Pfiorochemisrry and Phorobiology 1975, Vol. 22, pp. 213 216. Pergamon Press. Printcd in Great Britain
`
`RESEARCH NOTE
`PYRENE FLUORESCENCE LIFETIME AS A PROBE
`FOR OXYGEN PENETRATION OF MICELLES
`
`MARGARET W. GEIGER and NICHOLAS J. TURRO
`Department of Chemistry, Columbia University, New York, New York 10027, USA.
`
`(Received 14 April 1975; accepted 4 June 1975)
`
`solubilized in HDTBr and in sodium dodecyl sulfate
`(SDS) which are slower than the quenching rate con-
`stant in water. The effect of oxygen upon the fluores-
`cence lifetime of a probe solubilized in micelles pre-
`sented in this paper provides further evidence that
`micelles are penetrated by oxygen. We have studied
`the pyrene: HDTBr, HDTCl (hexadecyltrimethyl
`ammonium chloride) and SDS systems in oxygen, air
`and nitrogen saturated, as well as degassed solutions.
`
`INTRODUCTION
`When bound to proteins and membranes, fluorescence
`probes can provide valuable information about the
`environment of the binding site (Edelman & McClure,
`1968; Brand & Gohlke, 1972). Recently much interest
`has been shown in aromatic hydrocarbons as fluores-
`cence probes. Because they are non-polar molecules,
`they should not interact as strongly with charged
`groups as the amino-napthalene sulfonate probes do.
`Aromatic hydrocarbons exhibit environment depen-
`dent fluorescence lifetime and polarization (e.g.
`Hautala et a!., 1973; Shinitsky et al., 1971; Gratzel &
`Thomas, 1973). These properties can be used to
`characterize the probe environment in biological sys-
`tems. Additionally, the well-known quenching behav-
`ior of aromatic hydrocarbons has been exploited to
`obtain information about the permeability of mem-
`brane-like systems (Gratzel & Thomas, 1973 ; Infelta
`et al., 1974; Wallace & Thomas, 1973; Pownall &
`Smith, 1974; Chen et al., 1974) and the accessibility
`of protein environments (Vaughan & Weber, 1970;
`Lakowicz & Weber, 1973).
`Synthetic micelles have been particularly useful in
`characterizing the fluorescence and quenching behav-
`ior of aromatic hydrocarbons in various environments
`(e.g. Hautala et al., 1973; Infelta et al., 1974; Pownall
`& Smith, 1974; Patterson & Vieil, 1974; Chen et al.,
`1974 Soutar et al., 1974; Cheng et al., 1974). Although
`oxygen quenching of aromatic hydrocarbon fluores-
`cence has been studied in proteins (Vaughan &
`sions were analyzed on a system that included a i / 4 m
`Weber, 1970; Lakowicz & Weber, 1973), the question
`Jarrell-Ash monochrometer, Amperex 56 AVP phototube,
`Hewlett-Packard 5614L preset counter, and a Northern
`of oxygen permeability in micelles has not been
`Econ I1 multichannel pulse height analyzer (calibrated at
`resolved. Previous work in this group suggests that
`1.55 or 3.12 ns per channel). Fluorescence spectra were
`oxygen is soluble in the micelle interior and that
`recorded on a Perkin-Elmer MPF-2A or MPF-3L spectro-
`oxygen solubility is one of the factors which deter-
`photometer.
