J . Phys. Chem. 1990, 94, 8457-8463
8457
Unimodai Lorentzian Lifetime Distributions for the 2-Aniiinonaphthalene-6-sulfonate-~-Cyclodextrin Inclusion Complex Recovered by Multifrequency Phase-Modulation Fluorometryt Jingfan Huang and Frank V. Bright* Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14214 (Received: April IO, 1990)
The inclusion complex of 2-anilinonaphthalene-6-sulfonicacid (2,6-ANS) and @-cyclodextrin(@-CD) was studied by frequency-domain fluorometry. Fluorescence lifetimes were measured in the presence of various concentrations of dynamic quenchers (Cu2+,acrylamide, and I-). Frequency-domain data were analyzed by global analysis linked by the Stern-Volmer equation using both discrete and distributed models. A unimodal Lorentzian distribution model was determined to be the best fit to the experimental data because the quenching constants recovered matched those from steady-state quenching measurements and x 2 values were relatively small. This conclusion was also confirmed by measuring the lifetime of the inclusion complex (2,6-ANS-P-CD) at different temperatures. The quenching constants are discussed for these three diverse quenchers in solutions of 2,6-ANS in water, ethanol, and p-CD measured at temperatures ranging from 20 to 50 O C . The effects of different solutes on the inclusion complex formation constant are also reported.
Cyclodextrins (CDs) have received much attention in recent years because they can modify chemical pathways and catalyze chemical reactions. In aqueous media, CDs can selectively include various molecules into their cavities, which have microheterogeneous structures with unique sizes. The nature of the noncovalent interactions involved in inclusion between guests and CDs has been well characterized. Hydrophobic effects and van der Waals interactions are considered to be the major forces that drive the inclusion c~mpIexation.'-~Proton nuclear magnetic resonance ('H NMR) experiments indicate that inclusion complexes are not static species.6 Rather, it has been reported that guest molecules can have different orientations about the axis parallel to the cavity and/or are rapidly spinning about this axis within the cavity.' Guest molecules also exchange rapidly between free and bound forms. The recombination and dissociation rate constants have been determined to be on the order of 107-109and 104-107M-I s-I by using temperature-jump8 and ultrasonic relaxation9 techniques, respectively. Because these exchange rates are so fast, little effort has been expended to distinguish between the guest molecules included to different extents within the cavity. Moreover, an investigation of the different stages of inclusion complexation is severely restricted by the time resolution of the particular spectroscopic technique used. For example, NMR techniques can be used to about s, which is much slower than the exchange rate between free and bound guest molecules. Accordingly, only the time-averaged information about free and cyclodextrin-bound complexes are determined by NMR. Electron spin resonance (ESR) can explore a relatively short time scale (-10" s).Io It is capable of identifying free and bound guest species but fails to differentiate bound species in various stages of inclusion which alternate at rates greater than the time resolution of the ESR experiment. For this reason, ESR can only detect the average signal of, for example, discrete 1:1 (guest:host) inclusion complexes. Fluorescence techniques have been used widely to examine the microenvironment of fluorescent guests included within a-, p- and y-cyclodextrin cavities. Because of earlier limitations of both instrumentation and data analysis, fluorescence decays for 1: 1 inclusion complexes were all described as discrete monoexponential decays.I'-l6 Only recently has a 1:l p-CD inclusion complex been identified to have two different conformations by picosecond time-resolved fluorescence spe~troscopy.~~ Fluorescence lifetime studies from this laboratory, on p-CD complexed with various anilinonaphthalenesulfonate (ANS) probes, suggest that a uni-
modal lifetime distribution may actually be the best model to describe the fluorescence decay.I8 The conclusion was based on analyzing only one set of data for each ANS compound and comparing the xz goodness of tit for different models. However, it is rather difficult to recover the underlying distribution lifetime from phase-modulation data if data precision is not very high. Therefore, more solid evidence is demanded to support further our preliminary results.18 In this paper, extensive new results from steady-state intensity and time-resolved fluorescence lifetime data are reported. The influence of temperature on fluorescence lifetime and quenching rate is also discussed. To improve the precision and accuracy of the recovered kinetic parameters, multiple sets of frequency-domain data are linked together and analyzed by using global a n a l y ~ i s . ' ~All the experimental results strongly support the Lorentzian lifetime distribution model for the fluorescence decay of 2,6-ANS-@-CD inclusion complex. We attribute this lifetime distribution to the complexational distribution within the 2,6ANS-0-CD inclusion complexes.
