planar probe molecule had only a small effect on the sizes of the aggregates. Also, these calculations included an estimated thickness for the hydrated shell. For the dimer, if 25 water molecules are included in the shell, the Stokes radius increases from 7 to 7.5 A. If 40 or 80 HzO molecules were included in the hydrodynamic volume, the radius would be 7.8 or 8.5 A, respectively. For larger aggregates, a simple first water shell with 1.5-A thicknesswas included. The corresponding rotational correlation times for the various micelles were calculated from the Debye-Stokes-Einstein equation (eq 3) and are also listed in Table I. The bulkviscositiesof the NaTC and NaTDC solutions were assumed to be the same as pure water sinceviscositiesaround 1CPhave been reported for solutionsof bile salts at concentrations below 100 mM at room t e m p e r a t ~ r e . ~ * ~ ~ ~ * ~ Compared with conventional detergent micelles, which have radii in the range 25-35 A and internal viscosities in the range 15-30cP, bilesalt aggregatesaresmall (7-20A) and their internal viscosities are high (100 CP I 9 I 675 cP).27-29A steady-state fluorescenceanisotropyexperimentshowed that the microviscosity experienced by 2-methylanthracene in NaTC micelles is as high as 675 cP.27,28 In a recent paper, the microviscosity was conservatively reestimated as 1100 CP by introduction of a correction in the restricted rotation modelz9but is nonetheless indicative of high restricted rotation. The most recent dynamic anisotropy measurementsof perylene in viscous solutions yielded correlation times of 5 and 43 ns when T (OK)/9 (cP) = 1.0 (Le., 9 = 300 CP).~OAlso, it was reported that perylene in phosphate lipid vesicles undergoes restricted rotation with three correlation times; the ratio of the two smallest correlation times was approximately seven, which is similar to the ratio in homogeneous viscous solution,and the third correlation time was so large that it can be treated as a constant term of essentially infinity (r-).I8 The two smallest correlation times in a heterogeneous system could therefore be estimated as in a homogeneous system, with the smaller of the two reflecting primarily the in-plane rotation of perylene.1417-31 From the high internal viscosity in bile salt micelles and the results in Table I, it was estimated that the in-plane rotational correlation time of perylene should be an order of magnitude larger than that of the bilesalt dimer. Therefore, thecontribution of perylene rotation to the smallest reduced correlation time ($1) recovered from dynamic anisotropy studies should be negligible. Fluorescence Lifetime of Perylene in NaTC. Fluorescence lifetimes were determined for use in the anisotropy analysis. These experiments also investigated the possibility that the lifetime of perylene in the bile salt solutions may not be a discrete, unique value as assumed in the anisotropy analysis software, as well as the possibility of energy transfer or other nonradiative mechanisms that would complicate the anisotropy experiment. Table I1 shows the fluorescence lifetimes of perylene in the NaTC solutions. At total NaTC concentrations below 20.0 mM, a two-component fit is indicated. The shorter lifetime component, which accounts for only a small fraction of the total fluorescence intensity, may result from energy transfer among partially solubilized perylene molecules, either in microcrystalline aggregates or associated with the bile salt. At higher total NaTC concentrations, the shorter lifetime component is no longer observed, leaving a single, discrete component of 6.15 ns. This is significantly larger than the lifetimes of 4.14 and 5.35 ns observed for perylene in benzene and cyclohexane, re~pectively.~~ The lifetime of perylene is not easily measured in water due to its low solubility (- 1.6 nMZ1sS3). The lifetime results indicate that formation of microcrystalline suspensions of perylene in the bulk aqueous phase is negligible,

6748 The Journal of Physical Chemistry, Vol. 97, No. 25, 1993

TABLE II: Fluorescence Lifetimes ( 7 ) and Fractional Intensity Contributions ( f ) of Perylene (10.0 fiM) in Aqueous NaTC S o l ~ t i ~ ~

