RAYMOND F. CHEN AND JAY R. KNUTSON. Laboratory qf ..... time-resolved fluorescence, F(t), is given by ..... by X' according to Parker and Rees (54) be-.
ANALYTICAL
BIOCHEMISTRY
172,61-77
(1988)
Mechanism of Fluorescence Concentration Quenching of Carboxyfluorescein in Liposomes: Energy Transfer to Nonfluorescent Dimers RAYMOND
F.
CHEN
AND
JAY
R.
KNUTSON
Laboratory qf Technical Development. Nationul Heart, Lung. and Blood Institute, Bethesda. Maryland 20892 Received
February
16. I988
When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98s of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system ‘is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years, Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. Forster critical transfer distances were calculated to be 5 I and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CFemission. No direct evidence for collisional self-quenching of 6CF could be found. although a model compound. salicylate, did quench weakly. For xanthene dyes. the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition ofdimerization in liposomes, (iii) partial protection of dye from quenching by KI. (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid. and (v) enhancement of fluorescence lifetime in the presence of Triton X- 100. C, 1988 Academic PXSS. IK KEY WORDS: fluorescence: fluorescence quenching; carboxyfluorescein: liposomes: energy transfer.
Liposomes containing concentrated 6carboxyfluorescein (6CF)’ were introduced by Weinstein et al. (1) to follow endocytosis. When encapsulated at high concentration (0.2 M), the dye was virtually nonfluorescent, but when liposomes were taken up by cells and lysed, the dye was released and diluted,
with regain of fluorescence. Concentrationquenched dye-liposome systems have wide applicability in following liposome fusion with cells or other membranous organelles, as well as liposome integrity itself. For a review, see Weinstein et al. (2). The nature of the concentration quenching in liposomes is not well understood. For instance, 6CF in liposomes is up to 97-98% quenched; but why is it not 100% quenched? Concentration quenching occurs even in simple dye solutions and has been known for about 100 years (3) but the mechanisms of
’ Abbreviations used: 6CF. a mixture of 5- and 6carboxyfluorescein, unless stated to be the pure 6isomer: 5CF. 5-carboxyfluorescein; FWHM, full width at half maximurn; DPPC, dipalmitoylphosphatidylcholine. 61
0003-2697/88
$3.00
Copyright (0 1988 by Academic Press. Inc. All rights of reproducf~on 8” any form reserved.
62
CHEN
AND
the quenching are not widely understood. It is instructive to consider briefly some of the highlights in the study of this phenomenon over the last century. In 1888 Walter (3,4) noted that fluorescence seemed to increase with dilution for solutions of fluorescein, Magdala red, and eosin. Using early spectroscopic equipment, it was shown that the ratio F/A, the fluorescence divided by the absorption, continuously increased with dilution even at the highest dilutions at which observations were practical. It was even demonstrated that the optical absorption characteristics of dilute and concentrated solutions of these dyes differed (5). Walter (5) advanced the idea that aggregation was the cause of quenching, and that fluorescence arose only from “single molecules” (monomers). This concept was supported by finding that concentrated dye solutions showed a paradoxical increase in fluorescence on heating, presumably because aggregation was inversely proportional to temperature. The observations by Walter seemed to provide an obvious reason for the changes in optical properties of concentrated dye solutions. A number of investigators in the early part of this century showed that xanthene dyes such as rhodamine B and eosin aggregated (see Ref. (6)). Dimerization in dyes other than xanthenes is well known, for instance, in cyanines (7) thionines (8,9), and acridines (10-12). Levshin (13) noted the decrease in fluorescence of dyes in both viscous and nonviscous solutions (where collisional events were unlikely) and attributed this to dimer formation. Rabinowitch and Epstein (9) studied the polymerization of thionine and methylene blue, and again explained fluorescence quenching as due to the formation of nonfluorescent dimers. The dimerization theory has never been completely satisfactory, because it does not explain the fact that concentration quenching is accompanied by a decrease in the fluorescence lifetime, as was found by Gaviola (14) in 1927 using one of the first instruments for the direct measurement of life-
KNUTSON
time. Szymanowski (15) made direct lifetime measurements and reported an apparent proportionality between quantum yield and lifetime for fluorescein and rhodamine B solutions of different concentrations. Nonfluorescent dimer formation alone would not change the lifetime, so a dynamic quenching mechanism was evidently present. Quenching by collisional interaction between dye molecules was postulated as early as 1924 by Pen-in ( 16). Simple calculations showed that at high concentrations, there were a significant number of dye collisions during the excited state lifetime. To complicate the situation further, when concentrational depolarization of fluorescence was discovered in 1924 ( 17-20) yet another phenomenon, energy transfer, was added to those factors which could be causing concentration quenching. It was apparent that depolarization involved molecular interactions over distances considerably larger than those kinetically feasible for collisional interactions. Vavilov (21) developed a theory of concentration quenching involving both dynamic and static factors, but the nature of his “quenching transfers” was unclear. Energy was somehow lost in the process of resonance energy migration. Fiirster (22) developed the theory of resonance energy transfer and developed a method for quantitating intermolecular distances based on spectral data. He and Konig (23) studied concentration quenching of dyes and postulated that it was a combination of dimerization and energy transfer to dimers which explained the phenomenon. They obtained the dimer spectra and dimerization constants for fluorescein, rhodamine B, and eosin by measuring absorption spectra in cuvettes having path lengths of several micrometers. Rohatgi and Singhal (24) and Arbeloa (25) have also affirmed the likelihood of quenching by energy transfer to nonfluorescent dimers. Levshin and Bocharov (26). examining different dyes, concluded that the degree of quenching due to dimer formation or due to “transfer extinction” varied with different dyes. Lev-
CARBOXYFLUORESCEIN
shin and Baranova (27,28) found that more than one mechanism was involved in concentration of several dyes and showed how separate factors could be probed by changing parameters such as temperature and viscosity. Collisional quenching by dye-dye interaction is one of the mechanisms proposed in concentration quenching. Aside from the evidence of shortened fluorescence decay times mentioned above, support for collisional quenching rests on data suggesting that quenching occurs even when dye concentration is below that required for dimer formation. Thus, Rohatgi and Singhal (29), Bud6 and Ketskemety (30), and Arbeloa (3 1) obtained data which seemed to show that fluorescence decreases with concentration even at low concentrations. Data presented as Stern-Volmer plots (24,25) for fluorescein yielded a collisional quenching rate nearly equal to the bimolecular collision rate. Formation of the nonfluorescent excimer during collision was postulated (24). However, such data are obtained from difficult measurements of fluorescence yields of concentrated dye solutions. ‘The experimental problems in working with such optically dense solutions have been pointed out by various authors (29-3 1). Melhuish (32) indicated that if front surface geometry is used, one must apply corrections for self-absorption of excitation and emission, reemission of reabsorbed emission, changes in refractive index with concentration, etc. These corrections may be huge. His recalculation of data published by Bud6 et al. (33) showed little if any quenching in dilute fluorescein solutions. For completeness, it should also be mentioned that self-absorption of fluorescence in solutions of significant path length can masquerade as self-quenching (34). Such “trivial” quenching of emitted radiation occurs only in the spectral region where the absorption and emission spectra overlap and would not account for loss of fluorescence in short path length systems such as liposomes.