( I ) Matsui, Y.; Fujie, M.; Hanaoka, K. Bull. Chem. SOC.Jpn. 1989, 62, 1451. ( 2 ) Eftink, M. R.; Andy, M. L.; Bystrom, K.; Perlmutter, H. D.; Kristol, D. J . A m . C h e m . S o c . 1989, 111,6765. ( 3 ) Cromwell, W. C.; Bystrom, K.; Eftink, M. R. J . Phys. Chem. 1985, 89, 326. ( 4 ) Gelb, R. I.; Schwartz, L. M.; Laufer, D. A. J . Chem. Soc., Perkin Trans. 2 1984, 15. ( 5 ) Harrison, J. C.; Eftink, M. R. Biopolymers 1982, 21, 1153. ( 6 ) Demarco, P. V.; Thakkar, A. L. Chem. Commun. 1970, 2. ( 7 ) Bender, M. L.; Komiyama, M. Cyclodexfrin Chemistry; SpringerVerlag: Berlin, 1978. (8) Cramer, F.; Saenger, W.; Spatz, H.-ch. J . A m . Chem. SOC.1967.89, 14. ( 9 ) Rohrbach, R. P.; Rodriguez, L. J.; Eyring, E. M.; Wojcik, J. F. J . Phys. Chem. 1977, 81, 944. (IO) Kotake, Y.; Janzen, E. G. J. A m . Chem. SOC.1988, 110, 3699. (1 1 ) Kano, K.; Takenoshita, I.; Ogawa, T. Chem. Lett. 1980, 1035. ( 1 2 ) Kano, K.; Takenoshita, I.; Ogawa, T. J . Phys. Chem. 1982,86, 1833. ( 1 3 ) Nelson, G.;Patonay, G.; Warner, 1. M. Appl. Spectrosc. 1987, 41, 1235. ( 1 4 ) Kobashi, H.; Takahashi, M.; Muramatsu, Y.; Morita, T. Bull. Chem. SOC.Jpn. 1981, 54, 2815. (15) Turro, N . J.; Okubo, T.; Chung, C. J . A m . Chem. Soc. 1982, 104, 3954. ( 1 6 ) Hamai, S. Bull. Chem. Soc. Jpn. 1982, 55, 2721. (17) Durenech, G.L.; Sitzmann, E. V.; Eisenthal, K. B.; Turro, N. J. J . Phys. Chem. 1989, 93, 7166. ( 1 8 ) Bright, F. V.; Catena, G.C.; Huang, J. J . A m . Chem. Soc. 1990, 112, 1343. ( 1 9 ) Beechem, J. M.; Gratton, E. Proc. SPIE 1988, 909, 7 0 .
'A preliminary account of this work has been presented at the 1989 FACSS meeting, Chicago, IL, paper No. 114. 'Author to whom all correspondence should be addressed.
0022-3654/90/2094-8457$02.50/0, I
,
~
1
0 1990 American Chemical Society
8458 The Journal of Physical Chemistry, Vol. 94, No. 22, 1990
Huang and Bright
Materials and Methods
Materials. All reagents were used as received without further purification. The following chemicals were used: 8-cyclodextrin (Sigma Chemical Co.); 2-anilinonaphthalene-6-sulfonicacid (2,6-ANS) (Molecular Probes); acrylamide (99+% purity) (Aldrich): CuSO4.5H20 (J. T. Baker, Inc.); NaI, NaCI, and Na2S203.5H20(Fisher Scientific Co.); and EtOH (absolute, 200 proof, Aaper Alcohol and Chemical Co.). 1,4-Bis(4-methyl-5phenyl-2-oxazolyl)benzene ( Me2POPOP) (Aldrich) in methanol was used as the reference lifetime standard. Its lifetime was assigned a value of 1.45 ns.m All aqueous solutions were prepared in doubly distilled-deionized water. Methods. Fresh sample solutions were used in both steady-state and dynamic fluorescence experiments. Determination of binding constants for 2,6-ANS-p-CD were made as described previously.21 Steady-state quenching experiments were performed by measuring the fluorescence intensity of I O pM 2,6-ANS in H 2 0 , ethanol, and 15 mM 0-CD with various quencher concentrations. A Perkin-Elmer LS-3 fluorometer was used for steady-state intensity measurements whenever fluorescence signals were strong and higher imprecision could be tolerated. More precise measurements were obtained with an SLM 48000 (SLM-Aminco) using an arc lamp as an excitation source. The concentration ranges for quenchers Cu2+,acrylamide, and I- were 0.0-20.0 mM, 0.0-0.8 M, and 0.0-0.5 M, respectively. In the iodide quenching experiments, NaCl was added to maintain the ionic strength of each sample at 0.5 M. In addition, I O mM Na2S203was added to all solutions to prevent the oxidation of iodide. Blank corrections were made for each sample measurement. Equilibrium data were analyzed by a nonlinear least-squares program (ENZFITTER).~~ Fluorescence lifetimes were determined with an SLM 48000 multifrequency phase-modulation fluorometer. An argon ion laser (Coherent, Model 90-6) operating at 363.8 nm was used as the excitation source. Emission was observed through a 420 long-pass filter (Oriel). Magic angle polarization was used for all sample measurements.23 Ambient oxygen dissolved in the samples was not eliminate before lifetime measurements. The cuvette holder was temperature-regulated to *O.l "C via a Lauda RLS-6 temperature circulator. Lifetime data were both individually and globally analyzed by using single or double exponentials, Lorentzian distribution, Gaussian distribution, and other theoretical decay model^.'*^^^ The average experimental phase and modulation variances were utilized for minimization of x2 values. In cases where multiple experiments were analyzed, the largest experimental phase and modulation variances within the particular set of experiments were used for calculating x2. The most adequate fitting model meets the following criteria: simplest model (i.e., having the minimum number of total floating parameters) with relatively small x2 and concordance with results from steady-state experiments.