100.0

0.917 0.953 0.975 1.OOO 1.000 1.OOO 1.000 1.ooO 1.000

5.97 6.10 6.21 6.09 6.14 6.14 6.19 6.20 6.10

10.0 12.0 15.0 20.0 30.0 40.0 50.0 60.0 100.0

0.920 0.953 0.992 1.000 1.OOO 1.000 1.000 1.000 1.OOO

6.24 6.26 6.19 6.15 6.17 6.20 6.31 6.33 6.30

20.0

10.0 12.0 15.0 20.0 30.0 40.0 50.0

60.0 40.0

0.083 0.047 0.025

1.10 1.37 1.78

0.080

1.14 1.71 1.60

0.047 0.008

TABLE IIk Reduced RotPtio~lCorrelation Times ($I) and Their Preexponential Factors (y) for Perylene (10.0 pM) in Aqueous NaTC Solutionsa T,OC C N ~ T C , ~ M YI h n s 7 2 42,ns 7 3 Q3,ns X ~ R 20.0

20.0 30.0 40.0 50.0 60.0b 100,ob

40.0

20.0 30.0 40.0 50.0 60.0

100.0

0.68 0.65 0.66 0.60 0.60 0.58 0.57 0.56

0.35 0.38 0.35 0.37 0.40 0.38 0.42 0.41

0.32 0.35 0.34 0.40 0.40 0.19 0.43 0.22

2.7 2.9 2.7 2.6 2.7 2.2 3.0 2.4

0.82 0.82 0.83 0.72 0.70 0.67

0.23 0.25 0.25 0.24 0.23 0.25

0.18 0.18 0.17 0.28 0.30 0.33

1.7 2.2 2.0 1.5 1.5 1.5

0.23

3.0

0.22

3.4

0.12 0.22 0.31 0.96 1.9 2.0 1.1 1.1 0.24 0.46 0.22 1.6 2.0 7.4

The limiting anisotropy ro = 0.35 and 7 in Table I1 were used in the anisotropy analysis. The data at these concentrationscan also be fit by three correlation time fit. and solubilizationof more than one perylene moleculeper micelle is unlikely at NaTC concentrations 120.0 mM. This conclusion is supported by the absenceof excimer emission in the fluorescence spectra of the solutions. Therefore, perylene molecules appear to be completely and individually solubilized in the bile salt aggregates and isolated from the aqueous solution at NaTC concentrations above 20 mM, which suggests that each perylene molecule must be surrounded by at least two NaTC monomers. Dynamic Anisotropy of Perylene in NaTC. The reduced correlation time includes contributions from all rotations which contribute to the reorientation of the transition dipoles of the fluorophore, from both the probe and the micelle. The dynamic anisotropy data for perylene in NaTC aqueous solutions were best fitted by either two-component or three-component correlation time models, as shown in Table 111. Three-component fits were not used if the third preexponential factor was 1%or less. It is important that the first reduced correlation time (41)is independent of NaTC concentration while its normalized preexponential factor (n)decreases as NaTC concentration is increased. Moreover, $I1 is very similar to the estimated rotational correlation timeof the bile salt dimer (Table I). These two results strongly suggest that 41corresponds to the rotation of the bile salt dimer with no significant contributions from rotation of the perylene or larger NaTC aggregates. The temperature dependence of $I1 follows the behavior predicted for the dimer by the Debye-Stokes-Einstein equation (eq 3), which yields a mean value of 7.2 f 0.1 A for the hydrodynamic radius of NaTC dimer using the $I1 values from Table 111.