QUENCHING
63
MECHANISM
In summary, the principal theories of the mechanism of concentration quenching are dimerization of the dye, energy transfer to nonfluorescent dimers, and collisional quenching interactions between dye monomers. A very recent paper by Plant (35) addresses the question of concentration quenching of liposome-encapsulated sulforhodamine IO 1, a xanthene dye. The main cause of the quenching was thought to be collisional, although some evidence for static quenching was found. We have investigated the mechanism of concentration quenching of 6CF, which is the most commonly used dye for liposome encapsulation studies, using timeresolved and steady-state fluorescence measurements. 6CF is also a xanthene dye, whose fluorescence properties should be similar to those of sulforhodamine 101 (35). However, our results do not support the presence of significant collisional quenching. The quantum yield and lifetime data do not fit the Stern-Volmer relation. Energy transfer between monomers and finally to nonfluorescent dimers is sufficient to explain concentration quenching. In addition, the inability to quench fluorescence completely in liposomes is attributed to dye interaction with the lipid, thus shielding it from quenching. A preliminary report of this work has been given (36). MATERIALS
AND
METHODS
Sulforhodamine B, sulforhodamine 10 1, and 5(6)-carboxyfluorescein were purchased from Eastman Chemicals; the latter was recrystallized from ethanol-water mixtures ( 1). 5(6)-Carboxyfluorescein was also obtained from Sigma Chemical Co. and Molecular Probes and gave the same results as the Eastman material. The separate isomers, 5carboxyfluorescein and 6-carboxyfluorescein, were obtained from Calbiochem and examined for spectral differences in solution. Although the dry materials differed in color, the solutions were indistinguishable. Fluo-
64
CHEN
AND
rescence lifetime measurements showed no difference (see Table 2). Most of the data were therefore obtained with the mixed isomer preparations obtained from Eastman. The liposomes were made from dipalmitoy1 DL-a-phosphatidylcholine (DPPC) or Type V egg yolk lecithin obtained from Sigma Chemical Co. and used without further purification. Typically 10 mg of DPPC was added to 2 ml of 6CF solution and sonicated for 5 min with a Heat Systems-Ultrasonics, Inc. Model W-225 sonicator. The sonicate was passed through a Sephadex G-25 PD-IO column developed with 0.2 M Tris-Cl buffer, pH 8.5, in order to remove unencapsulated dye. The initial colored band, containing the liposomes, was passed through another column in the same way. Lifetime measurements on liposomes were performed within 5 min of sample collection and usually required photon counting times of 2 min. Absorption spectra were recorded on a Cary spectrophotometer in 1-cm cells except for some samples examined with a lo-pm path length cell which was kindly lent to us by Dr. W. Hagins. Steady-state fluorescence was recorded on an Aminco-Bowman spectrofluorometer and corrected for detector response nonlinearities (37). Steady-state polarization, P, is defined by
where Z,, and Z, are the fluorescence intensities observed at right angles to the excitation beam, with the emission analyzer oriented parallel and perpendicular to the direction of polarization of the excitation. Fluorescence decay data were obtained by the time-correlated single photon counting method using pulsed excitation. The light source was a Spectra Physics tunable dye laser system utilizing the doubled output of a mode-locked Nd:YAG laser for synchronous pumping. The dye used was rhodamine 6G, which yielded cavity-dumped pulses used to
KNUTSON
excite sulforhodamine 101 or were KDPdoubled to excite 6CF at 3 10 nm. The light pulses had a FWHM of under 20 ps. The system is similar to those used by others (e.g., (38,39)), but also has a microprocessor-controlled sample changer and emission monochromator to facilitate running of controls, alternate measurement of lamp and sample, and collection of wavelength-resolved data for decay-associated spectra (40). Excitation for decay times was with light polarized at 54.7” to avoid polarization artifacts (41). The ratio of TAC stops/starts was l/30 in order to avoid pileup error. Data were accumulated in a Tracer Northern multichannel analyzer, and decay curves were analyzed by nonlinear least-squares fitting to multiexponential models (42) using a dedicated Hewlett-Packard A900 minicomputer. The time-resolved fluorescence, F(t), is given by F(t) = K C a;exp(-t/T,) where K is a constant, and CY,and T, are the preexponentials and lifetimes. Goodness of fit was evaluated with the standard test (42). Fluorescence anisotropy decays were analyzed as described by Ross et al. (43). The anisotropy r is defined by r-
4-z1 3 I,, + 21,
where Z,, and Z1 have the meanings assigned above. In both steady-state polarization and time-resolved anisotropy measurements, a correction was added for instrumental polarization artifacts (4 1). All measurements were made at 23°C. RESULTS
Evidence for dye dimerization. The absorption spectra of liposomes containing 6CF and sulforhodamine B in different concentrations are shown in Fig. 1. It can be seen that as the concentration is raised, a shoulder appears at about 468 nm. The absorption peak is at 492 nm, the same as that for fluorescein itself. With increasing concentration,
CARBOXYFLUORESCEIN
QUENCHING
65
MECHANISM
isosbestic points are noted at 474 and 5 15 pounds differ by one carboxyl group, the nm. The appearance of isosbestic points, and spectra of the monomers are similar, and it might be expected that the dimer spectra also the tendency for the single peak of monomeric 6CF to split at high concentration, is correspond. The fact that the extrapolated spectrum and the dimer spectrum do not suggestive of dimer formation as described for fluorescein itself (23). An attempt was match may be explained by inhibition of dimade to obtain the dimer spectrum of 6CF merization by interaction of the dye with by extrapolating the data of the three curves phospholipid. In fact, we have noted that the of encapsulated 6CF to infinite dye concen- amount of dimerization, as indicated by the absorption spectra, depends on the type of tration. The data from the curves for liposoused to form the liposomes ma1 6CF at 5-nm intervals were plotted as phospholipid (unpublished observations). 1/absorbance vs 1/[6CF] and extrapolated to The effect on the spectra of concentration infinite dye concentration. The resulting is difficult to observe in free 6CF solution curve in Fig. 1 (open squares) does not show because of its relatively low tendency to dithe amount of hypochromism and splitting merize, coupled with the high dye extinction which would be expected on the basis of the coefficient. However, the effect can be seen dimer spectrum of fluorescein itself, as determined by Forster and Konig (23). For with the dye sulforhodamine B, which seems comparison, their fluorescein dimer spec- to dimerize more readily. We have found trum, reduced in intensity by multiplying by that sulforhodamine B, like 6CF, can be inthe ratios of the peak absorptions of 6CF and corporated into sonicated liposomes and exfluorescein (i.e., 74,900/89,000), is also hibits lysis-relieved concentration quenchshown in Fig. 1. Although the two coming. Figure 2A shows how the absorption spectrum of encapsulated 0.05 M dye exhibits the splitting characteristic of dimeriza*I tion. The degree of dimerization of 0.05 M sulforhodamine B appears to be higher when dye is free in solution than when encapsulated in liposomes, as shown by the absorption spectrum measured in a IO-pm path length cell (Fig. 2B). This difference is consistent with inhibition of dimerization due to a competitive interaction of dye with phospholipid. (In the case of 6CF, concentrations higher than those used in Fig. 2 are needed to 0 L------J I produce significant dimerization and have 430 450 470 490 510 530 WAVELENGTH, nm too high an absorbance to be measured even FIG. 1. Absorption spectra of 6CF in liposomes. 6CF with a IO-pm cell.) was encapsulated in egg phosphatidylcholine hposomes Dimerization constunt for 6CF. Dye diat concentrations of 0.067 M (- - -). 0.133 M ( * * * ), and merization can be represented by 0.2 M (---). The spectra were recorded and the liposomes lysed with 0.03% Triton X-100. The spectra of the encapsulated dye solutions were normalized by comparing the heights ofthe spectra after lysis. (--) The spectrum of free 6CF. (0) The apparent dimer spectrum calculated by extrapolation to infinite concentration of dye (see text). (--. -) The fluorescein dimer spectrum of Forster and KSnig (23) normalized as described in the text. The extinction shown for the dimer is based on monomer content.