Results and Discussions Formation Constant Experiments. The formation constants ( K J of the following equilibrium were determined when quenchers were present or absent: 2,6-ANS p-CD F! 2,6-ANS-P-CD (1)
+
It was found that both acrylamide and I- reduced the inclusion complex formation constant (Figure 1). The formation constant in the presence of 0.8 M acrylamide and 0.5 I- is only 23% (panel A) and 35% (panel B) of that in the absence of quenchers, respectively. Also, K f in the absence of I- was 20% larger than in the absence of acrylamide. This is because, in the former case, 2,6-ANS-@-CD is in a 0.5 M NaCl solution, whereas in the latter case the equilibrium was studied in neat aqueous solutions. In order to fully understand the effect of CI- on the inclusion process, (20) Lakowicz, J. R.; Cherek, H.;Baiter, A. J. Biochem. Biophys. Methods 1981, 5. 131. (21) Catena,
G.C.; Bright, F. V. Anal. Chem. 1989, 61, 905. (22) Leatherbarrow, R. J. Emjitter: A Non-linear Regression Data Anulysis Program: Elsevier Biosoft: Cambridge, UK, 1987. ( 2 3 ) Spencer, R. D.; Weber, G. J . Chem. Phys. 1970, 52, 1654.
0.20
0.00
0.40
0.60
I
0.80
[acrylamide] (M)
h
r
I
z 2-
v
500
1
0 0.00
5.10
5.20
5.30
0.40
0
IO
[No11 (MI
Figure 1. 2,bANS-P-CD inclusion complex formation constant (KJ at 20 O C in the presence of different concentrationsof acrylamide (A) and iodide (B). Various concentrations of NaCl are present in (B) to maintain the ionic strength of each solution at 0.5 M.
-,
y' 3000'* I 2000
c v
1000 4 3.000
cJ /g 0
3.100
3.200 l/r
3.300
3.400
3. 00
(IO-~K-~)
Figure 2. van't Hoff plots of formation constants of 2,6-ANS-@-CD complex at various ionic strengths ( a ) in NaCl solution and ( b ) in Na2S0, solution: 0.0 (O), 0.2" ( O ) , 0.3b (A),0.5" (A),and 1.0 Ma(0). TABLE I: Thermodynamic Constants for the Formation of 2,6-ANS-D-CD Inclusion Complex in Solutions at Different Ionic Strengths ( I , ) IC, M AH', kJ/mol ASo,J/(mol K) AGO, kJ/mol 0.0 -19.1 f 1.4 -1.6 f 4.7 -18.6 0.2" -19.3 f 0.9 -1.6 f 2.8 -18.8 0.3b -19.1 f 0.5 -0.67 f 1.5 -18.9 0.5" -21.0 f 1.0 -5.9 f 3.4 -19.2 1 .O' -18.3 f 0.4 5.1 f 1.3 -19.8 a
In NaCl solution.
Na2S04solution.
the formation constants were measured in the presence of different over a temperature range of 20-50 concentrations of CI-and "C. van't Hoff plots are shown in Figure 2, and the standard enthalpy ( A H " ) and entropy (AS") changes are summarized in Table I. The effect of Cuz+ was not studied because Cu2+ has been reported not to affect the e q u i l i b r i ~ m . ~ ~ (24) Hashimoto, S.; Thomas, J. K. J . A m . Chem. SOC.1985, 107, 4655.
2,6-ANS-P-CD Inclusion Complex
1.80
The Journal of Physical Chemistry, Vol. 94, No. 22, 1990 8459
t
0.00
5.00
10.00
15.00
20.00
[CUSO~](mM)
Figure 3. Stern-Volmer (intensity) plots for 2,6-ANS-j3-CD quenched by Cu2+at 20 (-), 25 (--), 30 35 (---), and 40 OC (---). [2,6ANSI = IOpM, [p-CD] = 15 mM. (e-),
Obviously, the observed decrease of fluorescence intensity by quenchers is a result of both quenching of fluorescence and the disruption of the inclusion complex. It has been known that Ibinds with o-CD very weakly, with a binding constant less than 20 M-1.9 However, at I- concentrations as high as 0.5 M, more than 90% of the 8-CD will be complexed with iodide. The competition between I- and 2,6-ANS shifts the equilibrium (eq I), which makes the apparent 2,6-ANS-@-CD formation constant smaller. To elucidate the reason for a decreasing formation constant with the addition of acrylamide, ‘HNMR spectra of samples containing acrylamide alone, only @CD, and their 1 :2 and 2: 1 molar mixtures were studied.25 The spectra of the mixtures were simple linear combinations of the individual spectra (not shown). That is, there were neither new nor shifted resonances, which suggests minimal interaction between acrylamide and j3-CD. We believe the decrease in Kr is due to the acrylamide lowering the surface tension of the solvent. The free energy change associated with the formation of inclusion complex 2,6-ANS-&CD is decreased, which is supported by the arguments of Orstan and Ross.26 On the other hand, CI- is an antichaotropic ion like SO?-, which does not bind with o-CD.’v9 However, both CI- and S042-have an indirect effect on the inclusion process. An increase in the concentration of CI- or S042-increases the solution ionic strength, which leads to more 2,6-ANS molecules being ion-paired with counterions such as Na+. This apparently neutral species can be sequestered within the p-CD hydrophobic cavity more effectively, and Kr concomitantly increases. Also, the addition of the electrolytes elevates the surface tension of the solvent, which further increase the formation constant. Steady-State Quenching Experiments. Steady-state fluorescence intensities of 26-ANS-0-CD quenched by Cu2+,acrylamide, and I- were studied at temperature ranging from 20 to 45 OC. In every case, the intensity data were well fit by the Stern-Volmer equation
[acrylomide] (M)
Figure 4. Stern-Volmer (intensity) plots for 2,6-ANS-&CD quenched by acrylamide at 20 (-), 25 (--), 30 35 and 40 O C (---). [2,6-ANS] = 10 pM, [p-CD] = 15 mM. (-a),
(-e-),
1.80
1.60
I
“13 ( M I Figure 5. Stern-Volmer (intensity) plots for 2,6-ANS-@-CDquenched by iodide at 20 (-), 25 (--), 30 35 40 (---),and 45 OC [2,6-ANS] = IO pM, [&CD] = 15 mM, [NaI] [NaCI] = 0.5 M, [Na2S203]= IO mM. (e-),
(-e-),
(-ma-).