-

Li and McGown In order for the above interpretation of the reduced correlation time to be valid, the smaller correlation time of perylene, which largely reflects the in-plane rotation, must be at least 10 times greater than $1 (eq 6b). This assumption is supported by reports elsewhere of highly rigid binding of probe molecules in bile salt micelle^.^^-^^ If, on the other hand, we consider a case in which there are no dimers and the smallest aggregates are trimers ( 4 ~ a 1.0 ns) or tetramers ($M c 1.4 ns), then in order to get a reduced correlation time of -0.38 ns at 20.0 OC, the in-plane rotational correlation time of perylene would have to be 0.6-0.5 ns, which roughly corresponds to a viscosity of less than 70 CP?' This is not a realistic model since the reported microviscosities in NaTC micelles are considerably h i g l ~ e r . * ~ - ~ ~ The results in Table I11can be explained in terms of the reduced correlation time concept for a polydisperse system of NaTC micelles. If we assume that there is an equal probability for perylene to be located in any of the different size aggregates and negligible probability of two or more probes to be in the same aggregate, which is consistent with the lifetime data and emission spectra, then the first normalized preexponential factor (vj, which is approximately equal to , 3 in~ this ~ case-see Theory) reflects the proportion of the aggregates that are dimers. The results then indicate that, within the examined concentration range, dimers account for 6846% of the NaTC aggregates in aqueous solutions at 20 OC and 82474%at 40 OC. Note that the results are expressed in terms of the dimer as a percent of the total number of aggregates rather than as a percent of the total bile salt mass. This is necessary because the dynamic anisotropy experiment does not identify or resolve the higher order aggregates, and so the distribution of total mass between dimer and higher order aggregates is unknown. The percent dimer decreases as the bile salt concentration increases or temperature decreases (Table 111) because both conditions favor the formation of larger aggregates. It should be noted that there is an apparent decrease in percent dimer around CN~TC = 50.0 mM and the temperature effect becomes smaller above this concentration. Previous observations that changes occur around 45.0-50.0 mM, based on light scattering meas u r e m e n t ~and ~ ~ NMR mea~urements,~~ could therefore corre spond to the decrease in percent dimer observed here. The second reduced correlation time ($2) is attributed to a combination of the correlation times of perylene and all of the larger micellar aggregates. This correlation time can be divided among two components (42 and $13)in three-component fitting models at higher NaTC concentrations at 20.0 OC, while the smallestcorrelation time ($1) and its preexponential factorsremain approximatelythe same for the two- and threecomponent models and is attributed to dimer alone. This indicates that the two larger reduced correlation times in the threecomponent fits correspond to the same rotations that are combined in 42 in the two-component fits and reflect an increase in micellar polydispersity and average micellar size as the total NaTC concentration is increased. A third component is not observed at 40.0 OC, indicating that higher temperatures do not favor increased polydispersity in this case. Using the two-component fits to the anisotropy data and assumingthat theratioof thefmt twocorrelationtimeaofperylene and the preexponential factors of those correlation times are similar to those in lipid vesicles,18a mean radius of 12-1 5 A is estimated for the larger micelles. The radius is slightly concentration dependent, as seen from the increases in $3 in the three-component fits at higher concentrations (Table 111). This range corresponds to aggregation numbers of 6-10 (Table I). If, from the above discussion, 60% of the aggregates are dimer and the remaining aggregates range from all hemmer to all dccamer, a range of 9-10 A by number-average or 10-12 A by weightaverage is obtained for the mean radius of all aggregates at 20.0 "C. This agrees with the weight-average radius of 10 A obtained

The Journal of Physical Chemistry, Vol. 97, No. ZS, 1993 6749

Polydispersity Studies of NaTC and NaTDC Fhnnwmnce lifetimes ( T ) iad hctiollfl TABLE Integity coatdbuth# (1)of Perylene (10.0 p M ) in Aqaeom N8TC WOM with 3.0 M N8Cl Tv0c 20.0

40.0

CNnTGmM

20.0 30.0 40.0 50.0 60.0 100.0 20.0 30.0 40.0 50.0 60.0 100.0

A 1.OOO 1.O00 1.OOO 1.O00 1.OOO 1.OOO 1.OOO 0.985 0.984 0.991 0.985 0.982

7bns 5.81 5.80 5.82 5.87 5.92 5.97 5.70

f2

5.75 5.74 5.81 5.80 5.81

0.015 0.016 0.009 0.015 0.018

28

‘I/

(A)