M+M=D,
PI
Kd=[M12’
where h4 and D are monomer and dimer, and Kd is the dimerization constant. Although the spectral features of fluorescein and 6CF are very similar, the dimerization constants, &, are not likely to be the same
66
CHEN
AND
1 .O
0.5
450
500
550 WAVELENGTH
500 WAVELENGTH
600
650
(nm)
KNUTSON
for 6CF, using the absorption at 492 nm of free 6CF, 0.2 M 6CF in liposomes, and dimer (Fig. 1). This value is likely to be lower than the true dimerization constant, because the interaction of dye with phospholipid decreases the amount of dimerization. Kd is therefore estimated to be between 3.3 and 24 M-‘. Titration oj‘6CF. This dye is known to change spectral characteristics upon ionization, so it was necessary to define the pK,. Figure 3A shows the titration curve based on absorption spectra which yielded, from the computer fit shown, a pK, of 6.45. This agrees with the value obtained by Babcock and Kramp (47) from changes in absorbance. Their values of the ionization constant obtained by fluorescence titration were 0.2-0.4 pK unit lower, suggesting facilitated proton transfer in the excited state. To exam-
600 (nm)
FIG. 2. Spectra of sulforhodamine B. (A) Spectra of sulforhodamine B encapsulated in egg phosphatidylcholine liposomes at 0.05 M before and after (curves 2 and 1) lysis with Triton X-100. (B) Absorption spectrum of a 0.05 M solution in a IO-pm path cell in 0.1 M Tri-Cl buffer, pH 8. 4
5
6
7
8
9
PH 50
since charge repulsion is enhanced by the presence of an additional carboxyl group in 6CF. Forster and Kiinig (23) report Kd = 24 Mm’ for fluorescein, while others report 5 to 7.7 (44,45). In order to calculate the dimerization constant for 6CF, we assume that its dimer spectrum is that shown in Fig. 1. (The fluorescein dimer spectrum was directly observed by Fijrster and Kijnig (23) using a micrometer-controlled cell. Dimer spectra obtained by extrapolative methods (44-46) differ somewhat and are probably less reliable.) With this assumption an apparent dimerization constant of 3.3 M-’ is calculated
4.6
FIG. 3. Titration cence. Excitation and 520 nm.
5.6
6.6 PH
7.6
6.6
of 6CF. (A) Absorption. (B) Fluoresand emission wavelengths were 360
CARBOXYFLUORESCEIN
QUENCHING
ine this point, we performed fluorometric titration of 6CF, using excitation at 462 nm, where the absorptions of di- and trianionic forms are equal, as shown in Fig. 3B. It was not possible to fit the data in the same manner as that shown in Fig. 3A, as the curve showed no plateau representing the fluorescence of the pure dianion. Were the fluorometric titration performed with excitation at some other wavelength, the data would reflect the change in absorption in addition to the change in fluorescence. While fluorescence titration does not provide a good estimate of the ionization constant of the hydroxyl group of 6CF, the titration curve of Fig. 3B does not indicate a large change in the trianion-dianion in the excited state. The p& inclicates that when experiments using 6CF are done at “physiological” pH values near 7, 6CF is present in two fluorescent forms. Interpretation of data such as fluorescence decay times may be made more complicated as the lifetime of the more acidic form i:s shorter (see Table 2). The present study was carried out at pH 8.5 to isolate the fully ionized form. Fluorescence quenching in fiposomes: A “Stern- Volmer plot “? It has been observed that the amount of quenching of 6CF varies with the dye concentration (1) and this is illustrated in the data shown in Fig. 4. The data were derived from liposomes containing 6CF at various concentrations. Fluorescence was measured before and after lysis of the liposomes by a nonionic detergent. The data are presented in the form of a Stern-Volmer plot, as has been done in other studies (24.45) and a best-fit straight line is drawn through the points. The Stern-Volmer equation is (48)
F,,IF = 1 + &dQl,
ill
where FO and Fare the fluorescence intensities in the absence and presence of quencher, Ksv is the Stern-Volmer quenching constant, and Q is quencher. Further, &iv = rkq,
PI
67
MECHANISM
0
0.03
0.06
[6CFl,
0.09
0.12
M
FIG. 4. “Stern-Volmer plot” for 6CF. Different concentrations of dye, as shown on the abscissa, were encapsulated in liposomes and the fluorescence was measured before and after lysis with T&on X- 100 to yield F and FO. The curve is the best least-squares fit straight line.
where T is the fluorescence lifetime in the absence of quencher, and kq is the quenching rate constant. The straight line of Fig. 4 results in KSv = 412 M-l, and k, = 9.3 X lOlo, assuming T = 4.4 ns (Table 2). The value of kq is impossibly high, being considerably greater than the bimolecular collision rate in aqueous solution under these conditions (6.5 X 109. Ref. (49)). Plant (35) obtained a similarly high rate when presenting data on sulforhodamine 10 1 in Stern-Volmer fashion. The reason for the inordinately high kg is that the data of Fig. 4 do not follow the Stern-Volmer relation, as shown by the poor fit of the straight line to the experimental points. Our impression from repeated runs is that such data do not describe a straight line. For instance, at the lowest concentrations, no significant quenching is observed. The data of Fig. 4 could perhaps best be described by a sigmoidal curve, but it is difficult to define such a curve since the points at high Fe/F where fluorescence is over 97% quenched are subject to large uncertainties. In any case, the data do not support simple collisional quenching. Evidence that dimerization results in quenching. It is well established that aggrega-
68
CHEN
IO0
200 CHANNEL
100
300 NUMBER
400
AND
KNUTSON
500
FIG. 5. Fluorescence decay curves for solutions of 6CF. The concentrations of dye were 0.01, 0.02, 0.03. and 0.043 M for curves 1-4, respectively, in 0.1 M TrisCl buffer, pH 8.5. The lifetimes are listed in Table 1. Time base. 46 ps/channel.