`mines the lifetime of solubilized probes (Hautula et
`Concentrated stock solutions of detergent were added
`to gas saturated solutions of pyrene (- lo-' M ) in water to
`al., 1973). However, Dorrance & Hunter (1972) failed
`obtain the nitrogen and oxygen saturated solutions. Long-
`to obtain any increase in fluorescence quantum yield
`necked 1 cm fluorescence cells with Teflon stopcocks were
`of pyrene upon deoxegenation of aqueous hexadecyl-
`used. Solutions were degassed using three freeze, pump,
`trimethylammonium bromide (HDTBr) solutions
`thaw cycles (mechanical pump, oil diffusion pump)
`containing this hydrocarbon. They suggest that the
`and transferred under vacuum to the cell containing a pre-
`weighed amount of detergent. The cell was sealed with a
`lack of fluorescence quenching results because a bar-
`teflon vacuum stopcock (Kontes). After 12 h, the lifetime
`rier exists to oxygen penetration of micelles contain-
`of a degassed pyrene solution dropped from 328 to 298 ns,
`ing pyrene molecules. Wallace and Thomas (1973)
`showing that the leakage of air back into the cell is slow.
`report rate constants for oxygen quenching of pyrene
`The detergent concentration in the degassed samples is
`273
`
`MATERIALS AND METHODS
`HDTBr (ethonol-ether, mp 25 1- 252") HDTCI (acetone),
`and SDS (ethanol) were purified by recrystallization (> 5 x ,
`norite 2 x ). Critical micelle concentrations (eosin dye,
`Corrin & Harkins, 1947) of 0.0008M
`(HDTBr) and
`0.003 M
`(HDTCI) were obtained
`(literature values
`0.0009 M and 0.0015 M , Mukerjee & Mysels, 1971). Water
`was redistilled from potassium permanganate. The purified
`detergents in solution showed no absorption or emission
`in the spectral regions of interest.
`Pyrene (Aldrich, 99%) was recrystallized (ethanol, norite,
`3 x ) until colorless crystals were produced. Material
`obtained by this procedure displayed absorption and emis-
`sion spectra (cyclohexane) which agree with published
`spectra (Berlman, 1965). Pyrene fluorescence lifetime in
`water was found to depend on its purity. The fluorescence
`lifetime, T ~ , in aqueous solution was -. 125 ns for pyrene
`purified in this manner.
`The fluorescence lifetimes were determined using single
`lamp (Tao, 1969) gave a pulse of half-width - 2 ns. Emis-
`photon counting techniques (Ware, 1971). An air spark
`
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`274
`
`MARGARET W. GEIGER and NICHOLAS J. TURRO
`
`only approximate since the amount of water transferred
`to the cell is not known exactly.
`Conductivity measurements were done on a model 16B2
`Industrial Instruments conductivity bridge. Critical micelle
`concentrations were obtained in the usual manner from
`plots of reciprocal resistance vs detergent concentration
`(Mukcrjee & Mysels, 1971).
`
`RESULTS AND DISCUSSION
`The emission spectra of pyrene ( - lo-‘ M ) in water
`and in detergent solutions are shown in Fig. 1. Even
`at low concentrations of pyrene, it might be expected
`that aggregation occurs in water. However, the emis-
`sion spectra obtained showed pyrene monomer fluor-
`escence with no evidence of excimer emission
`
`(A - 470nm) present. On the average, there is less
`
`than one pyrene molecule pcr micelle in detergent
`solutions. It is, therefore, not surprising that the
`excimer emission is absent. Pyrene is thought to be
`solubilized in the micellar phase in detergent solu-
`tions because of its hydrophobicity (Pownall & Smith,
`1973; Dorrance & Hunter, 1972). Results from pulsed
`radiolysis experiments (Wallace & Thomas, 1973) and
`NMR studies (Gratzel et al., 1974) support the solu-
`bilization of pyrene in the micellar phase.
`In aerated solutions, the lifetime of pyrene in pure
`water is shorter than in aqueous solutions of HDTCl
`or SDS (Table 1). We interpret this result to suggest
`either (a) a lesser solubility of oxygen in the micelle
`relative to water, (b) a rate constant for oxygen
`quenching of solubilized pyrene slower than diffusion
`controlled in bulk solution, or (c) a quenching of pyr-
`ene fluorescence by water. The first possibility would
`be rather surprising. Since oxygen is approximately
`20 times more soluble in air-saturated hydrocarbon
`
`0.0055 M HDTBr
`
`0.052 MHDTCI
`
`0.0097 M SDS
`
`Figure 1. The fluorescence spectra of pyrene in water and
`detergent solutions; Aexcir = 320 nm.