721

117 60.7 67.9 50.8 16.9

8 d

t-----$ml

TABLE V Reduced Rotational Correlation Tiwe (4) and Their Reexponential Factors ( 7 ) for Perylest (10.0 p M ) in Aqueous NaTC Solutiom dtb 3.0 M NaCl at 20.0 O c a CNnWBmM 20.0 30.0

40.0 50.0 60.0 1OO.ob

Yl 63,ns X2R 611m YZ d&ns 7 3 7.7 0.06 2.4 0.34 0.35 0.32 0.31 0.33 0.30 0.29 2.1 0.38 8.0 0.09 8.6 0.11 2.4 0.38 0.34 0.32 0.28 2.0 0.29 0.22 0.31 0.40 9.5 0.08 0.34 0.35 0.28 2.5 0.38 10.4 0.29 0.32 0.31 0.29 2.8 0.39 13.2 0.09 i0.03 i0.03 10.01 10.2 i0.03 i0.6

a The limiting anisotropy ro = 0.35 and 7 in Table IV were used in the anisotropy analysis. The data are an average of three replicates, and the standard deviation is given under each mean value.

using QEU8and ‘H-NMR9experiments. As mentioned above, the sizeof the bile salt dimer is on the edge of the QELS detection limit, and the percent dimer might therefore be underestimated from the mean size by this technique. This could account for some of the discrepanciesamong the reported values for the mean radii of the trihydroxy bile salt m i c e l l e ~ . ~ J l . ~ ~ Flsorcscenee Lifetimes and Dynamic Anisotropy of Perylene in NaTC with NaCL The fluorescence lifetimes of perylene in NaTC in the presence of 3.0 mM NaCl at two temperatures is shown in Table IV. At T = 20.0 OC, a single, discrete lifetime was recovered over the entire NaTC concentration range. It is reasonable that the lifetime is shorter in the presence of a high concentration of sodium and chloride ions. At T = 40.0 OC, two lifetime components were recovered, indicating heterogeneous microenvironments for perylene in the bile salt solutions. The anisotropy data for the perylene/NaTC/NaCl system at T = 20.0 OC were fitted best by a three-component correlation time model (Table V). The 100.0 mM NaTC solution was measured in triplicate in order to ascertain the uncertainty of the measurements, and relative standard deviations of 5-1096 were found for the three rotational correlation times as well as their corresponding preexponential factors. Figure 2 shows a representative example of the fit between the experimental data and three-component model. The lifetime heterogeneityexhibited at 40.0 O C precludes anisotropy analysis using our current methods, which assume a single, discrete lifetime for the probe. The small decrease in 41that occurs in the presence of 3.0 mM NaCl may result from a reduction of the contribution from the in-plane rotation of perylene andf or the bulk viscosity of the bile salt solution. A reduction in viscosity was reported for NaTDC solutions (dimer) aggregatesof NaTC and may therefore largely reflect the in-plane rotation of perylene.

*ad 5

I

I I I I

10

I

20

I

I

I

I 1 1 1 1

80

40

150

Modulation Frequency (MHz) Figure 2. Three-component fit of the experimental dynamic anisotropy data for 10.0 pM perylene in 20.0 mM NaTC with 3.0 M NaCI: (A) residuals for PAD,(B) residuals for MAR,(C) the experimental curves and the curves from the fits for PAD and M A R vmus modulation frequency.