tion of dyes such as fluorescein results in quenching (5,13). We have confirmed this observation for 6CF by noting the following: (i) At high concentrations where dimer formation occurs, no new emission band which might arise from the dimer occurs. (ii) The excitation spectrum of any emission which remains is the same as that of the monomer. Decrease in fluorescence lifetime with concentration in 6CF. We noted that increasing the concentration of 6CF results in a shortening of the fluorescence lifetime (Fig. 5). This finding would be surprising if dimerization were the only mechanism of quenching, because the fluorescence lifetime is not decreased in static quenching. The mean lifetimes found with different 6CF concentrations are plotted in Fig. 6. The data describe a curve whose shape would be totally inconsistent with a straight-line Stern-Volmer plot such as that shown in Fig. 4. The lifetime is constant until the concentration reaches about 0.01 M, and then falls precipitously. Propylene glycol had a marked effect on the lifetimes of 6CF in concentrated solutions. This solvent would be expected to diminish the tendency of 6CF to dimerize. Table 1 shows that when propylene glycol is added to 0.04 M 6CF, the lifetime increases. Conversely, when propylene glycol is kept constant at 40% (v/v), the lifetimes are longer for different concentrations of 6CF than they
0
1
2
3 -LOG
4 [6CF]
5
6
J 7
FIG. 6. Dependence of lifetime on 6CF concentration. The lifetimes are best single exponential fits to data such as those in Fig. 5.
are in buffer alone. These results suggest that dimerization is crucial for the shortening of lifetime with increasing concentration (and hence for concentration quenching). It is argued below that dimers act as energy transfer sinks, resulting in quenching. A summary of other 6CF decay times is given in Table 2. Note that the fluorescence decay of 6CF is well described by a single exponential, and that the lifetimes of the pure 5- and 6-carboxyfluorescein isomers are indistinguishable. Also, at pH 5, 6CF exhibits a faster fluorescence decay, which is also at least biexponential as would be exTABLE
I
EFFECTOFPROPYLENEGLYCOLONLIFETIMES OFCONCENTRATED~CF Percentage propylene 0 20 40 60 40 40 40
(v/v) glycol
16CFl
7
(M)
(ns)”
0.04 0.04 0.04 0.04 0.01 0.02 0.06
0.43 0.92 1.71 2.13 5.23 3.85 0.80
No&. The solutions also contained 0.04 M Tris-Cl buffer, pH 8.5. ’ Fit to monoexponential model, as in Fig. 6.
CARBOXYFLUORESCEIN
QUENCHING
petted for the coexistence of more than one ionic form. In the presence of Triton X- 100, which is used here to lyse liposomes, 6CF had a lifetime of 4.85 ns, an increase of lo%, indicative of interaction with this detergent. Fluorescence decay kinetics of 6CF in liposomes. Nonexponential decay was observed from 6CF encapsulated in liposomes at concentrations of 0.01 M or higher. The data were fit only by curves having at least two exponentials. Typical results are shown in Fig. 7A and are summarized in Table 2. In order to reduce the possibility that a significant amount of free dye was present, care was taken to separate liposomes from free dye by double passage through Sephadex, and the lifetime measurements were made immediately. The data indicate that the fluo-
rescence of concentrated encapsulated 6CF, which may be up to 98% quenched, has a major lifetime component of 3 to 4 ns, plus shorter components. It would be tempting to attribute the 3- to 4-ns component to free 6CF in the liposome preparation, but if this were the case, the addition of 0.5 M KI should eliminate that component. We have found that KI is a diffusion-limited quencher of 6CF fluorescence with a Stern-Volmer constant of about 30 M-‘. At 0.5 M KI, the quantum yield and lifetime of 6CF would be expected to be reduced to l/16 of their original values. Figure 7B shows that KI does not eliminate the long component. KI reduces the long lifetime component by only lo-30% (Table 2). The simplest explanation of the long component
TABLE FLUORESCENCE Sample Sulforhodamine Sulforhodamine 6CF” 6CF” 6CF” 6CF” 5CFh 6CFh 6CFh 6CFh
69
MECHANISM
2 DECAY DATA
Conditions lo-’
M. free
1.545
1.34
B 0.05 M, liposomal
0.213
0.899
0.726
1.549
I .23
0.858
4.58
0.186
4.40
6.74 0.139
0.084 0.800
0.355 0.018
4.19 3.07
0.039 0.29 I 0.266 0.067 0.218 0.822 0.363 0.483 0.140 0.248 0.197
0.880 0.774 0.796 1.04 0.728 1.07 0.902 1.61 1.44 2.21 1.01
0.023 0.088 0.045 0.025 0.040 0.185 0.096 0.579 0.140 0.724 0.180
3.89 3.65 3.01 3.95 3.47 3.32 2.73 3.50 3.22 4.05 3.11
B 1om6 M, free 10e6 M, free 10m6 M, free, pH 10mh M, free, pH 10e6 M, free, pH 1om6 M, free O.;! M, liposomes O.;! M, liposomes O.;! M, liposomes 0.12 M, hpOSOmeS 0.10 M, liposomes 0.10 M, liposomes 0.08 M liposomes
0.04 M
0.214 5 5 8.5
0.878
+ 0.5 M KI + Triton
+ 0.5
M
-12.15 0.385 0.293 0.39 I 1.05 1.61 1.48 1.43 1.23 1.03 0.333 0.718 0.352
KI
1ipOSOmeS
0.02 M liposomes 0.02 M liposomes + 0.5 M KI 0.0 1 M liposomes 0.01 M liposomes + 0.5 M KI 0.(105 M hpOSOmeS 0.005 M Iiposomes + 0.5 M KI Note. The liposomes a Mixed 5(6)-isomers. b Pure isomer.
were made with
DPPC.
Unless
otherwise
4.42 3.58 3.50 3.26 4.4 1 4.42 0.005 0.187 4.85 0.096 0.125 0.120 0.110 0.179 0.162 0.128 0.146 0.376 0.149 0.184
stated, the pH was 8.5.