+
[acrylamide] (M)
Here Fo and F represent fluorescence intensities in the absence and presence of quenchers, respectively. K , is the Stern-Volmer constant, k, is the bimolecular quenching constant, [Q]is the molar concentration of quencher, and T~ is the fluorescence lifetime in the absence of quencher. The greatest K,, values are observed for Cu2+quenching and the smallest for I-. Also, Cu2+quenching is enhanced significantly at higher temperatures (Figure 3), whereas acrylamide quenching is far less dependent upon temperature (Figure 4). I-quenching behaves in an opposite fashion. Instead of an increased quenching effect with temperature, we actually observe a slight decrease in ( 2 5 ) Experiments are performed using a Varian VXR-S 400-MHZ NMR spectrometer. Samples were maintained at 18 OC, and D20(99.996%. Aldrich) was used a solvent. Resolution of the experiment is 0.5 Hz. (26) Orstan, A,; Ross, J. B. A. J . Phys. Chem. 1987,91, 2739.
Figure 6. Comparison of Stern-Volmer plots from observed fluorescence intensity (--) and after being corrected for the quencher disruption effect
(-1. Ksv as temperature is increased (Figure 5 ) . In order to quantitatively calculate an accurate Stern-Volmer constant, the decrease in fluorescence caused by quencher disruption of the inclusion complex was corrected. This was done by multiplying the observed F o / F values by a/ao to obtain the corrected Fo/F values. Here a and a. are the fraction of inclusion complex in solution in the presence and absence of quencher, respectively. In the presence of 0.8 M acrylamide, the ratio a/ao is 0.91. Figure 6 illustrates the effect of disruption for the acrylamide quenching of 26-ANS-o-CD. In this particular case, a 16% increase in Ksv results when the data are corrected. Much smaller deviation in K , originates from I-. (a/aois 0.954 at an I- concentration of 0.5 M.) Consequently, the deviation in the
8460
The Journal of Physical Chemistry, Vol. 94, No. 22, 1990
Huang and Bright
TABLE 11: Comparison of Fluorescence Quenching Constants (K,) for 2,6-ANS in Different Environments Recovered from Intensity (F) and Lifetime ( T ) Experiments medium quencher H20 Cu” acrylamide
KS”(FO/R M-’ 0.000 0.681
0.085
1-
EtOH p-CD
3
acrylamide
6.27 22.5 2.35 1.05
CU”’ acrylamide 1-
y;p
k p 1 . y ns 0.350 0.0 0.350 1.96 X IO9 0.350 0.24 X IO9 7.41Ib 0.846 X lo9 0.847 X IO9 5.952( 3.78 X IO9 3.70 X IO9 6.016( 0.391 X IO9 0.367 X IO9 5.659d 0.186 X lo9 0.186 X IO9 io,
“$(F,/F) values are calculated from K,,(F,/F) and lifetime io listed in the table. *Lifetime recovered from global linked analysis using the monoexponential decay model. Lifetime recovered from global linked analysis using Lorentzian distribution (6-CD-bound)combined with a monoexponential decay (free 2,6-ANS) model. dLifetime recovered from global linked analysis using the unimodal Lorentzian distribution model.
K,, values because of the quencher disruption can be essentially
ignored for Cu2+ and I- under our experimental conditions. The recovered k, values for different quenchers are compiled in Table 11. I n the presence of p-CD, the bimolecular quenching constants follow the sequence kq(Cu2+)> k,(acrylamide) > k,(I-). The difference in quenching arises, partially, from the local charge effects. At neutral pH, 2.6-ANS-P-CD carries a net negative charge and would tend to attract Cu2+and repel I-. However, this alone cannot fully account for the IO-fold enhancement in k,(Cu2+) compared to k,(acrylamide). An important reason for Cu2+having the highest k, value is that Cu2+quenches fluorescence through electron t r a n ~ f e r . ~ ~This . ~ ’ process can occur over a distance larger than the collisional distance.24 The mechanism of acrylamide and 1- quenching is collisional energy transfer, and contact with the excited-state 2,6-ANS is required for efficient quenching. The difference in the quenching mechanism thus allows 2,6-ANS to be better protected by p-CD from acrylamide and I- than Cu2+. From the results presented in Figures 3-5 one can see that K,, and thus k , vary with temperature. The temperature dependence of k, can be explained by the following quenching scheme28 F*
+
ko
ki
(F-Q)*
F
+ Q + heat
r i+ hu
(3)
F
k, = k0k2/(kl + k,) = kor
(4)
where (F-Q)* is the excited-state 2,6-ANS-@CD-quencher complex and k, and k , are its association and dissociation rate constants, respectively. Both these rates are diffusion-controlled and therefore are proportional to the temperature-to-viscosity ratio ( T / v ) . The quenching efficiency ( r ) is given by r = k 2 / ( k ,+ k,). For an efficient quencher, quenching occurs immediately after (F-Q)* is formed, so k2 is much greater than k , and r approaches unity. Under these conditions, k, is equivalent to ko and is also a function of T / q . However, for a less efficient quencher, k, is comparable to k,. Thus, a marked enhancement in quenching efficiency ( r ) will result if the viscosity of the solution is increased. The contributions from both ko and r cancel the effect of T / ? to some extent, and therefore less T / q dependence of k , will be observed. This is the case for the acrylamide quenching process. In more extreme cases, e.g., iodide quenching, k, may even appear to decrease slightly as temperature is increased. 2,6-ANS in H 2 0 and EtOH quenched by the same quenchers was also studied to compare these simple systems with the more complicated P-CD system. A linear Stern-Volmer relationship was again observed. The K,, values from these experiments are (27) Atherton. S. J.: Beaumont. P. C. J . Phvs. Chem. 1986. 90. 2252. (28) Lakowicz. J. R Principles of Fluorescence Spectroscopy: Plenum Press New York. 1983
10
100
FREQUENCY (MHz)
Figure 7. Multifrequency phase and modulation data (points) for 2.6ANS-P-CD inclusion complex quenched by acrylamide. Acrylamide concentrations are 0.0 (O,.), 0.2 (A,A),0.4 (n,.), and 0.8 M (v,V). Open and filled symbols signify phase angle and demodulation factor, respectively. The solid lines are recovered global fits utilizing a unimodal Lorentzian distribution (P-CD-bound 2,6-ANS) decay plus a monoexponential (free 2,6-ANS) decay.