The third reduced correlation time increases sisnificantly when the total NaTC concentration is increased, indicating enhanced aggregation in the presence of 3.0 M NaCl since 43 comprises the longer correlation times of perylene and those of the larger NaTC micllles. If we assume from the above discussion that 42 approximates the first correlation time of perylene and that the second correlation time of perylene is 7 times larger than the fir~t,~’J* Le., 4p.2 = 7 X Qp.1 PJ 7 X 2.4 17 ns, then the contributions of perylene rotation and micellar rotation to 43 can be determined and the mean radius (RM) of the larger (>dimer) NaTC micelles can be estimated. Values determined in this manner increase from 24 to 39 A as the NaTC concentration is increased from 20.0 to 100.0 mM. The value of 39 A for 100.0 mM NaTC is in close agreement with the value of 38.5 A that was obtained from a QELs experiment.8 As shown in the above calculations,it is important to be careful in estimating micellar size from the anisotropy data because the measured correlation times may contain contributions from rotation of both the probe and the micelles. If 43 were used directly to calculate the mean micellar radius without correction for probe contributions, similar to the approach taken in a study of conventional detergent micelles,39then an incorrect value of 20-23 A would be obtained for the mean radius of the larger (>dimer) NaTC micelles. The anomalous value of 41 at CN~TC = 50.0 mM in Table V may arise from a true discontinuity in the micellization proccss, but it is more likely that the anomaly is an artifact of the fitting algorithm in the anisotropyanalysis. Certain patterns of random noise in the PAD and MAR measurements can give rise to a change in the chi-square ( x z ~surface ) and lead the minimum

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Li and McGown

6750 The Journal of Physical Chemistry, Vol. 97, No. 25, I993 TABLE VI: Fluorescence Lifetimes ( T ) , Reduced Rotationnl Correlation Times (4), and Their Preexponential Factors (y) for Perylene (10.0 pM) in Aqueous NaTC (20.0 mM) Solutions with Different Concentrations of Terbium (HI) Nitrate at 20.0 O c a

TABLE M: F l u o ~ ~ ~ LifetimcS eme ( 7 ) , R&cd RotaCorrelation Ti" (#), aad Tbeir PrecxpowatklFWra ( y ) for Perylene (10.0 fiM) in Aqpeous NaTDC SoMozm at 20.0 OC' C N I T D G ~ M~ , n s YI d1,ns YZ d2,ns 73 Q3,ns X ~ R 1O e .

5.0 10.0 20.0 25.0 50.0 100.0 250.0 500.0 750.0

6.06 6.08 6.01 6.07 6.05 6.02 5.95 5.76 5.63

0.48 0.45 0.44 0.48 0.44 0.45 0.45 0.41 0.38

0.43 0.39 0.40 0.37 0.34 0.35 0.35 0.30 0.30

0.46 0.47 0.48 0.46 0.43 0.43 0.45 0.36 0.36

2.7 2.7 2.8 2.7 2.3 2.5 2.7 2.2 2.2

0.06 0.08 0.08 0.06 0.13 0.12 0.10 0.23 0.26

46.3 27.6 27.5 11.9 7.7 7.9 9.9 6.5 7.4

0.09 0.06 0.06 0.07 0.08 0.07 0.09 0.05 0.06

20.0 35.0 50.0 75.0 100.0

5.96 0.50 0.45 0.28 0.24 6.05 0.43 0.39 6.06 0.42 0.42 6.12 0.42 0.44 6.01 0.37 0.39 6.07 0.35 0.39

3.3 1.0 2.1 2.4 2.3 2.7 2.8

0.42 0.38 0.35 0.39 0.31 0.33

3.6 4.0 4.6 5.0 7.2 8.4

0.20 0.12 0.20 0.35 0.56 0.27 0.55

a The limiting anisotropy ro = 0.35 and T from this table were used in the anisotropy analysis. This data can be fit by both two and three correlation time fits.

The limiting anisotropy ro = 0.35 and T from this table were used in the anisotropy analysis.