`
`Table 1. Pyrene fluorescence lifetime (in ns) in environ-
`ments of various ,oxygen concentration
`
`02
`saturated
`
`Air
`
`N2
`Saturated
`
`Degassed
`
`Naphthalene
`A , -
`
`water
`
`6gf4
`
`1 d 3
`
`226tlO
`
`HDTBP(.0055M)
`
`62%
`
`lz2t8
`
`165t5
`
`HDTCl( 0065M)
`
`67t8
`
`15712
`
`281z22
`
`SDS(.OO97Mi
`
`5Lif3
`
`158z4
`
`314110
`
`Cycloherane
`
`20
`
`202
`
`201
`206
`175
`158
`
`328
`268
`314
`304
`
`370
`
`39
`
`11
`
`23
`
`60
`
`17
`degassed 108
`
`*From Hautala et al. (1973).
`
`solvents than in water, it is expected that oxygen solu-
`bility would be greater in the ‘hydrocarbon-like’
`micelle interior than in water. There is some evidence
`to support the second possibility. The only reported
`rates for oxygen quenching of solubilized pyrene
`(Wallace & Thomas, 1973) are slower than the rates
`observed in water. Fluorescence quenching of several
`aromatic hydrocarbons by water has been reported
`(Hautala et a]., 1973; Stevens & Strickler, 1973;
`Vaughan & Weber, 1970) and pyrene may also
`be quenched in aqueous solutions.
`Upon oxygen saturation, the lifetime of pyrene de-
`creases in water and in detergent solutions.. These life-
`times are essentially the same (Table 1) and the de-
`crease of pyrene lifetime in micelles is not significantly
`greater than the decrease in water. These results sug-
`gest that oxygen is at least as soluble in micelles as
`in water. Since we expect that water quenching of
`
`pyrene is slow (k,(H,O) - lo5 mol-’ s-’ for naph-
`
`thalene) in comparison to the rate of oxygen quench-
`ing in saturated solutions, only the latter process is
`considered.
`Nitrogen saturation increases the lifetime of pyrene
`in water, HDTC1, and SDS solutions due to the re-
`moval of oxygen. Vacuum degassing the solutions does
`not siginifcantly alter the lifetime of pyrene from that
`measured in nitrogen saturated solutions.
`Pyrene’s lifetime is shorter in air saturated HDTBr
`than in HDTCl or SDS and more importantly, its
`lifetime in degassed or nitrogen saturated solutions
`is shortest in HDTBr. Although the lifetime of pyrene
`increases with nitrogen saturation or degassing, the
`increase is small relative to the changes observed in
`HDTCI, SDS and water. This result is consistent with
`the Dorrance and Hunter report (1972) that pyrene
`fluorescence yield in HDTBr was not changed upon
`deoxygenation. Anomalous behaviour of aromatic hy-
`drocarbon fluorescence in HDTBr has been reported
`and explained previously (Hautala et al., 1973;
`Gratzel and Thomas, 1973; Patterson and Vieil, 1973.
`Bromide ion is known to quench aromatic hydrocar-
`bon fluorescence (Watkins, 1974). The high local con-
`centration of Br- around the micelle enhances this
`quenching effect, causing shorter pyrene lifetimes to
`
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`Research Note
`
`275
`
`X
`
`,
`
`,
`
`,
`
`s
`
`.
`
`s
`
`0 01
`
`
`
`0 005
`Detergent concentration, M
`Figure 2. The effect of micelle formation on pyrene fluores-
`indicate
`the approximate
`cence lifetime. The arrows
`CMC's. " x " refers to HDTCl and "0" to SDS.