1.36 1.30 1.52 1.26 1.23 1.34 1.34 1.56 1.51 1.26 1.94 1.23 2.66 2.19 1.74 1.33 1.67 1.49 1.23 1.17
70
CHEN
AND
indicate that Fiirster energy transfer is negligible at concentrations as high as 10-j M, but becomes highly significant above that. Figure 8B compares the time-dependent anisotropies of sulforhodamine 10 1 in glycerol at different concentrations. The same amount of anisotropy decay would be expected at the two concentrations shown because of rotational motion. The more concentrated solution has, in addition, a component of anisotropy decay due to energy transfer between dyes. The initially photoselected dipoles become increasingly randomized with time due to energy transfer. The same phenomenon has been observed on a much slower time scale for uranium glasses by Sevchenko (50).
B F
4.0
-
5
1
0 u
--.
^.
E -J
0
100
200
CHANNEL
300
400
KNUTSON
0.5
500
,-
NUMBER
FIG. 7. (A) Fluorescence decay curves for 6CF encapsulated in DPPC liposomes at these concentrations: 0.12 M (curve 2). 0.08 M (curve 3) and 0.04 M (curve 4). Curve 1, the decay after addition of 0.003% Triton to sample of curve 2. (B) Decay curves for 0.2 M 6CF encapsulated as in (A) before (curve 2) and after (curve 3) addition of 0.05 M KI or 0.003% Triton (curve I). Time base, 46 ps/channel.
is that it is due to dye molecules associated with the bilayer and only partly accessible to quenching by iodide. Energy transfer in 6CF detected by polarization. Because of the strong overlap of excitation and emission bands in xanthene dyes, energy transfer in concentrated solutions can be observed. Depolarization in 90% glycerol solutions of 6CF is shown in Fig. 8. Under these conditions, depolarization due to Brownian rotation is negligible. The energy transfer responsible for the observed depolarization can be both radiationless and due to trivial reabsorption of emission. Shown in Fig. 8A are data for the same solutions obtained by right angle and front surface geometries. The front surface method results in less trivial energy transfer, although complete elimination is unlikely. The data
p 0.4 aN
0.3
E 4
0.2
0 a
0.1
0
..’
n zz 20’
‘,
5-
1
2
4
6
TIME
6
10
12
(nsec)
FIG. 8. Effect of concentration on polarization of dye emission. (A) Steady-state polarization of 6CF in 97% (v/v) glycerol observed by right angle (A) or front surface (0) geometry. (B) Anisotropy decay of sulforhodamine 101 emission in 97% glycerol. Dye concentrations were 10e6 M (curve 1) and 10m4 M (curve 2). The curves have not been deconvolved for the lamp profile, which is shown. Left ordinate refers to the lamp; right-hand scale shows anisotropy values. After deconvolution, r0 = 0.3 18 and @J= 50 ns for curve I, and r,, = 0.323 and + = 13 ns for curve 2.
CARBOXYFLUORESCEIN
QUENCHING
71
MECHANISM
The rotational correlation time + can be calculated from the time dependence of anisotropy (4 1) from r(r) = rOexp(-t/+),
131
where r. is the limiting anisotropy in the absence of motion. The data of Fig. 8B yield apparent correlation times of 50 and 13 ns for the dilute: and concentrated solutions, even though the rotation rates should be the same in the two solutions. The faster anisotropy decay in the more concentrated solution reflects the effect of monomer-monomer energy transfer. (The experiment of Fig. 8B was performed with sulforhodamine 10 1 rather than with 6CF because the 310-nm excitation available for us to excite 6CF would have yielded only a small absolute value of the limiting anisotropy, whereas 570-nm light from the dye laser could be used to excite into the lowest energy band of sulforhodamine 10 1 to yield a high r. .) Natural and measured lifetime of 6c’F and quantum yield. In basic solution, we found the relative quantum yield of 6CF compared with that of fluorescein to be 0.8 1. If we accept that the absolute yield is 0.92 for fluorescein dianion (5 I), the quantum yield for 6CF trianion is 0.74. In the case of dynamic quenching, the quantum yield (4) is related to the lifetime by d, = T/TO, where 7 and 7. are the actual and natural lifetimes. It is instructive to calculate 7. in order to see whether there is any gross deviation from this relationship. Strickler and Berg (52) developed an equation which takes into account the different wavenumbers covered by the absorption and emission bands of organic molecules in solution: L = 2.88
X
10-9n2(iy3),;’ 2 s td In ;,
[4]
70
where n is the average refractive index over the emission band, (VY~)~” is the average value of ; -3 over this band, Z is the spectral position in wavenumbers, and G, and G2 are
Ol--7. WAVENUMBER
(CM-‘)
22
18
WAVEtkBER
X IO-‘:
(CM-‘)
FIG. 9. Spectra for energy transfer calculations. (A) Absorption and emission spectra of 6CF. (B) Absorption spectrum of sulforhodamine B (1) and emission spectrum of 6CF (2).
the degeneracies of the lower and upper states. Strickler and Berg (52) obtained 7. = 4.37 ns for fluorescein. Since the absorption and emission spectra are essentially identical for 6CF and fluorescein, except that the extinction coefficient is lower for 6CF (74,900 instead of 89,000 at the maximum), the natural lifetime for 6CF should be 89,000/74,900 X 4.37 = 5.24 ns. We have also carried out the calculations according to Eq. [3], using the absorption and emission spectra of 6CF shown in Fig. 9A, and arrived at 7. = 5.17 ns. Taking 7. = 5.2 ns and 4 = 0.74, one would expect a fluorescence lifetime 7 of 3.88 ns, while our measured value is 4.3 ns, a deviation of 10%. This difference is well within the range found by Strickler and Berg (52) for a variety of compounds. The good agreement between calculated and
72
CHEN AND KNUTSON
experimental lifetimes suggests that there is no ground state static quenching or gross configurational changes in the excited state of 6CF. Calculation of critical transfer distances for monomer-monomer and monomer-dimer energy transfer. The distance between donor and acceptor at which energy transfer is 50% efficient, RO, the critical transfer distance of Fijrster (22) can be represented in the following manner (53): Rb = 8.8 x lo-*5r& 0 n4
J
[51
and
where n is the refractive index, K* is an orientation factor taken to be 3 for rapidly rotating, randomly oriented donor and acceptor molecules, 4n is the fluorescence quantum yield for the donor in the absence of acceptor, FD and tA are the fluorescence and absorption spectra of the donor and acceptor, J is the overlap integral, and v is the spectral wavenumber. The directly determined value of tin for 6CF was 0.74. Relevant spectra for energy transfer from 6CF monomer are shown in Fig. 9. The emission spectrum of 6CF, originally recorded as intensity vs wavelength X with a grating instrument, was corrected for detector response and divided by X’ according to Parker and Rees (54) before replotting as relative quanta vs wavenumber. Figure 9A shows the absorption and emission spectra of 6CF. The peak extinction of 74,900 M-’ agrees with reported values for the anionic form (55). The overlap integral J was found to be 1.27 X lo-l3 cm3 M-l, and R. was 50.83 A for monomer-monomer energy transfer. The latter value is similar to the critical transfer distance calculated by Rohatgi and Singhal (24) for fluorescein homotransfer, namely, 57 A. The difference is due to the greater extinction coefficient and quantum yield of fluorescein.