point further away from the true minimum. As a matter of fact, the x 2surface ~ for this particular data is flat around the minimum point. Fluorescence Lifetime and Dynamic Anisotropy of Perylene in NaTC with Tb3+. Experimental results are shown in Table VI for fluorescence lifetime and dynamic anisotropy of perylene in 20.0 mM NaTC in the presence of different concentrations of = 100.0 Tb3+. The decrease in fluorescencelifetime above mM is attributed to fluorescencequenching by Tb3+at such high concentrations, while the constancy of the lifetime at lower concentrationssuggests that the probe is protected from theTb3+. Previous studies have demonstrated fluorescence enhancement by Tb3+of probes that are solubilized in N ~ T C , Z 'and S ~ ~it has been shown that lanthanide ions affect the aggregation of NaTC through complexation with the taurine tail group.40 As the concentration of Tb3+increases, 41 decreases from 0.43 and 0.30ns and the percentage of dimer decreases from 48% to 38%. Compared with the result for 20.0 mM NaTC at 20 OC in the absence of Tb3+(Table 111), the addition of 5.0 mM Tb3+ significantly reduced the percent dimer. This supports the view that fluorescence enhancement by Tb3+ that was observed in previous ~ t u d i e s ~is'the ,~~ result of complexationwith the NaTC.40 The effects of high concentrations of Tb3+are very similar to the effects of 3.0 M NaCl solution and are attributed to bulk, ionic strength effects. As shown in Table VI, the three reduced correlation times and the first two preexponential factors decrease with increasing of Tb3+concentration, while the third preexponential factor increases. The behavior of 43 and its preexponential factor is particularly informative. At low concentrationsof Tb3+, 43 is very large but the preexponential factor is relatively small, suggesting enhanced aggregation of NaTC that is attributed to complexation with the Tb3+.40At high concentrations of Tb3+, bulk ionic strength effects play the most important role in the aggregation process, and the resolved correlation times and preexponential factors are very close to those obtained in the presence of 3.0 M NaCI. Fluorescence Lifetime and Dynamic Anisotropy of Perylene in NaTDC. Results of fluorescence lifetime and anisotropy experiments for perylene in NaTDC micellar solutions are shown in Table VII. The lifetime of perylene in NaTDC is similar to that in NaTC. The single lifetime is recovered even at 10.0 mM because the critical micelle concentration (cmc) of NaTDC is 3 4 mM; this is in contrast to the lifetime heterogeneity observed for perylene at low concentrations of NaTC (Table 11), which has a higher cmc of 8-12 mM.22 The anisotropy data were fit best by a three-component model at CNaTDC 2 20.0 mM. At 10.0 mM NaTDC it is difficult to choose between the two-component and three-component fits, although the two-component fit is more consistent with the rest of the results. The preexponential factor of 41, which is again attributed to rotation of the dimer, decreases from 0.43 to 0.35 as CN~TDC is increased; as was the case for NaTC, a significant

0.50 0.30 0.19 0.23 0.19 0.32 0.32

OH

OH

?H

'RR '

R' = NHCH2CH2S03Na+ Figure 3. Sandwichdimer model in which peryleneis sandwichedbetween two NaTC monomers.

decrease is observed above 50.0 mM. A mean value of 7.3 f 0.1 A was calculated for the hydrodynamic radius of NaTC dimer using the 41 values from Table VII. The 42 and 43 are again attributed to rotations of perylene and of the larger (>dimer) micelles. Both 42 and 43 increaseas CN~TDC increases, suggesting an increase in average aggregate size and a high polydispersity for NaTDC micelles. The NaTDC aggregates are larger and more polydispersethan the NaTC aggregates, based on the lower fraction of dimer, the need for a three-component model for the correlation times, and the dramatic increases in 42 and 43 with increasing CN~TDC. This is consistent with numerous other studies that have shown that the aggregates of dihydroxy bile salts are larger than those of trihydroxy bile salts.'-12 Unlike NaTC, the size of the larger (>dimer) aggregates of NaTDC increases dramatically with increasingtotal monomer concentration. The mean radius of the larger aggregates could vary from 16 to 25 A Over the concentration range examined. Although the mean radius of 18.5 A that was reported for NaTDC at 20 OC from QELS experiments* is within this range, the concentration dependence of the radius is more consistent with the dependence suggested in a later QELS study.' A Sandwich Dlmer Aggregation Model for Perylene in NaTC. As discussed earlier, the dimer is the minimum size of NaTC aggregate that is required to solubilize perylene in aqueous solution. Since dimer accounts for more than half of the total aggregatesin NaTC solution over the concentrationrange studied here, it is necessary to propose a model for solubilization in which perylene is solubilized in the NaTC dimer. Figure 3 shows a "sandwich" dimer model for perylene in NaTC. This model is supported by NMR studies that show that CISand C19 of the methyl groups of bile salt molecules (Figure 1B)are most affected by solubilized aromatic compound^.^' In the sandwich dimer model, perylene is in contact with the hydrophobic surfaces of