`
`s C '
`
`be observed in air and N, saturated HDTBr solu-
`tions. After the removal of oxygen, Br- quenching
`dominates the deactivation of excited state pyrene in
`HDTBr and limits its lifetime.
`Fluorescence lifetime studies (Hautala et ul., 1973).
`absorption spectroscopy (Riegelman et al., 1958), and
`pulsed radiolysis experiments (J. Fendler, unpub-
`lished) indicate that naphthalene is solubilized on the
`micellc-water interface. Pyrene, due to its lower solu-
`bility in water, is expected to be solubilized further
`from the surface, which is supported by pulse radiolysis
`experiments (Wallace & Thomas, 1973) and NMR
`studies (Gratzel er al., 1974). A comparison of pyrene
`and naphthalene fluorescence lifetimes in micelles also
`suggests a deeper penetration of pyrene into the
`mieelle. since the ratio of pyrene lifetirnc in micclles
`relative to water or degassed cyclohexane is larger
`than for naphthalene
`lifetime. In addition,
`the
`quenching effects in HDTBr are not as great for pyr-
`ene compared to naphthalene. The rate constant, k,,
`for bromide quenching of naphthalene fluorescence
`in water is approximately 2 x lo* mol- ' s- ' (Hau-
`tala et al., 1973). We have obtained k, ,. l x
`for pyrene from quenching of fluores-
`lo7 mol-'
`cence intensity (Stern-Volmer analysis) and from life-
`time quenching studies (I). Values of the effective Br-
`concentration can be obtained from (1) for
`= t - 1 + k,[Br-]
`(1)
`naphthalene and pyrene, solubilized in HDTBr. We
`can use the lifetimes in HDTCl (air) for to, since
`anion quenching is negligible for C1-
`(Watkins,
`1974). The t values are the lifetimes in HDTBr
`s--' and
`(air). Thus, k,[Br-]
`pyrene = 1.8 x
`naphthalene = 4.8 x IO-'s-'.
`Substitut-
`k,[Br-]
`ing in the measured values of kq, we obtain [Br-]
`pyrene = 0.18 M and [Br-] naphthalene = 0.24M.
`The smaller effective Br concentration at
`the
`same detergent concentration for pyrene suggests a
`larger probe to counterion distance. This is consis-
`tent with pyrene being solubilized further from
`micelle surface than naphthalene.
`The oxygen effects reported here are known to be
`micellar since the lifetime of pyrene increases upon
`micelle formation (SDS and HDTCI). A plot of fluor-
`escence lifetime vs detergent concentration (Fig. 2)
`exhibits a break characteristic of micelle formation.
`This method gives critical micelle concentrations
`(CMC) for HDTCl 2 0.004 M and SDS = 0.0035 M
`which
`compare
`favorably with
`values
`for
`HDTCl 2 0.005 M and SDS 2 0.0035 M obtained by
`
`values:
`(literature
`conductivity measurements
`HDTCl = 0.001 M , SDS = 0.009 M ; Mukerjee &
`Mysels, 197 I).
`Conductivity measurements of SDS and HDTCl
`gave identical values of the CMC in the presence and
`absence of pyrene implying that pyrene does not sig-
`nificantly perturb the micelle. Saturation by oxygen
`or nitrogen also does not change the CMC. Upon
`standing for a week, the lifetime of pyrene in oxygen
`and nitrogen saturated solutions returned to the
`values measured in air indicating that no reaction
`had taken place to cause the observed changes in life-
`time. The shape of the fluorescence spectra in the
`presence of oxygen and nitrogen was unperturbed. In
`all cases, the lifetime was observed as a single
`exponential decay confirming the absence of excimer
`formation. Also, pyrene lifetime in SDS does not vary
`with the age of the solution, suggesting that the aging
`effect reported for SDS (Gratzel and Thomas, 1973)
`does not affect the lifetime measurement.