Similar calculations were made for energy transfer from monomer to dimer, using the putative dimer spectrum shown in Fig. 1 and the 6CF emission spectrum of Fig. 9A. The integrated absorption band of the dimer, on a molar basis, is roughly twice that of the monomer, as expected because of the doubling of molecular weight, and the overlap is somewhat increased. The overlap integral J was found to be 2.69 X lOpI3 cm3 M-‘, and R. = 57.38 A. Overlap between 6CF emission and sulforhodamine B absorption is also high (Fig. 9B). J for this system was 2.94 X lo-l3 cm3 M-l, and R. = 58.26 A. As the critical transfer distance is about the same as that for 6CF transfer to its dimer, sulforhodamine B could serve as a model for quenching by 6CF dimer. In experiments in which DPPC liposomes were made containing 0.01 M 6CF and sulforhodamine B at concentrations from 0.0 1 to 0.05 M, about 90% quenching of 6CF emission which was released upon lysis with ‘Triton was noted. Therefore, if for some reason it is necessary to use concentrations of 6CF in liposomes where self-quenching does not occur, the addition of an energy acceptor dye such as sulforhodamine B can afford the desired quenching. Can dimerization alone explain concentration quenching? The amount of dimerization is plotted against dye concentration for various values of Kd in Fig. 10. The dimeri-
TOTAL
DYE CONCENTRATION,
M
FIG. 10. Degree of dimerization as a function of concentration, for different dimerization constants (K).
CARBOXYFLUORESCEIN
zation constants include those for rhodamine B (1500 M-l), eosin (400 M-l), and fluorescein (24 M-‘) reported by Fijrster and Konig (23). Note that if one assumes a Kd of 3.3 M-’ for 6CF in liposomes, at 0. I and 0.2 M only 30 and 45% of the molecules have dimerized, and yet quenching is 90-98% complete (1). Even if the Kd were as high as 24 M-t, as it is for fluorescein (23), only 70% of 6CF is quenched at 0.2 M. Thus, for 6CF, dimerization, although significant, cannot be the only quenching mechanism. On the other hand, for a dye such as rhodamine B with a much higher dimerization constant, dimerization is sufficient to explain quenching at these concentrations. Is there evidence for collisional selfquenching o~tSCF? Collisional deactivation is normally analyzed by measuring fluorescence as a function of quencher concentration. However, in the case of self-quenching of 6CF, the measurement is made difficult because of the high extinction coefficient. For instance, if 6CF were a highly efficient, diffusion-limited collisional quencher, 10% quenching would be observed only at a concentration somewhat higher than 0.002 M, where the peak absorbance at 492 nm is about 150 in a standard 1-cm cuvette. Artifacts would arise due to inner filter absorption of the excitation and self-absorption of emission. We have attempted to circumvent this problem by using a microcell with an effective path length of 1.5 mm, by exciting into the absorption minimum at 400 nm, and by observing emission at 560 nm where absorption is negligible. The only corrections applied were for self-absorption at 400 nm, using directly measured absorbance values and then multiplying the observed fluorescence signal by the antiloglo of 0.15 X OD’ cm (56). The results for 6CF concentrations up to 0.002 M are shown in Fig. 11A. No significant self-quenching is noted at these concentrations. It is conceivable that 6CF is such a weak self-quencher that a decreased yield would be experimentally observable only at higher concentrations. However, it is hard to
QUENCHING
73
MECHANISM
0
40
[GCF]
.‘M” x
104
FIG. 11. Constancy of 6CF quantum yield up to 0.002 (A) Corrected fluorescence readings vs concentration. (B) Correction factor, calculated as described in the text. Dotted line: the maximum correction of 3.5 used in part A, for 0.002 M 6CF. Inset: the correction factors required for solutions up to 0.0 I M. M.
justify attempting to measure the fluorescence yield of higher concentrations because of the large correction factors needed. Figure 11B shows the magnitude of the correction as a function of concentration. At 0.002 M, the highest concentration attempted in the experiment of Fig. 11A, the correction factor is already 3.5. Significantly, in assessing the corrected data presented in the literature to support collisional self-quenching of xanthene dyes, one is not always able to determine the size of the correction factors. Depending on the geometrical characteristics of the system used, Fig. 11 suggests that where measurements up to 0.1 M have been reported, the correction factors which were used may have been as high as 10’ or 103. Such results should therefore be cautiously evaluated.
74
CHEN AND KNUTSON
[SALICYLATE],
FIG. 12. Stern-Volmer orescence by salicylate.
M
plot for quenching of 6CF flu-
Salicylate was chosen as a model for 6CF as a quencher, because it does not absorb in the visible and yet, like 6CF, has aryl hydroxyl and carboxyl groups. Salicylate was found to quench 6CF fluorescence, as shown in the Stern-Volmer plot of Fig. 12.Ksv and kq were 4.64 M-’ and 1.05 X lo9 s-‘. This indicates that only one in six or seven collisions results in quenching. If 6CF were of similar efficiency, 10% quenching would be observed at 0.022 M, and 50% quenching would occur at 0.2 M, which is the concentration commonly used in liposomes. Collisional quenching in combination with dimerization would thus be insufficient to account for the 98% quenching of 6CF in liposomes. DISCUSSION
Concentration quenching of dye fluorescence is a complex phenomenon. Although it has been about 100 years since Walter (3-5) showed that xanthene dyes formed nonfluorescent aggregates, a static quenching mechanism cannot explain the shortening of fluorescence lifetime with concentration (14,15). Fiirster and Kijnig (23) suggested that quenching also resulted from energy transfer from monomers to nonfluorescent dimers. Others have postulated some sort of collisional interaction between dyes to explain
the lifetime results. Levshin and Baranova (57) point out that a combination of factors rather than one mechanism underlies most self-quenching. In the present studies on 6CF, the evidence shows that dimerization and energy transfer to dimer are sufficient to explain the concentration quenching. Energy transfer between monomers facilitates the eventual transfer to the nonfluorescent dimer. Energy transfer calculations showed a critical transfer distance of 51 A for monomer-monomer transfer and 57 A for monomer-dimer transfer. Direct evidence for the former was the steady-state and time-resolved decrease in polarization with concentration in 6CF and sulforhodamine 101. The larger R. for monomer-dimer transfer makes this even more feasible than the demonstrated monomer-monomer transfer. In addition, sulforhodamine B acts as a model for quenching by dimer, as shown by the quenching of 6CF emission when both are incorporated into liposomes. To appreciate the effectiveness of transfer to dimers as a quenching mechanism, consider the intermolecular distances in a 0.2 M solution of 6CF. Assuming a & of 3.3 M-’ as calculated above for the liposome system, some 40% of the dye will be dimerized. Therefore, the monomer concentration is 0.12 M and the dimer concentration is 0.04 M. The mean distance between molecular centers can be taken as twice the radius of the average spherical volume occupied by one molecule. The intermonomer distance is thus 30 A and that between dimers is 40 A. The efficiency E for transfer of an absorbed photon is given by E = l/[l
+ (Ro/R)6].