The Journal of Physical Chemistry, Vol. 97, No. 25, 1993 6751

Polydispersity Studies of NaTC and NaTDC the NaTC monomers and is fairly well protected from contact with the bulk solution. This would account for the experimental observation made here that the fluorescence lifetime of perylene in the NaTC solutions is much higher than in simple solvents and does not change much as the total NaTCconcentration is increased (Table 11). It has previously been shown by fluorescenceprobe experiments that different polycyclic aromatic hydrocarbons experience different microenvironments in NaTC micellar solution due to their different sizes and s t r u c t ~ r e s .Results ~ ~ suggestedthat larger probes experiencea higher microenvironmentalpolarity because they protrude into the aqueous solution. The dimer model supports this view since the two bile salt molecules would not be able to completely surround the larger probes. A “two-step” model for aggregation of bile salts has been this model, a primary proposed by several g r o u p ~ . ~ - ~ .In *J~ micellar unit, which is formed by hydrophobicinteraction between monomers, is the basic building block of larger, secondarymicelles. It has been reported elsewhere that a critical region in NaTC aggregation occurs around 3-5 mM, which is below the “cmc” of NaTC.22 It is possible that this premicellar critical region corresponds to the formation of dimer and that the higher cmc region at 8-1 2 mM corresponds to the beginning of larger micelle formation. Concentrations of NaTC below 20 mM could not be studied using the dynamic anisotropy technique in this work because of the fluorescence lifetime heterogeneity exhibited by perylene in these solutions. The decrease in percent dimer observed at 50.0 mM NaTC may reflect yet another critical region in the aggregation process as well. Analogous conclusions could be drawn for NaTDC, although the case for primary-secondary micellization is less compelling for the dihydroxy salt because of its lower aqueous solubility and the resulting drive to form larger micelles at lower concentrations. Additional Considerations. The shape of the probe-dimer complex shown in Figure 3 is not the perfect sphere that was assumed in the calculations of its correlation time. A rigorous, theoretical treatment in the case of an ellipsoid is complicated because the overall rotation of the complex can no longer be completely separated from the internal rotation of the probe and eq 2 does not then apply.I6 However, an upper limit of the ratio between the correlation times of a nonspherical micelle and a spherical micelle with the same hydrodynamic volume can be estimated. If it is assumed that the sandwich dimer is an oblate ellipsoid with p = 0.5 (the ratio of two main diffusion axes) and that the probe is fixed with its long axis oriented within the disk plane, then the upper limit of the ratio is estimated to be at most 1.1-1.2. If the orientation of the probe is random as the result of in-plane rotation, then both p and the orientation effect will make the ellipsoid behaue closer to a sphere, and the effect of a nonspherical shape will lead to an overestimate of at most 10% in the correlation time. On the other hand, when the difference between the internal rotational rate of the probe and the overall rotational rate of the dimer complex is only 10-fold,interference of the internal rotation may result in underestimation of the correlation time of the complex (41) by as much as 10%. Thus, the two effects tend to cancel each other, and the final result for the radius of the bile salt dimer would not be significantlychanged. Even if the effects do not cancel, they are not large enough to reassign the correlation time 41 to either a monomer or a trimer complex, and the dimer model still stands. Another important considerationis the effect of the fluorescent probe on the aggregation of the bile salt. If the probes themselves induced the formation of dimers or higher-order aggregates in the solubilization of the probe, then we would expect to observe solubilization of probe at every bile salt concentration, and the probe signal should therefore increase linearly with increasing bile salt concentration even below the cmc. This is not the case. Previous studies have shown that insoluble probes are not