`
`CONCLUSIONS
`The fluorescence lifetime of solubilized pyrene has
`been used to show that micelles can be oxygenated
`and deoxygenated. The results suggest that oxygen
`is at least as soluble in micelles as in water and that
`oxygen moves across the micelle-water interface. The
`measured fluorescence lifetime in micelles (especially
`in HDTBr) is consistent with pyrene being solubilized
`further from the micelle surface than naphthalene.
`
`authors wish to thank the Air
`Acknowledgements-The
`Force Ofice of Scientific Research (Grant AFOSR-
`74-2589B) and the National Science Foundation (Grant
`NSF-GP-2660x and NSF-GP-40330~) for their generous
`support of this research. The authors would like to thank
`Dr. Walter H. Waddell for helpful discussions.
`
`REFERENCES
`(1965) Handhook of' Fluorescence Spectra of Aromatic Molrculrs, p. 173, Academic Press,
`
`Berlman, I.
`N.Y.
`Brand. L.. and J. Gohlke (1972) Ann. Rer. Rior-hem. 41. 843-868.
`Chen, M., M. Gratzel and I . K . Thoinas
`(1974)
`Chem. Phys. Ler1er.s 24. 65-68.
`Cheng, S., J. K. Thomas and C. F. Culpa
`(1974) Biochemistry 13. 1135-1 139.
`Corrin, M., and W. Harkins (1947)
`J . Am. Chem. SOC. 69, 679-683.
`Dorrance, R., and T. Hunter
`(1972) J.C.S. Faraduy 168. 1312-1321.
`
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`276
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`MARGARET W. G r i r c ~ ~ and NICHOLAS J. TURRO
`
`Edelman, G. M., and W. 0. McClure (1968) Accouirts Ciiern. Research 1. 65-70.
`Gratzel, M., and J. K. Thomas (1973) J . Am. Chem. Soc. 95. 688556889,
`Gratzel, M., K. Kalyanasundaram and J. K. Thomas (1974) J . Am. Chem. Soc. 96. 7869-7874.
`Hautala, R. R., N. E. Schore and N. J. Turro (1973) J . Am. Chrm. SOC. 95. 5508-5514.
`Infelta, P. P., M. Gratzel and J. K. Thomas
`(1974) J . Phys. Chem. 78. 19c195.
`Lackowicz, J. R., and G. Weber
`(1973) Biochemistry 12. 4161, 41714179.
`Mukerjee, P., and K. Mysels
`(1971) Critical Micelle Concentrations of Aqueous Surjiactants, Nat.
`Stand. Ref. Data. Ser.
`Patterson, L. K., and E. Vied
`(1973) J. Phys. Chem. 77. 1191-1 192.
`Pownall, H., and L. Smith
`(1974) Biochemistry 13. 25942597.
`Ricgclman, S., N. A. Allawak, M. K. Hrenoff and L. A. Strait (1958) J. Colloid Sci. 13. 208-217.
`Shinitsky, M., A. C. Dianoux, C. Gitler and G. Weber
`(1971) Biochemistry 10. 21062113.
`Soutar, A. K., H. J. Pownall, A. S. Hu and L. C. Smith
`(1974) Biochemistry 13. 2828-2836.
`Stevens, C. G., and S. J. Strickler
`(1973) J . Am. Chem. SOC. 95, 3922-3928.
`Tao, T. (1969) PhD Thesis, Columbia University.
`Vaughan, W. M., and G. Weber
`(1970) Biochemistry 9. 464473.
`Wallace, S. C., and J. K. Thomas
`(1973) Radiation Res. 54. 49-62.
`Ware, W. A.
`(1971) In Creation and Detection of the Excited State (Edited by A. A. Lamola),
`Vol. IA, pp. 213-302. Marcel Decker, New York.
`Watkins, A. R. (1974) J . Phys. Chem. 78. 1885, 2555-2558.
`
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