[71
The efficiencies are thus 96 and 90% for monomer-monomer and monomer-dimer transfer. In other words, 90% of the absorbed photons are immediately quenched by transfer to dimers, and 96% of the remainder are transferred to other monomers, where 90% are transferred to dimers, and so forth.
CARBOXYFLUORESCEIN
QUENCHING
Lavorel (58) has calculated the amount of energy migration for fluorescein as a model for photosynthetic pigments. He concluded that the excited state could migrate over 300 dye molecules in a nanosecond when the fluorescein concentration was 0.05 M. Because of the spectral similarities of 6CF and fluorescein, the effect of energy migration in 0.2 M 6CF would be essentially complete quenching by the dimers, acting as energy sinks. The above calculations could even have underestimated the amount of energy transfer, since they do not take into consideration the enha.ncement of transfer due to diffusion in fluid media (59-6 1). In order to compare the effects of concentration on 6CF parameters, the polarization and lifetime data have been plotted in Fig. 13 along with calculated curves for dimerization and energy transfer. Note the rapid decrease in polarization with concentration starting when only a small proportion of initially absorbed photons are transferred. The polarization decreases, as expected, before the lifetime is shortened, because homotransfer of energy does not obviously result in a shortening of lifetime or a decrease in quantum yield. In the range 0.01 to 0.05 M, both dimerization and energy transfer become sig-
Y J!?
-5
-4
-5
-2
-1
LOG [~cF]
FIG. 13. Variation of 6CF parameters with concentration. The curves are: I. efficiency of monomer-monomer transfer. for initially absorbed photons only, in percent; 2. percentage dimerization, based on Kd = 5; 3. mean fluorescence lifetime X 10, in ns: 4, polarization in 97% glycerol. X 100.
MECHANISM
75
nificant, and lifetime drops to near zero. If the lifetime can be assumed to be proportional to the quantum yield, fluorescence should be completely quenched at 0.2 M, the concentration normally used in liposomes (1). That there some fluorescence still remains is evidence for 6CF-lipid interaction. The time-resolved fluorescence studies on 6CF in liposomes consistently detected a relatively long lifetime component when the data were fitted to two or three exponentials. The long component is attributed to mostly unquenched dye. Although unencapsulated dye could produce this phenomenon, the rate of leakage of dye from liposomes in buffer has been studied (62), and at 23°C is negligible in the few minutes required between purification and measurement. Free dye was also ruled out by the small effect of 0.5 M KI, which would have reduced the lifetime to picoseconds. The exact nature of the 6CF-lipid interaction is unclear, but the unquenched 6CF molecules would also have to be protected from energy transfer to dimers. In a unilamellar liposome with an internal diameter of 200 A. encapsulation of 0.2 M 6CF results in only about 480 dye molecules surrounded by 2000 phosphatidylcholine molecules (63). Electrostatic interaction of 6CF monomers with phospholipid polar headgroups would result in perpendicular orientation of the dyes relative to the membrane plane. Dyes trapped within the membrane parallel to its plane then would be both protected from collisional quenching by KI and oriented unfavorably for energy transfer to other dye molecules. In the egg phospholipid liposomes containing 0.2 M 6CF used in the present study, the dye molecules can be broken down as follows: 40% are dimerized, about 58% are nearly completely quenched by energy transfer to dimers. and about 2% are membrane associated and largely unquenched. It has been suggested that liposomes constitute a convenient vehicle by which the optical properties of dyes at high concentration can be observed (35). The high optical ab-
76
CHEN
AND
sorption of dyes makes it impossible to measure spectra and quantum yields in cuvettes of conventional path length. If liposomes could be considered neutral spherules, one would be able to make these measurements in effective path lengths of no more than 200 A. Unfortunately, this approach is complicated by dye-lipid interaction such as that detailed in this work. Also, the small volume of the liposomes means that only a few dye molecules may be present, depending on the concentration. At 0.001 M, for instance, an average of 2.4 molecules of dye would be present in a sphere of 200 A diameter. The optical properties observed would be for an ensemble of liposomes and a distribution of concentrations centered at 2.4 molecules per liposome. Does collisional interaction cause concentration quenching of 6CF fluorescence? Collisional quenching has been postulated by some (e.g., (24,25)) for fluorescein to explain the apparent decrease in quantum yield at concentrations where dimerization is negligible. The mechanism of such quenching was thought by Rohatgi and Singhal (24) to be excimer formation, since it was known that the dimer was nonfluorescent. In the present work no quenching was observed for 6CF at concentrations as high as 0.002 M; and salicylate, as a model quencher, had a low Stern-Volmer constant for quenching of 6CF. Formation of the fluorescein or 6CF dimer during the excited state seems improbable because of the strict geometric requirements (45) and the unlikelihood that two molecules would collide with the exact orientations needed. For fluorescein, the rates of energy migration have been calculated for different concentrations (24,58) and, at 0.2 M, are orders of magnitude greater than the collision rate. The same result probably holds for 6CF, which is spectrally very similar. Ksv for collisional quenching is proportional to the lifetime, which in turn is vanishingly small at 0.2 M 6CF due to energy transfer to dimers.
KNUTSON
Concentration quenching has been noted to involve more than one quenching mechanism; not only do these mechanisms vary depending on which dye is being studied, but also they may change depending on the concentration range (24,57). When a dye like rhodamine 6G is self-quenched, the mechanism is almost entirely dimer formation (64). Similarly, dyes sensitive to membrane potentials have been found to enter cells and form nonfluorescent aggregates (65). Conversely, highly charged dyes may have little tendency to dimerize, and any concentration quenching would be due mostly to collisions. Such may be the case for a fluorescein derivative containing one more charge than 6CF (66). Fluorescein, which has one less charge than 6CF, leaks too rapidly from liposomes (2). The usefulness of 6CF derives from the fact that the molecule has enough polarity that it does not leak rapidly from liposomes, and yet retains the ability to dimerize for effective self-quenching. REFERENCES I. Weinstein, J. N.. Yoshikami, S., Henkart, P., Blumenthal, R.. and Hagins, W. A. (1977) Science 195,489-492. 2. Weinstein, J. N.. Ralston. E., Leserman. L. D., Klausner. R. D., Dragsten, P.. Henkart, P.. and Blumenthal, R. (1984) in Liposome Technology (Gregoriadis, G., Ed.) Vol. 3, pp. 183-204, CRC Books, Boca Raton. FL. 3. Walter. B. ( 1888) Ann. Php. (Leip:ig) 34, 3 16-326. 4. Walter, B. ( 1888) Ann. Phys. (Leipzig) 34, 502-5 17. 5. Walter, B. (1988) Ann. Phys. (Leipzig) 34, 518-5333. 6. Pringsheim. P. ( 1949) Fluorescence and Phosphorescence. p. 349 ff, Interscience, New York. 7. West, W., and Pearce, S. ( 1965) J. Phys. Chern. 69, 1894-1903. 8. Havemann, R., Nutsch, E., and Pietsch, H. ( 1962) Z. Phys. Chem. (Leipzig) 219, 171-179. 9. Rabinowitch, E., and Epstein, L. F. (194 1) J. Amer. Chem. Sot. 63,69-78. 10. Zanker, V. (1952) Z. Phys. Chem. 199,255-258. 11. Zanker. V. (1952) Z. Phys. Chem. 200,250-292. 12. Lamm, M. E., and Neville, D. M., Jr. (1965) J. Phys. Chem. 69,3872-3877. 13. Levshin, W. L. (1927) Z. Phys. 43,230-253. 14. Gaviola, E. (1927) Z. Phys. 42, 862-869. 15. Szymanowski, W. (1935) Z. Phys. 95,460-465.