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solubilized in NaTC solutions below the cmc, and the cmc is independent of which insoluble probe is used.z2 Moreover, the same cmc that was found for NaTC using insoluble fluorescent probes was also observed by surface tension measurements in the absence of probe.” It has also been observed that scattered light intensity of bile salt solutions is essentially the same in the presence and absence of probe above concentrations required to completelysolubilizealloftheprobeinthesolution.u Bulkeffects of the probes on the bile salt aggregation process are unlikely in these experiments because the probe is at such a low concentration (less than 1/1OOO that of the bile salt). Based on these previous results, it is reasonable to expect that the probe studies described here provide an accurate model for bile salt aggregation in the absence of probe.

Conclusions The results of this work provide a strong case for a primarysecondary model for NaTC and NaTDC aggregation in aqueous solution in which the basic building block is a dimer. Although it is not possible to determine directly from these experiments whether the perylene probe influences the aggregation process, the good agreement between the rotational correlation times recovered for the dimer from the experiments and the values calculated from molecular models indicates that the experiments do provide an accurate view of NaTC aggregation in the absence of probe. Moreover, the large proportion of dimer even at high bile salt concentrations has important implications for the development of models decribing, and analytical techniques based on, the solubilizationof molecules by bile salts in aqueous solution, in which the probes represent solutes and are an integral part of the system. A critical aspect of this work is the introduction of the reduced correlation time and its ability in this case to isolate the rotation of the dimer from both the rotations of the larger aggregates and the internal rotations of the probe itself. This is a new approach to studying polydispersity in micellar systems that will be particularly important for resolution of contributionsfrom smaller aggregates that may not be resolvable by other techniques.

Acknowledgment. This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy Research, United States Department of Energy (Grant DE-FG0588ER1393l), and by the Environmental Protection Agency (Grant R8 17127-01). References and Notes (1) Small, D. M. Adu. Chem. Ser. 1968, 84, 31. (2) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971; Vol. 1, Chapter 8. (3) OConnor, C. J.; Wallace, R. G. Adu. Colloid Inter-ace Sci. 1985, 22, 1. (4) Campanelli, A. R.;Candeloro De Sanctis, S.;Chiessi, E.; DAlagni, M.; Giglio, E.; Scaramuzza, L. J . Phys. Chem. 1989, 93, 1536. (5) Smith, W. B.; Barnard, G. D. Can. J . Chem. 1981, 59, 1602. (6) Barnes, S.;Geckle, J. M. J. Lipid Res. 1982, 23, 161. (7) Fung, B. M.; Peden, M. C. Biochem. Biophys. Acra 1976,437,273. (8) Mazer, N. A.; Carey, M. C.; Kwasnick, R. F.;Bencdek, G. B. Biochemistry 1979, 18, 3064. (9) Schurtenberger, P.; Lindman, B. Biochemistry 1985, 24, 7161. (10) Mukerjee, P.; Cardinal, J. R. J. Pharm. Sci. 1976, 65, 882. (11) Kratohvil, J. P.; Hsu,W. P.; Jacobs, M. A,; Aminabhavi, T. M.; Mukunoki, Y. Colloid Polym. Sci. 1983, 261, 781. (12) Kratohvil, J. P. Hepatology 1984, 4, 85s. (13) Kawamura, H.; Murata, Y.; Yamaguchi, T.; Igimi, H.;Tanaka, M.; Sugihara, G.; Kratohvil, J. P. J . Phys. Chem. 1989, 93, 3321. (14) Kinosita, Jr., K.; Kawato, S.;Ikegami, A. Biophys. J. 1977,20,289. (15) Zannoni, C.; Arcioni, A.; Cavatorta, P. Chem. Phys. Lipids 1983,32, 179. (16) Szabo, A. J . Chem. Phys. 1984,81, 150. (17) Barkley, M. D.; Kowalczyk, A. A.; Brand, L. J . Chem. Phys. 1981,

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