also listed in Table 11. Cu2+ was found not to quench aqueous 2,6-ANS. In ethanol the fluorescence of 2,6-ANS is quite strong, and the rate acrylamide quenches 2,6-ANS is between that in H 2 0 and (3-CD (Table 11). Additionally, there was a more profound increase in the quenching constant than in the case of 2,6ANS-(3-CD as experimental temperature was elevated from 20 to 50 OC. Therefore, p-CD protects 2,6-ANS from acrylamide and Iquenching (Table 11). Such observation suggests indirectly that ternary complexes of fluorophore, quencher, and cyclodextrin are not formed in the ground state. This is contradictory to inclusion complexation of hydrophobic fluorophores such as pyrene and naphthalene quenched by aliphatic amines.”J2 In these experiments, quenching is accelerated in the presence of p-CD and ternary complexes are formed.”-12 To confirm or refute ternary complex formation in the ground state, we investigated the UV-vis absorbance spectra of 2,6ANS-P-CD when quenchers were present and absent. Essentially, identical spectra (not shown) are observed under all conditions. This result argues against static quenching; Le., no higher order complexes are formed. In summary, based on steady-state measurements only a dynamic quenching mechanism is involved in the quenching processes studied, considering the good correlation with the steady-state Stern-Volmer equation, the temperature dependence of quenching process, and the evidence provided by absorbance spectroscopy. Fluorescence Lifetimes in the Presence of Quenchers. In order to more completely understand the 2,6-ANS-P-CD inclusion complex, multifrequency phase and modulation fluorescence was used.18 Specifically, experiments were performed at 20 OC in the presence of various quencher concentrations. Typical results of acrylamide quenching experiments are shown in Figure 7. These illustrate that the fluorescence lifetime decreases as the quencher concentration is increased. Similar results were also obtained with Cu2+and I- (not shown). The fluorescence lifetime of 2,6-ANS in ethanol quenched by acrylamide was also investigated, and it is fit best by a single-exponential decay model (Table 111). Unimodal distribution models yield higher x 2 values and widths of only several picoseconds or even negative numbers. Similarly, 2,6-ANS in H 2 0 was determined to have a lifetime of approximately 350 ps at 20 O C . The frequency-domain data were fit both individually and linked globally by the Stern-Volmer equation, using the procedure described in the Methods section. The x2 values in the latter case were comparable to the former when the same theoretical decay model was used. In all cases, very similar lifetimes and K, values were recovered from both methods. Nevertheless, a linked global analysis used fewer total floating parameters. For this reason, it was considered to be a more rigorous method. In this paper,
2,6-ANS-P-CD Inclusion Complex
The Journal of Physical Chemistry, Vol. 94, No. 22, 1990 8461
TABLE 111: Stern-Volmer Constant (K,,,M-I) and Global x 2 Values Recovered from Global Analysis Using Different Theoretical Decay Models 2,6-ANS in
2.6-ANS-B-CD
cu2+ model'
nb
A
2
B
2 3 3 3 9 9
C D
E F G
KW 19.8 20.2
21.1 21.4 18.3 18.5 22.1
acrylamide
EtOH quenched by acrylamide
I-
X2
n
K,"
X2
n
KW
X2
n
K."
x2
30.3 13.7 3.4 4.4 3.6 1.1 1.8
2 2 3 3 3
1.81 1.76 1.59 1.66 1.59 2.22 2.22
31.2 25.8 18.9 17.6 18.0 6.2 4.8
2 2 3 3 3 9 9
1.12 1.10 1.16 0.99 0.97 1 .05 1.45
42.8 33.4 11.8 13.3 8.8 3.1 5.0
2
6.28
13.3
3 3
5.94 6.00
15.9 15.2
9
6.29
14.0
11 11
' A = monoexponential decay, B = double-exponentialdecay, C = Lorentzian distribution with the same half-width, D = Gaussian distribution with the same standard deviation, E = Lorentzian distribution with the same half-width plus a monoexponential decay, F = Lorentzian distribution with different half-widths, and G = Lorentzian distribution with different half-widths plus a monoexponential decay. b n = the number of floating parameters used in the fit.