CARBOXYFLUORESCEIN 16. Perrin, F. (1924) Compt. Rend. 178, 1978-1980. 17. Gaviola, E., and Pringshim, P. (1924) Z. Pl~.rs. 24, 24-36. 18. Levshin, V. L. (1924) Z. Phys. 26, 274-284. 19. Weigert, F., and Klppler, G. (1924) Z. f’/z>x. 25, 99-117. 20. Vavilov, S. I. (1925) Z. Php. 31, 750-764. 2 1. Vavilov, S. I ( 1942) C. R. Acud. Sci. URSS 35, 100-106. 22. Forster. T. (195 1) Fluoreszenz Organischer Verbindung, Vandenhoeck u Ruprecht, Gottingen. 23. Forster. T., and Konig. E. (1957) Z. Elektrochrm. 61,344-348. 24. Rohatgi, K. K., and Singhal. G. S. ( 1972) J. Phys. Chem. 69, 1894- 1903. 25. Arbeloa, I. L. (1981) J. Chern. Sot. Faraday Trans. 2,77, 1735-1742. 26. Levshin, L. V., and Bocharov. V. G. (1960) Op/. Spekfrosk. 10, 330-333. 27. Levshin, L. V., and Baranova, E. G. (1958) Izv. Akad. Nauk SSR Ser. Fiz. 22, 1038-1042. 28. Levshin, L. V.. and Baranova, E. G. (1959) Opt. Spektrosk. 6. 55-64. 29. Rohatgi, K. K.. and Singhal, G. S. (1968) Photochern. Photobiul. 7, 36 l-367. 30. Bud& A., and Ketskemety. I. (1956) J. Chem. Phys. 25,595-596. 31. Arbeloa, 1. L. (1980) J. Photochem. 14, 97-105. 32. Melhuish, H. (1961) J. Chem. Phys. 65, 229-235. 33. Bud& A., Dombi. J., and Sz6116sy. L. (1956) Actu Phvs. Chon. 2, 18-27. 34. Rollefson, Gt. K., and Dodgen, H. W. ( 1944) J. Chem. Phys. 12, 107-l 1 I. 35. Plant, A. L. (1986) Photochem. Phofobiol. 44, 453-459. 36. Chen, R. F., .and Knutson, J. R. (1987) Biophyx J. 51, 539a. 37. Chen, R. F. (1967) Anal. Biochem. 20, 339-357. 38. Small, E. W.. Libertini. L. J., and Isenberg, 1. (I 984) Rev. Sci. Insrrum. 55, 879-885. 39. Ludescher. R.. D.. Volwerk. J. J., de Haas, G. H.. and Hudson, B. S. (1985) Biochemistry 24, 7240-7249. 40. Knutson. J. R., Walbridge, D. G., and Brand, L. (1982) Biochemisrry 21,4671-4679. 41. Badea, M. G., and Brand, L. (1979) in Methods in Enzymology (Hirs. C. H. W., and Timasheff, S. N., Edr,.), Vol. 61, pp. 378-425. Academic Press, New York.
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MECHANISM
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42. Grinvald, A., and Steinberg, I. Z. (1974) Anal. Biothem. 59, 583-598. 43. Ross, J. B. A., Rousslang, K. W.. and Brand, L. ( I98 I) Biochemistry 20,436 l-4369. 44. Rohatgi, K. K., and Mukhopadhyay, A. K. (1972) J. Phys. Chcm. 76. 3970-3975. 45. Arbeloa, I. L. (1977) J. Chem. Sot.. Furuduy Trans. 2,77,1725-1733. 46. Lavorel. J. (1957) J. Phys. Chem. 61, 1600-1605. 47. Babcock, D. F.. and Kramp. D. C. ( 1983) J. Biol. Chem. 258, 6389. 48. Stern, 0.. and Volmer, M. ( 19 19) Phys. Z. 20, 183-188. 49. Parker, C. A. (1968) Photoluminescence of Solutions, p. 75. Elsevier, New York. 50. Sevchenko. A. N. (1944) J. Ph.vs. (USSR) 8, 163-171. 5 1. Weber, G., and Teale, F. W. J. (1957) Trans. Furada?: Sot. 53, 646. 52. Strickler, S. J., and Berg. R. A. (1962) J. Chem. Phys. 37, 8 14-822. 53. Lamola, A. A. (1968) Photochem. Photobiol. 8, 601-616. 54. Parker, C. A., and Rees. W. T. (1960) Analyst 85, 587-600. 55. Haugland, R. (1985) Handbook of Fluorescent Probes and Research Chemicals, obtained from Molecular Probes, Junction City, OR. 56. Chen, R. F.. Edelhoch. H.. and Steiner, R. F. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., Ed.). Part A, pp. 17 I-244. Academic Press, New York. 57. Levshin. V. L., and Baranova. E. G. (1958) J. Chim. Phvs. 55. 869-877. 58. Lavorel. J. (1957) J. Phys. Chern., 864-869. 59. Feitelson, J. (1966) J. Chem. Phys. 44, 1497-1500. 60. Feitelson, J. (1966) J. Chern. Phvs. 44, 500-I 504. 6 I. Elkana. Y., Feitelson, J., and Katchalski, E. (1968) J. (‘hem. Phys. 48,2399-2404. 62. Lelkes. P. I., and Tandeter. H. B. (1982) Biochim. Biophys. Actu 716, 410-419. 63. Tanford, C. (1978) Science 200, 1012-1018. 64. Baranova, E. G. (1965) Opt. Spektrosk. 18, 230-234. 65. Sims, P. J., Waggoner, A. S., Wang, C.-H., and Hoffman. J. F. (1974) Biochemistry 13, 3315-3330. 66. Rink, T. J., Tsien, R. Y ., and Pozzan, T. (1982) J. Cell. Biol. 95, 189-196.