only the results from linked global analyses are presented (Table 111) and discussed. In Table 111, models B, E, and G contain two components. The major component corresponds to the fluorescence decay of the inclusion complex, and the minor component was assumed to be from free 2,6-ANS. In all the fits the lifetime of the free 2,6-ANS in the absence of quencher was fixed at 350 ps. Its bimolecular quenching constants were fixed also at k , values determined previously by steady-state measurements. In this way the total number of floating parameters is minimized and a physically significant fit protocol thus employed. Because sample solutions contained a large excess of p-CD ( I 500-fold), its concentration does not change significantly after complexation with 2,6-ANS. The fraction of inclusion complex v) in the sample solution can be calculated from eq 1 as
f = KfC/(l
+ KfO
I
om4
0.0
I
8.0
4.0
12.0
16.0
[ C U S O ~(mM)
20.0
.
(5)
where C i s the analytical concentration of p-CD. This fraction can also be calculated from29 where A designates the preexponential factor of the fluorescence decay. The subscripts 1 and 2 denote the 0-CD-bound and free 2,6-ANS, respectively. f values calculated from eq 5 using the formation constant obtained from steady-state experiments (Figure 1) were fixed in the global analysis. In this way, we use physically meaningful information and solve simultaneously (globally) for a self-consistent set of kinetic parameters. Seven and nine frequency files were linked globally for Cu2+ and acrylamide quenching, respectively. A Lorentzian distribution with variable half-widths combined with a monoexponential decay model (model G, Table 111) is the best-fit model for both Cu2+ and acrylamide quenching. We arrive at this conclusion because of a lower x 2 value and the closeness of the recovery K,, values to those from steady-state experiments (Table 11). The best model for I- quenching, observed from global analysis of seven experiments, is a unimodal Lorentzian distribution with different half-widths (model F, Table 111). The fluorescence lifetimes of 2,6-ANS-@-CD in pure H 2 0 recovered from Cu2+and acrylamide quenching experiments are 5.95 and 6.02 ns, respectively. A 70-ps lifetime difference is surprisingly small, considering the substantially dissimilar nature of these two quenchers. In contrast, a 300-ps difference is observed for lifetimes in the presence of 0.5 M NaCI. Another important feature of the recovered fluorescence lifetime distribution is, in all cases, that it broadens as the quencher concentration increases (Figure 8). This is because 2,6-ANS is very sensitive to its local environment; i.e., its lifetime will be affected if another solute is able to interact with it either directly or indirectly. Although, the p-CD cavity can protect 2,6-ANS from the influence of other solutes to some extent, the conformation of the inclusion complex can still be affected. Thus, it (29) Nelson, G.;Patonay, G.;Warner, I . M. Anol. Chem. 1988,60, 274.
40 0.000.0
3.00
0.4
0.2
0.6
Ob
[acrylamide] (M)
-
zoo-1.S-
O.S--
0.00 '1 0.0
0.1
02
0.3
0.4
0.5
[Nail (MI
Figure 8. Half-width of Lorentzian distribution of 2,6-ANS-P-CD at different concentrations of Cu2+ (A), acrylamide (B), and I- (C).
is not surprising that both the center and the width of the lifetime distribution are dependent on the solute concentration. The interactions between solutes and inclusion complexes lead to more diverse environments for 2,6-ANS and a broader distribution results. Furthermore, different quenchers change the width of 2,6-ANS-P-CD lifetime distribution to different degrees. Specifically, the order of influence on width, on a per mol quencher basis, is Cu2+> acrylamide > I-. Interestingly, this trend parallels the interactions between quenchers and the 2,6-ANS-&CD inclusion complex (Table 11). As a result, even though acrylamide modifies the surface tension of the bulk solvent and I- competes with 2,6-ANS for the available 8-CD cavities (other than their ability to quench fluorescence), they do not lead to further changes in the lifetime distribution width. The rationale for this is that 2,6-ANS (included inside the p-CD cavity) is insensitive to the
The Journal of Physical Chemistry, Vol. 94, No. 22, 1990
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Huang and Bright
TABLE IV: Recovered Fluorescence Lifetime of 2,6-ANS-&CD in the Presence of 0.5 M NaCI’ A B temp, O C 20 25 30 35 40 45 “A =
I,
ns
5.54 5.47 5.39 5.34 5.28 5.22
X2
31
15 14 4.4 1.6 4.4
r I . ns
rlr ns
ffl
X2
5.95 5.70 5.58 5.45 5.88 5.42
2.24 1.73 1.95 1.99 4.58 3.52
0.85 0.9 1 0.92 0.94 0.5 1 0.88
2.6 2. I 2.3 I .5 4.4 2.5
T
C
(hw),ns
5.38 5.39 5.32 5.28 5.24 5.20
(1.27) (1.01) (0.88) (0.66) (0.55) (0.54)
D X2
r (std), ns
X2
4.1 2.8 2.4 1.6
5.45 (1.40) 5.41 (1.17) 5.34 (1.02) 5.29 (0.78) 5.25 (0.66) 5.20 (0.66)
4.5 3.3 2.9 1.7
1.6 2.5
1.6 2.5
single-exponential decay, B = double-exponential decay, C = Lorentzian distributed decay, and D = Gaussian distributed decay
bulk solvent surface tension and I- is sequestered in a P-CD cavity different from one occupied by 2,6-ANS. It should be pointed out that global analysis of multiple experiments linked by the Stern-Volmer equation is only an approximate treatment at high quencher concentrations. Specifically, we noticed that at high quencher concentrations (especially above 0.5 M for acrylamide) the individual x2 values for each multifrequency data set are larger than they are at lower quencher concentrations. The residuals of the fit are more systematic, too. As seen in Figure 7 , there is some deviation between the theoretical calculation (solid line) and the experimental data at an acrylamide concentration of 0.8 M. We attribute this to transient effects which occur immediately following excitation of the fluorophore and result in complex rather than simple exponential fluorescence decay kinetic~.’~Although transient effects affect the fluorescence decay kinetics most significantly in a high-viscosity solution, they have also been detected by the frequency-domain method in solutions with low vi~cosities.~’Models such as the Smoluchowski decay law32and the radiation boundary conditions’’ have to be used to more accurately describe fluorescence decay in the presence of quencher. These more complicated theoretical models have not been applied in our present studies. However, the simultaneous analysis of lifetime experimental data at lower acrylamide concentrations (less than 0.5 M), using the combination of Lorentzian distribution and single-exponential model, recovered the same lifetimes and quenching constant. Therefore, we believe our conclusions regarding the fluorescence decay model for 2,6ANS-P-CD are still valid in spite of neglecting transient effects at higher quencher concentrations. Fluorescence Lifetimes at Different Temperatures. The lifetimes of 2,6-ANS-P-CD in pure H 2 0 and 0.5 M NaCl solutions were also studied over a temperature range from 20 to 45 “C. There were only subtle differences in phase and modulation values when temperature was changed. Results from individual lifetime analyses of 2,6-ANS-P-CD in 0.5 M NaCl are listed in Table IV. Comparing the fits from different models, the monoexponential decay was excluded due to its high x2 at low temperature. Gaussian distributions had similar or higher x 2 values than the corresponding Lorentzian distribution and always resulted in wider distribution width. Throughout the experimental temperature range, double-exponential fits appeared to have the lowest x2,but neither the lifetime of the minor component nor its fractional contribution followed any trend as temperature was adjusted. The fractional contribution and lifetime from this component changed randomly from 0.51 to 0.94 and 1.73 to 4.58 ns, respectively. Moreover, the fractional contributions recovered from doubleexponential fits differed appreciably from those calculated by using the equilibrium information (eq 5). With this in mind, we considered the Lorentzian distribution to be the best fitting model because it was continuous over the entire temperature range studied and resulted in a self-consistent set of parameters. It also used fewer floating parameters yet had a x2 values comparable to, and in some cases even less than, those of the two-component fits. Similarly, the Lorentzian distribution was the best model (30) Smoluchowski, V. M . Phys. 2. 1914, 17, 585. (31) Lakowicz, J. R.; Johnson, M . L.; Joshi, N.: Grycznski, I.; Laczko, G. Chem. Phys. Lett. 1986, 131, 343. (32) Nemzek, T. L.; Ware, W . R. J . Chem. Phys. 1975, 62, 477. (33) Joshi, N.; Johnson, M . L.; Gryczynski, I.: Lakowicz, J. R. Cbem. Phys. Lett. 1987. 135, 200.
*- -*- -*- -*- -*- -*
0-0-0-0-0-0 4.0
2.0
t
_ _10.0
20.0
30.0
40.0
50.0
60.0
50.0
60.0
TEMPERATURE (OC)
. 10.0
20.0
30.0
40.0
TEMPERATURE
(OC)
Figure 9. The center (A) and the half-width (B) of Lorentzian lifetime distributions of 2,6-ANS-@-CDat different temperatures in H20(0) and 0.5 M NaCl ( 0 ) .
for the same inclusion complex in water in the absence of NaCI. Under both circumstances, the center of the Lorentzian distribution was only decreased slightly (Figure 9A), but the width changed significantly at different temperatures (Figure 9B). Clearly, as solution temperatures increase, the half-width decreases and then levels off. At 20 OC the lifetime is shorter and the distribution is broader in the presence of 0.5 M NaCl than in its absence. This agrees with the results from the quenching experiments (Table 11, Figure 8). The temperature dependence of the distribution width is similar to lifetime distributions recovered for tryptophan emission in a pr~tein.’~.’~ It may be rationalized by the effect of temperature on the “interconversion” kinetics. The rate of association and dissociation is increased at elevated temperatures. That is, the interconversion between different conformations of 1:1 inclusion complex is accelerated. In contrast, the average (central) lifetime of the inclusion complex is hardly affected by the temperature. Therefore, at higher temperatures, it is more likely for a fluorophore to interconvert among different conformations while it is still in the excited state. The lifetime measured is thus the ensemble average of the lifetimes in different environments. Consequently, less ground-state heterogeneity will be dete~ted.’~*’~ On a per mole basis, the effect of CI- on the lifetime distribution width is less than I-. Chloride does not interact strongly with the (34) Alcala, J. R.; Gratton, E.; Prendergast, F. G. Eiophys. J. 1987, 51, 925. (35) Alcala, J. R.;Gratton, E.; Prendergast, F. G. Eiophys. J . 1987, 5 1 , 597.
2,6-ANS-P-CD Inclusion Complex fluorophore 2,6-ANS.’.9 Therefore, a high degree of correlation between quencher and temperature-dependent experiments (Table 11, Figure 9) is found. However, there is a 0.3-11s difference in recovered lifetime values between these two systems. Because the temperature data are fit individually (there is not an appropriate set of global linking parameters), we expect the lifetimes recovered from our quenching experiments to be more accurate. Also, both the lifetime and its fraction contribution have physical meaning (via Kf)rather than being only empirical parameters. For the sake of comparison with the 2,6-ANS-p-CD results, the lifetime of 2,6-ANS in ethanol was studied at different temperatures. In all cases, a monoexponential decay was always the best fitting model and the lifetime was unaffected by temperature. Both the quenching and temperature studies on the lifetime of 2,6-ANS in EtOH unambiguously demonstrate that its fluorescence decays monoexponentially. Thus, the lifetime distribution observed for 2,6-ANS-p-CD is generated by the heterogeneity of the 0-CD cavity probed by 2,6-ANS rather than the inherent fluorescent property of the probe. The fluorescence lifetime of the 2,6-ANS-j3-CD decay modeled by a unimodal Lorentzian distribution is supported by all our experimental results. (1) The Lorentzian distribution model has the smallest x2 for all three quenchers studied. (2) The SternVolmer constants recovered from the lifetime experiments are very close to those determined from steady-state experiments. (3) Identical lifetime values of 2,6-ANS-j3-CD in H 2 0 are obtained from different series of quenching experiments by using this model. (4) This simpler model yields x2 values comparable to those of double-exponential decay from our temperature-dependent experiments. ( 5 ) The lifetimes recovered are essentially the same as those from quenching experiments. It is unlikely that all this agreement is just coincidence. Nonunique lifetime values for a probe sequestered within a CD cavity have already been p r o p o ~ e d ; ’ however, ~ * ~ ~ the specific lifetime distribution has not been retrieved unambiguously due to the limitation of the precision of the experiments. The application of simultaneous analysis of multiple data sets (global analysis) offers us the advantage of improving the precision and accuracy of the recovered kinetic parameters. Moreover, by matching the parameters recovered from frequency-domain fluorescence measurements with those from steady-state intensity studies, the most adequate fitting model is not merely empirical-it has physical significance. Using simple CPK molecular models and other researchers’ studies on the inclusion complexes,l-” we are able to postulate the following scheme to account for the distributed lifetime of 2,6-ANS-j3-CD complexation. In the first step the apolar benzene ring enters the @-CDcavity3’ from the secondary hydroxyl end, Le., the bigger end.’** As the benzene ring extends further into (36) James, D. R.; Liu, Y.; Siemiarczuk, A.; Wagner, B. D.; Ware, W. R. Proc. SPIE 1988, 909, 90. (37) Bergeron, R.; Rowan, R. 111. Bioorg. Chem. 1976,5, 425.
The Journal of Physical Chemistry, Vol. 94, No. 22, 1990 8463
the 8-CD cavity, the naphthalene ring starts to be included. This process continues until ultimately the whole naphthalene ring is included. This is the most stable form of the inclusion complex, and hence it is the most populated conformation. Further movement in the same direction is prohibited by the sulfonate group. The sulfonate group will not enter the hydrophobic cavity of p-CD because it is negatively charged. The reverse process can also take place for the dissociation of the inclusion complex. When the interconversion process is slower than the fluorescence decay, a continuous distribution of lifetimes results. Otherwise less heterogeneity of the environment will be reported by the probe molecule; consequently, the distribution becomes narrower and could even appear to be a simple exponential
Conclusions The quenching by the dynamic quenchers Cu2+, acrylamide, and I- on 2,6-ANS is inhibited in the presence of p-CD, and there are no ternary complexes formed between 0-CD, 2,6-ANS, and these quenchers. In addition, Cu2+ is the strongest quencher, acrylamide is intermediate, and I- is the weakest. Some of these quenchers also shift or modify the equilibrium of @-CDincluding 2,6-ANS by actively competing for 0-CD cavities or altering bulk solvent properties (e.g., surface tension). By investigating the steady-state and dynamic fluorescence with various quenchers, we have found that the fluorescence decay of 2,6-ANS-&CD is described best by a unimodal Lorentzian lifetime distribution. This distribution is not due to the intrinsic characteristics of the fluorophore 2,6-ANS. (The same studies with 2,6-ANS in EtOH show clearly that it decays monoexponentially.) The distribution of lifetimes suggests the coexistence of 2,6-ANS molecules included inside the @-CDcavity to different extents. That is, at any time, there is an ensemble of 2,6-ANS molecules in different stages of complexation within the 8-CD cavity. These 2,6-ANS molecules are excited simultaneously and stay in the excited state for different lengths of time.’* Consequently, the recovered lifetime information is distributed rather than a simple exponential. Our experimental results show also that the lifetime distribution width is related to both solute (quencher) concentration and sample temperatures. Acknowledgment. This work was supported in part by BRSG SO7 07066 awarded by the Biomedical Research Support Grant Program, Division of Resources, National Institutes of Health, a Non-Tenured Faculty Grant from 3M, Inc., the Health Care Instruments and Devices Institute at SUNY-Buffalo, the National Institute of Mental Health, and the National Science Foundation. Registry No. 2,6-ANS-@-CD,125 172-39-6; Cuz+, 15 158- 1 1-9; I-, 20461-54-5; acrylamide, 79-06- 1.
(38) Bergeron, R.; Channing, M . A. Bioorg. Chem. 1976, 5, 437. (39) Bergeron, R.; Channing, M. A.; Gibeily, G.J.; Phillor, D. M. J . Am. Chem. SOC.1977, 99, 5146. (40) Harata, K. Bull. Chem. Soc. Jpn. 1976, 49, 2066.
